Cell Stem Cell

Article

Directed Differentiation and FunctionalMaturation of Cortical Interneuronsfrom Human Embryonic Stem CellsAsif M. Maroof,1,2,8 Sotirios Keros,4,5 Jennifer A. Tyson,3 Shui-Wang Ying,4 Yosif M. Ganat,1,2 Florian T. Merkle,8

Becky Liu,1,2 Adam Goulburn,3 Edouard G. Stanley,6 Andrew G. Elefanty,6 Hans Ruedi Widmer,7 Kevin Eggan,8

Peter A. Goldstein,4 Stewart A. Anderson,3,9,* and Lorenz Studer1,2,*1Center for Stem Cell Biology2Developmental Biology Program

Sloan-Kettering Institute for Cancer Research, 1275 York Ave, New York, NY 10065, USA3Department of Psychiatry4Department of Anesthesiology5Division of Pediatric Neurology, Department of Pediatrics

Weill Cornell Medical College, 1300 York Ave, New York, NY 10021, USA6Monash Immunology and StemCell Laboratories, MonashUniversity, Building 75, STRIP1,West Ring Road, Clayton, Victoria 3800, Australia7Department of Neurosurgery, University of Bern, Inselspital, CH-3010 Bern, Switzerland8Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA9Department of Psychiatry, Children’s Hospital of Philadelphia and University of Pennsylvania Medical School, Philadelphia, PA 19104, USA

*Correspondence: andersons3@email.chop.edu (S.A.A.), studerl@mskcc.org (L.S.)

http://dx.doi.org/10.1016/j.stem.2013.04.008

SUMMARY

Human pluripotent stem cells are a powerful toolfor modeling brain development and disease. Thehuman cortex is composed of two major neuronalpopulations: projection neurons and local interneu-rons. Cortical interneurons comprise a diverse classof cell types expressing the neurotransmitter GABA.Dysfunction of cortical interneurons has been impli-cated in neuropsychiatric diseases, including schizo-phrenia, autism, and epilepsy. Here, we demonstratethe highly efficient derivation of human cortical inter-neurons in an NKX2.1::GFP human embryonic stemcell reporter line. Manipulating the timing of SHHactivation yields three distinct GFP+ populationswith specific transcriptional profiles, neurotrans-mitter phenotypes, and migratory behaviors. Furtherdifferentiation in amurine cortical environment yieldsparvalbumin- and somatostatin-expressing neuronsthat exhibit synaptic inputs and electrophysiologicalproperties of cortical interneurons. Our study definesthe signals sufficient for modeling human ventralforebrain development in vitro and lays the founda-tion for studying cortical interneuron involvement inhuman disease pathology.

INTRODUCTION

Human pluripotent stem cells (hPSCs) are a powerful tool for

the study of human development and disease and for applica-

tions in regenerative medicine. The use of hPSCs differentiated

toward CNS lineages has been of particular interest given the

lack of effective therapies for many neurodegenerative and

neuropsychiatric disorders and the availability of protocols for

efficiently directing neuronal specification in vitro (Chambers

et al., 2009). Early studies using hPSCs have been primarily

geared toward neurodegenerative disorders, which are known

to affect specific neuron types such as midbrain dopamine

neurons in Parkinson’s disease (PD; Kriks et al., 2011) or motor

neurons in amyotrophic lateral sclerosis (ALS; Dimos et al.,

2008) and spinal muscular atrophy (SMA; Ebert et al., 2009).

More recent studies suggest the possibility of tackling complex

neuronal disorders such as schizophrenia (Brennand et al.,

2011) or autism-related syndromes (Marchetto et al., 2010;

Pasxca et al., 2011). Unlike in PD, ALS, and SMA, the neurontypes critical for modeling schizophrenia or autism are less

well defined, and no attempts have been made to direct

neuron subtype identity in those studies. There has been

considerable progress in establishing protocols for the deriva-

tion of human embryonic stem cell (ESC)-derived cortical

projection neurons (Espuny-Camacho et al., 2013; Shi et al.,

2012). However, inhibitory neurons such as cortical inter-

neurons may be particularly important in schizophrenia or

autism (Insel, 2010; Lewis et al., 2005). We have previously

demonstrated the derivation of cortical interneurons using an

Lhx6::GFP reporter mouse ESC line (Maroof et al., 2010). How-

ever, the efficiency of cortical interneuron generation was low,

and it was uncertain whether those conditions would apply for

generating human cortical interneurons from PSCs. Modeling

the development of human cortical interneurons in vitro is

of particular interest, because their developmental origin is

controversial: studies suggest considerable differences across

mammalian species (Letinic et al., 2002; Yu and Zecevic,

2011). Furthermore, the protracted in vivo development of

several cortical interneuron types (Anderson et al., 1995) repre-

sents an additional challenge for modeling their differentiation

using human cells in vitro.

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Here, we present a small-molecule-based strategy for the

efficient induction of human cortical interneurons. Exposure to

the tankyrase inhibitor XAV939 enhances forebrain differentia-

tion. Using an NKX2.1::GFP hESC reporter line, we demonstrate

the selective derivation of three distinct ventral forebrain precur-

sor populations by combining XAV939 treatment with the timed

activation of sonic hedgehog (SHH) signaling. Importantly,

results in both hPSCs and human embryos reveal differences

between mouse and human forebrain development, such as

the human-specific, yet transient, FOXA2 expression within the

ventral forebrain. Finally, mature functional properties and

the expression of late cortical interneuron markers, including

parvalbumin (PV) and somatostatin (SST), demonstrate the

feasibility of studying human cortical interneuron differentiation

from hPSCs in vitro despite their protracted development in vivo.

RESULTS

Combined Inhibition of Wnt and Activation of SHHSignaling Triggers Efficient Induction of NKX2.1+ NeuralPrecursorsThe first step in modeling cortical interneuron development is

the robust induction of forebrain precursors. Specification of

anterior and forebrain fate is considered a default program

during neural differentiation of hPSCs (Chambers et al., 2009;

Eiraku et al., 2008; Espuny-Camacho et al., 2013; Gaspard

et al., 2008). However, not all cell lines adopt anterior neural fates

at equal efficiencies (Kim et al., 2011; Wu et al., 2007). Patterning

factors secreted within differentiating cultures such as fibroblast

growth factors (FGFs), Wnts, or retinoids can suppress forebrain

induction and trigger the induction of caudal cell fates. We

recently reported that early, high-dose SHH treatment also

suppresses forebrain markers such as FOXG1 via inhibition of

DKK1 induction (Fasano et al., 2010). This is a concern for

deriving cortical interneurons that are thought to require strong

SHH activation at the NKX2.1+ progenitor stage (Xu et al.,

2010). Here, we tested whether pharmacological inhibition of

WNT signaling can improve FOXG1 induction and subsequently

enable the controlled, SHH-mediated ventralization toward

NKX2.1+ forebrain progenitor fates (Figure 1A).

Neural differentiation of hESCs via the dual SMAD-inhibition

protocol (Chambers et al., 2009) using Noggin + SB431542

(NSB) robustly induced FOXG1+/PAX6+ precursors. Replace-

ment of Noggin with the ALK2/3 inhibitor LDN-193189 (LSB)

induced PAX6 expression equally well but showed a trend to-

ward lower percentages of FOXG1+ cells (Figures 1B and 1C).

Adding recombinant DKK1 or the tankyrase inhibitor XAV939

(Huang et al., 2009), inhibitors of canonical Wnt signaling,

enhanced FOXG1 expression in LSB-treated cultures (Figures

1B–1D). Importantly, the effect of XAV939 on FOXG1 induction

was consistent (Figure 1E) across multiple independent hESC

(HES-3 and WA-09) and human induced PSC (iPSC) lines (C72

line, Papapetrou et al., 2009; SeV6, Kriks et al., 2011). Therefore,

the use of three small molecules (XAV939, LSB, and SB431542;

termed XLSB) enables rapid and robust induction of forebrain

fates across human ESCs and iPSC lines.

We next wanted to test our ability to induce ventral fates using

XLSB in the presence of SHH activators (Figure 1A). The tran-

scription factor NKX2.1 is a marker of ventral prosencephalic

progenitor populations (Sussel et al., 1999; Xu et al., 2004). Using

a previously established NKX2.1::GFP knockin reporter hESC

line (Goulburn et al., 2011), we observed GFP induction by day

10 following activation of the SHH pathway by recombinant

SHH (R&D Systems, C-25II) and the smoothened activator

purmorphamine (Figure 1F). We found maximal induction of

NKX2.1::GFP at day 18 upon treatment with 1 mM purmorph-

amine and 5nM SHH (Figure 1G), a condition referred to as

‘‘SHH’’ for the remainder of the manuscript. Our past studies

on the derivation of hESC-derived floor plate cells demonstrated

that early treatment with SHH (day 1 of differentiation) is critical

for the efficient induction of FOXA2 (Fasano et al., 2010). In

contrast, we observed here that cells exposed to SHH at late

differentiation stages (day 10 of the XLSB protocol) remain

competent for inducing NKX2.1::GFP as measured by fluores-

cence-activated cell sorting (FACS) (Figure 1H). Therefore,

XLSB+SHH treatment represents a strategy for generating

NKX2.1+ progenitors at high efficiencies.

Timing of SHH Exposure Induces Distinct VentralProgenitor PopulationsThe efficient induction of NKX2.1 largely independent of the

timing of SHH exposure raised the question of whether timing

impacts the subtype of NKX2.1+ neural progenitors generated.

Analogous to rodents, NKX2.1 is expressed during early neural

human development in both the FOXG1+ telencephalon and

the FOXG1-negative ventral diencephalon (Figure 2A; Kerwin

et al., 2010; Rakic and Zecevic, 2003). During embryonic

mouse development, NKX2.1 expression in the telencephalon

(FOXG1+) is restricted to the ventral (subcortical) domain. In

contrast, reports in human fetal tissue suggested that NKX2.1

may not be restricted to the ventral forebrain, instead extending

into the dorsal forebrain and cortical anlage (Rakic and Zecevic,

2003; Yu and Zecevic, 2011). However, those studies were

largely based on the analysis of second-trimester human

fetuses, making it difficult to distinguish whether NKX2.1+ cells

were induced in the dorsal forebrain or migrated dorsally after

induction in the ventral domain (Fertuzinhos et al., 2009). By

performing immunocytochemical analyses in early-stage human

embryos (Carnegie stage 15 [CS15],�38 days post conception),we observed restriction of NKX2.1 expression to the ventral

forebrain (Figures 2B–2E) comparable to the developing mouse

CNS. The NKX2.1 domain in the human embryonic ventral

forebrain was further subdivided into an OLIG2+ ganglionic

eminence and an OLIG2-negative preoptic-area anlage (Figures

2C and 2D).

To address whether in vitro timing of SHH exposure affects the

regional identity of the resulting hPSC-derived NKX2.1::GFP+

cells, we compared the identities of sorted cells at day 18 of

differentiation (Figure 2F). In agreement with our previous results

on floor-plate induction (Fasano et al., 2010), we observed that

early SHH exposure (days 2–18) suppressed FOXG1 induction

(Figure 2G, left panel) despite robust induction of NKX2.1::GFP

(see Figure 1H). In contrast, both day 6–18 and day 10–18

treatment groups showed expression of FOXG1 but differed

in OLIG2 expression (Figure 2G, middle and right panels).

We hypothesize that differential expression of FOXG1 and

OLIG2 reflect the distinct NKX2.1+ precursor domains observed

during early human development (Figures 2A–2E) and mimic the

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Olig2+ domains during mouse forebrain development (Flames

et al., 2007). More than 90% of the NKX2.1::GFP+ cells in the

6–18 and 10–18 protocols coexpressed FOXG1, and nearly

80% of the GFP+ cells in the 10–18 protocol coexpressed

OLIG2 (Figure 2H).

The impact of the timing of SHH exposure on the derivation of

specific ventral precursor populations was further examined by

global transcriptome analysis (Figures 2I–2K; Table S1 available

online; all raw data are available in theGene ExpressionOmnibus

[GEO], http://www.ncbi.nlm.nih.gov/geo/). A direct comparison

of day 10–18 versus untreated cultures (no SHH) confirmed

the robust induction of ventral specification markers such as

NKX2.1, NKX2.2, ASCL1, SIX6, OLIG2, and NKX6.2, which

were induced at the expense of dorsal forebrain markers such

Figure 1. Wnt Inhibition and Activation of SHH Signaling Yields Highly Efficient Derivation of Forebrain Fates and NKX2.1 Induction

(A) Schematic of the differentiation protocol in the dual-SMAD inhibition paradigm for generating anterior neural progenitors.

(B–E) When either DKK1 or XAV939, both Wnt-signaling antagonists, was added to the dual-SMAD inhibition protocol (DLSB or XLSB), there was a significant

increase in the percentage of FOXG1+ cells (B) without a loss of PAX6 expression (C): **p < 0.01; ***p < 0.001; n.s., not significant; using ANOVA followed by

Scheffe test. (D) Representative immunofluorescent image for FOXG1 (red) and PAX6 (green) expression at day 10 following XLSB treatment. Single-channel

fluorescent images of the marked region are shown in the three right panels. (E) Robust telencephalic specification using XLSBwas also observed at comparable

efficiencies in hiPSCs (lines SeV6 and C72; n = 4).

(F–H) Addition of SHH signaling to the XLSB protocol significantly enhanced the production of NKX2.1::GFP-expressing progenitors. (F) Added from day 4, 5 nM

SHH (C24II) and 1 mm purmorphamine showed synergistic effects in inducing NKX2.1::GFP expression at day 10 (***p < 0.001, compared to SHH). A range

of concentrations of SHH and purmorphamine are compared at day 18 in (G), and again, cotreatment was greatly superior to quite high concentrations of

either SHH or purmorphamine alone (***p < 0.001, compared to no SHH, using ANOVA followed by Scheffe test). (H) Delaying the timing of SHH exposure

between 2 and 10 days of differentiation did not dramatically affect the efficiency of NKX2.1::GFP induction measured at day 18 (***p < 0.001 compared to 0–18,

using ANOVA followed by Scheffe test). P, purmorphamine; S, sonic hedgehog.

Data are from hESC line HES-3 (NKX2.1::GFP) in (B), (C), and (F)–(H); from hESC line WA-09/H9 in (D); and from hESC line WA-09/H9 and hiPSC lines SeV6

and C72 in (E). The scale bar in (D) represents 125 mm. Data in (B), (C), and (E)–(H) are represented as the mean ± SEM.

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http://www.ncbi.nlm.nih.gov/geo/

Figure 2. Timing of SHH Exposure Determines the Regional Identity of NKX2.1::GFP-Expressing Progenitors

(A) Model of human prosencephalon (sagittal view at CS14) with expression of forebrain patterning markers based on published data (Kerwin et al., 2010).

(B–E) Coronal (oblique) hemisection of the human prosencephalon at CS15 demonstrates expression of NKX2.1, OLIG2, and PAX6. NKX2.1 and OLIG2 are

expressed in various regions throughout the ventral prosencephalon, whereas PAX6 is restricted to the dorsal prosencephalon and the eye. The expression of

these proteins is nonoverlapping, except in the ganglionic eminence (C), where OLIG2 and NKX2.1 are coexpressed. The scale bar in (B) represents 200 mm.

(F) Schematic illustration of the distinct time periods of SHH and purmorphamine treatment used in combination with the XLSB protocol.

(legend continued on next page)

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as EMX2 and PAX6 (Figure 2I). There were no significant differ-

ences in the expression of general anterior markers such as

FOXG1 and OTX2 (Figure 2I), supporting the notion that both

populations are of a forebrain identity. Comparison of day 10–

18 and day 2–18 treated cultures (Figure 2J) illustrated differ-

ences in the expression of anterior markers such as FOXG1

versus diencephalic markers such as RAX. In contrast, day 10–

18 versus day 6–18 treated cultures showed less pronounced

differences in forebrain marker expression (Figure 2K). Overall,

our data demonstrate that the window of SHH treatment signifi-

cantly impacts the identity of hPSC-derived NKX2.1 precursors.

One surprising finding from the gene-expression data was the

induction of FOXA2 in all SHH-treated cultures (Figure 2I).

Expression of FOXA2, a marker thought to be largely restricted

to the floor plate within the developing CNS, was confirmed at

day 18 of differentiation in NKX2.1::GFP+ cells of all three SHH

treatment paradigms (Figures S1A and S1B). To address

whether FOXA2 expression represents an in vitro artifact

following strong extrinsic activation of SHH signaling or whether

FOXA2 expression occurs in the telencephalon during in vivo

development, we performed histological analyses in the devel-

oping mouse and human forebrain. In mouse embryos (embry-

onic day 11.5 [E11.5]), FOXA2 expression within the mouse

prosencephalon was segregated from the FOXG1 and NKX2.1

domains (Figure S1C). However, immunohistochemical analysis

for FOXA2 in primary human forebrain tissue (CS15 embryo)

confirmed expression in the telencephalic NKX2.1+ ganglionic

eminence (Figures S1D–S1F). These in vivo data are in agree-

ment with the in vitro hPSC differentiation data demonstrating

(transient) FOXA2 expression during human ventral forebrain

development.

Neuronal Differentiation and Migratory Properties ofhESC-Derived NKX2.1+ PrecursorsCortical interneurons are important for balancing excitation and

inhibition within cortical circuitry and play critical roles in control-

ling developmental plasticity. Cortical interneuron dysfunction

has been implicated in various pathological conditions such as

epilepsy, autism, and schizophrenia (Baraban et al., 2009; Insel,

2010; Lewis et al., 2005; Peñagarikano et al., 2011). Our data

demonstrate that day 10–18 treated cells express cortical inter-

neuron progenitor markers such asOLIG2,MASH1, andNKX6.2.

At day 18 of differentiation, NKX2.1+ cells exhibit progenitor cell

properties based on Nestin and Ki67 expression. Prolonged cul-

ture in the absence of extrinsic SHH activation led to a gradual

decrease in Ki67 expression (Figures 3A–3C, upper panels)

and increased percentages of cells expressing the neuronal pre-

cursor markers DCX and TUJ1 (Figures 3A–3C, lower panels). In

addition, many GFP+ cells in the 10–18 and 2–18 conditions ex-

press g-aminobutyric acid (GABA) and subsequently calbindin

(Figures 3A–3C, lower panels), a marker of tangentially migrating

cortical interneuron precursors (Anderson et al., 1997). Impor-

tantly, western blot analysis at day 32 showed that LHX6 protein,

a marker of postmitotic, migratory cortical interneuron precur-

sors in the mouse, was selectively enriched in the 10–18 condi-

tion. In contrast, the hypothalamic marker RAX was selectively

enriched in the 2–18 condition (Figure 3D). Additional immunocy-

tochemical data showed expression of DLX2 and ASCL1 in most

neuronal progeny derived from the day 10–18 NKX2.1::GFP+

progenitors (Figures 3E–3G). These data indicate that following

withdrawal of extrinsic SHH activators, NKX2.1::GFP+ progeni-

tors give rise to region-specific postmitotic neurons including

immature cortical interneurons.

We next asked whether hESC-derived putative cortical inter-

neuron precursors (day 10–18 group) exhibit the characteristic

migratory potential observed for primary cortical interneurons

in the mouse (Anderson et al., 1997) and human (Letinic et al.,

2002) brain. In both species, major subclasses of cortical inter-

neurons undergo tangential migration on their way from the

ventral telencephalon into the cortex (Anderson et al., 1997; Fer-

tuzinhos et al., 2009; Letinic et al., 2002). NKX2.1::GFP+ cells at

day 32 of differentiation were collected via FACS and injected

into forebrain slices isolated from E13.5 mouse embryos. The

cell injection was carefully targeted to the medial ganglionic

eminence under microscopic visual guidance (Figure 3H). The

migratory potential of NKX2.1::GFP+ cells and their ability to

reach the cortex (see zone 2 in Figure 3H) were assessed for

each treatment group (2–18, 6–18, and 10–18 of SHH treatment;

Figures 3I–3L). At day 2 (Figure 3M), and more pronounced at

day 6 after injection (Figure 3N), GFP+ cells were observed

migrating from the injection site toward the cortex (zone 2).

Remarkably, human cells from the 10–18 treatment, but not

the two other treatment groups, showed a robust propensity

formigration into the neocortical portions of themurine slice (Fig-

ures 3I–3N). These data suggest that day 10–18 treated cells

exhibit migratory properties previously described for interneuron

precursors derived from the primary mouse medial ganglionic

eminence (MGE) (Sussel et al., 1999; Wichterle et al., 1999).

To further probe the migratory capacity of the day 2–18

versus 10–18 treated cells, we transplanted FACS-isolated

NKX2.1::GFP+ cells into the neonatal neocortex of genetically

immunocompromised mice. Four weeks after transplantation

we observed extensive migration in both the radial and tangen-

tial planes within the mouse cortex, but only for the day 10–18

group (Figures 3O and 3P). Similar results were obtained upon

transplantation into the adult cortex (Figure S2), though overall

migration of NKX2.1::GFP+ cells by day 30 in vivo was less

extensive compared to the neonatal grafts.

(G andH) Immunofluorescence for OLIG2 and FOXG1 in theNKX2.1::GFP line at day 18 of differentiation. Treatmentwith SHH after day 6 (6–18 and 10–18 groups)

significantly increases the percentage of NKX2.1::GFP+ cells that coexpressed FOXG1, as quantified in (H). The scale bar in (G) represents 50 mm. Treatment

with SHH after day 10 (10–18 group) enhanced the derivation of NKX2.1::GFP+ cells coexpressing OLIG2 (data are the mean ± SEM; *p < 0.05, ***p < 0.001,

compared to 2–18 using ANOVA followed by Scheffe test). Expression of FOXG1, NKX2.1, and OLIG2 indicates a pattern characteristic of the ganglionic

eminence (Tekki-Kessaris et al., 2001).

(I–K) Microarray data from cells sorted for NKX2.1::GFP expression at day 18 of differentiation, comparing gene-expression levels between the SHH day 10–18

protocol versus no SHH (I), the day 10–18 versus the day 2–18 protocol (J), and the day 10–18 versus day 6–18 protocol (K). Red bars indicate genes expressed at

higher levels in the SHH 10–18 protocol; blue bars indicate genes expressed at lower levels in the day 10–18 protocol. All changes are significant at p < 0.001.

See also Figure S1 and Table S1.

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To determine the extent of interneuron precursor maturation

in vivo, we assessed morphological appearance and the expres-

sion of interneuron-related markers in the grafted cells from the

day 10–18 group (Figure S3). At 1–2 months after grafting into

the neonatal cortex most human cells retained undifferentiated

bipolar or unipolar morphologies and NKX2.1 expression.

Figure 3. Conversion of Cycling Neural Progenitors to Neuronal Precursors and Assessment of Their Migratory Potential

(A–C) At day 18, cells from the three indicated protocols were subjected to FACS forNKX2.1::GFP expression, then replated and evaluated for colabeling with the

markers indicated. For all three protocols there was a decline in colabeling with markers of progenitors (upper panels: red line, nestin; blue line, Ki67) and an

increase in markers of neuronal differentiation (lower panels: green, GABA; yellow, TUJ1; purple, doublecortin [DCX]; pink, calbindin). Data are the mean ± SEM.

(D) Western blotting showed an increase in the hypothalamic-enriched protein RAX in the 2 to 18 condition and an increase in the MGE-enriched protein LHX6 in

the 10 to 18 condition. Cells were sorted for NKX2.1::GFP prior to analysis.

(E–G) At day 32, many of the NKX2.1::GFP+ cells from the 10 to 18 condition also expressed DLX2 and ASCL1.

(H–N) Grafting of day 32-sortedNKX2.1::GFP+ cells into theMGE of E13.5 coronal mouse slice cultures. (H) Schematic of coronal hemisection demonstrating the

site of transplantation and the zones for quantification of migration. (I and J) In both the 2 to 18 and the 6 to 18 conditions, very few cells migrated into zone 1, and

fewer into zone 2. (K and L) Only the 10 to 18 condition demonstrated significant and robust migration into the cortical and striatal regions, with many GFP+ cells

exhibiting bipolar morphologies consistent with a migratory cell (L). The regions where GFP+ cells were detected were quantified 2 (M) and 6 (N) days post

transplantation (DPT). Data are the mean ± SEM; *p < 0.05, **p < 0.01 using ANOVA followed by Scheffe test.

(O and P) Transplantation of day 32-sorted NKX2.1::GFP+ cells (day 10 to 18 protocol) into the neocortex of neonatal mice followed by their evaluation in fixed

sections at postnatal day 30. In marked contrast to the MGE-like cells from the SHH 10–18 protocol (P), neither the SHH 2–18 protocol (O) nor the SHH 6–18

protocol (data not shown) resulted in extensive migration from the graft site. The scale bar in (P) represents 200 mm.

See also Figures S2 and S3.

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Furthermore, essentially all grafted cells expressed the neuronal

precursor marker DCX, including those with multipolar morphol-

ogies (Figure S3). As in rodents, the large majority of human

interneuron precursors expressed GABA (46/51 GFP+ cells

located at the section surface; Figure S3). In contrast, there

was no colabeling for PV or SST, two proteins marking the

major subclasses of Nkx2.1-lineage interneurons. These data

suggest that even by 6 or 8 weeks posttransplant, the Nkx2.1-

GFP+ cells had not yet differentiated into mature interneurons

(Figure S3).

One explanation for the apparent lack of differentiation of

the grafted GFP+ cells in the mouse host cortex could be that

NKX2.1 is downregulated following maturation, as occurs in

mouse cortical interneurons (Marin et al., 2000), resulting in the

loss of GFP expression. For testing this possibility, sections

were examined for expression of SC121, a human-specific pan-

neuronal marker (Kelly et al., 2004). As expected, all GFP+ cells

also expressed SC121 (Figure S3). At 6 weeks after transplan-

tation, less than 10% of human cells were single positive for

SC121 and lacked GFP expression (25 out of 372 SC121+ cells).

In sum, perhaps due to the protracted rate of cortical inter-

neuron maturation in vivo, transplantation studies did not result

in mature cortical interneurons within the first 2 months of

transplant.

Phenotypic and Synaptic Maturationof NKX2.1+ Neurons In VitroWe have previously studied cortical interneuron development

using a coculture system in which mouse embryonic MGE-

derived progenitors are plated over a ‘‘feeder culture’’ com-

posed mainly of mouse cortical pyramidal neurons and glia (Xu

et al., 2004). To determine whether aspects of human inter-

neuron maturation would be accelerated in this system relative

to the xenografts, NKX2.1::GFP+ cells were collected via FACS

at day 32 and replated onto cultures of dissociated embryonic

mouse cortex (Figure 4A). The cortical cells were isolated

from E13.5 mouse embryos, a stage prior to the immigration of

the ventrally derived interneurons into the dorsal neocortex

(Anderson et al., 1997). Thus, this coculture system mimics

aspects of normal development with human NKX2.1+ cells

developing in the presence of the glutamatergic cortical pyrami-

dal neurons. Studies were performed in parallel using GFP+ cells

derived from each of the three SHH treatment regimens (Figures

4B–4E). Roughly 80% of the GFP+ cells from the 10–18 treated

‘‘MGE-like’’ cultures, versus 40% from the 2–18 ‘‘hypothalam-

ic-like’’ culture and 15% from the 6–18 culture, expressed

GABA. Likewise, the 6–18 culture that is enriched for telence-

phalic (FOXG1+) cells but lacks expression of the interneuron

precursor marker LHX6 (Figure 3D) was enriched for choline

acetyltransferase (ChAT) neurons.

We next determined whether GFP+ cells from day 10–18

treated cultures (enriched for GABA neurons) undergo

GABAergic synaptic transmission. Whole-cell patch-clamp

electrophysiological studies (Figures 4F–4K) of identified GFP+

cells showed spontaneous firing in both day 10–18 and day 6–

18 treated cultures (Figures 4H and 4I). However, only the day

10–18 group showed modulation of the firing rate of NKX2.1::

GFP+ cells in response to the GABA type A (GABA-A) receptor

antagonist bicuculline (Figure 4J). Because the cortical feeders

contained very few murine interneurons (in both day 10–18

and day 6–18 coculture conditions), these results indicate that

hPSC-derived GABAergic neurons mediate the inhibitory synap-

tic output. Accordingly, in the day 6–18 cultures in which

GABAergic neurons are more rare (Figure 4C), the firing rates

following bicuculline exposure remained unchanged (Figure 4K).

To further analyze synaptic inputs onto the NKX2.1::GFP+

cells from the day 10–18 protocol, we examined spontaneous

postsynaptic currents and localization of inhibitory or excitatory

synaptic markers. GFP+ cells cultured on the mouse cortical

feeder for 30 days expressed high levels of vesicular GABA

transporter (VGAT), present within the presynaptic terminal

of GABAergic synapses. The subcellular localization of VGAT

within GFP+ cells closely matched expression of the GABAergic

postsynaptic marker gephyrin (Figures 5A–5E). Whole-cell

patch-clamp analyses demonstrated that NKX2.1::GFP+ cells

receive spontaneous inhibitory postsynaptic currents (sIPSCs;

Figure 5F), which are reversibly blocked by the addition of the

GABA-A receptor antagonist bicuculline. In addition, NKX2.1::

GFP+ cells also receive excitatory inputs, as demonstrated

by the presence of vesicular glutamate transporter 1 (VGLUT1)

expression adjacent to GFP+ putative dendrites and colabeling

with the postsynaptic excitatory synapse marker PSD-95

(Figure 5G–5K). Consistent with the presence of glutamatergic

synaptic inputs, spontaneous excitatory postsynaptic currents

(sEPSCs; Figure 5L) were also readily detected in the NKX2.1::

GFP+ neurons. Additional analysis of spontaneous synaptic

activity in NKX2.1::GFP+ cells is presented in Figure S4 and

Table S2.

Cortical interneurons include a diverse set of neuron types

with distinct roles in cortical development and function (Ba-

tista-Brito and Fishell, 2009). The relatively rapid pace of matura-

tion in our mouse cortical feeder system allowed us to assess

coexpression of more mature interneuron markers and to

define neurotransmitter phenotypes beyond GABA and ChAT.

In the day 2–18 group we observed a large percentage of

GFP+ cells expressing tyrosine hydroxylase (TH) (Figure 6A),

consistent with the dopaminergic nature of NKX2.1-lineage

hypothalamic neurons (Yee et al., 2009). Immunohistochemistry

showed high percentages of neuronal nitric oxide synthase

(nNOS) cells in GFP+ cells from the day 2–18 and Calbindin in

those from the day 10–18 group (Figures 6B and 6C). We also

observed expression of markers characteristic of differentiated

cortical interneurons, including SST and PV (Figures 6D and

6E), again mainly in the day 10–18 group. Representative

images for each subgroup marker, coexpressed together with

NKX2.1::GFP, are presented in Figures 6F–6J. No vasoactive

intestinal polypeptide (VIP)- or calretinin-expressing neurons

were observed, suggesting a lack of cells exhibiting features of

the caudal ganglionic eminence (Xu et al., 2004). The derivation

of NKX2.1+/PV+ neurons was particularly remarkable given the

late developmental expression of this marker in cortical inter-

neurons in vivo (Zecevic et al., 2011). Only a subset of the cells

counted as GFP+/PV+ by the automated image analyses

(Operetta high-content scanner, see Experimental Procedures)

showed high levels of PV expression and exhibited inter-

neuron-like morphologies (about 5% of total GFP+ cells, Fig-

ure 6J). Our results point to the need for further optimization

of the derivation of selective cortical interneuron subtypes.

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However, the fact that PV+ hPSC-derived neurons could be

generated at all suggests that the mouse cortical coculture

strategy facilitates expression of cortical interneuron subtype

markers.

Given the protracted maturation of Nkx2.1-GFP+, putative

interneuron precursors in vivo, we were surprised to obtain

morphological, marker-based, and physiological evidence of

interneuron-like differentiation in the coculture system. We next

tested whether similar results can be obtained by growing the

same Nkx2.1::GFP+ cells on cultures enriched for human

cortical projection neurons. The human cortical feeders were

obtained upon differentiation of hESCs (modified XLSB condi-

tions) followed by FACS for CD44�/CD184+/CD24+ neurons(Yuan et al., 2011) (Figures S5A–S5D). The cortical identity of

the hESC-derived neurons was confirmed by the expression

of VGLUT1, MAP2, CTIP2, and SATB2. Only very few GFAP+

or Nestin+ cells were present under those conditions (Figures

S5E–S5T).

Figure 4. Maturation of NKX2.1+ Cells into Physiologically Active Neurons

(A) Preparation of cortical excitatory neuron cultures from E13.5 mice, onto which human NKX2.1::GFP+ cells (after FACS at day 32) are plated. The coculture

system was critical for promoting neuronal maturation given the protracted in vivo maturation rates of NKX2.1+ cells.

(B–E) After 30 DIV, cultures from the SHH 10–18 conditions were enriched for NKX2.1::GFP+ cells that coexpress GABA (B), quantified in (C) (mean ± SEM;

*p < 0.05, **p < 0.01 using ANOVA followed by Scheffe test). In contrast, only the SHH 6–18 condition was enriched for NKX2.1::GFP colabeling with ChAT (D),

quantified in (E) (mean ± SEM; *p < 0.05 using ANOVA followed by Scheffe test).

(F and G) Spiking patterns of SHH day 10–18 (F) and SHH day 6–18 (G) neurons recorded at 28 DIV. Action potentials were initiated by protocols shown at

the bottom.

(H–K) Spontaneous spiking was recorded from cultures enriched for GABAergic (SHH day 10 to 18) and cholinergic (6 to 18) neurons in the absence (H and I) and

the presence (J and K) of the GABA-A receptor antagonist bicuculline. Bicuculline had little effect on the spontaneous firing activity in the 6 to 18 condition,

consistent with the lack of GABAergic cells from either the mouse feeder or the human NKX2.1::GFP+ cells generated by this protocol.

The scale bars in (B) and (D) represent 50 mm.

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Interestingly, we observed a consistent difference in electro-

physiological maturation of NKX2.1+ cells plated on mouse

versus human cortical feeders as shown in Table S2. Recordings

were obtained at either 14–16 days in vitro (DIV) or 20–30 DIV

for the mouse feeder condition and 20–30 DIV for the human

cortical feeder condition. Analysis of the basic electrophysio-

logical properties of both the 10– 18 and 6–18 groups plated

on mouse feeders showed more mature characteristics at

later time points of differentiation. There was significant hyper-

polarization of the resting membrane potential (RMP) with time,

as well as a decrease in the input resistance (Ri). In addition,

the action potentials became faster and narrower, as evidenced

by the decrease in the rise time and half-width. In contrast, GFP+

neurons from the day 10–18 condition that were recorded

20–30 days after plating on the human feeder layer retained

relatively immature characteristics, comparable to the mea-

surements seen in the younger 14–16 DIV neurons plated onto

a mouse feeder layer. The RMP, action potential rise time, action

potential half-width, and Ri were significantly different in the

cortical feeder condition compared to the 10–18 neurons on

mouse feeders recorded at the same DIV range. Sample traces

depicting qualitative firing properties under the various condi-

tions are shown in Figure S6.

These preliminary findings suggest that the pace of functional

maturation is influenced by the local environment, though the

corresponding mechanisms remain to be determined. Although

it is possible that species-specific maturation rates trigger

the timing of cortical interneuron maturation (e.g., by regulating

activity), it is also possible that there are simply more astrocytes

in mouse versus human feeders (Johnson et al., 2007). Future

studies should include coculture with primary cortical neurons

of human embryonic origin for further corroboration of our

Figure 5. NKX2.1::GFP+ GABAergic Interneurons Receive Both Excitatory and Inhibitory Synaptic Inputs

(A–E) Collapsed z stack confocal image showing NKX2.1::GFP+, the vesicular GABA transporter (VGAT; red in A), and the postsynaptic GABAergic marker

gephyrin (blue in A). The dendrites of this GFP+ cell that colabel with gephyrin are receiving VGAT-expressing presynaptic terminals (arrows). In addition, a GFP+

axonal process formed a VGAT+ presynaptic terminal adjacent to a GFP-negative, gephyrin-expressing postsynaptic process (asterisk).

(F) Whole-cell patch clamp reveals sIPSCs recorded from an NKX2.1::GFP+ neuron (SHH 10 to 18 protocol), which are reversibly blocked by the addition of the

GABA-A receptor antagonist bicuculline.

(G–K) Collapsed z stack confocal image showingNKX2.1::GFP, VGLUT1 (red in G), and the postsynaptic marker PSD-95 (blue in C). This GFP+ cell has dendrites

that colabel with PSD-95 and are adjacent to VGLUT1-expressing presynaptic terminals. Note the presence of a GFP-negative cell expressing VGLUT1 (red in G,

arrowheads), confirming the presence of excitatory glutamatergic neurons in the culture. (L) Consistent with the apparent presence of glutamatergic synaptic

inputs, sEPSCs were detected in theNKX2.1::GFP+ neurons (10 to 18). All cells were plated on mouse cortical feeder following FACS forNKX2.1::GFP at day 32.

See also Figure S4.

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findings. Independent of the mechanism, our data demonstrate

that the mouse coculture conditions enhance maturation of

hESC-derived cortical interneurons and enable functional

in vitro studies.

DISCUSSION

The use of WNT-inhibitory molecules such as DKK1 has previ-

ously been proposed for the derivation of telencephalic and optic

progenitors during mouse and human ESC differentiation

(Lamba et al., 2006; Watanabe et al., 2005). We demonstrate

that the tankyrase inhibitor XAV939 (Huang et al., 2009) can

replace recombinant DKK1 for enhancing forebrain induction

using the dual-SMAD inhibition protocol. The use of only three

small molecules (XLSB protocol) renders our induction condi-

tions well defined, cost effective, and robust across multiple

cell lines. XLSB thus represents a general platform for generating

forebrain lineages including cortical interneurons.

A recent paper demonstrated the importance of SHH dosage

during in vitro ventral forebrain specification toward GSX2+,

NKX2.1-negative progenitors capable of generating medium

spiny striatal neurons (Ma et al., 2012). In addition to the SHH

dose, timing can also affect the efficiency of ventral cell-fate

specification (Fasano et al., 2010). A remarkable feature of the

current study is that the difference in timing of SHH activation

is sufficient for triggering the generation of distinct ventral

progenitors of divergent anterior-posterior identity. We have

previously reported the derivation of hESC-derived progenitors

expressing markers of the hypothalamic anlage (Kriks et al.,

2011) following early activation of SHH signaling in the presence

of FGF8. Our day 2–18 data suggest that addition of FGF8 is not

critical for directing hypothalamic progenitor fate. Cholinergic

neuron derivation from NKX2.1::GFP+ progenitors of the 6–18

group should be of great value for modeling and treating disor-

ders associated with loss of cognitive function. For example, in

Alzheimer’s disease basal forebrain cholinergic neurons are

among the neurons most vulnerable during early stages of the

disease (Whitehouse et al., 1982). However, the main focus of

the current study is the derivation of human cortical interneuron

lineages triggered in the day 10–18 protocol.

Our study points to several surprising differences in human

versus mouse forebrain development, such as human-specific

expression of FOXA2 in hESC-derived ventral forebrain progen-

itors and in CS15 human forebrain tissue. We speculate that

transient FOXA2 expression reflects a prolonged requirement

for SHH signaling during human development given the obvious

differences in brain size and extended proliferation of SHH-

dependent progenitors. To further address this hypothesis, it

will be important to establish a time course of FOXA2 expression

during human ventral forebrain development and to determine

whether FOXA2 is linked to SHH expression as commonly

observed in other FOXA2+ CNS domains such as the floor plate

(Placzek and Briscoe, 2005).

Past studies have reported species differences in cortical

interneuron development, based on the presence of ASCL1+

cells within the second-trimester human fetal cortex (Letinic

et al., 2002). Those data led to the hypothesis that many, and

perhaps most, human cortical interneurons are born within the

cortex itself (Letinic et al., 2002). In contrast, studies in mice

have demonstrated a subcortical origin of most, if not all, cortical

Figure 6. Neurochemical Profiling of NKX2.1::GFP+ Cells Grown on Mouse Cortical Feeders for 30 DIV

Cells were labeled by immunofluorescence for the markers indicated, and the results were quantified in graphs (A–E: mean ± SEM; *p < 0.05, **p < 0.01, ***p <

0.001 using ANOVA followed by Scheffe test: (*) p < 0.05 when compared directly to the 6–18 group; the p value did not reach significance in the standard Scheffe

test: p = 0.08). Representative images of cellular labeling are shown in (F–J). In the SHH 2 to 18 condition, most of the cells colabeled with TH (A and F) and nNOS

(B and G). In the 10 to 18 condition, many of the GFP+ cells colabeled with calbindin (Calb; C and H), somatostatin (SST; (D and I), and parvalbumin (PV; E and J),

each of which is present in subpopulations of mature cortical interneurons in humans. All cells were plated on mouse cortical feeder following FACS for

NKX2.1::GFP at day 32. The scale bars in (F)–(J) represent 50 mm. See also Figures S5 and S6 and Table S2.

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interneurons (Fogarty et al., 2007; Xu et al., 2004). Although

our study was not specifically geared toward addressing this

issue, we found restricted NKX2.1 expression within the ventral

forebrain in CS15 human embryo and a complete lack of

expression in the dorsal forebrain (see also the Allen Brain Atlas,

http://human.brain-map.org/). Furthermore, the ability of hESC-

derived cortical interneurons to migrate in our slice culture

assay and in the developing murine cortex in vivo suggests

that human cortical interneuron precursors have a capacity for

tangential migration similar to that of their mouse counterparts.

However, a distinctive feature in hESC-derived cortical inter-

neurons was the persistent expression of NKX2.1. Nkx2.1 is

extinguished in mouse cortical interneuron precursors by the

time they leave the MGE prior to entering the cortex (Marin

et al., 2000). Our in vitro data are consistent with findings in the

human neocortex in vivo showing the presence of postmitotic

NKX2.1+ neurons (Fertuzinhos et al., 2009). However, our data

do not rule out the possibility that a subset of our human

ESC-derived cells correspond to striatal interneurons, given

that those share markers of cortical interneurons in the mouse

while retaining NKX2.1 expression (Marin et al., 2000).

Current paradigms of modeling human psychiatric disease

such as schizophrenia feature the use of patient-specific iPSC-

derived neurons (Brennand et al., 2011; Marchetto et al., 2010;

Pasxca et al., 2011). However, those published studies were per-formed in mixed neural cultures of unclear neuronal subtype

identity and with limited characterization of subtype-specific

synaptic and functional properties. There is a convergence of

postmortem findings that attempt to link genetic defects to psy-

chiatric disorders, such as the interneuron-associated Erbb4

receptor in schizophrenia (Fazzari et al., 2010). Therefore, it is

essential to have access to purified populations of mature

cortical interneurons for modeling human disease. Our results

demonstrate that the highly efficient derivation of cortical

interneurons is possible following timed exposure to develop-

mental cues.

We report that putative hESC-derived GABAergic inter-

neurons receive synaptic inputs fromboth other human interneu-

rons and from excitatory mouse projection neurons. In addition,

cells with neurochemical properties of cortical interneurons

adopt fairly mature physiological properties within 30 days of

coculture. Future studies will be required to address the mecha-

nisms of accelerated in vitro maturation of the NKX2.1::GFP+

neurons on mouse cortical cultures and to determine whether

species-specific timing factors are involved, as suggested

from our preliminary studies using hESC-derived cortical

feeders. However, our data demonstrate that the proposed

culture system can yield synaptically active cortical interneurons

in vitro, a key prerequisite for modeling cortical interneuron

pathologies in psychiatric disorders such as schizophrenia

and autism. The generation of hESC-derived PV-expressing

neurons and the presence of relatively rapid spiking, nonaccom-

modating neurons in these cultures is of particular interest given

the implications of PV interneuron dysfunction in schizophrenia

(Lewis et al., 2005). Fast-spiking PV+ cortical interneurons are

observed late during primate prenatal development and

continue their maturation into early adulthood (Anderson et al.,

1995; Insel, 2010). Given the important role of PV+ neurons

under various pathological conditions our data suggest that

disease modeling of such states may be feasible using our

differentiation strategies. Another key for the future will be the

development of protocols that allow the enriched generation of

specific cortical interneuron subgroups, such as sSST+ versus

PV+ cells. Our previous results in MGE progenitors indicate

that this decision may yet again be under the control of SHH

signaling, with higher levels promoting SST+ and lower levels

promoting PV+ neurons (Xu et al., 2010).

Finally, future studies will be required to address the full in vivo

potential of human in vitro-derived cortical interneurons for ap-

plications in regenerative medicine. Our current study illustrates

the remarkable migratory potential of the hESC-derived cortical

interneurons upon transplantation into the neonatal mouse

cortex. Transplantation studies into several adult CNS models

of disease will be of particular interest given the potential use

of cortical interneuron grafts in modulating pathological seizure

activity (Baraban et al., 2009), treating aspects of Parkinson’s

disease (Martı́nez-Cerdeño et al., 2010), and inducing learning

and plasticity within the postnatal brain (Southwell et al., 2010).

One specific challenge for such transplantation studies is the

protracted maturation of grafted hPSC-derived cortical inter-

neuron precursors in vivo. Preliminary longer-term transplanta-

tion studies into the adult mouse cortex confirm their continued

slow maturation rate, with NKX2.1+ putative interneuron pre-

cursors retaining immature growth cones and showing limited

integration at 3 months post grafting (data not shown). Those

data are in contrast to our in vitro coculture work, wherein rapid

functional integration and phenotypic maturation was readily

achieved. In sum, this study provides a framework for the gener-

ation of distinct ventral prosencephalic neuron types that can

be used to study various aspects of development and serves

as a powerful in vitro platform for studying the dysfunction of

specific neuron types implicated in a myriad of neuropsychiatric

diseases.

EXPERIMENTAL PROCEDURES

hESC Culture and Neural Differentiation

hESCs (WA-09, HES-3 (NKX2.1::GFP), and iPSCs (C72 and SeV6) were

maintained on mouse embryonic fibroblasts and dissociated with accutase

(Innovative Cell Technologies) for differentiation or dispase for passaging

(Chambers et al., 2009). Differentiation media were described previously

(Kriks et al., 2011): knockout serum replacer (KSR) and N2 medium for neural

induction, and neurobasal medium + B27 (GIBCO) and N2 supplements

(Invitrogen) for neuronal differentiation. Small-molecule compounds were as

follows: XAV939 (2 mM; Stemgent;), LDN193189 (100 nM; Stemgent),

SB431542 (10 mM; Tocris Bioscience), purmorphamine (1–2 mM; Calbiochem),

ascorbic acid (200 mM), and dibutyryl-cyclic AMP (200 mM; both from

Sigma-Aldrich). Recombinant growth factors were as follows: SHH (C25II;

50–500 ng/ml), Noggin (125 ng/ml), DKK1 (250 ng/ml), BDNF (10 ng/ml), all

from R&D Systems, and FGF2 (5–10 ng/ml; Promega).

E13.5 Slice Migration Analysis

Coronal telencephalic slices (250 mm) were prepared as described previously

(Anderson et al., 2001) andmaintained in neurobasal medium/B27 with 5 ng/ml

FGF2 (Promega). hESC-derived NKX2.1::GFP+ cells were isolated by FACS

and carefully injected (�5,000 cells) into the periventricular region of theMGE using an oocyte microinjector (Nanoject II; Drummond Scientific). After

2–6 days, cultures were fixed and immunostained. For cell counting, zone 1

was defined as the region from the ventricular zone of the lateral ganglionic

eminence extending to the pallial-subpallial border, and zone 2 was defined

as the region from the lateral cortex through the neocortex to the cortical hem.

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http://human.brain-map.org/

Mouse and Human Cortical Culture Preparation

The cortical feeder cultures were prepared by dissociating dorsal cortices

from 250mm coronal slices of E13.5 mouse brain and cultured as described

previously (Xu et al., 2010). For human cortical feeder preparation, cells were

subjected to XLSB-mediated neural induction in the presence of cylcopamine

and purified for CD24+/CD44�/CD184+ at day 32 (see Supplemental Experi-mental Procedures for details).

Gene-Expression Profiling

Total RNA was isolated at day 18 of differentiation from FACS-sorted

NKX2.1::GFP+ cells from three varying differentiation conditions and a

‘‘No SHH’’ condition that did not contain any GFP-expressing cells (Trizol,

Sigma-Aldrich). Samples in triplicate for each group were processed for

Illumina bead arrays (Illumina HT-12) by the Memorial Sloan-Kettering

Cancer Center genomics core facility according to the specifications of the

manufacturer.

Immunocytochemical Analyses

Cultures were fixed in 4% paraformaldehyde and processed for immunocyto-

chemistry. Secondary antibodies were species-specific Alexa-dye conjugates

(Invitrogen). The use of the primary antibodies is summarized in Table S3.

For the automated quantification (Operetta, PerkinElmer), see Supplemental

Experimental Procedures.

Transplantation Studies

Transplantation into the neonatal cortex of NOD-SCID IL2RG�/� mice wasconducted as described previously (Wonders et al., 2008). In brief, �50 3103 cells were injected at following coordinates from bregma (2.0mm A,

2.5mm L, 1.0mm D), targeting cortical layers 3–6. All animal experiments

were carried out in accordance with institutional and National Institutes of

Health guidelines.

Human Tissue Analysis

Tissue derived from aborted human fetuses was obtained from the Depart-

ment of Gynecology, University Bern, Switzerland. The study was approved

by the ethics committee of the Medical Faculty of the University of Bern,

Switzerland, and the ethics committee of the state of Bern, Switzerland

(no. 52/91, 71/94, and 188/95). Written consent was given by the women

seeking abortion.

Statistical Analysis

For all experiments, analysis was derived from at least three independent ex-

periments. Statistical analysis was performed using ANOVA followed by a

Scheffe test to for significance among individual groups (SYSTAT v.13).

FACS

All cells were dissociated using accutase (Stem Cell Technologies) for 30 min,

then resuspended in neurobasal medium/B27 with Y27632 (10 mM; Tocris

Bioscience) and sorted on a BD FACSAria II (gating for side scatter [SSC],

forward scatter [FSC], and DAPI exclusion). For postsort analysis, data was

processed using FlowJo (Tree Star) software.

Electrophysiology

Whole-cell voltage and current clamp recordings were performed as des-

cribed previously (Ying et al., 2007). Details are provided in Supplemental

Experimental Procedures.

ACCESSION NUMBERS

The raw data reported in this paper have been deposited in the GEO under

accession number GSE46098.

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures, three tables, and Supplemental

Experimental Procedures and can be found with this article online at http://dx.

doi.org/10.1016/j.stem.2013.04.008.

ACKNOWLEDGMENTS

Wewould like to thank J. Hendrikx (Sloan-Kettering Institute [SKI] FlowCytom-

etry Core), A. Viale (SKI Genomics Core laboratory), M. Leversha (Molecular

Cytogenetics Core), and E. Tu and M. Tomishima (SKI Stem Cell Facility) for

excellent technical support. We further thank the Department of Gynecology

(directors: M. Mueller and D. Surbek), University of Bern, Switzerland for

providing us with the human fetal tissue. Studies using the NKX2.1::GFP

hESC line were supported by grants from NYSTEM (S.A.), the European Com-

mission project NeuroStemcell (L.S.), and the C.V. Starr Foundation (S.A. and

L.S.). Experiments using hESC line WA-09 or iPSC lines were supported by

NIMH grants 2RO1 MH066912 (S.A.) and 1RC1MH089690 (S.A. and L.S.)

and by the Swiss National Science Foundation grant no. 31003A_135565

(H.R.W.).

Received: May 8, 2012

Revised: March 2, 2013

Accepted: April 12, 2013

Published: May 2, 2013

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572 Cell Stem Cell 12, 559–572, May 2, 2013 ª2013 Elsevier Inc.

Cell Stem Cell, Volume 12

Supplemental Information

Directed Differentiation and Functional

Maturation of Cortical Interneurons

from Human Embryonic Stem Cells

Asif M. Maroof, Sotirios Keros, Jennifer A. Tyson, Shui-Wang Ying, Yosif M. Ganat, Florian T. Merkle,

Becky Liu, Adam Goulburn, Edouard G. Stanley, Andrew G. Elefanty, Hans Ruedi Widmer Kevin Eggan,

Peter A. Goldstein, Stewart A. Anderson, and Lorenz Studer

Figure S1. FOXA2 Is Transiently Expressed in the hPSC-Derived NKX2.1+ Lineages and in the Human Ganglionic Eminence, Related to Figure 2 A) Time course of FOXA2 expression after sorting NKX2.1::GFP+ cells at day 18, followed by culture in the absence of SHH for two additional weeks. Data represent mean ± SEM. B) Representative immunocytochemistry image of day 10-18 treated cultures stained for FOXG1, FOXA2 and GFP (day 18). C) Embryonic day 11.5 coronal section of a developing mouse brain showing the expression of FOXG1 (red), NKX2.1 (green), and FOXA2 (D'Amato et al.). There is no expression of FOXG1 in the diencephalon (Di) and no FOXA2 expression in the telencephalon (Tel). NKX2.1 is expressed in the medial ganglionic eminence (MGE) and the

hypothalamus (HYPO), but is not expressed in the floor plate (FP) and does not co-label with FOXA2. D) Carnegie stage 15 (CS15; approximately 5.5 gestational weeks) human embryo sectioned for immunohistochemical analysis. E,F) Adjacent coronal (oblique) hemisections of the human CS15 prosencephalon demonstrating the expression of FOXA2 and NKX2.1. While FOXA2 appears to be specifically expressed in the ganglionic eminence (E) NKX2.1 is expressed in various regions of the ventral prosencephalon (F).

This figure demonstrates differences in FOXA2 expression between human and mouse, suggestive of an alternative mechanism to generate NKX2.1 progenitors in the ventral forebrain. However, FOXA2 expression in these NKX2.1 cells may be transient as shown in vitro (A) and as suggested by in situ hybridization analyses of later stage human embryos (Allen Brain Atlas, http://human.brain-map.org/).

http://human.brain-map.org/

Figure S2. NKX2.1::GFP+ Neuronal Precursors Distribute within the Adult Mouse Cortex, Related to Figure 3 NKX2.1::GFP expressing cells were sorted at day 32 of differentiation and grafted into adult IL2RG immunocompromised mice. While the 2 to 18 (A) and the 6 to 18 (B) condition produced regions of GFP expression in the cortex after 30 days, the GFP cell bodies did not leave the graft core. Only in the 10 to 18 condition (C) did GFP expressing cells leave the graft core and distribute throughout the cortical parenchyma. In addition the GFP expressing cells from the 10 to 18 condition distribute throughout the cortical layers and display neuronal morphologies (D).

Figure S3. Slow Pace of Differentiation by Nkx2.1::GFP+ Cells Following Transplantation into Neonatal Mouse Neocortex, Related to Figure 3 Shown are examples of transplants of the Nkx2.1::GFP line, differentiated to day 32 with the “MGE-like protocol (SHH 10-18), then subjected to FACS for GFP, transplanted into the neonatal neocortex of genetically immune-compromised mice, and evaluated in fixed sections after 6 weeks (see methods). A) Low-magnification view of GFP immunofluorescence labeling. Scale bar=100µm. The boxed area shows three cells at higher magnification in panel (A1). Note that even after 6 weeks of differentiation in vivo most of these cells continue to have relatively undifferentiated appearance with bipolar or unipolar processes, often tipped by growth cones

(arrow). Scale bar=20µm. B) shows immunofluorescence for the human specific cell marker SC121 (B,B1,B3) and GFP (B,B1,B2). All GFP+ cells express SC121, and most but not quite all SC121 cells at this age express GFP (arrows; ). As we see similarly low percentages of SC121+/GFP(-) cells two weeks after transplantation (not shown), it is not clear whether these cells represent downregulation of Nkx2.1 and GFP or represent GFP(-) cells that came through the FACS. B) Scale bar=35µm, B1-3 scale bar=40µm. C, C1) shows the same view of a section with immunofluorescence for GABA (red) and GFP. Two strongly co-labeled cells are indicated by the arrows. As GABA immunodetection penetrates poorly into sections, co-labeling was quantified by selectively counting only those GFP+ cells with cell bodies located within 5 microns of the cut edges of the section. By this approach, 46 of 51 cells (90.2%) from two 6-week transplants were strongly co-labeled. Scale bar=40µm. D) shows immunofluorescence for GFP, Nkx2.1 (red) and the marker of neuronal precursors DCX (blue; pseudocolored Cy5 signal). All the GFP+ cells have Nkx2.1-expressing nuclei (arrows), and also co-label for DCX. Scale bar=40µm.

Figure S4. Electrophysiological Recordings from NKX2.1::GFP+ Neurons of the Day 10–18 Group, Related to Figure 5 A) Examples of spontaneous and miniature IPSCs recorded from a day 10-18 group neuron. B) Overlay of the averaged sIPSCs (gray; n=150) and mIPSCs (black; n=56). Neurons were recorded on day 26-30 on mouse cortical feeders. C) Cumulative probability histogram of IPSCs (gray) and mIPSCs (black). D) Grouped data (mean ± SEM) comparing the frequency of sIPSPs (gray; n=\ markers (MAP2), astrocytic markers (GFAP), GABA, and cortical markers (SATB2, TBR1) in human ESC derived cortical precursors at day 30 of differentiation. All cells were plated on mouse or human ESC-derived cortical feeder following FACS for NKX2.1::GFP at day 32.

Figure S5

Figure S5. Phenotypic Characterization of Human ESC-Derived versus Primary Mouse Cortical Feeder Cells, Related to Figure 6

A-D) FACS-mediated isolation of cortical neuron precursors based on expression of CD24 and lack of expression of CD44 and CXCR4 (CD184). E-L) Immunocytochemical analysis for expression of neuronal markers (MAP2), astrocytic markers (GFAP), GABA, and cortical markers (SATB2, TBR1) in hESC-derived cortical precursors at day 30 of differentiation. M-T) Immunocytochemical analysis for expression of neuronal markers (MAP2), astrocytic markers (GFAP), GABA, and cortical markers (SATB2, TBR1) in mouse E13.5 cortical cultures. Overall, we observed comparable marker expression between primary mouse and hPSC derived cortical feeders.

Figure S6. Representative Current-Clamp Recordings from NKX2.1+ Cortical Interneurons, Related to Figure 6 A-C) Representative tracings from GFP-positive neurons from the day 10-18 treated group recorded on mouse feeders (A,B) or human ES-derived neurons enriched for putative cerebral cortical projection neurons (C). Depolarized voltage responses were initiated using protocols shown below each set of traces. The holding potential was –60 mV for all recordings. All cells were plated on mouse or human ESC-derived cortical feeder following FACS for NKX2.1::GFP at day 32. These data are related to Figure 6.

Table S1. Fold Changes and Significance of Differentially Expressed Transcripts from Figure 2I, Related to Figure 2

day 10-18 versus no SHH

day 10-18 versus day 2-18

day 10-18 versus day 6-18

Probeset F-Change p-value

Probeset

F-Change p-value

Probeset

F-Change p-value

SIX6 7.88 6.35E-09

ZIC2 9.25 1.56E-09

SOX21 3.14 3.37E-07

NKX2-1 6.92 3.40E-10

FOXG1 6.70 6.70E-05

SP8 2.81 1.40E-08

LIPA 6.47 8.53E-09

PCSK5 3.39 3.35E-05

PCDH19 2.65 2.29E-06

IFIT1 5.73 5.21E-09

ZIC3 3.19 2.25E-04

FOXG1 2.39 6.69E-05

SNORD3A 5.37 4.69E-07

NDP 3.12 2.76E-04

OTX2 2.38 5.20E-08

OLIG1 4.70 2.51E-07

WNT5B 2.66 2.90E-04

C14ORF23 2.37 5.84E-06

NKX2-2 4.62 8.69E-09

PLCH1 2.56 1.52E-05

SHISA2 2.36 6.38E-05

PTCH1 3.96 2.07E-06

KCNK12 2.54 2.74E-04

PCSK5 2.19 1.67E-05

OLIG2 3.90 1.15E-05

CDH11 2.44 4.13E-05

MYCN 2.19 1.14E-05

NKX6-2 3.74 2.12E-07

MYCN 2.41 2.18E-06

FRZB 2.12 6.43E-05

HES1 3.46 2.42E-09

REC8 2.08 1.04E-04

DOCK11 2.11 2.29E-04

ASCL1 3.00 1.42E-06

HCRT 2.08 3.20E-04

FOXO1 2.08 1.74E-04

FOXA2 2.63 6.86E-07

LMO2 2.06 2.05E-05

ALCAM 2.08 3.61E-04

SEMA3A -2.00 4.75E-04

KLF6 -2.21 4.29E-06

DCX -2.11 2.73E-04

SEMA3C -2.14 4.49E-06

EFNB2 -2.24 2.35E-04

CHRNA3 -2.69 2.69E-06

MEIS1 -2.14 1.05E-04

APOE -2.41 8.27E-05

GAP43 -2.96 5.08E-06

DLX5 -2.22 2.04E-05

RAX -2.57 2.70E-05

CTGF -3.06 1.94E-05

NR2F2 -2.64 2.63E-06

LAMB1 -2.64 1.10E-05

ANKRD1 -3.60 1.35E-06

EVI1 -2.68 3.30E-06

GAP43 -2.76 2.45E-04

POU3F1 -3.70 3.79E-07

PDGFC -2.80 4.38E-06

CTGF -4.43 9.83E-07

SYT4 -4.35 4.97E-08

DCT -3.52 9.43E-11

ANKRD1 -4.46 1.28E-07

GRP -4.59 4.02E-08

PAX6 -4.64 2.32E-08

HEY1 -4.48 2.55E-04

EMP1 -4.68 5.10E-06

GAS1 -5.43 9.25E-08

LEFTY2 -4.67 2.27E-06

NKX2-2 -4.74 7.66E-09

CCL2 -5.85 7.78E-08

EPCAM -4.93 6.11E-05

STMN2 -5.50 2.16E-09

EMX2 -6.08 1.00E-10

COL1A1 -4.98 4.33E-08

VGF -5.86 1.97E-09

ALDH1A1 -8.99 9.36E-11

EMP1 -6.14 8.08E-06

MMP7 -6.62 4.37E-09

Table S2. Electrophysiological Properties of hESC-Derived NKX2.1::GFP+ Neurons on Mouse versus Human Cortical Feeders and Averaged Properties of GABAergic Synaptic Currents in GFP+ GABAergic Interneurons, Related to Figure 6 A) Electrophysiological properties of hESC-derived NKX2.1::GFP+ neurons (day 10-18 and day 6-18 group) on mouse versus human cortical feeders (related to Figure 6).

DIV: days in vitro; RI: Input resistance; AP: action potential; AHP: after-hyperpolarization. *: P < 0.01, unpaired t-test, compared to corresponding 14-16 DIV group **: P < 0.001, unpaired t-test, compared to corresponding 14-16 DIV group †: P < 0.001, unpaired t-test, compared to 10 to 18 mouse (20-30 DIV) ††: P < 0.0001, unpaired t-test, compared to 10 to 18 mouse (20-30 DIV) Action potential properties were measured at threshold as elicited by current injection.

10 to 18 on mouse feeder 6 to 18 on mouse feeder 10 to 18 on human feeder

DIV 14-16 20-30 14-16 20-30 20-30

RMP (mV) -38.6 ± 2.4 -65.6 ± 1.6** -36.1 ± 2.4 -62.6 ± 3.6** -44.8 ± 6.6†

AP Amp (mV) 57.4 ± 8.3 66.2 ± 4.1 56.2 ± 7.3 64.2 ± 5.1 62.5 ± 5.1

AP Rise time

(ms)

1.472 ± 0.07 1.08 ± 0.03** 1.52 ± 0.08 1.11 ± 0.09* 1.9 ± 0.38†

AP ½ Width

(ms)

3.05 ± 0.06 2.42 ± 0.12* 3.02 ± 0.05 2.5 ± 0.11* 4.8 ± 0.32††

AHP peak

(mV)

10.1 ± 3.5 15.6 ± 2.4 11.2 ± 3.5 16.3 ± 2.6 8.8 ± 3.72

RI (GΩ) 1.5 ± 0.1 0.72 ± 0.05** 0.94 ± 0.05 0.68 ± 0.12 1.66 ± 0.2††

No. of cells 8 15 8 12 5

B) Averaged properties of GABAergic synaptic currents in GFP-positive GABAergic interneurons (related to Figure 6).

sIPSC mIPSC

Amplitude (pA) 62.6 ± 5.2 35.1 ± 2.3*

Rise time (ms) 2.4 ± 0.3 2.3 ± 0.4

Decay time (ms) 66.2 ± 5.4 51.2 ± 3.6 *

½ width (ms) 54.5 ± 5.3 41.3 ± 3.2*

No. of cells 8 4

Recordings were made at 26-30 DIV. *:P < 0.05, unpaired t-test

Table S3. List of Primary Antibodies and Their Sources and Concentrations Used in the Current Study, Related to Figures 1–6

Antibody Species Dilution Source Cat. No.

ASCL1 Mouse 1/250 BD Pharmingen 556604 Calbindin Rabbit 1/3000 Swant CB38a Calretinin Mouse 1/2000 Swant 6B3

ChAT Goat 1/100 Millipore AB144P DLX2 Rabbit 1/200 gift from J. Rubenstein n/a

Doublecortin Goat 1/100 SCBT sc-8067 FOXA2 Goat 1/200 SCBT sc-6554 FOXG1 Rabbit 1/500 Neuracell NC-FAB FOXG1 Rabbit 1/1000 gift from Eseng Lai n/a GABA Rabbit 1/4000 Sigma Aldrich A2052

GAPDH Rabbit 1/10000 Thermo Scientific PA1-16780 Gephyrin Mouse 1/2000 Synaptic Systems 147011

GFAP Goat 1/300 SCBT sc-6170 GFP Chicken 1/1000 Abcam ab13970 KI67 Rabbit 1/1000 Thermo Scientific RM-9106 KI67 Mouse 1/200 DAKO MIB-1 LHX6 rabbit 1/500 Abcam ab22885 MAP2 Chicken 1/10000 Abcam AB5392 Nestin Mouse 1/500 Neuromics MO15012

Nkx2.1 (TTF1) Rabbit 1/1000 Epitomics 2044-1 Nkx2.1 (TTF1) Goat 1/100 SCBT sc-8762

NOS Rabbit 1/1000 Immunostar 24287 OLIG2 Rabbit 1/1000 Millipore AB9610

Parvalbumin Mouse 1/2000 Millipore MAB1572 Parvalbumin Rabbit 1/2000 Swant PV25

PAX6 Mouse 1/250 DSHB PAX6 PAX6 Rabbit 1/1000 Covance PRB-278P

PSD-95 Rabbit 1/2000 Zymed 51-6900 RAX Mouse 1/250 Abnova H00008575-B01

SATB2 Mouse 1/100 Abcam ab51502 SC121 Mouse 1/1000 Stemcells AB-121-U-050

Somatostatin Rat 1/250 Millipore MAB354 TBR1 Rabbit 1/1000 Abcam ab31940

TH Mouse 1/1000 Immunostar 22941 TuJ1 Mouse 1/500 Covance MMS-435P VGAT Rabbit 1/2000 Synaptic Systems 131003

VGLUT1 guinea pig 1/5000 Millipore AB5905 VIP Rabbit 1/1000 Immunostar 20077

SUPPLEMENTAL EXPERIMENTAL PROCEDURES Western blot Cells were lysed with Ripa Buffer (50mM Tris- HCl pH 8.0, 120mM NaCl, 5mM EDTA, 0.5% NP-40, and a tablet of protease inhibitor). Protein concentration was measured by the Bradford Assay (Bio-Rad). For immunoblot, whole cell extract (4 x 106 cells per sample) were boiled for 5 minutes in Laemmli sample buffer before separation by electrophoresis on 4-12% Bis- Tris gel in MOPS SDS running buffer at 100V for 2 hours using a iBlot electroblotter (Invitrogen). Protein was transferred to nitrocellulose membrane using the iBlot gel transfer device (Invitrogen). Non- specific protein binding was prevented by blocking the membrane with 3% BSA and PBS-T (0.1% Tween- 20). Membrane was incubated at 4 degrees overnight in 3% BSA and PBS-T with primary antibodies (LHX6 (1:500; Abcam), RAX (1:250; Abnova), GAPDH (1:10,000; Thermo Scientific). After five washes with PBS-T (0.1% Tween-20), the blot was incubated with respective secondary antibodies (Rabbit (1:10,000), Mouse (1:20,000) at room temperature. ECL kit, Western Lightning Plus- ECL, was used for detection according to manufacturer's instruction (PerkinElmer). Secondary antibodies were all purchased from Jackson Immunoresearch. ECL kit, Western Lightning Plus- ECL, was used for detection according to manufacturer's instruction (PerkinElmer). Electrophysiology Whole-cell voltage and current clamp recordings were performed as described previously (Ying et al., 2007), and the methods were slightly modified for this study. Briefly, neurons were visualized using a Nikon microscope (ECLIPSE FN1) equipped with a 4x objective and a 40x water immersion objective and GFP-positive neurons were identified using epifluorescence optics. Neurons were recorded at 23 – 24 °C. Input resistance was measured from voltage response elicited by intracellular injection of a small current pulse (-2 or 5 pA, 500 ms). Electrical signals were obtained using a Multiclamp 700B amplifier connected to an interface using Clampex 10.2 software (Molecular Devices, Foster City, CA). Liquid junction potentials were calculated and corrected off-line (Ying and Golds