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Cell Stem Cell Volume 10

Supplemental Information

Erosion of Dosage Compensation

Impacts Human iPSC Disease Modeling Shila Mekhoubad Christoph Bock A Sophie de Boer Evangelos Kiskinis Alexander Meissner and Kevin Eggan

Supplemental Information Inventory

Supplemental Figure 1 Figure is related to Figure 1 and expands upon the examination of Lesch-Nyhan and wildtype fibroblasts for HPRT activity Supplemental Figure 2 Figure is related to Figure 1 and shows the characterization of the newly derived hiPSC lines in this work Supplemental Figure 3 Figure is related to Figure 2 and shows further characterization of the neuronal cultures and LNS neuronal phenotype reported in this work Supplemental Figure 4 Figure is related to Figure 2 and depicts the validation of the HPRT neuronal rescue lines generated for LNS HPRT deficient hiPSC lines Supplemental Figure 5 Figure is related to Figure 3 and provides further characterization of lines that have undergone irreversible erosion of dosage compensation Supplemental Figure 6 Figure is related to Figure 4 and shows how erosion of dosage compensation and HPRT expression from the Xi restores normal neuronal phenotype Supplemental Figure 7 Figure is related to Figure 4 and provides additional information on hiPSC lines with distinct patterns of XCI states and their differentiation into neurons

Supplemental Table 1 (available online) Table is related to Figure 5 and provides further information on the specific genes subjected to erosion of dosage compensation in cohort of hESC and hiPSC lines examined (provided in an Excel spreadsheet)

Supplemental Experimental Procedures Supplemental References

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Figure S1 Examination of Lesch-Nyhan Syndrome (LNS) and wildtype fibroblasts for HPRT activity (Related to figure 1) (A-D) Bright field images of wild type male and female fibroblasts mutant HPRT LNS male fibroblasts and female LNS carrier fibroblasts after 6 days of treatment with the drugs HAT or 6TG (A-B) Wild type male and female fibroblasts with an active HPRT (XHPRT+Y XHPRT+XHPRT+) are resistant to HAT medium which blocks the de novo purine pathway and are sensitive to the toxic purine analogue 6TG (C) Male LNS cells (XHPRT-Y) without HPRT activity are sensitive to HAT media while resistant to 6TG media (D) Female LNS carrier cells (XHPRT+XHPRT-) with a mixture of cells with the functional HPRT allele on the active or inactive X chromosome can expand under both drugs Cells that have selected the functional HPRT for inactivation (XaHPRT-XiHPRT+) are resistant to 6TG and sensitive to HAT Conversely cells that have inactivated the mutant HPRT allele (XaHPRT+XiHPRT-) and express the functional HPRT allele are sensitive to 6TG and resistant to HAT As a result treatment of HPRT +- fibroblasts with 6TG results in some cell death followed by expansion of fibroblast population homogenously composed of cells that have inactivated the functional HPRT (XaHPRT-XiHPRT+) Treatment with HAT medium selects for population of fibroblasts that homogenously express the functional HPRT allele (XaHPRT+XiHPRT-) (E) Quantification of cell survival for wildtype and LNS fibroblast lines after HAT or 6TG drug selection by measure of crystal violet stained cells absorbance at 590microm

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Figure S2 Characterization of newly derived hiPSC lines (Related to Figure 1) (A) Representative images of staining for the expression of the pluripotency markers NANOG OCT4 TRA1-60 TRA1-80 SSEA3 and SSEA4 in the newly derived hiPSC line CR-6TG-2 (B) Representative images of staining for the 3 germ layer markers TUJ1 SMA and AFP following EB differentiation of hiPSC line CR-6TG-2 (C) Chart displaying all the newly derived hiPSC lines reported in this work and the pluripotency and differentiation assays they were subjected to

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Figure S3 Characterization of neuronal cultures (Related to Figure 2) (A) TUJ1-positive neurons for wildtype hiPSC line 20b HPRT positive LNS carrier female line CR-HAT-1 and HPRT negative male LNS-2 also stain for the mature neuronal markers

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MAP2A and SYN1 and for the dopaminergic specific marker Thyrosine Hydroxylase (TH) which are implicated to be severely defected in Lesch-Nyhan affected individuals (B) Representative neuronal cultures stained for TUJ1 from the isogenic lines HPRT positive CR-HAT-1 HPRT negative CR-6TG-3 (low passage XIST+) as well as the HPRT negative male LNS-2 The neuronal phenotype seen in HPRT negative cultures with less number of surviving neurons is not rescued when neurons are plated on mouse glia cells that provide more neural support Scale bars=100microm (C) Quantification of number of TUJ1-positive neurons three days post EB dissociation and plating on glass slides with and without mouse brain derived glia show supportive glial cells do not rescue the HPRT negative neuronal phenotype

Figure S4 Validation of HPRT rescue for LNS lines (Related to Figure 2) (A) Alkaline Phosphatase staining of hiPSC colonies from LNS-2 iPSC line that was transduced with an empty lenti virus or an HPRT carrying lenti-virus and then treated with HAT containing media HPRT negative cells transduced with empty vector remain HAT sensitive while cells transduced with the HPRT vector were now HAT resistance and grew in HAT media This change in drug resistance confirms the expression of functional HPRT from the viral vector in this transduced line (B) Full field reprehensive images of neuronal cultures derived in parallel from diseased hiPSC LNS-2 wildtype iPSC 20b LNS-2-vecCNTL and two independently derived HPRT rescue lines transduced with virus carrying the intact HPRT LNS-2-HPRTrescA and B While LNS-

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2vecCNTL neurons are similar in number and morphology to their parental LNS-2 neurons neurons derived from the two rescue lines are similar in number and morphology to wildtype lines Scale bars=100microm

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Figure S5 Characterization of lines that have lost XCI marks (Related to Figure 3) (A) Diagram depicting decrease in XCI marks with time in culture for HPRT positive carrier hiPSC lines with the mutated HPRT on the inactive X chromosome Crystal Violet staining of hiPSC line CR-HAT-2 after selection for the drugs HAT or 6TG over 16 passages in culture

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display no changes in drug resistance Quantification of H3K27me3 foci in NANOG-positive cells for hiPSC lines CR-HAT-1 CR-HAT-2 show a decline in proportion of cells with proper XCI over extended culture (B) Quantitative RT-PCR (qPCR) analysis with primers specific for viral (v=red bars) and endogenous (e=blue bars) expression of the reprogramming genes OCT4 SOX2 KLF4 and cMYC in control fibroblasts wildtype hESC lines (HUES 51 and HUES 53) wildtype established iPSC lines (iPSC 20b iPSC 18c) newly derived male LNS iPSC (LNS-2) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines reported in this work (CR-HAT-1 CR-6TG-2 and CR-6TG-3) All hiPSC lines at both low and high passage are free from viral expression of OCT4 SOX2 and cMYC and express the endogenous genes indicative of full reprogramming Some low to moderate levels of viral KLF4 was detected in all of our iPSC lines However this exogenous expression was observed in similar levels at both low and high passage lines suggesting this expression is passage independent and not related to loss of XCI marks in higher passage female lines (C) Quantification of percentage of surviving Alkaline Phosphatase positive pluripotent colonies after treatment of control hESC lines (HUES 51 and HUES 53) established iPSC lines (18c and 20b) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines (CR-HAT-1 CR-6TG-2 and CR-6TG-3) with FGF or TGF-β inhibitors All pluripotent lines regardless of passage number appeared to be similarly sensitive to these inhibitors This indicates that the dependency on these pathways for pluripotency and self-renewal is maintained in these iPSC lines regardless of time in culture and XCI status (D) Representative images and quantification of XIST clouds in teratomas derived from low passage (XIST +) iPSC lines (CR-HAT-1 and CR-6TG-3) and high passage (XIST-) iPSC (CR-6TG-2 p29) Lines with proper XCI at low passage generated teratomas with high proportion of cells with XIST clouds However iPSC line CR-6TG-2 at passage 29 which has lost XCI marks generated a teratoma without any XIST clouds Scale bars=10microm (E) Representative images and quantification of XIST clouds and H3K27me3 foci in NANOG positive cells for iPSC 29A and 29e at around the same passages used for whole genome exome sequencing in (Gore et al 2011) At these passages both these lines have lost marks of XCI in nearly all their cells Scale bars=10microm (F) Examination of genomic mutations in iPSC 29A and 29e (Gore et al 2011) indicate no common somatic mutations between the two lines and no X-linked mutations at all

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Figure S6 Erosion of the Xi and functional HPRT expression restores normal neuronal phenotype (Related to Figure 4) (A) Neuronal cultures stained for TUJ1 and H3K27me3 for the HPRT positive dosage compensated CR-HAT-1 line Neurons from this line show normal neuronal morphology with clear foci of H3K27me3 indicative of the inactive X in the majority of the neurons (B) Neuronal cultures from the line CR-6TG-2 at passage 13 that has lost XCI marks in at least a portion of its cells (as seen in Fig 3C) Neurons at this passage show a mix phenotype with a portion of neurons with normal neuronal morphology and a portion of neurons with abnormal neuronal morphology Staining of H3K27me3 shows clear foci of H3K27me3 in neurons with abnormal morphology that have maintained XCI and lack expression of HPRT Conversely neurons with normal morphology lack clear foci of H3K27me3 indicative of erosion of dosage compensation and the expression of the functional HPRT allele from the Xe Arrows point to foci of H3K27me3 indicative of an Xi Scale bars=50microm

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Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

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Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

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Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

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Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

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Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

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Figure S1 Examination of Lesch-Nyhan Syndrome (LNS) and wildtype fibroblasts for HPRT activity (Related to figure 1) (A-D) Bright field images of wild type male and female fibroblasts mutant HPRT LNS male fibroblasts and female LNS carrier fibroblasts after 6 days of treatment with the drugs HAT or 6TG (A-B) Wild type male and female fibroblasts with an active HPRT (XHPRT+Y XHPRT+XHPRT+) are resistant to HAT medium which blocks the de novo purine pathway and are sensitive to the toxic purine analogue 6TG (C) Male LNS cells (XHPRT-Y) without HPRT activity are sensitive to HAT media while resistant to 6TG media (D) Female LNS carrier cells (XHPRT+XHPRT-) with a mixture of cells with the functional HPRT allele on the active or inactive X chromosome can expand under both drugs Cells that have selected the functional HPRT for inactivation (XaHPRT-XiHPRT+) are resistant to 6TG and sensitive to HAT Conversely cells that have inactivated the mutant HPRT allele (XaHPRT+XiHPRT-) and express the functional HPRT allele are sensitive to 6TG and resistant to HAT As a result treatment of HPRT +- fibroblasts with 6TG results in some cell death followed by expansion of fibroblast population homogenously composed of cells that have inactivated the functional HPRT (XaHPRT-XiHPRT+) Treatment with HAT medium selects for population of fibroblasts that homogenously express the functional HPRT allele (XaHPRT+XiHPRT-) (E) Quantification of cell survival for wildtype and LNS fibroblast lines after HAT or 6TG drug selection by measure of crystal violet stained cells absorbance at 590microm

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Figure S2 Characterization of newly derived hiPSC lines (Related to Figure 1) (A) Representative images of staining for the expression of the pluripotency markers NANOG OCT4 TRA1-60 TRA1-80 SSEA3 and SSEA4 in the newly derived hiPSC line CR-6TG-2 (B) Representative images of staining for the 3 germ layer markers TUJ1 SMA and AFP following EB differentiation of hiPSC line CR-6TG-2 (C) Chart displaying all the newly derived hiPSC lines reported in this work and the pluripotency and differentiation assays they were subjected to

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Figure S3 Characterization of neuronal cultures (Related to Figure 2) (A) TUJ1-positive neurons for wildtype hiPSC line 20b HPRT positive LNS carrier female line CR-HAT-1 and HPRT negative male LNS-2 also stain for the mature neuronal markers

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MAP2A and SYN1 and for the dopaminergic specific marker Thyrosine Hydroxylase (TH) which are implicated to be severely defected in Lesch-Nyhan affected individuals (B) Representative neuronal cultures stained for TUJ1 from the isogenic lines HPRT positive CR-HAT-1 HPRT negative CR-6TG-3 (low passage XIST+) as well as the HPRT negative male LNS-2 The neuronal phenotype seen in HPRT negative cultures with less number of surviving neurons is not rescued when neurons are plated on mouse glia cells that provide more neural support Scale bars=100microm (C) Quantification of number of TUJ1-positive neurons three days post EB dissociation and plating on glass slides with and without mouse brain derived glia show supportive glial cells do not rescue the HPRT negative neuronal phenotype

Figure S4 Validation of HPRT rescue for LNS lines (Related to Figure 2) (A) Alkaline Phosphatase staining of hiPSC colonies from LNS-2 iPSC line that was transduced with an empty lenti virus or an HPRT carrying lenti-virus and then treated with HAT containing media HPRT negative cells transduced with empty vector remain HAT sensitive while cells transduced with the HPRT vector were now HAT resistance and grew in HAT media This change in drug resistance confirms the expression of functional HPRT from the viral vector in this transduced line (B) Full field reprehensive images of neuronal cultures derived in parallel from diseased hiPSC LNS-2 wildtype iPSC 20b LNS-2-vecCNTL and two independently derived HPRT rescue lines transduced with virus carrying the intact HPRT LNS-2-HPRTrescA and B While LNS-

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2vecCNTL neurons are similar in number and morphology to their parental LNS-2 neurons neurons derived from the two rescue lines are similar in number and morphology to wildtype lines Scale bars=100microm

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Figure S5 Characterization of lines that have lost XCI marks (Related to Figure 3) (A) Diagram depicting decrease in XCI marks with time in culture for HPRT positive carrier hiPSC lines with the mutated HPRT on the inactive X chromosome Crystal Violet staining of hiPSC line CR-HAT-2 after selection for the drugs HAT or 6TG over 16 passages in culture

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display no changes in drug resistance Quantification of H3K27me3 foci in NANOG-positive cells for hiPSC lines CR-HAT-1 CR-HAT-2 show a decline in proportion of cells with proper XCI over extended culture (B) Quantitative RT-PCR (qPCR) analysis with primers specific for viral (v=red bars) and endogenous (e=blue bars) expression of the reprogramming genes OCT4 SOX2 KLF4 and cMYC in control fibroblasts wildtype hESC lines (HUES 51 and HUES 53) wildtype established iPSC lines (iPSC 20b iPSC 18c) newly derived male LNS iPSC (LNS-2) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines reported in this work (CR-HAT-1 CR-6TG-2 and CR-6TG-3) All hiPSC lines at both low and high passage are free from viral expression of OCT4 SOX2 and cMYC and express the endogenous genes indicative of full reprogramming Some low to moderate levels of viral KLF4 was detected in all of our iPSC lines However this exogenous expression was observed in similar levels at both low and high passage lines suggesting this expression is passage independent and not related to loss of XCI marks in higher passage female lines (C) Quantification of percentage of surviving Alkaline Phosphatase positive pluripotent colonies after treatment of control hESC lines (HUES 51 and HUES 53) established iPSC lines (18c and 20b) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines (CR-HAT-1 CR-6TG-2 and CR-6TG-3) with FGF or TGF-β inhibitors All pluripotent lines regardless of passage number appeared to be similarly sensitive to these inhibitors This indicates that the dependency on these pathways for pluripotency and self-renewal is maintained in these iPSC lines regardless of time in culture and XCI status (D) Representative images and quantification of XIST clouds in teratomas derived from low passage (XIST +) iPSC lines (CR-HAT-1 and CR-6TG-3) and high passage (XIST-) iPSC (CR-6TG-2 p29) Lines with proper XCI at low passage generated teratomas with high proportion of cells with XIST clouds However iPSC line CR-6TG-2 at passage 29 which has lost XCI marks generated a teratoma without any XIST clouds Scale bars=10microm (E) Representative images and quantification of XIST clouds and H3K27me3 foci in NANOG positive cells for iPSC 29A and 29e at around the same passages used for whole genome exome sequencing in (Gore et al 2011) At these passages both these lines have lost marks of XCI in nearly all their cells Scale bars=10microm (F) Examination of genomic mutations in iPSC 29A and 29e (Gore et al 2011) indicate no common somatic mutations between the two lines and no X-linked mutations at all

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Figure S6 Erosion of the Xi and functional HPRT expression restores normal neuronal phenotype (Related to Figure 4) (A) Neuronal cultures stained for TUJ1 and H3K27me3 for the HPRT positive dosage compensated CR-HAT-1 line Neurons from this line show normal neuronal morphology with clear foci of H3K27me3 indicative of the inactive X in the majority of the neurons (B) Neuronal cultures from the line CR-6TG-2 at passage 13 that has lost XCI marks in at least a portion of its cells (as seen in Fig 3C) Neurons at this passage show a mix phenotype with a portion of neurons with normal neuronal morphology and a portion of neurons with abnormal neuronal morphology Staining of H3K27me3 shows clear foci of H3K27me3 in neurons with abnormal morphology that have maintained XCI and lack expression of HPRT Conversely neurons with normal morphology lack clear foci of H3K27me3 indicative of erosion of dosage compensation and the expression of the functional HPRT allele from the Xe Arrows point to foci of H3K27me3 indicative of an Xi Scale bars=50microm

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Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

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Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

3

Figure S2 Characterization of newly derived hiPSC lines (Related to Figure 1) (A) Representative images of staining for the expression of the pluripotency markers NANOG OCT4 TRA1-60 TRA1-80 SSEA3 and SSEA4 in the newly derived hiPSC line CR-6TG-2 (B) Representative images of staining for the 3 germ layer markers TUJ1 SMA and AFP following EB differentiation of hiPSC line CR-6TG-2 (C) Chart displaying all the newly derived hiPSC lines reported in this work and the pluripotency and differentiation assays they were subjected to

4

Figure S3 Characterization of neuronal cultures (Related to Figure 2) (A) TUJ1-positive neurons for wildtype hiPSC line 20b HPRT positive LNS carrier female line CR-HAT-1 and HPRT negative male LNS-2 also stain for the mature neuronal markers

5

MAP2A and SYN1 and for the dopaminergic specific marker Thyrosine Hydroxylase (TH) which are implicated to be severely defected in Lesch-Nyhan affected individuals (B) Representative neuronal cultures stained for TUJ1 from the isogenic lines HPRT positive CR-HAT-1 HPRT negative CR-6TG-3 (low passage XIST+) as well as the HPRT negative male LNS-2 The neuronal phenotype seen in HPRT negative cultures with less number of surviving neurons is not rescued when neurons are plated on mouse glia cells that provide more neural support Scale bars=100microm (C) Quantification of number of TUJ1-positive neurons three days post EB dissociation and plating on glass slides with and without mouse brain derived glia show supportive glial cells do not rescue the HPRT negative neuronal phenotype

Figure S4 Validation of HPRT rescue for LNS lines (Related to Figure 2) (A) Alkaline Phosphatase staining of hiPSC colonies from LNS-2 iPSC line that was transduced with an empty lenti virus or an HPRT carrying lenti-virus and then treated with HAT containing media HPRT negative cells transduced with empty vector remain HAT sensitive while cells transduced with the HPRT vector were now HAT resistance and grew in HAT media This change in drug resistance confirms the expression of functional HPRT from the viral vector in this transduced line (B) Full field reprehensive images of neuronal cultures derived in parallel from diseased hiPSC LNS-2 wildtype iPSC 20b LNS-2-vecCNTL and two independently derived HPRT rescue lines transduced with virus carrying the intact HPRT LNS-2-HPRTrescA and B While LNS-

6

2vecCNTL neurons are similar in number and morphology to their parental LNS-2 neurons neurons derived from the two rescue lines are similar in number and morphology to wildtype lines Scale bars=100microm

7

Figure S5 Characterization of lines that have lost XCI marks (Related to Figure 3) (A) Diagram depicting decrease in XCI marks with time in culture for HPRT positive carrier hiPSC lines with the mutated HPRT on the inactive X chromosome Crystal Violet staining of hiPSC line CR-HAT-2 after selection for the drugs HAT or 6TG over 16 passages in culture

8

display no changes in drug resistance Quantification of H3K27me3 foci in NANOG-positive cells for hiPSC lines CR-HAT-1 CR-HAT-2 show a decline in proportion of cells with proper XCI over extended culture (B) Quantitative RT-PCR (qPCR) analysis with primers specific for viral (v=red bars) and endogenous (e=blue bars) expression of the reprogramming genes OCT4 SOX2 KLF4 and cMYC in control fibroblasts wildtype hESC lines (HUES 51 and HUES 53) wildtype established iPSC lines (iPSC 20b iPSC 18c) newly derived male LNS iPSC (LNS-2) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines reported in this work (CR-HAT-1 CR-6TG-2 and CR-6TG-3) All hiPSC lines at both low and high passage are free from viral expression of OCT4 SOX2 and cMYC and express the endogenous genes indicative of full reprogramming Some low to moderate levels of viral KLF4 was detected in all of our iPSC lines However this exogenous expression was observed in similar levels at both low and high passage lines suggesting this expression is passage independent and not related to loss of XCI marks in higher passage female lines (C) Quantification of percentage of surviving Alkaline Phosphatase positive pluripotent colonies after treatment of control hESC lines (HUES 51 and HUES 53) established iPSC lines (18c and 20b) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines (CR-HAT-1 CR-6TG-2 and CR-6TG-3) with FGF or TGF-β inhibitors All pluripotent lines regardless of passage number appeared to be similarly sensitive to these inhibitors This indicates that the dependency on these pathways for pluripotency and self-renewal is maintained in these iPSC lines regardless of time in culture and XCI status (D) Representative images and quantification of XIST clouds in teratomas derived from low passage (XIST +) iPSC lines (CR-HAT-1 and CR-6TG-3) and high passage (XIST-) iPSC (CR-6TG-2 p29) Lines with proper XCI at low passage generated teratomas with high proportion of cells with XIST clouds However iPSC line CR-6TG-2 at passage 29 which has lost XCI marks generated a teratoma without any XIST clouds Scale bars=10microm (E) Representative images and quantification of XIST clouds and H3K27me3 foci in NANOG positive cells for iPSC 29A and 29e at around the same passages used for whole genome exome sequencing in (Gore et al 2011) At these passages both these lines have lost marks of XCI in nearly all their cells Scale bars=10microm (F) Examination of genomic mutations in iPSC 29A and 29e (Gore et al 2011) indicate no common somatic mutations between the two lines and no X-linked mutations at all

9

Figure S6 Erosion of the Xi and functional HPRT expression restores normal neuronal phenotype (Related to Figure 4) (A) Neuronal cultures stained for TUJ1 and H3K27me3 for the HPRT positive dosage compensated CR-HAT-1 line Neurons from this line show normal neuronal morphology with clear foci of H3K27me3 indicative of the inactive X in the majority of the neurons (B) Neuronal cultures from the line CR-6TG-2 at passage 13 that has lost XCI marks in at least a portion of its cells (as seen in Fig 3C) Neurons at this passage show a mix phenotype with a portion of neurons with normal neuronal morphology and a portion of neurons with abnormal neuronal morphology Staining of H3K27me3 shows clear foci of H3K27me3 in neurons with abnormal morphology that have maintained XCI and lack expression of HPRT Conversely neurons with normal morphology lack clear foci of H3K27me3 indicative of erosion of dosage compensation and the expression of the functional HPRT allele from the Xe Arrows point to foci of H3K27me3 indicative of an Xi Scale bars=50microm

10

Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

11

Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

4

Figure S3 Characterization of neuronal cultures (Related to Figure 2) (A) TUJ1-positive neurons for wildtype hiPSC line 20b HPRT positive LNS carrier female line CR-HAT-1 and HPRT negative male LNS-2 also stain for the mature neuronal markers

5

MAP2A and SYN1 and for the dopaminergic specific marker Thyrosine Hydroxylase (TH) which are implicated to be severely defected in Lesch-Nyhan affected individuals (B) Representative neuronal cultures stained for TUJ1 from the isogenic lines HPRT positive CR-HAT-1 HPRT negative CR-6TG-3 (low passage XIST+) as well as the HPRT negative male LNS-2 The neuronal phenotype seen in HPRT negative cultures with less number of surviving neurons is not rescued when neurons are plated on mouse glia cells that provide more neural support Scale bars=100microm (C) Quantification of number of TUJ1-positive neurons three days post EB dissociation and plating on glass slides with and without mouse brain derived glia show supportive glial cells do not rescue the HPRT negative neuronal phenotype

Figure S4 Validation of HPRT rescue for LNS lines (Related to Figure 2) (A) Alkaline Phosphatase staining of hiPSC colonies from LNS-2 iPSC line that was transduced with an empty lenti virus or an HPRT carrying lenti-virus and then treated with HAT containing media HPRT negative cells transduced with empty vector remain HAT sensitive while cells transduced with the HPRT vector were now HAT resistance and grew in HAT media This change in drug resistance confirms the expression of functional HPRT from the viral vector in this transduced line (B) Full field reprehensive images of neuronal cultures derived in parallel from diseased hiPSC LNS-2 wildtype iPSC 20b LNS-2-vecCNTL and two independently derived HPRT rescue lines transduced with virus carrying the intact HPRT LNS-2-HPRTrescA and B While LNS-

6

2vecCNTL neurons are similar in number and morphology to their parental LNS-2 neurons neurons derived from the two rescue lines are similar in number and morphology to wildtype lines Scale bars=100microm

7

Figure S5 Characterization of lines that have lost XCI marks (Related to Figure 3) (A) Diagram depicting decrease in XCI marks with time in culture for HPRT positive carrier hiPSC lines with the mutated HPRT on the inactive X chromosome Crystal Violet staining of hiPSC line CR-HAT-2 after selection for the drugs HAT or 6TG over 16 passages in culture

8

display no changes in drug resistance Quantification of H3K27me3 foci in NANOG-positive cells for hiPSC lines CR-HAT-1 CR-HAT-2 show a decline in proportion of cells with proper XCI over extended culture (B) Quantitative RT-PCR (qPCR) analysis with primers specific for viral (v=red bars) and endogenous (e=blue bars) expression of the reprogramming genes OCT4 SOX2 KLF4 and cMYC in control fibroblasts wildtype hESC lines (HUES 51 and HUES 53) wildtype established iPSC lines (iPSC 20b iPSC 18c) newly derived male LNS iPSC (LNS-2) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines reported in this work (CR-HAT-1 CR-6TG-2 and CR-6TG-3) All hiPSC lines at both low and high passage are free from viral expression of OCT4 SOX2 and cMYC and express the endogenous genes indicative of full reprogramming Some low to moderate levels of viral KLF4 was detected in all of our iPSC lines However this exogenous expression was observed in similar levels at both low and high passage lines suggesting this expression is passage independent and not related to loss of XCI marks in higher passage female lines (C) Quantification of percentage of surviving Alkaline Phosphatase positive pluripotent colonies after treatment of control hESC lines (HUES 51 and HUES 53) established iPSC lines (18c and 20b) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines (CR-HAT-1 CR-6TG-2 and CR-6TG-3) with FGF or TGF-β inhibitors All pluripotent lines regardless of passage number appeared to be similarly sensitive to these inhibitors This indicates that the dependency on these pathways for pluripotency and self-renewal is maintained in these iPSC lines regardless of time in culture and XCI status (D) Representative images and quantification of XIST clouds in teratomas derived from low passage (XIST +) iPSC lines (CR-HAT-1 and CR-6TG-3) and high passage (XIST-) iPSC (CR-6TG-2 p29) Lines with proper XCI at low passage generated teratomas with high proportion of cells with XIST clouds However iPSC line CR-6TG-2 at passage 29 which has lost XCI marks generated a teratoma without any XIST clouds Scale bars=10microm (E) Representative images and quantification of XIST clouds and H3K27me3 foci in NANOG positive cells for iPSC 29A and 29e at around the same passages used for whole genome exome sequencing in (Gore et al 2011) At these passages both these lines have lost marks of XCI in nearly all their cells Scale bars=10microm (F) Examination of genomic mutations in iPSC 29A and 29e (Gore et al 2011) indicate no common somatic mutations between the two lines and no X-linked mutations at all

9

Figure S6 Erosion of the Xi and functional HPRT expression restores normal neuronal phenotype (Related to Figure 4) (A) Neuronal cultures stained for TUJ1 and H3K27me3 for the HPRT positive dosage compensated CR-HAT-1 line Neurons from this line show normal neuronal morphology with clear foci of H3K27me3 indicative of the inactive X in the majority of the neurons (B) Neuronal cultures from the line CR-6TG-2 at passage 13 that has lost XCI marks in at least a portion of its cells (as seen in Fig 3C) Neurons at this passage show a mix phenotype with a portion of neurons with normal neuronal morphology and a portion of neurons with abnormal neuronal morphology Staining of H3K27me3 shows clear foci of H3K27me3 in neurons with abnormal morphology that have maintained XCI and lack expression of HPRT Conversely neurons with normal morphology lack clear foci of H3K27me3 indicative of erosion of dosage compensation and the expression of the functional HPRT allele from the Xe Arrows point to foci of H3K27me3 indicative of an Xi Scale bars=50microm

10

Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

11

Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

5

MAP2A and SYN1 and for the dopaminergic specific marker Thyrosine Hydroxylase (TH) which are implicated to be severely defected in Lesch-Nyhan affected individuals (B) Representative neuronal cultures stained for TUJ1 from the isogenic lines HPRT positive CR-HAT-1 HPRT negative CR-6TG-3 (low passage XIST+) as well as the HPRT negative male LNS-2 The neuronal phenotype seen in HPRT negative cultures with less number of surviving neurons is not rescued when neurons are plated on mouse glia cells that provide more neural support Scale bars=100microm (C) Quantification of number of TUJ1-positive neurons three days post EB dissociation and plating on glass slides with and without mouse brain derived glia show supportive glial cells do not rescue the HPRT negative neuronal phenotype

Figure S4 Validation of HPRT rescue for LNS lines (Related to Figure 2) (A) Alkaline Phosphatase staining of hiPSC colonies from LNS-2 iPSC line that was transduced with an empty lenti virus or an HPRT carrying lenti-virus and then treated with HAT containing media HPRT negative cells transduced with empty vector remain HAT sensitive while cells transduced with the HPRT vector were now HAT resistance and grew in HAT media This change in drug resistance confirms the expression of functional HPRT from the viral vector in this transduced line (B) Full field reprehensive images of neuronal cultures derived in parallel from diseased hiPSC LNS-2 wildtype iPSC 20b LNS-2-vecCNTL and two independently derived HPRT rescue lines transduced with virus carrying the intact HPRT LNS-2-HPRTrescA and B While LNS-

6

2vecCNTL neurons are similar in number and morphology to their parental LNS-2 neurons neurons derived from the two rescue lines are similar in number and morphology to wildtype lines Scale bars=100microm

7

Figure S5 Characterization of lines that have lost XCI marks (Related to Figure 3) (A) Diagram depicting decrease in XCI marks with time in culture for HPRT positive carrier hiPSC lines with the mutated HPRT on the inactive X chromosome Crystal Violet staining of hiPSC line CR-HAT-2 after selection for the drugs HAT or 6TG over 16 passages in culture

8

display no changes in drug resistance Quantification of H3K27me3 foci in NANOG-positive cells for hiPSC lines CR-HAT-1 CR-HAT-2 show a decline in proportion of cells with proper XCI over extended culture (B) Quantitative RT-PCR (qPCR) analysis with primers specific for viral (v=red bars) and endogenous (e=blue bars) expression of the reprogramming genes OCT4 SOX2 KLF4 and cMYC in control fibroblasts wildtype hESC lines (HUES 51 and HUES 53) wildtype established iPSC lines (iPSC 20b iPSC 18c) newly derived male LNS iPSC (LNS-2) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines reported in this work (CR-HAT-1 CR-6TG-2 and CR-6TG-3) All hiPSC lines at both low and high passage are free from viral expression of OCT4 SOX2 and cMYC and express the endogenous genes indicative of full reprogramming Some low to moderate levels of viral KLF4 was detected in all of our iPSC lines However this exogenous expression was observed in similar levels at both low and high passage lines suggesting this expression is passage independent and not related to loss of XCI marks in higher passage female lines (C) Quantification of percentage of surviving Alkaline Phosphatase positive pluripotent colonies after treatment of control hESC lines (HUES 51 and HUES 53) established iPSC lines (18c and 20b) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines (CR-HAT-1 CR-6TG-2 and CR-6TG-3) with FGF or TGF-β inhibitors All pluripotent lines regardless of passage number appeared to be similarly sensitive to these inhibitors This indicates that the dependency on these pathways for pluripotency and self-renewal is maintained in these iPSC lines regardless of time in culture and XCI status (D) Representative images and quantification of XIST clouds in teratomas derived from low passage (XIST +) iPSC lines (CR-HAT-1 and CR-6TG-3) and high passage (XIST-) iPSC (CR-6TG-2 p29) Lines with proper XCI at low passage generated teratomas with high proportion of cells with XIST clouds However iPSC line CR-6TG-2 at passage 29 which has lost XCI marks generated a teratoma without any XIST clouds Scale bars=10microm (E) Representative images and quantification of XIST clouds and H3K27me3 foci in NANOG positive cells for iPSC 29A and 29e at around the same passages used for whole genome exome sequencing in (Gore et al 2011) At these passages both these lines have lost marks of XCI in nearly all their cells Scale bars=10microm (F) Examination of genomic mutations in iPSC 29A and 29e (Gore et al 2011) indicate no common somatic mutations between the two lines and no X-linked mutations at all

9

Figure S6 Erosion of the Xi and functional HPRT expression restores normal neuronal phenotype (Related to Figure 4) (A) Neuronal cultures stained for TUJ1 and H3K27me3 for the HPRT positive dosage compensated CR-HAT-1 line Neurons from this line show normal neuronal morphology with clear foci of H3K27me3 indicative of the inactive X in the majority of the neurons (B) Neuronal cultures from the line CR-6TG-2 at passage 13 that has lost XCI marks in at least a portion of its cells (as seen in Fig 3C) Neurons at this passage show a mix phenotype with a portion of neurons with normal neuronal morphology and a portion of neurons with abnormal neuronal morphology Staining of H3K27me3 shows clear foci of H3K27me3 in neurons with abnormal morphology that have maintained XCI and lack expression of HPRT Conversely neurons with normal morphology lack clear foci of H3K27me3 indicative of erosion of dosage compensation and the expression of the functional HPRT allele from the Xe Arrows point to foci of H3K27me3 indicative of an Xi Scale bars=50microm

10

Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

11

Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

6

2vecCNTL neurons are similar in number and morphology to their parental LNS-2 neurons neurons derived from the two rescue lines are similar in number and morphology to wildtype lines Scale bars=100microm

7

Figure S5 Characterization of lines that have lost XCI marks (Related to Figure 3) (A) Diagram depicting decrease in XCI marks with time in culture for HPRT positive carrier hiPSC lines with the mutated HPRT on the inactive X chromosome Crystal Violet staining of hiPSC line CR-HAT-2 after selection for the drugs HAT or 6TG over 16 passages in culture

8

display no changes in drug resistance Quantification of H3K27me3 foci in NANOG-positive cells for hiPSC lines CR-HAT-1 CR-HAT-2 show a decline in proportion of cells with proper XCI over extended culture (B) Quantitative RT-PCR (qPCR) analysis with primers specific for viral (v=red bars) and endogenous (e=blue bars) expression of the reprogramming genes OCT4 SOX2 KLF4 and cMYC in control fibroblasts wildtype hESC lines (HUES 51 and HUES 53) wildtype established iPSC lines (iPSC 20b iPSC 18c) newly derived male LNS iPSC (LNS-2) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines reported in this work (CR-HAT-1 CR-6TG-2 and CR-6TG-3) All hiPSC lines at both low and high passage are free from viral expression of OCT4 SOX2 and cMYC and express the endogenous genes indicative of full reprogramming Some low to moderate levels of viral KLF4 was detected in all of our iPSC lines However this exogenous expression was observed in similar levels at both low and high passage lines suggesting this expression is passage independent and not related to loss of XCI marks in higher passage female lines (C) Quantification of percentage of surviving Alkaline Phosphatase positive pluripotent colonies after treatment of control hESC lines (HUES 51 and HUES 53) established iPSC lines (18c and 20b) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines (CR-HAT-1 CR-6TG-2 and CR-6TG-3) with FGF or TGF-β inhibitors All pluripotent lines regardless of passage number appeared to be similarly sensitive to these inhibitors This indicates that the dependency on these pathways for pluripotency and self-renewal is maintained in these iPSC lines regardless of time in culture and XCI status (D) Representative images and quantification of XIST clouds in teratomas derived from low passage (XIST +) iPSC lines (CR-HAT-1 and CR-6TG-3) and high passage (XIST-) iPSC (CR-6TG-2 p29) Lines with proper XCI at low passage generated teratomas with high proportion of cells with XIST clouds However iPSC line CR-6TG-2 at passage 29 which has lost XCI marks generated a teratoma without any XIST clouds Scale bars=10microm (E) Representative images and quantification of XIST clouds and H3K27me3 foci in NANOG positive cells for iPSC 29A and 29e at around the same passages used for whole genome exome sequencing in (Gore et al 2011) At these passages both these lines have lost marks of XCI in nearly all their cells Scale bars=10microm (F) Examination of genomic mutations in iPSC 29A and 29e (Gore et al 2011) indicate no common somatic mutations between the two lines and no X-linked mutations at all

9

Figure S6 Erosion of the Xi and functional HPRT expression restores normal neuronal phenotype (Related to Figure 4) (A) Neuronal cultures stained for TUJ1 and H3K27me3 for the HPRT positive dosage compensated CR-HAT-1 line Neurons from this line show normal neuronal morphology with clear foci of H3K27me3 indicative of the inactive X in the majority of the neurons (B) Neuronal cultures from the line CR-6TG-2 at passage 13 that has lost XCI marks in at least a portion of its cells (as seen in Fig 3C) Neurons at this passage show a mix phenotype with a portion of neurons with normal neuronal morphology and a portion of neurons with abnormal neuronal morphology Staining of H3K27me3 shows clear foci of H3K27me3 in neurons with abnormal morphology that have maintained XCI and lack expression of HPRT Conversely neurons with normal morphology lack clear foci of H3K27me3 indicative of erosion of dosage compensation and the expression of the functional HPRT allele from the Xe Arrows point to foci of H3K27me3 indicative of an Xi Scale bars=50microm

10

Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

11

Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

7

Figure S5 Characterization of lines that have lost XCI marks (Related to Figure 3) (A) Diagram depicting decrease in XCI marks with time in culture for HPRT positive carrier hiPSC lines with the mutated HPRT on the inactive X chromosome Crystal Violet staining of hiPSC line CR-HAT-2 after selection for the drugs HAT or 6TG over 16 passages in culture

8

display no changes in drug resistance Quantification of H3K27me3 foci in NANOG-positive cells for hiPSC lines CR-HAT-1 CR-HAT-2 show a decline in proportion of cells with proper XCI over extended culture (B) Quantitative RT-PCR (qPCR) analysis with primers specific for viral (v=red bars) and endogenous (e=blue bars) expression of the reprogramming genes OCT4 SOX2 KLF4 and cMYC in control fibroblasts wildtype hESC lines (HUES 51 and HUES 53) wildtype established iPSC lines (iPSC 20b iPSC 18c) newly derived male LNS iPSC (LNS-2) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines reported in this work (CR-HAT-1 CR-6TG-2 and CR-6TG-3) All hiPSC lines at both low and high passage are free from viral expression of OCT4 SOX2 and cMYC and express the endogenous genes indicative of full reprogramming Some low to moderate levels of viral KLF4 was detected in all of our iPSC lines However this exogenous expression was observed in similar levels at both low and high passage lines suggesting this expression is passage independent and not related to loss of XCI marks in higher passage female lines (C) Quantification of percentage of surviving Alkaline Phosphatase positive pluripotent colonies after treatment of control hESC lines (HUES 51 and HUES 53) established iPSC lines (18c and 20b) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines (CR-HAT-1 CR-6TG-2 and CR-6TG-3) with FGF or TGF-β inhibitors All pluripotent lines regardless of passage number appeared to be similarly sensitive to these inhibitors This indicates that the dependency on these pathways for pluripotency and self-renewal is maintained in these iPSC lines regardless of time in culture and XCI status (D) Representative images and quantification of XIST clouds in teratomas derived from low passage (XIST +) iPSC lines (CR-HAT-1 and CR-6TG-3) and high passage (XIST-) iPSC (CR-6TG-2 p29) Lines with proper XCI at low passage generated teratomas with high proportion of cells with XIST clouds However iPSC line CR-6TG-2 at passage 29 which has lost XCI marks generated a teratoma without any XIST clouds Scale bars=10microm (E) Representative images and quantification of XIST clouds and H3K27me3 foci in NANOG positive cells for iPSC 29A and 29e at around the same passages used for whole genome exome sequencing in (Gore et al 2011) At these passages both these lines have lost marks of XCI in nearly all their cells Scale bars=10microm (F) Examination of genomic mutations in iPSC 29A and 29e (Gore et al 2011) indicate no common somatic mutations between the two lines and no X-linked mutations at all

9

Figure S6 Erosion of the Xi and functional HPRT expression restores normal neuronal phenotype (Related to Figure 4) (A) Neuronal cultures stained for TUJ1 and H3K27me3 for the HPRT positive dosage compensated CR-HAT-1 line Neurons from this line show normal neuronal morphology with clear foci of H3K27me3 indicative of the inactive X in the majority of the neurons (B) Neuronal cultures from the line CR-6TG-2 at passage 13 that has lost XCI marks in at least a portion of its cells (as seen in Fig 3C) Neurons at this passage show a mix phenotype with a portion of neurons with normal neuronal morphology and a portion of neurons with abnormal neuronal morphology Staining of H3K27me3 shows clear foci of H3K27me3 in neurons with abnormal morphology that have maintained XCI and lack expression of HPRT Conversely neurons with normal morphology lack clear foci of H3K27me3 indicative of erosion of dosage compensation and the expression of the functional HPRT allele from the Xe Arrows point to foci of H3K27me3 indicative of an Xi Scale bars=50microm

10

Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

11

Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

8

display no changes in drug resistance Quantification of H3K27me3 foci in NANOG-positive cells for hiPSC lines CR-HAT-1 CR-HAT-2 show a decline in proportion of cells with proper XCI over extended culture (B) Quantitative RT-PCR (qPCR) analysis with primers specific for viral (v=red bars) and endogenous (e=blue bars) expression of the reprogramming genes OCT4 SOX2 KLF4 and cMYC in control fibroblasts wildtype hESC lines (HUES 51 and HUES 53) wildtype established iPSC lines (iPSC 20b iPSC 18c) newly derived male LNS iPSC (LNS-2) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines reported in this work (CR-HAT-1 CR-6TG-2 and CR-6TG-3) All hiPSC lines at both low and high passage are free from viral expression of OCT4 SOX2 and cMYC and express the endogenous genes indicative of full reprogramming Some low to moderate levels of viral KLF4 was detected in all of our iPSC lines However this exogenous expression was observed in similar levels at both low and high passage lines suggesting this expression is passage independent and not related to loss of XCI marks in higher passage female lines (C) Quantification of percentage of surviving Alkaline Phosphatase positive pluripotent colonies after treatment of control hESC lines (HUES 51 and HUES 53) established iPSC lines (18c and 20b) and low (XIST + ple12) and high passage (XIST- pge27) female iPSC lines (CR-HAT-1 CR-6TG-2 and CR-6TG-3) with FGF or TGF-β inhibitors All pluripotent lines regardless of passage number appeared to be similarly sensitive to these inhibitors This indicates that the dependency on these pathways for pluripotency and self-renewal is maintained in these iPSC lines regardless of time in culture and XCI status (D) Representative images and quantification of XIST clouds in teratomas derived from low passage (XIST +) iPSC lines (CR-HAT-1 and CR-6TG-3) and high passage (XIST-) iPSC (CR-6TG-2 p29) Lines with proper XCI at low passage generated teratomas with high proportion of cells with XIST clouds However iPSC line CR-6TG-2 at passage 29 which has lost XCI marks generated a teratoma without any XIST clouds Scale bars=10microm (E) Representative images and quantification of XIST clouds and H3K27me3 foci in NANOG positive cells for iPSC 29A and 29e at around the same passages used for whole genome exome sequencing in (Gore et al 2011) At these passages both these lines have lost marks of XCI in nearly all their cells Scale bars=10microm (F) Examination of genomic mutations in iPSC 29A and 29e (Gore et al 2011) indicate no common somatic mutations between the two lines and no X-linked mutations at all

9

Figure S6 Erosion of the Xi and functional HPRT expression restores normal neuronal phenotype (Related to Figure 4) (A) Neuronal cultures stained for TUJ1 and H3K27me3 for the HPRT positive dosage compensated CR-HAT-1 line Neurons from this line show normal neuronal morphology with clear foci of H3K27me3 indicative of the inactive X in the majority of the neurons (B) Neuronal cultures from the line CR-6TG-2 at passage 13 that has lost XCI marks in at least a portion of its cells (as seen in Fig 3C) Neurons at this passage show a mix phenotype with a portion of neurons with normal neuronal morphology and a portion of neurons with abnormal neuronal morphology Staining of H3K27me3 shows clear foci of H3K27me3 in neurons with abnormal morphology that have maintained XCI and lack expression of HPRT Conversely neurons with normal morphology lack clear foci of H3K27me3 indicative of erosion of dosage compensation and the expression of the functional HPRT allele from the Xe Arrows point to foci of H3K27me3 indicative of an Xi Scale bars=50microm

10

Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

11

Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

9

Figure S6 Erosion of the Xi and functional HPRT expression restores normal neuronal phenotype (Related to Figure 4) (A) Neuronal cultures stained for TUJ1 and H3K27me3 for the HPRT positive dosage compensated CR-HAT-1 line Neurons from this line show normal neuronal morphology with clear foci of H3K27me3 indicative of the inactive X in the majority of the neurons (B) Neuronal cultures from the line CR-6TG-2 at passage 13 that has lost XCI marks in at least a portion of its cells (as seen in Fig 3C) Neurons at this passage show a mix phenotype with a portion of neurons with normal neuronal morphology and a portion of neurons with abnormal neuronal morphology Staining of H3K27me3 shows clear foci of H3K27me3 in neurons with abnormal morphology that have maintained XCI and lack expression of HPRT Conversely neurons with normal morphology lack clear foci of H3K27me3 indicative of erosion of dosage compensation and the expression of the functional HPRT allele from the Xe Arrows point to foci of H3K27me3 indicative of an Xi Scale bars=50microm

10

Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

11

Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

10

Figure S7 Re-isolation of pure populations of hiPSC with a known XCI status (Related to Figure 4) (A) Diagram depicting experimental outline and results selection of iPSC cell lines for HAT6TG yields two clear populations of cells 6TG selected subline maintain high levels of XCI marks yet HAT selected subline have low levels of XCI marks and now ectopically express HPRT from the silent X chromosome (B) Crystal Violet staining of differentiated iPSC line CR-6TG-2 indicates the differentiated cells have irreversibly reactivated the functional HPRT allele (C) Quantification of XIST clouds in iPSC lines and differentiated 6TG selected sublines (D) Diagram depicting selection process differentiation into neurons and results (E) Neurons stained for TUJ1 from the HAT selected subline of the line CR-6TG-2 that reactivated the functional HPRT allele from the Xi This reactivation resulted in production of more neurons While neurons from of 6TG selected sub-population of the line CR-6TG-2 that express XCI marks and maintain a silent functional HPRT allele stained for TUJ1 produced very few neurons with short neurites Scale bars = 50microm (F) Quantification of TUJ1 in HAT6TG selected sub-populations of the line CR-6TG-2 Error bars represent mean +- SD Statistical significance was analyzed using 2-tailed Student Test P lt 005 Plt001 Plt0001

11

Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

11

Supplemental experimental procedures hiPSC derivation and culture Lesch-Nyhan human fibroblasts and wild type human fibroblasts were commercially obtained (Coriell Cell Repository GM02226 GM02227 ATCC BJ fibroblasts wi-38 fibroblasts) and grown in human fibroblast media containing DMEM (invitrogen) 10-15 Fetal Bovine Serum (Hyclone) 2mM Glutamax 1 non-essential amino acids 50unitsml penicillin and 50μgml streptomycin (Invitrogen) 0055mM β-mercaptoethanol (Invitrogen) The murine leukemia retroviral vector pMXs containing the human cDNAs for OCT4 SOX2 KLF4 and cMYC were modified to produce higher titer virus by including the Woodchuck Post-Transcriptional responsive Element (WPRE) of FUGW (Addgene plasmid 14883) downstream of the 25 cDNA VSV-g pseudotyped viruses were packaged and concentrated by the Harvard Gene Therapy Initiative at Harvard Medical School (Boston MA) To produce hiPSC colonies 50000-75000 human fibroblasts were transduced at an MOI of 10-15 with viruses containing OCT4 SOX2 KLF4 and 05-1 MOI of cMYC (OSKM) in standard hESC medium with 8ugmL polyprene Cells were incubated with virus for 24h before medium was changed to standard fibroblast medium for 48h Cells were subsequently cultured in standard hESC medium and iPSC colonies were manually picked based on morphology within 2-4 weeks All hiPSC lines were continuously cultured and manually picked throughout the work in this study All hiPSC were derived and maintained at 37degC 5 CO2 20 O2 unless noted otherwise hESChiPSC culture and differentiation hESChiPSC lines were maintained on irradiated mouse embryonic fibroblasts (MEFs) in standard hESC media containing KO-DMEM (Invitrogen) supplemented with 10 serum replacement (Invitrogen) 10 plasmanate (Talecris) 1 nonesesential amino acids (Invitrogen) 2 mM glutamax-I (Invitrogen) 50 unitsml penicillin and 50 1056768gml streptomycin (Invitrogen) 0055 mM 2-mercaptoethanol (Invitrogen) and 10-20 ngml bFGF (Millipore) All human pluripotent lines were passaged by mechanical dissociation every 6-8 days Differentiation of human pluripotent stem cells was carried out by embryoid body (EB) suspension in low-attachment six-well plates (Corning) in media containing 10 ndash15 FBS (Hyclone) in place of the serum replacement and plasmanate After 8-10 days cells were plated onto gelatin coated plates and grown for another 8 ndash10 days before analysis Generation and analysis of secondary hPSC Human pluripotent cell lines (hPSC) were differentiated as previously described (Maherali et al 2008) and examined for the X inactivation status Briefly hPSC were differentiated by EB suspension for 10 days in media containing 10FBS then plated on gelatin with media containing 20 FBS Cells were grown for 8-10 days then passaged 3-4 times every 4-5 days until homogeneous secondary fibroblast population was obtained Secondary fibroblasts were transduced with reprogramming factors (OSKM) as described above and iPSC colonies were allowed to form for 4 weeks For each line at least 30 morphologically looking hiPSC colonies were picked and expanded for 1 additional passage hiPSC colonies were then manually picked and equal number of clumps were added to wells for further analysis of HAT and 6TG survival assays as described in main experimental procedures Selected cultures were then stained for Alkaline Phosphatase and quantified A small portion of each cell population was also used for XISTH3K27me3 analysis

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

12

Immunocytochemistry Cells were stained for pluripotency marker and three germ-layer after fixation overnight in 4 paraformaldehyde at 4degC as previously described (Dimos et al 2008) Neuronal cultures were fixed in 4 PFA for 15 minutes at 4degC Fixed cultures were permeabilized with 02 Triton-X or 05 Tween in PBS for 30 minutes Cells were blocked in PBS-01 Tween with 10 donkey serum or 10 FBS for 30 minutes and then incubated in blocking solution containing primary antibody overnight Cells were incubated in blocking solution containing secondary antibodies for 1-2 hours then washed and mounted in Vectashield with Dapi (Vector Laboratories) and imaged on an inverted Olympus epifluorescence microscope or Zeiss Axiomax epifluorescence microscope Primary antibodies used SSEA-3 (12 DSHB) SSEA-4 (12 DSHB) TRA1-60 (1500 Millipore) TRA1-81 (1500 Millipore) Nanog (1500 RampD) Oct34 (1500 Santa Cruz) AFP (1500 DAKO) alpha-SMA (1500 Sigma) TUJ1 (1700 Covance) Thyrosin Hydroxylase (1500 Millipore) MAP2A (15000 Millipore) SYN1 (11000 Millipore) H2K27me3 (1300 Millipore) Secondary antibodies used in this study AlexaFluor 488 555 594 and 647 conjugated (1300 Invitrogen) Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector SK-5100) according to the manufacturersquos instructions qRT-PCR Total RNA was isolated using Trizol (Invitrogen) and treated with DNase (Invitrogen) 1 μg was subsequently used to synthesize cDNA with iScript (Bio-Rad) qRT-PCR was then performed using SYBR green (Bio-Rad) and the iCycler system (Bio-Rad) Quantitative levels for all genes were normalized to endogenous GAPDH expression For pluripotency genes levels were expressed relative to the levels in human ESC lines Standard curves were run to ensure equal efficiency of all primers and RNA from 293 cells transfected with the plasmids encoding the transgenes was used as a positive control for viral transgene detection Primer sequences can be provided upon request Treatment of hiPSC with FGF and TGFβ inhibitors To test the dependency of lower and higher passage hiPSC on FGF and TGFβ approximately equal number of manually dissected colony clumps were placed 3mm wells Two days after colonies were marked and scored to ensure equal survival and were then fed daily with hESC media containing TGFβ inhibitor SB431542 (Sigma) or in hESC media without FGF containing the FGF inhibitor PD173074 (Sigma) Treatment with each inhibitor was carried out for six days after which cells were stained for Alkaline Phosphatase (AP) (Vector SK-5100) and quantified Colonies were manually scored for each inhibitor treatment and proportion of AP positive colonies was compared to population of cells that were not treated with either drug Generation of HPRT positive rescue line Human cDNA for the HPRT gene (Open Biosystems) was cloned into a lenti viral vector under the control of EF1α promoter and packaged in 293T cells as previously described (Son et al 2011) The HPRT negaive hiPSC LNS-2 was plated on matrigel (FISHER) in mTESR media (Stem Cell Technologies) and transduced with virus containing the intact HPRT gene or the empty viral vector 24 hours following viral transduction virus was washed and replaced with fresh mTESR media After three days cells with HPRT activity were selected with mTESR media containing HAT for 5 days Cells that were now expressing the viral HPRT were used for neural differentiation

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

13

Teratoma Assay and Analysis Human pluripotent stem cells were trypsinized to single cell washed and resuspended in a hESC media At least 1x106 cells were injected into the kidney of SCID hairless outbred mice (SHO) (2-3 micecell line) Xenograft tissue masses formed within 8-10 weeks Masses were extracted fixed sectioned and HampE stained Cells representing all three germ layers were identified after careful examination under the microscope For XCI analysis teratoma was minced using a razor blade and then lightly titrated in trypsin washed and resuspended in human fibroblast media Teratoma cells were then plated as a monolayer for 48 hours and subsequently trypsinazed into single cells cytospan onto slides and treated for XIST FISH experiments as described above Genomic data analysis DNA methylation and gene expression data for 20 hESC lines 12 hiPSC lines and 6 fibroblast cell lines were obtained from the supplementary material of a recent study which reported a reference map of human pluripotent cell lines (Bock et al 2011) This DNA methylation dataset was obtained by reduced-representation bisulfite sequencing using an established protocol (Gu et al 2010) which has been shown to provide excellent accuracy for detecting DNA methylation differences between cell types (Bock et al 2010) For each gene promoter which was defined as the region ranging from 5 kilobases upstream to 1 kilobase downstream of Ensembl-annotated transcription start sites the mean DNA methylation level was calculated as a coverage-weighted average over single-basepair measurements The DNA methylation variation was calculated for each promoter region as the standard deviation of the average methylation levels across samples The gene expression data were obtained using Affymetrix microarrays and the mean expression levels of Ensembl-annotated genes were calculated by averaging over individual microarray probe intensities The gene expression variation was calculated for each gene as the standard deviation of the average methylation levels across samples Promoter regions and genes with insufficient coverage in the dataset were discarded using the same criteria as in the original publication (Bock et al 2011)

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218

14

Supplemental References Bock C Kiskinis E Verstappen G Gu H Boulting G Smith ZD Ziller M Croft GF Amoroso MW Oakley DH et al (2011) Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines Cell 144 439-452 Bock C Tomazou EM Brinkman AB Muller F Simmer F Gu H Jager N Gnirke A Stunnenberg HG and Meissner A (2010) Quantitative comparison of genome-wide DNA methylation mapping technologies Nat Biotechnol 28 1106-1114 Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Croft GF Saphier G Leibel R Goland R et al (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 321 1218-1221 Gore A Li Z Fung HL Young JE Agarwal S Antosiewicz-Bourget J Canto I Giorgetti A Israel MA Kiskinis E et al (2011) Somatic coding mutations in human induced pluripotent stem cells Nature 471 63-67 Gu H Bock C Mikkelsen TS Jager N Smith ZD Tomazou E Gnirke A Lander ES and Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution Nat Methods 7 133-136 Maherali N Ahfeldt T Rigamonti A Utikal J Cowan C and Hochedlinger K (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells Cell Stem Cell 3 340-345 Son EY Ichida JK Wainger BJ Toma JS Rafuse VF Woolf CJ and Eggan K (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons Cell Stem Cell 9 205-218