derivation of high-purity definitive endoderm from human...

Cell Transplantation, Vol. 21, pp. 217–234, 2012 0963-6897/12 $90.00 + .00Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368911X582723Copyright 2012 Cognizant Comm. Corp. E-ISSN 1555-3892

www.cognizantcommunication.com

Derivation of High-Purity Definitive EndodermFrom Human Parthenogenetic Stem Cells Using

an In Vitro Analog of the Primitive Streak

Nikolay Turovets,* Jeffrey Fair,† Richard West,‡ Alina Ostrowska,* Ruslan Semechkin,* Jeffrey Janus,*Li Cui,§ Vladimir Agapov,* Irina Turovets,* Andrey Semechkin,* Marie Csete,§ and Larissa Agapova*

*International Stem Cell Corporation, Oceanside, CA, USA†Cedars-Sinai Medical Center, Los Angeles, CA, USA

‡West Labs Scientific, Grand Rapids, MI, USA§Organovo, San Diego, CA, USA

Human parthenogenetic stem cells (hpSCs) are pluripotent stem cells with enormous potential as cell sourcesfor cell-based therapies: hpSCs may have histocompatibilty advantages over human embryonic stem cells(hESCs) and derivation of hpSCs does not require viable blastocyst destruction. For translation of all pluripo-tent stem cell-based therapies, derivation of differentiated cell products that are not contaminated with undif-ferentiated cells is a major technical roadblock. We report here a novel method to derive high-puritydefinitive endoderm (DE) from hpSCs, based on reproducing features of the normal human embryonicmicroenvironment. The method mimics the developmental process of transition through a primitive streak,using a differentiation device that incorporates a three-dimensional extracellular matrix (ECM) combinedwith a porous membrane. Treatment of undifferentiated hpSCs above the membrane results an epithelial-to-mesenchymal transition (EMT); thus, responsive cells acquire the ability to migrate through the membraneinto the ECM, where they differentiate into DE. Importantly, the resultant DE is highly purified, and is notcontaminated by undifferentiated cells, as assessed by OCT4 expression using immunocytochemistry andflow cytometry. The functional properties of the DE are also preserved by the process: DE differentiated inthe device can generate a highly enriched population of hepatocyte-like cells (HLCs) characterized byexpression of hepatic lineage markers, indocyanine green clearance, glycogen storage, cytochrome P450activity, and engraftment in the liver after transplantation into immunodeficient mice. The method is broadlyapplicable and we obtained purified DE using hESCs, as well as several hpSC lines. The novel methoddescribed here represents a significant step toward the efficient generation of high-purity cells derived fromDE, including hepatocytes and pancreatic endocrine cells, for use in regenerative medicine and drug discov-ery, as well as a platform for studying cell fate specification and behavior during development.

Key words: Human parthenogenetic stem cells (hpSCs); Human embryonic stem cells (hESCs);Differentiation; Definitive endoderm (DE); Extracellular matrix (ECM); Hepatocytes

INTRODUCTION antigen (HLA) homozygosity or alternate oocyte activationtechniques yield hpSCs (heterozygous at most loci exceptHLA or HLA homozygous at all loci). hpSCs thus haveThe first intentionally created human parthenogenetic

stem cells (hpSCs) were derived from the inner cell clinically relevant histocompatibilty advantages in signifi-cant segments of the human population, due to HLAmass of blastocysts of unfertilized oocytes activated by

chemical stimuli (41). These hpSCs, like human embry- homozygosity (25,40,41,51). These hpSCs carrying com-mon HLA haplotypes may reduce the risk of immuneonic stem cells (hESCs) (39,53), undergo extensive self-

renewal and have pluripotential differentiation capacity rejection (compared to hESC-derived cells) after transplan-tation of their differentiated derivatives into HLA-matchedin vitro and in vivo, giving rise to cells of all three germ

layers. Using the spontaneous or chemical activation of recipients. Moreover, creation of hpSCs is not associatedwith the ethical concerns associated with hESC derivation.an oocyte possessing the rare trait of human leukocyte

Received September 28, 2010; final acceptance April 3, 2011. Online prepub date: June 9, 2011.Address correspondence to Larissa Agapova, International Stem Cell Corporation, 2595 Jason Court, Oceanside, CA 92056, USA. Tel: 1-760-940-6383; Fax: 1-760-940-6387; E-mail: [email protected]

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218 TUROVETS ET AL.

Two promising applications of pluripotent stem (PI3K); however, pluripotency markers such as octamer-binding transcription factor 4 (OCT4) and NANOG werecells involve cell replacement therapy for diabetes

(9,12) or chronic liver diseases (3,4,9,20,46). Produc- detectable in the final differentiated cell product (54).All previous studies used a two-dimensional (2D)tion of high-purity definitive endoderm (DE) is a criti-

cal first step in the generation of therapeutically useful culture system (monolayer cultures on a flat plastic dish)and did not provide a substrate to promote mesendodermcells of the DE lineage, including hepatocytes and pan-

creatic endocrine cells. migration. 2D culture systems also cannot easily presenta physiologically relevant 3D extracellular matrix (ECM)DE is formed during gastrulation from epiblast cells

that undergo an epithelial-to-mesenchymal transition environment, which provides the crucial signals and sub-strate for migration during gastrulation. Some attempts to(EMT) and ingress through the embryonic primitive

streak (27,42). Upon differentiation signaling from the provide ECM signaling as part of protocols for deriva-tion of DE point to the importance of ECM in theseenvironment, epithelial-like cells of the epiblast undergo

multiple morphologic and biochemical changes that protocols (1), but results are far from optimized for effi-ciency and purity of derived DE.enable them to assume a mesenchymal cell phenotype.

That phenotype includes disruption of the intracellular We describe here a simple and novel 3D differentia-tion system that captures important features of the gas-adhesion complexes and loss of epithelial cell apical–

basal polarity (2,31,37). These cytoskeletal changes are trulation stage embryo, utilizing soluble growth factorsto induce differentiation, 3D ECM to promote cell–cellcritical for cells to leave the epithelium and begin migra-

tion (29,32). The completion of the EMT is signaled by and cell–ECM interactions, and a physical path (pores)for promoting migration. We show that application ofthe migration of mesenchymal cells away from the epi-

thelial layer of origin. Once formed, the primitive streak, the system to various pluripotent cell lines produceshigh-purity DE without contamination of OCT4-positiveacting via ingression, generates the mesendoderm,

which subsequently separates to form the mesoderm and cells, and the DE can be differentiated further into func-tional hepatocyte-like cells (HLCs). These studies areendoderm (19) (see Fig 1A). Thus, only cells that

undergo the EMT participate in this migration and con- also the first demonstration of differentiation of highlyenriched HLCs from hpSCs.tribute to the final population of DE.

In vitro, DE has been derived from hESCs (11,18,MATERIALS AND METHODS30), hpSCs (55), and human-induced pluripotent stem

cells (hiPSCs) (36,47,49,59), using high-level activin A All hpSC lines used in this study were previouslyderived by our research group (40,41) and are ownedand Wnt3a signals to mimic signaling received by cells

during ingress at the primitive streak (7,26,38,45). Knowl- by International Stem Cell Corporation. The hESC lineWA09 was provided by Dr. Mike West. Informed con-edge about the major differentiation signals directing

stem cells toward DE has not translated into methods to sent was obtained for generation of the cell lines fromthe donors and was approved by independent ESCROdifferentiate highly purified DE without undifferentiated

cell contamination in the cultures (5,44,54,55). For clini- Committee of the University of California Irvine.cal application, these residual undifferentiated cells are

Cell Culture and Differentiationa major safety concern since they can generate terato-mas. For example, 7 of 46 mice developed teratomas Undifferentiated hpSCs and hESCs were grown on

mouse embryo fibroblast feeder layers in KnockOut-after injection of unpurified pancreatic cultures of DEderivatives generated from hESCs (23). Moreover, undif- DMEM/F12 supplemented with 15% KnockOut serum

replacement, 0.05 mM nonessential amino acids, 2 mMferentiated cells that remain from the first stages of dif-ferentiation may significantly reduce efficacy of whole Glutamax-I, penicillin/streptomycin, 55 µM 2-mercap-

thoethanol (all from Invitrogen), supplemented with 5differentiation procedure. One of the most advancedprotocols to derive hepatocyte-like cells from hESCs ng/ml recombinant human FGF-basic (PeproTech) and

20 ng/ml recombinant human activin A (rh-activin A;resulted in an estimated efficiency of 18–26%, andenrichment of the differentiated hepatocytes required a R&D Systems). Cultures were manually passaged and

split at ratios of 1:4–1:6 every 5–7 days.flow cytometry step (yielding a population in which55% of cells expressed albumin) (3). HepG2 cells (ATCC) were cultured in 3D ECM

prepared with PureColTM (Advanced BioMatrix) asThe problem of cell purity of differentiated DE hasbeen addressed by several groups, recognizing the described below, in DMEM (Invitrogen) supplemented

with 10% fetal bovine serum (HyClone).importance of generating DE devoid of undifferentiatedcells. The best result was achieved by defined medium For differentiation procedures, hpSCs or hESCs were

plated at high density on top of the membrane of thecontaining high-dose activin A, bone morphogeneticprotein-4 (BMP4), fibroblast growth factor-2 (FGF2), differentiation device (see Fig. 1B). Control cultures

were plated on flat plastic dishes (cell culture treated;and a chemical inhibitor of phosphoinositide 3-kinase

IN VITRO ANALOG OF THE PRIMITIVE STREAK 219

Corning) pretreated with DMEM (Invitrogen) with 10% differentiation protocol described above. Cells were har-vested at days 0, 1, and 2 of the differentiation proce-fetal bovine serum (FBS) (HyClone), and were culti-

vated for a further 2–5 days until the start of the dif- dure. The insert was washed gently in PBS and cellswere removed from within the insert (on top of theferentiation procedure, in the hpSC growth medium

described above. membrane) using a dry cotton bud followed by twowashes in PBS. To isolate intact cells embedded in theThe differentiation device was based on a 25-mm tis-

sue culture insert (Nunc) with a synthetic membrane 3D ECM (or on the underside of the membrane in caseswhere the insert was used without the 3D ECM) thecontaining 8-µm pores (Whatman). For most experi-

ments, a layer of 3D ECM was applied to the underside device was washed twice with PBS and incubated in1000 U/ml collagenese solution (Invitrogen) at 37°C forof the porous membrane.

The ECM was prepared on ice from a mixture of 30 min. After incubation in collagenase solution the sus-pension of cell clumps was carefully collected from thePureColTM with 10× cell culture medium according to

the manufacturer’s instructions, with or without addition bottom of the membrane and centrifuged. To obtain asingle cell suspension the pellet was further dissociatedof human fibronectin (Sigma) to a final concentration of

100 µg fibronectin/ml ECM. (ECM with fibronectin was using 0.05% trypsin (Invitrogen) at 37°C for 1–2 min,then centrifuged, resuspended in PBS with 3% FBS, andonly used for cell migration assays.) To create a thin

layer of 3D ECM, 200 µl of the iced ECM mixture was counted with a hemacytometer.spread evenly on the underside of membrane of tissue Immunostaining and Morphologic Stainingculture inserts and incubated at 37°C for 60 min to

Cultures were fixed for 25 min at room temperatureinduce gelation. The cell culture medium was added

in 4% paraformaldehyde in PBS and permeabilized for(overnight) to each insert containing 3D ECM before

40 min in 0.1% Triton X-100 in PBS. Before immuno-cell seeding.

staining, the membrane with attached 3D ECM andFollowing published protocols (11,12) differentiation

embedded target cells were manually detached from theinto DE was carried out in RPMI-1640 (Invitrogen) sup-

tissue culture insert. Antibodies and dilutions used inplemented with Glutamax-I, penicillin/streptomycin, and

these studies are summarized in Table 1. The slides were0.5 mg/ml human serum albumin (Sigma). For the first

mounted in Vectashield mounting medium containing24 h, this medium was supplemented with 100 ng/ml rh-

4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories).activin A and 75 ng/ml recombinant mouse Wnt3a

To determine the histological and phenotypic charac-(R&D Systems). For the next 48 h, the medium was

teristics of the migrating cells, the ECM-coated filtersupplemented with 0.2% human AB serum (Fisher

membranes of tissue culture inserts were cut out intact,BioReagents) and 100 ng/ml rh-activin A. Wnt3a in

fixed in formalin, embedded in paraffin, and sectionedcombination with activin A increases the efficiency of

(5 µm). Following deparaffinization and rehydration, themesendoderm specification, a bipotential precursor of

sections were stained with hematoxylin and eosinDE and mesoderm, and improves the synchrony with

(H&E). For immunohistochemistry in situ on the mem-which hESCs (12) and hpSCs (55) undergo DE formation.

brane, antigen retrieval was performed with citrate bufferTo derive HLCs, DE cultures located in the 3D ECM

(pH 6.0). Then the sections were costained with anti-Oct4of the differentiation device were cultivated for 3 or 5

and anti-Sox17 [(sex determining region Y)-box 17] todays in KnockOut-DMEM/F12 supplemented with 20%

distinguish undifferentiated hpSC from DE (Table 1).KnockOut serum replacement, 30 ng/ml FGF4 (Pepro

After labeling with the appropriate secondary antibodies,Tech), and 20 ng/ml BMP2 (PeproTech). Then cells

and nuclear counterstain with DAPI, the sections werewere cultivated for 3 or 5 days in KnockOut-DMEM/

captured using a Zeiss fluorescence microscope.F12 supplemented with 20% KnockOut serum replace-

Real-Time Quantitative PCR (RT-qPCR)ment and 20 ng/ml hepatocyte growth factor (HGF;PeproTech) (instead of FGF4 and BMP2). Finally, the Total RNA was isolated using the QIAsymphony

automatic purification system, according to the manu-cells were cultivated for 5 days in hepatocyte culturemedium (HCM; Lonza) supplemented with SingleQuots facturer’s instructions (Qiagen). Total RNA (100–500

ng) was used for reverse transcription with the iScript(Lonza), 20 ng/ml oncostatin M (R&D Systems), and0.1 µM dexamethasone (Sigma). cDNA synthesis kit (Bio-Rad). PCR reactions were run

in duplicate using 1/40th of the cDNA per reaction andAll differentiation experiments were performed atleast in triplicate. Graphical data error bars all represent 400 nM forward and reverse primers or the QuantiTect

Primer Assay, together with Quantitest SYBR Greenstandard deviations.master mix (Qiagen). Real-time PCR was performed

Cell Migration Assay using the Rotor-Gene Q (Qiagen). Relative quantifica-tion was performed against a standard curve and quanti-hpSCs and hESCs were plated on top of the mem-

brane of the differentiation device, and put through the fied values were normalized against the input determined

220 TUROVETS ET AL.

Table 1. Source of Antibodies Used in Immunocytochemistry

Antibodies Host Dilution Producer/Reference

Anti-Sox17 rat 1:500 Chea et al. (7)Anti-Brachyury goat 1:100 R&D SystemsAnti-HNF-3β/FoxA2 goat 1:100 R&D SystemsAnti-α-1-Fetoprotein rabbit 1:500 DakoCytomationAnti-Cytokeratin-18 mouse 1:100 Santa Cruz BiotechnologyAnti-E-Cadherin mouse 1:100 InvitrogenAnti-N-Cadherin mouse 1:50 BD Transduction LaboratoriesAnti-Paxillin mouse 1:25 BD Transduction LaboratoriesAnti-Human albumin rabbit 1:100 AbcamAnti-α-Antitrypsin rabbit 1:1 AbcamAnti-Human Ki67 mouse 1:100 DakoCytomationAnti-Oct3/4 rabbit 1:50 Santa Cruz BiotechnologyAlexa Fluor 546-phalloidin 1:40 InvitrogenAlexa Fluor 488 anti-mouse IgG donkey 1:1000 InvitrogenAlexa Fluor488 anti-goat IgG donkey 1:1000 InvitrogenAlexa Fluor 546 anti-goat IgG donkey 1:1000 InvitrogenAlexa Fluor 546 anti-rabbit IgG donkey 1:1000 InvitrogenAlexa Fluor 546 anti-mouse IgG donkey 1:1000 InvitrogenAlexa Fluor 488 anti-rat IgG donkey 1:1000 Invitrogen

by one of the following housekeeping genes: cyclin G for 30 min at room temperature. Labeling was carried outwith anti-ATT (Invitrogen), anti-AFP (DakoCytomation),(CYCG), β-glucuronidase (GUSB), or TATA box bind-

ing protein (TBP). After normalization, the samples or anti-Oct-4 Alexa Fluor488 conjugate (Millipore).were plotted relative to the first sample in the data set

Cellular Uptake and Release of ICGand the standard deviation of the expression measure-ments was calculated. Primer sequences are reported in Indocyanine green (ICG) is eliminated exclusively by

hepatocytes, so uptake and elimination studies serve asTable 2. RNA isolated from cryopreserved primaryhuman hepatocytes (Invitrogen) was used as a compari- a marker of hepatocyte maturity. ICG (1 mg/ml, Sigma)

in DMEM was added to cell cultures (at late stage dif-son for gene expression of HLCs.ferentiation) and incubated at 37°C for 30 min. After

Flow Cytometry washing, cellular uptake of ICG was documented usinglight microscopy. Cells were then returned to the cultureCells were dissociated using trypsin-like enzyme

(TrypLE; Invitrogen) for 5 min, then pelleted and resus- medium and incubated for 6 h. The ICG was not detect-able inside the cells 6.5 h after its addition to the cul-pended in PBS with 3% FBS. Labeling was carried out

with CXC chemokine receptor 4-phycoerthyrin (CXCR4- tures (58).PE; BD Biosciences), 10 µl/1 × 106 cells for 30 min at

PAS Stain for Glycogenroom temperature. Isotype control was IgG2a, clone G155-178 (BD Biosciences). Cells were washed in buffer and Cultures of differentiated cells located in the 3D

ECM were fixed with 4% paraformaldehyde (or Car-resuspended in 1% paraformaldehyde (PFA). Sampleswere acquired on a Becton-Dickinson fluorescence acti- noy’s fluid) and stained using a commercial Periodic

acid-Schiff (PAS) staining system (Sigma) according tovated cell sorter (FACS) Calibur 4-color flow cytometerand data analyzed using Becton-Dickinson CellQuest the manufacturer’s instructions. Cultures of the cells

treated with 0.5% diastase (Sigma) before PAS stainingsoftware. Data were gated using forward versus sidescatter to eliminate debris and the resulting histograms were used as a control.plotted to reflect the mean fluorescence intensity of

PROD AssayCXCR-4 versus the IgG2a isotype control.For OCT4, α-fetoprotein (AFP), and α-1 antitrypsin The pentoxyresorufin o-dealkylase (PROD) assay is

a measure of cytochrome P450 CYP2B activity. Cul-(AAT) staining cells were fixed in 1% PFA in PBS for 1h at room temperature. Permeabilization was performed tures of differentiated cells located in the 3D ECM were

treated with phenobarbital sodium (Sigma) at a finalfor 30 min at room temperature in the permeabilization/wash buffer (R&D Systems). Antibody incubation was concentration 1 mM for 72 h. The phenobarbital was


Table 2. Real-Time PCR Primers

Gene Sequence/Cat. # Reference/Producer

Brachyury 5′-TGCTTCCCTGAGACCCAGTT-3′ Chea et al. (7)5′-GATCACTTCTTTCCTTTGCATCAAG-3′

CER1 5′-ACAGTGCCCTTCAGCCAGACT-3′ Chea et al. (7)5′-ACAACTACTTTTTCACAGCCTTCGT-3′

FOXA2 QT00212786 QuantiTect Primer Assay QiagenSOX17 QT00204099 QuantiTect Primer Assay QiagenCXCR4 QT00223188 QuantiTect Primer Assay QiagenOCT4 5′-TGGGCTCGAGAAGGATGTG-3′ Chea et al. (7)

5′-GCATAGTCGCTGCTTGATCG-3′SOX2 QT00237601 QuantiTect Primer Assay QiagenE-CAD 5′-AGGAATTCTTGCTTTGCTAATTCTG-3′ Chea et al. (7)

5′-CGAAGAAACAGCAAGAGCAGC-3′N-CAD 5′-CCCACACCCTGGAGACATTG-3′ Chea et al. (7)

5′-GCCGCTTTAAGGCCCTCA-3′AFP QT00085183 QuantiTect Primer Assay QiagenALB QT00063693 QuantiTect Primer Assay QiagenSOX7 5′-ACGCCGAGCTCAGCAAGAT-3′ Chea et al. (7)

5′-TCCACGTACGGCCTCTTCTG-3′SOX1 5′-ATGCACCGCTACGACATGG-3′ Chea et al. (7)

5′-CTCATGTAGCCCTGCGAGTTG-3′FOXF1 5′-GCCGAGCTGCAAGGCA-3′ Chea et al. (7)

5′-AACTCCTTTCGGTCACACATGC-3′BMP4 5′-GTGAGGAGCTTCCACCACGA-3′ Chea et al. (7)

5′-ACTGGTCCCTGGGATGTTCTC-3′MEOX1 5′-AGGCGGAGAAAGGAGAGTTCAG-3′ Chea et al. (7)

5′-CTCCGGCTTCCCTCTGTTC-3′FLK1 5′-ACTTTGGAAGACAGAACCAAATTATCTC-3′ Chea et al. (7)

5′-TGGGCACCATTCCACCA-3′HCG 5′-AAGGATGGAGATGTTCCAGGG-3′ Chea et al. (7)

5′-CCATGTCCCGCCCATG-3′CDX2 5′-GGGCTCTCTGAGAGGCAGGT-3′ Chea et al. (7)

5′-CCTTTGCTCTGCGGTTCTG-3′HNF4α QT00019411 QuantiTect Primer Assay QiagenAAT1 QT00077469 QuantiTect Primer Assay QiagenTTR QT00068110 QuantiTect Primer Assay QiagenPAH QT00049714 QuantiTect Primer Assay QiagenOTC QT00019509 QuantiTect Primer Assay QiagenCYP3A4 QT01672608 QuantiTect Primer Assay QiagenCYP3A7 QT00018662 QuantiTect Primer Assay QiagenCYP2B6 QT00000910 QuantiTect Primer Assay QiagenCYP7A1 QT00001085 QuantiTect Primer Assay QiagenCYP1B1 QT00209496 QuantiTect Primer Assay QiagenCYP1A1 QT00012341 QuantiTect Primer Assay QiagenCYP2D6 QT00036288 QuantiTect Primer Assay QiagenUGT2B7 QT01667554 QuantiTect Primer Assay QiagenCYCG 5′-CTTGTCAATGGCCAACAGAGG-3′ Chea et al. (7)

5′-GCCCATCTAAATGAGGAGTTGGT-3′GUSB 5′-ACGCAGAAAATATGTGGTTGGA-3′ Chea et al. (7)

5′-GCACTCTCGTCGGTGACTGTT-3′TBP 5′-TGTGCACAGGAGCCAAGAGT-3′ Chea et al. (7)

5′-ATTTTCTTGCTGCCAGTCTGG-3′ITG A1 QT00093723 QuantiTect Primer Assay QiagenITG A2 QT00086695 QuantiTect Primer Assay QiagenITG A5 QT00080871 QuantiTect Primer Assay QiagenITG B1 QT00068124 QuantiTect Primer Assay Qiagen

222 TUROVETS ET AL.

then washed away, and replaced with medium contain- that migrate through 8-µm pores, and 3D ECM on theunderside of the membrane, through which differentiat-ing the CYP2B substrate pentoxyresorufin (Sigma) at a

concentration of 10 µM. After 20 min, living cell cul- ing cells can migrate and embed. Details of use of thedevice are described in detail in Materials and Methods.tures were analyzed using fluorescence microscopy (24).

HLC Implantation in Mice Upon Undergoing EMT, Cells Acquire the Abilityto Migrate Into 3D ECMAnimal studies were performed in compliance with

institutional and NIH guidelines by Explora Labs (San Under the imposed growth factors, ECM, and surfacecues included in the differentiation protocol, the cellsDiego, CA). HLCs derived from hpSC line phESC-3

(41) were isolated from 3D ECM as described above and exhibited several hallmarks of EMT. Downregulation ofjunctional proteins is an essential part of EMT, and E-labeled with carboxyfluorescein diacetate, succinimidyl

ester (CFSE) using the Cell Trace CFSE Cell Prolifera- cadherin gene expression (Fig. 2A) in the differentiatedcells was accompanied by loss of cell surface immunolo-tion Kit (Invitrogen) according to the manufacturer’s

instructions. About 2 million cells in 50 µl Matrigel calization of the E-cadherin protein (Fig. 2B). N-cadherinis required for efficient migration and we showed upreg-diluted 1:1 with HCM (or diluted Matrigel without cells)

were injected into the spleen of the 4–6-week-old severe ulation of N-cadherin at the message (Fig. 2A) and pro-tein levels (Fig. 2C) in differentiated cells.combined immunodeficient (SCID)-beige (Bg) female

mice (Charles River). Experimental mice (n = 5) were Induction of EMT was also confirmed by characteris-tic structural rearrangement of the actin cytoskeleton.injected with labeled cells and three animals from a con-

trol group received injection of Matrigel only. Forty-two Undifferentiated stem cells had relatively few focaladhesions, a cortical arrangement of actin filaments, anddays later mice were euthanized and the livers were

either harvested for tissue sections or perfused to isolate a substantial cytoplasmic pool of paxillin (Fig. 2D). Incells that responded to the differentiation protocol, actinhepatocytes. Liver sections were embedded in optimal

cutting temperature compound (OCT; Tissue-TEK) and stress fibers replaced the cortical actin network and thefocal contact protein paxillin relocalized from a mainlysnap frozen until cryosectioning. Unfixed tissue sections

were further analyzed using fluorescence microscopy for cytoplasmic distribution to a predominantly focal adhe-sion localization at the end of well-organized actin stressthe presence of CFSE-positive cells, or fixed in 4% par-

aformaldyde and analyzed for human albumin expres- fibers (Fig. 2D). These structural rearrangements wereaccompanied by acquisition of another crucial behaviorsion using immunohistochemistry (Table 1).

To collect hpSC-derived HLCs from the grafted ani- needed for EMT—the ability to migrate. We assessedthe migration ability of undifferentiated and differenti-mals, animals were anesthetized with ketamine/xylazine

and the portal vein cannulated with a 24-gauge catheter ated cells by following the migration of cells through 8-µm pores in the differentiation device. Before differenti-(B Braun, Germany). The liver was perfused with Hanks’

balanced salt solution (Sigma) supplemented with ethyl- ation (day 0), no detectable numbers of cells wereobserved under the membrane, from the 0.6 million cellsene glycol tetraacetic acid (EGTA) (Sigma) for 3–4 min

followed by collagenase IV solution (Sigma) for 5–6 plated on top of the membrane. Under differentiationconditions, the number of cells detected under the mem-min. Perfused livers were further teased apart with nee-

dles, resuspended in Leibovitz (L-15) medium (Sigma) brane increased daily. By day 2 of differentiation about0.5 million cells reached the underside of the membranesupplemented with 10% FBS (Hyclone), and filtered

through 100-µm cell strainers (BD). Isolated hepatocytes if ECM was not applied to the system. Application of3D ECM to the underside of the membrane resulted inwere washed twice in ice-cold L-15 medium supple-

mented with 10% FBS and analyzed by flow cytometry. over 0.8 million migrated cells by day 2 (Fig. 2E). The3D ECM used in these studies was predominantly colla-

RESULTS gen I. Since basal lamina contains fibronectin, we alsotested a 3D ECM supplemented with fibronectin. With

Design of 3D Differentiation System fibronectin, the number of cells detected in 3D ECM byday 2 of differentiation was 1.5-fold higher than in theDuring vertebrate gastrulation, epiblast cells that have

acquired the mesenchymal phenotype migrate through the system with 3D ECM that did not contain fibronectin,and was 2.7-fold higher than the system with membraneprimitive streak to form DE and mesoderm (13,50,56)

(Fig. 1A). We built a differentiation device that sepa- alone (Fig. 2E). Finally, by the end of DE differentia-tion, the system containing the membrane together withrated DE from undifferentiated pluripotent stem cells

based on similar migration behavior in vitro. (Fig. 1B– 3D ECM supplemented with fibronectin promoted quitegood differentiation and migration efficacy: from 0.6D). Essential features of the device are a membrane on

which hpSCs can be cultured and segregated from cells million undifferentiated hpSCs plated, more than 1.6


Figure 1. Cell migration during definitive endoderm (DE) differentiation, in vivo and in vitro. (A) In vivo: Schematic of cellmigration through primitive streak during gastrulation. Epithelial-like cells of the epiblast (orange) undergo epithelial-to-mesenchy-mal transition (EMT) and acquire migration ability (green cells). These cells ingress through the primitive streak, replace hypoblastcells (yellow), then differentiate further to mesoderm and DE. (B) In vitro: Schematic of a 3D differentiation device that simulatesmigration through the primitive streak. Under differentiation signaling, pluripotent stem cells (orange) undergo EMT (green cells).These cells migrate through membrane pores into 3D extracellular matrix (ECM; yellow) and continue differentiation toward DEunder high-level activin A signaling. Thus, differentiated cells are separated from undifferentiated cells by the membrane and ahigh-purity population of DE is differentiated and physically isolated. (C) Hematoxylin and eosin stain of a section of paraffin-embedded, 3D differentiation system demonstrates two compartments of cells in 3D differentiation system after of 3 days ofdifferentiation, one population above and one below the membrane. (D) Immunofluorescent labeling of a section of paraffin-embedded, 3D differentiation system demonstrates identity of DE cells located below the membrane [(sex determining region Y) box17 (SOX17)-positive nuclei, green] distinct from the mixture of differentiated and undifferentiated [octamer-binding transcriptionfactor-4 (OCT4)-positive nuclei, red] cells located above the membrane. Sections were prepared after 3 days of DE differentiation.

224 TUROVETS ET AL.


million cells migrated through the membrane by day 3. 3D ECM demonstrated a temporal sequence of geneexpression similar to that which occurs in the course ofIn contrast, a negative control experiment indicated that

continued cultivation of hpSCs or hESCs using normal DE differentiation during vertebrate gastrulation.Compared to cells differentiated in the same mediagrowth medium did not produce detectable numbers of

cells below the porous membrane. in the 2D environment, the cells that migrated into 3DECM showed more rapid kinetics of downregulation ofUnder differentiation conditions, we also observed

decreased expression of integrins, the cell surface recep- pluripotency genes, significantly higher levels of endo-derm gene expression (SOX17, FOXA2, CER1, CXCR4),tors that mediate attachment of cells to the basal lamina

(Fig. 2F). This result is consistent with the observation higher peak levels of brachyury message at 24 h, andmore rapid reduction of brachyury expression by 48 hthat the cells acquired expression patterns that weakened

adherent junctions and facilitated active migration after (Fig. 3A).No consistent increases in transcript levels associatedundergoing EMT.

with extraembryonic endoderm (SOX7, AFP), mesoderm3D Differentiation System Allows Isolation [FOXF1, BMP4, mesenchyme homeobox 1 (MEOX1),of High-Purity DE fetal liver kinase-1 (FLK1)], ectoderm (SOX1, SOX2),

We characterized cells that migrated into the 3D ECM or trophectoderm [human chorionic gonadotropin (HCG),to determine their dynamic expression of DE-specific caudal type homeobox 2 (CDX2)] was observed in cellsgenes over the course of differentiation. Twenty-four embedded in 3D ECM by the end of activin A treatment.hours after the start of differentiation, brachyury, a prim- In 2D differentiation DE paradigms, hpSCs as wellitive streak marker, was expressed at high levels (Fig. as hESCs proceed through a gene expression sequence3A). Forkhead box A2 (FOXA2), cerberus 1, cysteine reminiscent of that occurring during gastrulation, as seenknot superfamily (CER1), and SOX17 transcripts, all when pluripotent stem cells undergo an EMT coincidentassociated with vertebrate DE, also exhibited a rapid with initiation of brachyury expression, and SOX17-increase in expression after the first 24 h. Expression of positive cells are derived from brachyury-positivethe chemokine receptor CXCR4 was delayed by 24 h precursors (11,55). To trace the origin of the SOX17-relative to the other DE markers, but was detectable at expressing cells in the population of cells that migrated48 h. The expression of these four DE markers was into the 3D ECM, we characterized SOX17 and brachy-maintained through day 3, but the high brachyury gene ury immunoreactivity over time. At 24 h there were aexpression was transient, and suppressed by day 2. The substantial number of brachyury-positive nuclei; by 36pluripotency genes SOX2 and OCT4 were rapidly down- h of differentiation more than half of the cells thatregulated during the 3-day differentiation (Fig 3A). expressed SOX17 were also brachyury immunoreactive,Thus, cells that migrated through the membrane into the and at 48 and 72 h the majority of cells expressed

FACING PAGE

Figure 2. Under differentiation signaling, pluripotent stem cells undergo an EMT and acquire ability to migrate. (A) RT-qPCRshows downregulation of E-cadherin and upregulation of N-cadherin expression during differentiation of human parthgenetic stemcells (hpSCs). d0 indicates results obtained from cells collected from above the porous membrane before induction of differentiation.d1, d2, d3 indicate results obtained from cells collected from the 3D ECM below the membrane, 24, 48, and 72 h after the start ofthe differentiation protocol. The y-axis indicates relative gene expression normalized to the d3 time point. Data in graphs ispresented using SD error bars. (B) Immunofluorescent labeling of undifferentiated and differentiated cultures demonstrates presenceof E-cadherin expression in undifferentiated cells before the application of differentiation signaling (0 h) and the lack of E-cadherinexpression in cells collected from the 3D-ECM, 72 h after the start of the differentiation protocol (72 h). Image is uncoupled intogreen plus blue channels (E-cadherin and DAPI). (C) Immunofluorescent labeling of differentiated cultures demonstrates expressionof N-cadherin in cells collected from the 3D ECM, 24 h after the start of the differentiation protocol. Image is uncoupled intogreen (N-cadherin, left) and green plus blue channels (N-cadherin and DAPI, right). (D) Phase contrast and indirect immunofluores-cence microscopy demonstrate cytoskeletal rearrangements characteristic of cells undergoing EMT. Each image is shown in fourversions: phase contrast (gray scale, far left), actin (red, middle left), paxillin (green, middle right), and superposition of actin,paxillin and DAPI (far right, DAPI in blue). Thirty-six hours after starting the differentiation protocol (36 h), actin stress fibershave replaced the cortical actin network present before differentiation (0 h), and the focal contact protein paxillin has relocalizedfrom the cytoplasm to focal adhesions at the ends of the actin stress fibers. Actin cytoskeleton is visualized using Alexa Fluor546 conjugated phalloidin. (E) Migration assay: Vertical bars indicate numbers of cells collected below the porous membranebefore differentiation (d0), 24 h (d1), and 48 h (d2) after the start of differentiation. Three different migration conditions are shown:membrane alone (“without 3D-extracellular matrix”), membrane with 3D ECM (“3D-extracellular matrix”), and membrane with3D ECM supplemented by fibronectin (“3D-extracellular matrix with FN”). Data in graphs are presented using SD error bars. (F)Temporal dynamics of integrin expression during differentiation of stem cells into DE determined by RT-qPCR. The y-axis showslevels of relative gene expression. d0 through d3 indicate days from the start of differentiation. Data in graphs are presented usingSD error bars.

226 TUROVETS ET AL.

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SOX17 but brachyury protein was no longer detectable determine the number of OCT4-positive cells in the pop-ulation of DE generated using the 3D differentiation sys-(Fig. 3B).

With the 3D differentiation system, we routinely tem. In three independent experiments we performedimmunohistochemical staining of the differentiated cul-observed that the overwhelming majority of cells in the

3D ECM were SOX17 positive by the end of activin A tures located on the underside of membrane usingOCT4-specific antibodies; cultures of undifferentiatedtreatment, as determined by immunocytochemistry. To

quantify the purity of the cell population, we performed cells were used as a positive control. At least 3,000nuclei were analyzed in each experiment. We did notflow cytometry analysis for the cell surface chemokine

receptor CXCR4. By the end of activin A treatment, observe a single OCT4-positive cell in the cultures iso-lated from the underside of the membrane in the 3Dmore than 90% of the cells in the 3D ECM were CXCR4

positive (Fig. 3C). In contrast, in a 2D system using the culture system. Absence of OCT4-positive cells in thefinal population of DE isolated from below the mem-same differentiation protocol, about half the cells

derived from hpSC were CXCR4 positive. brane by the end of day 3 of differentiation was con-firmed by FACS analysis (Fig. 3D).Recently published reports demonstrate that popula-

tions of DE that contain up to 80% CXCR4- or SOX17-DE Derived in the 3D System Can Be Differentiatedpositive cells can be derived from human pluripotentInto HLCstem cells using conventional 2D culture system

(5,44,54). These highly enriched DE cultures have sig- We tested the developmental competence of thederived DE cells by differentiating them further intonificantly reduced OCT4 expression (four- to fivefold)

compared to the original pluripotent cells, but undiffer- HLCs. Following activin A treatment, differentiatingcells were treated with FGF4 and BMP2, which supportentiated OCT4-positive cells remain (5,44,54), a poten-

tial source of teratomas after transplantation (23). The commitment of the ventral domain of the foregut to aliver cell fate (21,60). AFP and albumin gene expressionproblem of OCT4-positive cells that remain in the final

differentiated cultures is even more significant for became detectable on day 6 and increased continuouslyduring the course of the differentiation procedure (Fig.hpSCs using traditional 2D differentiation protocols:

after a 3-day course of DE differentiation, 50% or more 4A). AFP expression was not observed prior to day 5,as would be expected if substantial numbers of extraem-of the cells were OCT4 positive (55). Since undifferenti-

ated cells (like epithelial cells) have limited ability to bryonic endoderm cells were present in the culture. Bythe end of FGF4 and BMP2 treatment, the morphologymigrate, a major advantage of the membrane in our 3D

differentiation system is that it serves to isolate undiffer- of the cells in the 3D ECM resembled the cuboidalshapes typical of hepatocytes (Fig. 4B). Moreover, theentiated OCT4-positive cells from the population of DE

cells, confirmed by staining both cell populations on the majority of the cells from this population expressedAFP, cytokeratin 18 (CK18), and hepatic nuclear factordifferentiation device (Fig. 1C). Moreover, in the 3D

differentiation system we observed more than 11-fold 3β (HNF3β), detected by immunocytochemistry (Fig. 4C).To promote the maturation of early hepatic cellsreduction in OCT4 gene expression in the differentiated

cultures (Fig. 3A). These observations spurred us to derived in the 3D differentiation system, we used HGF

FACING PAGE

Figure 3. 3D differentiation system produces high-purity DE. (A) RT-qPCR shows temporal dynamics of marker gene expressionduring differentiation of stem cells into DE. The y-axis indicates relative gene expression in cells after migration and embeddingin 3D ECM of the device (gray bars), or from a flat plastic dish (white bars). d0 indicates results obtained from cells collectedfrom above the porous membrane or from flat plastic dish before the induction of differentiation. d1, d2, d3 data are from cellscollected from 3D ECM below the membrane, or flat plastic dish, 24, 48, and 72 h after differentiation. Data in graphs are presentedusing SD error bars. (B) Immunofluorescence labeling demonstrates coexpression of SOX17 and brachyury (BRACH) duringdifferentiation toward DE in the 3D differentiation system. After 24 h of differentiation (24 h), a majority of cells express brachyury(red). At 48 and 72 h, brachyury expression is undetectable and SOX17 expression (green) is increasing. At 36 h, the majority ofcells express both proteins (orange and yellow shades), reflecting the transition of brachyury-positive precursors into SOX17-positive DE. (C) Flow cytometry analysis of DE derived in 2D (“flat plastic dish”) and 3D (“3D-extracellular matrix”) systems.Plots show numbers of cells versus fluorescence intensity, at day 3 of differentiation, for cells collected from the 3D ECM of thedifferentiation device or from a flat plastic dish. Cells were dissociated and stained with anti-CXC chemokine receptor 4 (CXCR4)antibody. Isotype-matched control antibody staining was performed using the same cells to determine background fluorescence.(D) Flow cytometric analysis demonstrates absence of OCT4-positive cells in the DE cultures collected from the 3D ECM of thedifferentiation device at day 3 of differentiation. Undifferentiated cells cultivated under conditions that support pluripotency arepresented as positive control. Isotype-matched control antibody staining was performed using the same cells to determine back-ground fluorescence.

228 TUROVETS ET AL.

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treatment followed by oncostatin M and dexamethasone. CYP1B1 and CYP1A1 (absent or present at very lowlevels in human adult liver), at significantly higher lev-Upon addition of HGF to the culture medium, differenti-

ated cultures significantly increased AFP gene expres- els in comparison to human primary hepatocytes. HLCsexpressed very little CYP2B6, CYP2D6, CYP3A4, andsion (Fig. 4D). This increase was more than five times

higher in cells derived with the 3D system than in cells UDP glucuronosyltransferase 2 family, polypeptide B7(UGT2B7), normally expressed in adult hepatocytes.exposed to the same differentiation protocol in 2D. This

observation may be a result of higher HLC purity of the Expression of TTR, another marker of hepatocytes, wasmaintained at the same levels in all tested cells (Fig.3D cultures and/or the possibility that cells cultivated in

3D ECM system express liver-specific proteins at higher 5D). Overall, the results show that the cells are morefetal than adult in their expression profile. We observedlevels than monolayer cells differentiated on a flat plas-

tic dish. that HLCs derived in our system continue to proliferate,with up to 14% of nuclei staining for Ki67 protein, aThe HLCs derived in the 3D differentiation system

expressed a number of hepatic lineage genes including marker of the proliferating cells.To assess the ability of derived HLCs to survive inHNF4α, AAT, transthyretin (TTR), ornithine transacar-

bamylase (OTC), and phenylalanine hydroxylase (PAH) vivo we transplanted CFSE-labeled cells into immuno-deficient mice. Labeling with CFSE permits clear detec-(Fig. 4E, F). It is important to note that the levels of

expression of these hepatocyte markers were similar in tion of transplanted cells, correlates with cell function,and permits the visualization of these cells by fluores-HLCs derived from hpSCs and HLCs derived from

hESCs (Fig. 4E). These HLCs had functional character- cent microscopy (34,35). More than 40 days after trans-plantation, a significant population of CFSE cells wasistics of hepatocytes, including glycogen storage (shown

by PAS staining in Fig. 5A) and uptake and elimination detected in mice liver by flow cytometry. Moreover,three solid peaks on FACS histograms demonstrate atof IGC (Fig. 5B). A PROD assay demonstrated alkylox-

yresorufin hydrolyzed to resorufin by hpSC-derived least three successive generations of the inoculatedHLCs (Fig. 5E), consistent with the proliferative pheno-HLCs, confirming cytochrome CYP2B activity in the

cells (Fig. 5C). Real-time quantitative PCR (RT-qPCR) type. Clumps of viable CFSE-positive cells were alsoobserved in sections of the host liver (Fig. 5F). Immuno-also demonstrated CYP2B mRNA and three other P450

cytochromes, CYP3A7, CYP3A4, and CYP7A1 (Fig. histochemical analysis of these sections demonstratedpresence of the cells expressing human albumin (Fig.4E). To determine purity of the derived HLCs we per-

formed flow cytometry of the cultures located in the 3D 5G). These data indicate that HLCs derived from high-purity DE were able to migrate from the spleen, inte-ECM and stained for specific hepatocyte markers. FACS

analysis showed that the majority of cells express AFP grate into the liver, proliferate, and survive for at least42 days.and AAT; the channel increase over isotype control was

3.63-fold for AFP and 1.63-fold for AAT.3D Differentiation System Allows DerivationTo estimate the maturation stage of the HLCs, weof High-Purity DE From Different Pluripotentperformed comparative analysis of the expression levelsStem Cell Linesof genes associated with terminally differentiated pri-

mary adult human hepatocytes. As observed in Figure We tested the 3D differentiation system on five dif-ferent lines of human pluripotent stem cells, including5D, RT-qPCR analysis revealed expression of AFP (nor-

mally expressed in fetal, but not adult, hepatocytes), one line of hESCs (WA09), and four lines of hpSCs

FACING PAGE

Figure 4. Characterization of hepatocyte-like cells (HLCs) derived from DE in the 3D differentiation system. (A) RT-qPCRdemonstrates progressive upregulation of α-fetoprotein (AFP) and albumin (ALB) genes in cells collected from the 3D ECM duringdifferentiation of DE toward HLCs. The y-axis indicates relative gene expression. Days of differentiation are counted from the startof the initial differentiation from pluripotent cells toward DE. Data in graphs are presented using SD error bars. (B) Phase contrastimages show the cuboidal morphology of HLCs in the 3D ECM at day 8 of the differentiation protocol. (C) Immunofluorescentlabeling of cells located in the 3D ECM demonstrates expression of early hepatocyte markers at day 8 of differentiation. (D) RT-qPCR shows increasing AFP gene expression during differentiation toward HLCs. AFP expression is greater in cells collected fromthe 3D ECM of the differentiation device (solid line) than from a flat plastic dish (dotted line). The y-axis indicates relative geneexpression normalized to the d3 time point. Data in graphs are presented using SD error bars. (E) RT-qPCR demonstrates expressionof hepatocyte markers at the end of differentiation toward HLCs. The y-axis indicates relative gene expression in cells collectedfrom the 3D differentiation system (gray bars), normalized to that from the hepatic cell line HepG2 (white bars). Dark gray bars:HLCs derived from hpSC line phESC-3 (41); light gray bars: HLCs derived from human embryonic stem cell (hESC) line WA09.Data in graphs are presented using SD error bars. (F) Immunofluorescent labeling of cells located in the 3D ECM demonstratesexpression of albumin (ALB) and α-1-antitrypsin (AAT) at the end of the differentiation protocol.

230 TUROVETS ET AL.

Figure 5. Characterization of HLCs derived from DE in the 3D differentiation system. (A) Periodic acid Schiff (PAS) staining(pink) indicates that the derived HLCs store glycogen. Nuclei were counterstained with hematoxylin (violet). (B) Green indicatesindocyanine green (ICG) uptake by HLCs derived in the 3D differentiation system. (C) HLCs derived in the 3D differentiationsystem exhibit cytochrome P450 enzyme activity as evaluated by the pentoxyresorufin o-dealkylase (PROD) assay. Bright red inthis merged fluorescence/phase contrast image indicates nonfluorescent alkoxyresorufin has been hydrolyzed to fluorescent resoru-fin by the P450 cytochrome CYP2B. (D) RT-qPCR demonstrates expression of hepatocyte markers at the end of differentiationtoward HLCs. The y-axis indicates relative gene expression in cells collected from the 3D differentiation system (gray bars),normalized to those from human primary hepatocytes isolated from adult liver (white bars). Data in graphs are presented using SDerror bars. (E) Flow cytometric analysis demonstrates the presence of carboxyfluorescin diacetate succinimidyl ester (CFSE)-positive cells in the population of cells isolated from mouse liver 42 days after transplantation of CFSE-labeled HLCs derived in3D differentiation system (“HLC” plot). Population of cells isolated from the control liver (inoculated with a culture medium only)was analyzed to determine the background fluorescence. (F) Fluorescent microscopy analysis of frozen unfixed tissue sectionsdemonstrates the presence of CFSE-positive viable cells in mouse liver 42 days after transplantation of CFSE-labeled HLCs derivedin 3D differentiation system. (G) Immunofluorescent labeling of frozen tissue sections demonstrates the presence of cells expressinghuman albumin (ALB) in mouse liver 42 days after transplantation of HLCs derived in 3D differentiation system.


[phESC-1, phESC-3, phESC-5 (41) and hpSC-Hhom-1 ways. There may have been some direct (tropic) signal-ing from the ECM itself, promoting migration through(40)]. The results reported here were obtained using

phESC-3. However, all five stem cell lines gave similar the porous membrane, since the number of cells migrat-ing increased when ECM was added to the system, andresults, including production of high-purity DE with up

to 92% of cells positive for CXCR4, appropriate tempo- increased further still when fibronectin was added to theECM. This finding is consistent with earlier reports thatral dynamics of gene expression during differentiation

to DE, expression of appropriate DE markers, and abil- a collagen scaffold can be attractive for differentiatinghepatic cells (1).ity to differentiate further into HLCs that express hepa-

tocyte markers and perform hepatocyte functions. In addition, we speculate that the 3D cell distributionfacilitated by the 3D ECM may promote cell–cell sig-

DISCUSSION naling that approximates the interactions among cellsduring gastrulation, a theoretical advantage of 3D overThese in vitro experiments were designed to repro-

duce conditions and microenvironment encountered by 2D systems. A 3D environment, in which each cell issurrounded by similar cells, may reinforce the chemicalepiblast cells as they migrate through the primitive

streak and differentiate into DE during embryonic devel- signals that each cell experiences from its neighbors,helping to synchronize and promote differentiation ofopment. We used the migration capacity of mesendod-

erm to isolate a high-purity population of DE the entire cell population. This supposition is consistentwith our observation that, during differentiation to DE,differentiated from pluripotent hpSCs. The differentia-

tion device was straightforward, with the critical characteristic changes in gene expression were greaterin amplitude and narrower in time for the 3D systemarrangement of 3D ECM attached to the bottom of a

porous membrane. Pluripotent stem cells (hpSCs or than for the 2D system. The supposition is also consis-tent with a growing literature showing that many cellhESCs) were plated on top of the membrane, and

exposed to soluble growth factors known to direct dif- types have different secretory profiles when cultured in3D versus 2D (15).ferentiation toward DE. The cells underwent EMT by

gene expression, morphology, and behavioral criteria, Our results indicate that the cell type detected in the3D ECM by the end of activin A treatment was authen-and acquired migratory and invasive properties, as indi-

cated by mass migration of differentiated cells through tic DE. Marker analysis at the protein and RNA levelswas consistent with the formation of DE. Becausemembrane pores into 3D ECM on the underside of

membrane. The observed cell migration is very reminis- brachyury expression has not been identified in theprimitive endoderm lineage, the observation that SOX17cent of the physiological process that occurs during ver-

tebrate gastrulation, when epiblast cells ingress through expression is initiated in brachyury-positive precursors,together with the absence of SOX7 and AFP expression,the primitive streak.

Both the porous membrane and the 3D ECM appear further strengthens the conclusion that the SOX17-posi-tive cells were DE rather than primitive endoderm,important to the improved performance of the differenti-

ation device. The porous membrane was designed to which also can migrate (57). The purity of the derivedDE is very high, with flow cytometry showing moreexclude undifferentiated cells from the final cell popula-

tion. We chose a pore size smaller to the diameter of an than 90% of cells positive for CXCR4, for all stem celllines investigated. In similar studies, using 2D systemsundifferentiated cell, so that the membrane would only

be passable by cells that acquire the cytoskeletal the fraction of authentic DE cells is reportedly 50–80%for different hESC lines (11) and 50% or less forchanges necessary to migrate through the small opening,

as part of EMT. The success of the design was supported hpSCs (55).Further directed differentiation of DE cells within theby the nearly complete absence of cells below the mem-

brane before cells were exposed to differentiation cues, 3D ECM produced HLCs that stored glycogen, took upand eliminated IGC, and expressed active CYP2Beven after extended periods of cultivation under pluripo-

tency-maintaining conditions. Thus, the porous mem- enzyme. The cells also assumed the characteristic cuboi-dal shape of mature hepatocytes, and expressed a varietybrane contributed to the purity of the derived DE by

excluding undifferentiated cells throughout the growth of hepatocyte genes and proteins, including four mem-bers of the P450 cytochrome family. These results indi-and differentiation paradigms.

Numerous reports suggest that the ECM plays a criti- cate the differentiation competence of the DE cells, andthe effectiveness of the 3D ECM as an environment forcal role in regulating stem cell differentiation into differ-

ent lineages during embryonic development (6,8,14,16, cell differentiation. The full repertoire of adult cyto-chromes would be necessary for use of these cells in22,33,48), including the differentiation associated with

gastrulation. In our device, the 3D ECM may have toxicity studies, but the fetal hepatocyte phenotype maybe useful for clinical transplantation in selected pediatricenhanced the efficiency of cell differentiation in several

232 TUROVETS ET AL.

liver disease patients after further characterization. Human the yield and selectivity in any further differentiationtoward a final cell lineage. The virtual absence of OCT4-fetal hepatocyte transplantation is already practiced in

selected pediatric populations and under clinical study positive cells in the DE is an important step in develop-ing safe cell products from pluripotent stem cells.for chronic liver diseases in adults (http://clinicaltrials.

gov/ct2/show/NCT01013194). Finally, our results may help to establish hpSCs as auseful source of starting materials for stem cell technol-A major conclusion from our study is that 3D differ-

entiation conditions were superior to our own and oth- ogies. Parthenogenetic stem cells avoid some of the ethi-cal questions associated with hESCs. They may alsoers’ 2D culture systems to generate pure populations of

DE and for efficient HLC generation. During derivation reduce immunosuppression requirements for cell-basedtherapies, since they can be produced with HLA homo-of DE, the 3D system induced greater expression of

characteristic endoderm genes, better defined temporal zygosity to be histocompatible with a large segment ofthe human population. Until recently, very little waspeaks in gene expression, and a much higher percentage

of CXCR4-positive cells after activin A treatment. After known about the capacity of hpSCs for directed differ-entiation into desired cell lineages. Early studies offurther differentiation of DE to HLCs the vast majority

of cells in the 3D system performed some hepatocyte hpSCs only demonstrated their capacity for spontaneousdifferentiation in vitro and in vivo (25,28,40,41). Animalfunctions, while the 2D system produced only isolated

colonies of HLCs. studies have shown that parthenogenetic pluripotentcells can differentiate into functional cells (10,43).Our results may have several implications for future

work in stem cell and developmental biology. First, with Recently, we demonstrated the differentiation of hpSCsinto high-purity retinal pigment epithelium (RPE) thatits ability to exclude some cell types that respond differ-

entially to signaling, the 3D differentiation system may expresses appropriate RPE markers and is phagocytic(17). The RPE differentiation combined with results inallow us to derive high-purity cell populations from a

wide range of pluripotent stem cells. The consistent this report indicate that hpSCs can indeed be differenti-ated into high-purity, functional cell types.results across cell lines suggest that any pluripotent stem

cell capable of responding to direct DE differentiationSUMMARYsignaling will produce an isolated high-purity population

of DE cells in the 3D differentiation device. We describe a new differentiation device that adaptsbasic tissue engineering concepts (scaffold with pores,Second, the selectivity provided by migration through

a porous membrane, along with the physiological condi- signals from ECM, and culture in 3D) to improve thepurity of differentiated cells from hpSC and other pluri-tions provided by the 3D ECM, may be useful in a wider

range of applications, including isolation of various cell potent cells. The combination of pores and 3D ECM,in the presence of appropriate soluble signals, inducespopulations during differentiation of stem cells, isolation

of primary cell cultures from different tissues, or research migration and EMT, resulting in high-purity DE andHLCs from a range of human pluripotent stem cell lines.on cell migration and invasion, including cell ingress

into the primitive streak. The membrane pore size and Furthermore, the device isolates nonmigratory undifferen-tiated pluripotent stem cells from the final cell product.the composition of the 3D ECM can be varied to suit

the application, but the basic technique should be appli- These results also support the clinically relevant differen-tiation capacity of hpSCs. Finally, the protocols testedcable to any cell type that has migratory capacity, or to

populations of cells with different migratory capacities. here may be useful in a wide range of applications focusedon cell differentiation, and isolation of primary cells.Third, the composition of the ECM is an important

variable in cell differentiation, and this component of ACKNOWLEDGMENTS: We would like to thank Dr. Mikethe differentiation device deserves further optimization. West for providing hESC line WA09 for this research and Dr.

William Avrin for help with writing. Dr. Jeffrey Fair in partTeratani et al. (52) found that hepatocytes derived fromof the work that is connected with hESC research was sup-mouse embryonic stem cells are sensitive to ECM com-ported by NIH Award JHR R01HL082606.position, and that type I collagen may be optimal for

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