Rapid Induction and Long-Term Self-Renewal of Primitive Neural Precursors from Human Embryonic Stem Cells by Small Molecule Inhibitors

Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors

Wenlin Lia,b, Woong Sunc,d, Yu Zhanga, Wanguo Weia, Rajesh Ambasudhana, Peng Xiae, Maria Talantovae, Tongxiang Lina, Janghwan Kima, Xiaolei Wangc, Woon Ryoung Kimd, Stuart A. Liptone, Kang Zhangc,f,1, and Sheng Dinga,g,1

aDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; bDepartment of Cell Biology, Second Military Medical University, Shanghai 200433, China; cInstitute for Genomic Medicine and Shiley Eye Center, University of California, San Diego, CA 92093; dDepartment of Anatomy, Korea University College of Medicine, Brain Korea 21 Program, Seoul, 136-705, Korea; eDel E. Webb Center for Neuroscience, Aging, and Stem Cell Research, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; fMolecular Medicine Research Center and Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu 610065, China; and gGladstone Institute of Cardiovascular Disease, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158

Edited by Fred H. Gage, The Salk Institute, San Diego, CA, and approved March 28, 2011 (received for review September 20, 2010)

Human embryonic stem cells (hESCs) hold enormous promise for tion toward region-specific neuronal fates, but still could not be regenerative medicine. Typically, hESC-based applications would stably maintained (4). Recently, Koch et al. reported long-term require their in vitro differentiation into a desirable homogenous expansion of hESC-derived rosette-type NSCs (9). However, the cell population. A major challenge of the current hESC differenti- study used the conventional and undefined embryoid body (EB) ation paradigm is the inability to effectively capture and, in the differentiation strategy and required tedious mechanical isolation long-term, stably expand primitive lineage-specific stem/precursor of the overgrown neural rosettes from replated EBs. In addition, cells that retain broad differentiation potential and, more impor- under these conditions, NSCs could not maintain stable spatial fi properties and switch from forebrain to hindbrain identity after

tantly, developmental stage-speci c differentiation propensity. BIOLOGY Here, we report synergistic inhibition of glycogen synthase kinase prolonged expansion. DEVELOPMENTAL 3 (GSK3), transforming growth factor β (TGF-β), and Notch signal- In our attempts to convert conventional hESCs to a mESC-like ing pathways by small molecules can efficiently convert mono- naïve state by small molecules, we fortuitously created a homoge- layer cultured hESCs into homogenous primitive neuroepithelium nously converted cell population by combined treatment of human LIF (hLIF) and two small molecules, CHIR99021 and SB431542, within 1 wk under chemically defined condition. These primitive for about 10 d under chemically defined conditions. Remarkably, neuroepithelia can stably self-renew in the presence of leukemia β this population of cells, growing in colonies, appeared to self-renew inhibitory factor, GSK3 inhibitor (CHIR99021), and TGF- receptor and stably maintain their characteristics over numerous passages inhibitor (SB431542); retain high neurogenic potential and respon- under these defined conditions. CHIR99021 (referred to hereafter siveness to instructive neural patterning cues toward midbrain as CHIR) is a small molecule inhibitor of glycogen synthase kinase and hindbrain neuronal subtypes; and exhibit in vivo integration. 3 (GSK3) and can activate canonical Wnt signaling (10), which Our work uniformly captures and maintains primitive neural stem has been implicated in ES cell self-renewal (11). SB431542 (re- cells from hESCs. ferred to hereafter as SB) is a small molecule inhibitor of transforming growth factor β (TGF-β) and Activin receptors, and has uman embryonic stem cells (hESCs) hold enormous promise been implicated in the mesenchymal-to-epithelial transition and Hfor regenerative medicine (1). Typically, hESC-based appli- reprogramming (12, 13). Interestingly, these converted cells did cations require in vitro differentiation of hESCs into a desirable not express the pluripotency markers Oct4 and Nanog, but were homogenous cell population. Despite the enormous progresses positive for Sox2 and alkaline phosphatase (ALP). Subsequent made in differentiating hESCs into various functional cells, a studies revealed that this expandable cell population has features major challenge of the current hESC differentiation paradigm is of primitive neuroepithelium (and hereafter we refer them as the inability to effectively capture and stably expand primitive primitive neural stem cells/pNSCs). Interestingly, the self-renewal lineage-specific stem/precursor cells. These cells would ideally of pNSCs is dependent on LIF, which has been implicated in the retain broad differentiation potentials (e.g., have the ability to self-renewal of mESC-derived primitive NSCs (6, 14). Previous in serially repopulate the entire specific tissue) and, perhaps more vivo developmental studies have shown that bFGF-responsive importantly, the developmental stage-specific differentiation definitive NSCs first appear on embryonic day 8.5 (ED 8.5) in propensity, and would be devoid of tumorigenicity concerns. In mouse embryos (15, 16). However, at an earlier stage (ED 5.5–7.5), the case of neural induction of hESCs by various advanced primitive NSCs are LIF-dependent, and the in vivo generation of methods (2–5), there is still a lack of robust, chemically defined primitive NSCs was independent of Notch signaling. We reasoned conditions for the long-term maintenance of primitive neural that if pNSCs are analogous to primitive NSCs during de- epithelial precursor cells, which are highly neurogenic and can be patterned/regionalized by specific morphogens (6, 7). Under typically used growth factor conditions (including bFGF, EGF), Author contributions: W.L., K.Z., and S.D. designed research; W.L., W.S., Y.Z., W.W., R.A., neural stem cells (NSCs) “transition” in a few passages into a P.X., M.T., T.L., X.W., and W.R.K. performed research; W.W., T.L., and J.K. contributed new more glial-restricted precursor state (8), which is significantly less reagents/analytic tools; and W.L., W.S., R.A., P.X., S.A.L., K.Z., and S.D. analyzed data; and neurogenic. In addition, in vitro cultured NSCs respond poorly W.L. and S.D. wrote the paper. to patterning cues and exhibit a narrow repertoire for generating The authors declare no conflict of interest. specific neuronal subtypes. Previous studies in murine ESCs This article is a PNAS Direct Submission. (mESCs) have suggested the existence of leukemia inhibitory Data deposition: The data reported in this paper have been deposited in the Gene Ex- factor (LIF)-responsive primitive NSCs (6). However, these cells pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE28595). could not be maintained in culture. Recent studies in neural in- 1To whom correspondence may be addressed. E-mail: [email protected] or sheng.ding@ duction of hESCs have identified rosette-type NSCs that repre- gladstone.ucsf.edu. sent neural tube-stage precursor cells. These rosette NSCs were This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. capable of responding to patterning cues that direct differentia- 1073/pnas.1014041108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1014041108 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 velopment, temporarily inhibiting Notch signaling should not block the induction of pNSCs. Indeed, temporal treatment by another small molecule inhibitor of γ-secretase, Compound E (referred to hereafter as C-E; ref. 17), further accelerated neural induction and generated the homogenous self-renewing pNSC population within 1 wk. Even after long-term expansion and repeated passaging in the presence of hLIF, CHIR, and SB, pNSCs retain remarkably high neurogenic propensity, broad differentiation potential, responsiveness to extrinsic morphogens for subsequent development into subtype-specific neuronal identities, and the ability to integrate in vivo. Our work uniformly captures and stably maintains primitive neural stem cells from hESCs. Results Synergistic Inhibition of GSK3, TGF-β, and Notch Signaling Pathways Converts hESCs into Homogenous pNSCs. hESCs were cultured on X-ray inactivated CF-1 mouse embryonic fibroblasts (MEFs) in hESC growth media (DMEM/F12 containing 20% KSR and 10 ng/mL bFGF) or on Matrigel under feeder-free and chemically defined conditions as described (18). Primitive neuroepithelium was induced by switching from hESC growth media to neural induction media (1:1 Advanced DMEM/F-12:Neurobasal media supplemented with N2, B27, hLIF), supplemented with CHIR and SB, with or without C-E, for 7 d (a schematic representation of the differentiation process is shown in Fig. 1A). hESC differentiation was monitored by immunocytochemistry, flow cytometry and real-time PCR. As shown in images of the same visual field, the differentiated cells exhibited homogenous epithelial morphology during the entire differentiation process (Fig. S1A1–4). Real-time PCR analysis revealed that combined treatment with hLIF, SB, and CHIR (with or without C-E) induced a rapid loss of Oct4 and Nanog expression (Fig. 1B). However, the expression of Sox2, a pluripotency marker that is also a persistent marker of NSCs (19), remained largely unchanged. Pax6, an early marker of neural induction, was significantly up-regulated after 5 d in the presence of C-E (0.1 μM), whereas its up-regulation was first detected at the sixth day in the absence of C-E treatment (Fig. 1B). Consistent with this observation, immunocytochemistry confirmed the faster induction of Pax6 protein on the sixth day in the presence of C-E, as Pax6 protein only became detectable from the seventh day onwards in the absence of C-E (Fig. S1B7–12). In contrast, only a small fraction of cells were positive for Pax6 on day 7 when hESCs were treated with SB, C-E, and hLIF (Fig. S1C1). Similarly, no Pax6 positive cells could be detected at the Fig. 1. Real-time PCR and flow cytometry analysis of neural induction from same time point when hESCs were treated with CHIR, C-E, and hESCs treated with LIF, CHIR, and SB (with or without C-E). (A) Schematic hLIF (Fig. S1C2). These data suggest that inhibition of Notch representation of the neural induction process. (B) The expression of Pax6, signaling can enhance early neural induction. Interestingly, real- Sox2, Nanog, Oct4, BMP4, Noggin, Eomes, Brachyury, and Sox17 was ana- time PCR analysis showed that the induction of the Pax6 gene lyzed by real-time PCR. (C and D) Flow cytometry analysis was used to occurred in parallel with the suppression of BMP4 gene expres- quantify cells expressing Oct4, Sox2, or CD133 during neural induction. CDM, sion as well as induction of Noggin (BMP antagonist) expression chemically defined medium. (Fig. 1B), suggesting that endogenous mechanisms of BMP signaling inhibition may contribute to neural induction. Real-time PCR analysis also demonstrated that the differentiation is highly CD133-positive cell population would represent the primitive specific toward the neural lineage. Along with the induction of neuroepithelium. FACS analysis showed that more than 96% of Pax6, epiblast-associated nonneural genes such as Brachyury, undifferentiated hESCs were positive for both Oct4 and Sox2 Eomes, and Sox17, were repressed synchronously with pluri- (Fig. 1C). After treatment, FACS confirmed the rapid loss of Oct4 potency markers Oct4 and Nanog (Fig. 1B), suggesting the pres- expression. Especially Oct4-positive cell number dropped sub- ence of an intermediate cell type resembling differentiating stantially on day 5, when Pax6 was first induced, suggesting that epiblast cells before hESC neuralization. This highly directed day 5 was the turning point of neural induction. In addition, FACS neural induction was further confirmed by immunocytochemistry. analysis further showed that the addition of C-E induced a much Double staining of Oct4 and Nestin showed that Oct4 expression more rapid loss of Oct4 expression and consequent neural con- gradually diminished and was almost undetectable after 5 d of version. At day 5, only 13% of cells were still positive for Oct4 in treatment with hLIF, SB, CHIR, and C-E, whereas Nestin- the presence of C-E, whereas 33.9% were positive in its absence. expressing cells became the predominant population, comprising Despite the loss of Oct4 expression, cells persistently maintained ∼99% of the population on day 7 (Fig. S1B1–6). To further a high level of Sox2 expression (>96%) at all time points exam- quantify the efficiency of the neural induction, the expression of ined during differentiation, and >97% of cells were only positive Oct4, Sox2 and CD133, was analyzed by flow cytometry. In de- for Sox2 at day 7 with C-E treatment (Fig. 1C). In addition, FACS velopment, the neural plate and neural tube exhibit CD133 analysis showed that 98% of undifferentiated hESCs were positive (Prominin-1) immunoreactivity (20, 21). In vertebrate embryos, for CD133 and that small molecule treatment initially induced the Sox2 is one of the earliest markers for the neural plate. During loss of CD133. However, along with the induction of Pax6 from hESC differentiation, the earliest Oct4-negative, but Sox2/ day 5 onwards, the CD133-positive cells increased significantly,

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1014041108 Li et al. Downloaded by guest on October 1, 2021 with >98% cells being CD133-positive on day 9 (Fig. 1D). These homogenously differentiated neural cells could be stably expanded on MEF feeder cells or Matrigel coating in the presence of hLIF, CHIR, and SB, and are referred to hereafter as pNSCs. In the present study, pNSCs were regularly expanded on Matrigel. Taken together, these data suggested that the combination of hLIF/CHIR/SB/C-E directs the specific induction of primitive neuroepithelium within 7 d that can long-term homogenously self- renew under hLIF/CHIR/SB conditions without the need for any cell purification. Chambers et al. (2) recently demonstrated that dual inhibition of Smad signals by Noggin and SB431542 could convert >80% hESCs to neural fate in 13 d. However, the Noggin/ SB431542 condition (which also contains undefined serum products) generated heterogeneous neural populations containing cells of different developmental stages (e.g., nonpolarized neuroepithelia and polarized rosette-like structures). Most importantly, the dual Smad inhibition protocol cannot capture the NSCs and maintain their self-renewal. Our described neural induction process is much faster, more specific, and more efficient, representing a chemically defined single-step strategy for ob- taining self-renewing homogenous primitive NSCs from hESCs cultured in a monolayer. The results of our strategy are highly reproducible in multiple different hESC lines, including H1 (Fig. 1), HUES9, and HUES1 (Fig. S2A), under both feeder and feeder-free (Matrigel) culture conditions.

pNSCs Can Long-Term Self-Renew and Represent the Pre-Rosette

Stage NSCs. pNSCs can long-term self-renew over serial pas- BIOLOGY sages on Matrigel with SB, CHIR, and hLIF. pNSCs generated from HUES9 and H1 hESCs were routinely passaged 1:10 and DEVELOPMENTAL have been cultured for >30 passages without obviously losing proliferative capacity, which is equivalent to at least 94 population doublings. However, individual omission of hLIF, SB, or CHIR from the media compromised pNSCs’ long-term self- renewal. Single pNSC is clonogenic on Matrigel in the presence of hLIF/SB/CHIR (Fig. 2A). However, no colonies were observed under conditions including C-E, suggesting that Notch signaling Fig. 2. pNSCs stably self-renew and maintain a homogenous primitive NSC is critical to pNSC self-renewal. Consistently, treatment with C-E phenotype after long-term cultures. The pNSCs cultured on Matrigel for 48 h rapidly induced pNSCs to differentiate into Doublecortin exhibited characteristic epithelial morphology. (A) A single cell-derived pNSC (DCX)-positive neuronal precursors (Fig. S2B1and 2). Despite colony on Matrigel. (Inset) pNSCs were positive for ALP. (B–I) Immunocyto- their highly proliferative and clonogenic capacity, pNSCs are not chemistry showed that pNSCs (passage 6) expressed genes recently identified tumorigenic in SCID beige mice. We transplanted the early- as rosette-type NSC markers, including PLZF, ZO-1, and N-cad; CNS neural passage (passage 6, about 30 d in serial culture) and late-passage stem cell makers such as Nestin, Pax6, and Sox2; the cell proliferation marker (about passage 27) of HUES9- and H1-derived pNSCs (2 × 106 Ki-67; the anterior neural markers Forse1 and Otx2; and the midbrain marker cells suspended in Matrigel) into 24 SCID beige mice s.c. These Nurr1. (J) Flow cytometry analysis showed that pNSCs stably expressed NSC mice have been observed for as long as 6 mo with no sign of and cell proliferation markers after long-term in vitro expansion, including neoplasm formation, whereas the control animals transplanted Nestin, Pax6, CD133, Forse1, and Ki-67. (K and L) Nonneural lineage markers and genes associated with midbrain were analyzed by RT-PCR. with the parental hESCs produced teratomas within 6 wk. Remarkably, the long-term expanded pNSCs maintain a stable pNSC phenotype. The pNSCs cultured on Matrigel exhibited gained rosette-like structures with apical N-cad expression and typical epithelial morphology and positive ALP staining (Fig. 2A). interkinetic nuclear migration after being cultured in neural in- Immunostaining showed that both the early-passage (passage 6) duction media with 20 ng/mL bFGF for 4 d (Fig. S3C1and 2). and late-passage (passage 27) pNSCs stably expressed genes re- Consistent with their highly proliferative capacity, pNSCs uni- cently identified as rosette-type NSC markers (4), including PLZF formly expressed Ki-67 (Fig. 2F and Fig. S3A5). The stable phe- (promyelocytic leukemia zinc finger), ZO-1, and N-cad (N-cad- notype of pNSCs after extensive passaging was further confirmed herin); CNS (central nervous system) neural stem cell markers, by flow cytometry. Both early-passage and late-passage pNSCs such as Nestin, Pax6, and Sox2; anterior neural markers Forse1 exhibited nearly identical expression patterns for a set of NSC- and Otx2; and the midbrain marker Nurr1 (Fig. 2 B–I and Fig. specific markers, such as Nestin (98.2% positive), Pax6 (95.4% S3A1–8). Expression analysis by microarray confirmed the dra- positive), CD133 (93.9% positive), and the cell proliferation matic up-regulation of neural lineage genes such as Ascl1, Pax6, marker Ki-67 (98.3% positive; Fig. 2J), whose uniform expression Dach1, N-cad,andNestin, and down-regulation of pluripotency confirmed that pNSCs were a homogenous, expandable NSC gene Oct4 in pNSCs in comparision to hESCs. However, both population. Indeed, genes associated with non-neural lineages, such hESCs and pNSCs express ZO-1 and Sox2 at similar level. Even as Eomes, Brachyury, Sox17,orK15 were undetectable in pNSCs after long-term passaging, pNSCs uniformly expressed a panel of by RT-PCR (Fig. 2K). FACS analysis with propidium iodide primitive neuroepithelial genes, including Sox2, N-cad, PLAZ, revealed a very similar cell cycle profile for both early- and late- Dach1, ZO-1, Pax6, and proneuronal gene Ascl1, and both early- passage of pNSCs. The cell cycle distribution (G1, S, and G2/M) of and late-passage pNSCs demonstrated highly similar tran- early-passage pNSCs (P7) is 52.7%, 26.6%, and 16.5%; and the scriptome profile (Fig. S3B1and 2). Notably, N-cad and the tight cycle distribution of late-passage pNSCs (P28) is 51.7%, 32.4%, junction protein ZO-1 were expressed evenly on the surface of and 12.4%, respectively. Interestingly, FACS analysis showed that both early- and late-passage pNSCs, suggesting that pNSCs are the expression of Forse1, an anterior NSC marker, was not ho- primitive, nonpolarized prerosette NSCs (2). Indeed, pNSCs mogenous (53.6% of pNSCs were positive for Forse1; Fig. 2J).

Li et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Whether Forse1-negative pNSCs have a more posterior identity tional synapses. Next, to further examine pNSCs’ potential in needs to be further characterized. In addition, pNSCs did not vivo, they were transplanted into the lateral ventricle of neonatal express neural crest cell markers such as HNK1, Sox10, or p75. mice (P2-3). Histological analysis of GFP-expressing pNSC However, we did detect a small percentage (∼3%) of pNSCs (passage 27) grafts one month after transplantation revealed that positive for AP2, a premigratory neural crest gene initially engrafted cells were distributed in many brain areas, including the expressed throughout the neural plate border (22). To rule out the corpus callosum, the subcallosal zone, the caudate-putamen (Fig. possibility that pNSC cultures contained a separate (parallel or S6 A and B), and the hindbrain (Fig. S6 C–J). Most engrafted cells unrelated) neural crest cell population, we preformed clonal (>50% in the forebrain, and >80% in the hindbrain) express- analysis by immunostaining of single pNSC-derived colonies. ed differentiated neuronal markers such as MAP2 (Fig. S6A1–4). AP2-positive cells representing 2–5% of cells in each colony were In addition, we also detected DCX-positive engrafted cells in the detected in all examined colonies (n = 16), and they were mostly subcallosal zone (Fig. S6B1–4), where endogenous adult neural seen at the border of the colonies (Fig. S4), suggesting that they progenitor cells reside (24), but not in non-neurogenic environ- were derivatives of pNSCs. However, whether these AP2-positive ments such as the hindbrain, suggesting that their neuronal dif- cells possess neural crest potential remains to be confirmed. ferentiation was influenced by the host environment. Although To examine the multipotency of long-term expanded pNSCs, we failed to detect the mature neuronal marker NeuN in the both early- and late-passage pNSCs were plated at ultra-low subcallosal zone or caudate-putamen, a subset of GFP-express- density in six-well plates (200 cells per well) and cultured in dif- ing cells in the clusters near the aqueduct exhibited NeuN ex- ferentiation media for 2 wk. Among the single pNSC-derived cell pression (Fig. S6C1–4). We also failed to detect spontaneously clusters (n = 29), 100% contained both MAP2-positive neurons differentiated tyrosine hydroxylase (TH)-positive dopaminergic and GFAP-positive astrocytes (Fig. S5 A–C), but no cells positive (DA) neurons, but some engrafted cells (∼10%) appeared to for the oligodendrocyte marker O4 or the neural crest lineage have differentiated into GABA-expressing inhibitory neurons markers peripherin and α-SMA were detected at this time point. (Fig. S6D1–4). GFP-expressing cells in the hindbrain, closely Previous studies have shown that bFGF and/or EGF-expanded associated with presynaptic puncta labeled by synaptophysin, NSCs lose neurogenic propensity and become more gliogenic were also observed, indicating the synaptic contacts of the after long-term culture (23). However, pNSCs expanded under transplanted cells with the host mouse neurons (Fig. S6E1–4). our described conditions retained high neuronal differentiation All GFP positive cells also exhibited human nucleus antigen propensity. Flow cytometry analysis showed that pNSCs at pas- immunoreactivity (Fig. S6F1–4), further confirming their human sage 8 and passage 25 could give rise 73.9% and 77.6% MAP2- cell identity. In addition, some GFP-expressing hindbrain neu- positive, or 71.4% and 74.4% NeuN-positive neurons, respecti- rons also exhibited c-fos, a marker for neuronal excitation (Fig. vely (Fig. 3A1and 2). During CNS development, neurogenesis S6 G–I). On the other hand, we did not find any GFAP-positive largely precedes gliogenesis. NSCs from earlier stages generate engrafted cells (Fig. S6J), suggesting that pNSCs preferentially more neurons and have a lower propensity to produce glia than differentiate into the neuronal lineage in vivo. those from later stages. The remarkably high neurogenic potential and propensity of these long-term expanded pNSCs is con- pNSCs Possess Mesencephalic Regional Identity and Can Be Re- sistent with their self-renewal in the primitive state. Importantly, specified Toward Caudal Cell Fates. It is worthwhile to note that pNSCs could effectively differentiate and generate mature neu- pNSCs express the forebrain/midbrain gene Otx2 and the mid- rons that fired action potentials (5 cells in 7 tested cells; Fig. 3B), brain gene Nurr1 by immunostaining (Fig. 2 H and I). RT-PCR and produced fast inactivating inward Na+ currents (n =8of analysis confirmed the expression of Otx2 and Nurr1, and showed 8 cells recorded) that were sensitive to the Na+ channel blocker that pNSCs also express other midbrain genes, such as En-1, Tetrodotoxin (TTX; Fig. 3C). Furthermore, these differentiated Lmx1b, Pax2,andPitx3 (Fig. 2L). In contrast, the forebrain-re- neurons manifested spontaneous excitatory postsynaptic currents stricted transcription factors FoxG1 and Emx2 were barely de- (sEPSCs) and/or inhibitory postsynaptic currents (sIPSCs) in 4 of tectable, and anterior hindbrain transcription factors, such as 6 cells recorded (Fig. 3D), indicating that they can form func- Gbx2, HoxB2, and HoxA2, were expressed at low levels as in-

Fig. 3. pNSCs retain high neurogenic potential during long- term culture. (A) Flow cytometry analysis showed pNSCs at passage 8 and passage 25 could give rise to 73.9% and 77.6% MAP2-positive, or 71.4% and 74.4% NeuN-positive neurons, respectively. (B) Representative traces of evoked action potentials (whole-cell recording, current-clamp mode) generated by neurons after 4 wk of differentiation from pNSCs. Traces of Tetrodotoxin (TTX)-sensitive whole-cell currents recorded in voltage-clamp mode. (C) Cells were hyperpolarized to −90 mV for 300 ms before applying depolarizing pulses to elicit Na+ and K+ currents. (D) Traces of spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs), both recorded at a holding potential of −60 mV, indicated synapse formation. B–D represent the data recorded from pNSCs at passage 25 that had spontaneously differentiated to display neuronal properties.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1014041108 Li et al. Downloaded by guest on October 1, 2021 dicated by RT-PCR (Fig. S7A). pNSCs expressed the dorsal neural tube gene Pax3, the ventral neural tube genes Nkx2.2 and Nkx6.1, the NSC marker Dach1 and the Notch effector Hes5 (Fig. S7A). These observations suggested that in vitro-expanded pNSCs may possess a mesencephalic regional identity. To con- firm this, two different differentiation protocols were used to examine the potential of pNSCs to generate midbrain DA neurons: in the “induced” protocol, pNSCs were first treated with 100 ng/mL SHH (Sonic Hedgehog) and 100 ng/mL FGF8b for 10 d and were then further differentiated in the presence of 10 ng/ mL BDNF, 10 ng/mL GDNF, 10 ng/mL IGF1, 1 ng/mL TGF-β3, and 0.5 mM dibutyryl-cAMP (dbcAMP) for another ∼2-3 wk (Fig. 4A). In the “default” protocol, pNSCs were directly terminally differentiated in the presence of BDNF, GDNF, IGF1, TGF-β3, and dbcAMP for 3 wk without pre-patterning by morphogens (Fig. 4B). Both the “induced” and “default” differentiation conditions produced >50% TH-positive neurons that also exhibited aromatic L-amino acid decarboxylase (AADC), En-1, Lmx1a, Nurr1, FoxA2, and Pitx3 immunoreactivity (Fig. 4 A1–7 and B1–7). Notably, Pitx3 is a homeobox gene uniquely expressed in midbrain DA neurons (25). Real-time PCR confirmed the significant up-regulation of TH, AADC, En-1, Nurr1, and Pitx3 (Fig. 4 A8 and B8). Flow cytometry quantification demonstrated that the “induced” and “default” differentiation protocols produced 54.8% and 62.7% TH and MAP2 double positive neurons, respectively (Fig. 4 A9 and B9). These data demonstrate that pNSCs possess mesencephalic regional identity fi

and can differentiate into DA neurons with very high ef ciency. BIOLOGY Because they exhibit features of pre-rosette primitive NSCs, pNSCs were further examined for their responsiveness to in- DEVELOPMENTAL structive regional patterning cues. pNSCs were sequentially treated with caudalizing retinoic acid (RA, 1 μM) for 7 d, 100 ng/mL SHH and 0.1 μM RA for another 7 d, and then 50 ng/mL SHH and 0.1 μM RA for an additional 7 d. The cells were then terminally differentiated in the presence of 10 ng/mL BDNF and 10 ng/mL GDNF in differentiation media for about 7 d. Real-time PCR assays demonstrated significant induction of posterior genes, in- Fig. 4. pNSCs possess mesencephalic regional identity and can differentiate cluding HoxB4, HoxA5, and HoxC5 after treatment with 1 μMRA into DA neurons with high efficency. (A)Inthe“induced” protocol, pNSCs for 1 wk (Fig. S8A), suggesting that pNSCs are responsive to the were treated with SHH and FGF8b for 10 d before they were terminally caudalizing effect of RA. Under such conditions, immunocyto- differentiated in the presence of BDNF, GDNF, IGF1, TGF-β3, and dbcAMP for another ∼2–3 wk. (B)Inthe“default” protocol, pNSCs were directly termi- chemistry showed that pNSCs could differentiate into choline β acetyltransferase (ChAT)-positive neurons that are also positive nally differentiated in the presence of BDNF, GDNF, IGF1, TGF- 3, and B C dbcAMP for 3 wk. Under both protocols, pNSCs gave rise to TH positive for MAP2 and Isl-1 (Fig. S8 and ). Flow cytometry analysis neurons that also exhibited AADC, En-1, Lmx1a, Nurr1, FoxA2, and Pitx3 showed that 53.7% cells were double-positive for Isl-1 and MAP2 – – fi fi fi immunoreactivity (A17 and B17). Real-time PCR further con rmed the (Fig. S8D). Real-time PCR assays con rmed the signi cant in- significant up-regulation of TH, AADC, En-1, Nurr1, and Pitx3 (A8 and B8). duction of ChAT, HB9, Isl-1, and Lim3 after terminal differenti- Flow cytometry quantification demonstrated that the two differentiation ation (Fig. S8A), suggesting an induction of motor neurons. These protocols produced 54.8% and 62.7% TH and MAP2 double-positive neu- data indicated that pNSCs retain responsiveness to instructive rons, respectively (A9 and B9). cues promoting the induction of hindbrain neuronal subtypes.

Discussion layer lineages and undifferentiated hESCs. In the present study, To realize the potential of cell-based therapy for treating injuries our serendipitous observation led us to develop a robust chemi- and degenerative diseases, renewable sources of stem/progenitor cally defined condition using specific small molecules that rapidly cells need to be developed. Although hESCs indefinitely self- and uniformly converts hESCs into pNSCs, and, most impor- renew and have the differentiation potential to become any cell tantly, enables their long-term expansion without a loss of high type, they are practically inferior to lineage-restricted cells as they neurogenic propensity and regionalizable plasticity. To our are prone to causing teratomas and do not repopulate host tissues knowledge, this is the fastest and most efficient method so far to in vivo. However, significant challenges also remain in terms of produce neural stem cells from hESCs. In addition, pNSCs differ the isolation and long-term expansion of most tissue-specific from previously reported hESC-derived NSCs in that they rep- stem/progenitor cells from adults (e.g., even for the arguably most resent the primitive pre-rosette neuroepithelium that has never studied hematopoietic stem cells). Consequently, differentiation been long-term expanded in vitro before. Interestingly, pNSCs of hESCs into renewable tissue-specific cell types is highly desir- possess features of mesencephalic precursor cells and can dif- able for various biomedical applications. If achieved, cell pop- ferentiate into DA neurons spontaneously with high efficiency in ulations could be carefully quality controlled and serve as starting the absence of pre-patterning. Real-time PCR analysis showed materials, skipping hESCs that cannot be used directly. Further- the up-regulation of endogenous SHH, FGF8, and the ventral more, despite significant advances in development of various patterning gene Nkx6.1 under both “induced” and “default” dif- neural induction conditions for hESCs, most differentiation ferentiation protocols (Fig. S7B), suggesting the cells could be protocols use poorly defined culture conditions (e.g., going specified into DA neurons by an endogenous mechanism. These through EB formation, using undefined medium supplements/ observations are reminiscent of the previous in vivo studies that KSR), and usually yield mixed populations containing neural cells showed DA neurons originated from SHH-expressing domains of at different developmental stages, or even other embryonic germ the ventral midbrain (26). In addition, a mouse study demon-

Li et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 strated the antagonistic interaction between the activation of forebrain (32). The use of GSK3 inhibitor (which can activate Wnt/β-catenin and SHH (27). The activation of β-catenin in the canonical Wnt) and TGF-β receptor inhibitor may partly re- ventral midbrain promoted the expansion of early DA progeni- capitulate such in vivo self-renewal signals of midbrain NSCs. tors, but led to a reduced expression of SHH. The removal of the With an improved understanding of the signaling mechanisms GSK3 inhibitor (CHIR) during pNSC differentiation may lead to involved in lineage specification and maintenance of tissue-specific down-regulation of Wnt/β-catenin signaling and facilitate the up- stem cells, this strategy could also be generalized and applied to regulated SHH expression in turn. Considering the significance of developing renewable sources of DA neurons, it would be useful the capture of self-renewing stem cells from other germ layers, to examine whether pNSC transplantation could attenuate the such as endoderm or mesoderm. Finally, this protocol also pro- Parkinson’s symptoms in animal models in the future. vides a valuable tool with which to study the early molecular Recent studies suggest that GSK3 plays key roles in many events initiating human neural induction. fundamental processes, including mediating signaling downstream of Wnt, FGF, Hh, and Notch during neural development Materials and Methods (28–30). In our neural induction protocol, however, replacement For further details of cell cultures, neuronal differentiation, immunocyto- of CHIR with Wnt3a induced significant spontaneous differenti- chemistry, flow cytometry, quantitative and semiquantitative RT-PCR, elec- ation and could not generate a homogenous NSC population, trophysiological analysis, microarray analysis, teratoma assays, in vivo suggesting that GSK3 inhibition may coordinate multiple signals transplantation, and histology, see SI Materials and Methods. The antibodies besides canonical Wnt activation in the context of neural in- used in this study are shown in Table S1. For the primers of quantitative and duction under this condition. One possible explanation for this semi-quantitative RT-PCR, see Table S2. specific neural induction is that inhibition of TGF-β/Nodal signaling by SB431542 not only blocks the formation of mesen- ACKNOWLEDGMENTS. We thank our colleague Jem Efe for reading the doderm, but also engages in cross-talk with GSK3-mediated manuscript and for providing many insightful comments and suggestions, and signaling (for example FGF signaling) to enhance neural in- Jianwei Che (Genomics Institute of the Novartis Research Foundation, San duction, possibly by modulating a downstream component of Diego) for analyzing the microarray data. S.D. and K.Z. are supported by endogenous BMP signaling (2, 31). In addition, very recent National Institutes of Health (NIH) Director’s Transformative R01 Program (R01 studies showed that GSK3 is a master regulator of in vivo neural EY021374). S.D. is supported by funding from the National Institute of Child Health and Development, the National Heart, Lung, and Blood Institute, and progenitor homeostasis (28, 29). It is possible that neural in- the National Institute of Mental Health/NIH, the California Institute for Re- duction is also coupled with the capture/maintenance of primitive generative Medicine (CIRM), the Prostate Cancer Foundation, Fate Therapeu- NSCs through GSK3 inhibition. Specifically, the combination of tics, the Esther B. O’Keeffe Foundation, and The Scripps Research Institute. K.Z. GSK3 inhibitor, TGF-β receptor inhibitor, and hLIF is uniquely is supported by grants from the National Eye Institute/NIH, a Veteran Affairs required for long-term self-renewal of pNSCs under chemically Merit Award, the Macula Vision Research Foundation, Research to Prevent defined conditions. Recent in vivo studies demonstrated that Blindness, a Burroughs Wellcome Fund Clinical Scientist Award in Transla- β tional Research, the Dick and Carol Hertzberg Fund, and Chinese National TGF- pathway activation counteracts canonical Wnt and nega- 985 Project to Sichuan University and West China Hospital. S.A.L. is supported tively regulates self-renewal of midbrain neuroepithelial stem by grants from the National Eye Institute, the National Institute of Neurolog- cells in the developing mouse brain (32). Loss of TGF-β signaling ical Disorders and Stroke, the National Institute of Child Health and Develop- results in neuroepithelial expansion in the midbrain, but not the ment, the National Institute of Environmental Health Sciences, and CIRM.

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