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• W2938006872 abstract "In this study, we found that undifferentiated human pluripotent stem cells (hPSCs; up to 30% of total cells) present in the cultures of neural stem or precursor cells (NPCs) completely disappeared within several days when cultured under neural differentiation culture conditions. Intriguingly, the disappearance of undifferentiated cells was not due to cell death but was instead mediated by neural conversion of hPSCs. Based on these findings, we propose pre-conditioning of donor NPC cultures under terminal differentiation culture conditions as a simple but efficient method of eliminating undifferentiated cells to treat neurologic disorders. In addition, we could establish a new neural differentiation protocol, in which undifferentiated hPSCs co-cultured with NPCs become differentiated neurons or NPCs in an extremely efficient, fast, and reproducible manner across the hESC and human-induced pluripotent stem cell (hiPSC) lines. In this study, we found that undifferentiated human pluripotent stem cells (hPSCs; up to 30% of total cells) present in the cultures of neural stem or precursor cells (NPCs) completely disappeared within several days when cultured under neural differentiation culture conditions. Intriguingly, the disappearance of undifferentiated cells was not due to cell death but was instead mediated by neural conversion of hPSCs. Based on these findings, we propose pre-conditioning of donor NPC cultures under terminal differentiation culture conditions as a simple but efficient method of eliminating undifferentiated cells to treat neurologic disorders. In addition, we could establish a new neural differentiation protocol, in which undifferentiated hPSCs co-cultured with NPCs become differentiated neurons or NPCs in an extremely efficient, fast, and reproducible manner across the hESC and human-induced pluripotent stem cell (hiPSC) lines. The discovery of human embryonic stem cells (hESCs) has yielded new avenues for regenerative medicine, and somatic reprogramming of hESCs to human induced pluripotent stem cells (hiPSCs) offers further promise for patient-specific autologous cell transplantation and human disease modeling. Despite recent promising results from clinical trials using human pluripotent stem cell (hPSC)-based cell therapies, the risk of tumor formation arising from residual undifferentiated (undiff.) hPSCs remains a serious and critical hurdle for broader clinical implementation.1Brederlau A. Correia A.S. Anisimov S.V. Elmi M. Paul G. Roybon L. Morizane A. Bergquist F. Riebe I. Nannmark U. et al.Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma formation.Stem Cells. 2006; 24: 1433-1440Crossref PubMed Scopus (366) Google Scholar, 2Doi D. Morizane A. Kikuchi T. Onoe H. Hayashi T. Kawasaki T. Motono M. Sasai Y. Saiki H. Gomi M. et al.Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson’s disease.Stem Cells. 2012; 30: 935-945Crossref PubMed Scopus (127) Google Scholar, 3Katsukawa M. Nakajima Y. Fukumoto A. Doi D. Takahashi J. Fail-Safe Therapy by Gamma-Ray Irradiation Against Tumor Formation by Human-Induced Pluripotent Stem Cell-Derived Neural Progenitors.Stem Cells Dev. 2016; 25: 815-825Crossref PubMed Scopus (33) Google Scholar A variety of approaches to selectively ablate the undiff. cells and mitigate the tumor risks of hPSC-based cell therapy have been examined, including: (1) small-molecule targeting of cell death or survival pathways, (2) genetic engineering to introduce a suicide gene or microRNA switch, (3) antibodies targeting a surface-specific antigen (or antibody-guided toxins), and (4) induction of selective phototoxicity.4Jeong H.-C. Cho S.-J. Lee M.-O. Cha H.-J. Technical approaches to induce selective cell death of pluripotent stem cells.Cell. Mol. Life Sci. 2017; 74: 2601-2611Crossref PubMed Scopus (17) Google Scholar, 5Rodrigues G.M.C. Rodrigues C.A.V. Fernandes T.G. Diogo M.M. Cabral J.M.S. Clinical-scale purification of pluripotent stem cell derivatives for cell-based therapies.Biotechnol. J. 2015; 10: 1103-1114Crossref PubMed Scopus (21) Google Scholar, 6Ogura A. Morizane A. Nakajima Y. Miyamoto S. Takahashi J. γ-Secretase inhibitors prevent overgrowth of transplanted neural progenitors derived from human-induced pluripotent stem cells.Stem Cells Dev. 2013; 22: 374-382Crossref PubMed Scopus (28) Google Scholar All of these approaches induce selective elimination of hPSCs from the donor cells to be transplanted based on the unique molecular properties of hPSCs; however, none of these approaches are completely selective for hPSCs, and each invariably affects the viability of donor cells to some degree. It is evident that maintaining donor cell viability prior to grafting is critical for the success of cell transplantation. Another possible strategy to prepare tumor-free cultures could be complete induction of hPSC differentiation, in which all residual undiff. hPSCs are converted into the tissue-specific cell types associated with the disease. This approach could also prevent contamination of other tissue cell lineages in donor cell preparations, and thus eliminate associated side effects after transplantation. For hPSC application in neurologic disorders, various methods have been developed to induce hESC or hiPSC differentiation toward neural lineages, including those based on co-culture on stromal cell feeder layers,7Perrier A.L. Tabar V. Barberi T. Rubio M.E. Bruses J. Topf N. Harrison N.L. Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2004; 101: 12543-12548Crossref PubMed Scopus (857) Google Scholar embryoid-body formation,8Zhang S.-C. Wernig M. Duncan I.D. Brüstle O. Thomson J.A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells.Nat. Biotechnol. 2001; 19: 1129-1133Crossref PubMed Scopus (1552) Google Scholar and dual SMAD inhibition.9Chambers S.M.S.S.M. Fasano C.A. Papapetrou E.P. Tomishima M. Sadelain M. Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.Nat. Biotechnol. 2009; 27: 275-280Crossref PubMed Scopus (2431) Google Scholar However, a minority of cells positive for undiff. hESC or iPSC markers frequently remain after efficient neural induction,1Brederlau A. Correia A.S. Anisimov S.V. Elmi M. Paul G. Roybon L. Morizane A. Bergquist F. Riebe I. Nannmark U. et al.Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma formation.Stem Cells. 2006; 24: 1433-1440Crossref PubMed Scopus (366) Google Scholar, 10Ko J.Y. Lee H.S. Park C.H. Koh H.C. Lee Y.S. Lee S.H. Conditions for tumor-free and dopamine neuron-enriched grafts after transplanting human ES cell-derived neural precursor cells.Mol. Ther. 2009; 17: 1761-1770Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Vincent P.H. Benedikz E. Uhlén P. Hovatta O. Sundström E. Expression of Pluripotency Markers in Nonpluripotent Human Neural Stem and Progenitor Cells.Stem Cells Dev. 2017; 26: 876-887Crossref PubMed Scopus (9) Google Scholar which can potentially induce tumor formation in the grafted brain.1Brederlau A. Correia A.S. Anisimov S.V. Elmi M. Paul G. Roybon L. Morizane A. Bergquist F. Riebe I. Nannmark U. et al.Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma formation.Stem Cells. 2006; 24: 1433-1440Crossref PubMed Scopus (366) Google Scholar, 2Doi D. Morizane A. Kikuchi T. Onoe H. Hayashi T. Kawasaki T. Motono M. Sasai Y. Saiki H. Gomi M. et al.Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson’s disease.Stem Cells. 2012; 30: 935-945Crossref PubMed Scopus (127) Google Scholar, 3Katsukawa M. Nakajima Y. Fukumoto A. Doi D. Takahashi J. Fail-Safe Therapy by Gamma-Ray Irradiation Against Tumor Formation by Human-Induced Pluripotent Stem Cell-Derived Neural Progenitors.Stem Cells Dev. 2016; 25: 815-825Crossref PubMed Scopus (33) Google Scholar In addition, the neural induction efficiency of hESCs/hiPSCs varies considerably depending on the cell line,12Osafune K. Caron L. Borowiak M. Martinez R.J. Fitz-Gerald C.S. Sato Y. Cowan C.A. Chien K.R. Melton D.A. Marked differences in differentiation propensity among human embryonic stem cell lines.Nat. Biotechnol. 2008; 26: 313-315Crossref PubMed Scopus (647) Google Scholar, 13Zhang J. Perry G. Smith M.A. Robertson D. Olson S.J. Graham D.G. Montine T.J. Parkinson’s disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons.Am. J. Pathol. 1999; 154: 1423-1429Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar type of feeder culture, passage number of hESCs,14Ko J.-Y. Park C.-H. Koh H.-C. Cho Y.-H. Kyhm J.-H. Kim Y.-S. Lee I. Lee Y.S. Lee S.H. Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons.J. Neurochem. 2007; 103: 1417-1429Crossref PubMed Scopus (62) Google Scholar and other unidentified factors. Specifically, the neural differentiation propensities of hESCs are variable depending on the level of miR371-3 expression in the cells, and certain cell lines resist induction of differentiation by any of the available methods.15Kim H. Lee G. Ganat Y. Papapetrou E.P. Lipchina I. Socci N.D. Sadelain M. Studer L. miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells.Cell Stem Cell. 2011; 8: 695-706Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar Furthermore, the propensity for neural differentiation is thought to be more diverse among patient-derived hiPSCs,16Hu B.-Y. Weick J.P. Yu J. Ma L.-X. Zhang X.-Q. Thomson J.A. Zhang S.C. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.Proc. Natl. Acad. Sci. USA. 2010; 107: 4335-4340Crossref PubMed Scopus (811) Google Scholar because each of the cell lines is established from donor tissue obtained from patients with different disease backgrounds, and additional variation may arise during reprogramming processes, such as partial reprogramming, incomplete silencing of transgenes, or gene dysregulation resulting from insertional mutagenesis. As a result, researchers are frequently unable to induce neural induction of hESCs or hiPSCs efficiently. The development of protocols for efficient, complete, and reproducible neural induction across hESC or hiPSC lines without residual undiff. cells remains a critical issue in regenerative therapies for neuropsychiatric disorders. In this study, we found that undiff. hESCs or iPSCs mixed with neural stem or precursor cells (NPCs) completely differentiated toward neuronal cells within several days. Based on these findings, we propose that co-culture with NPCs can be utilized not only to remove residual undiff. cells, but also to develop a new protocol for neural differentiation of hPSCs in an efficient, fast, and reproducible manner across hESC and hiPSC lines. Differentiation of hPSCs toward proliferating neural precursor cells (NPCs) is induced by co-culture with MS5 stromal cell feeder layers.1Brederlau A. Correia A.S. Anisimov S.V. Elmi M. Paul G. Roybon L. Morizane A. Bergquist F. Riebe I. Nannmark U. et al.Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma formation.Stem Cells. 2006; 24: 1433-1440Crossref PubMed Scopus (366) Google Scholar, 7Perrier A.L. Tabar V. Barberi T. Rubio M.E. Bruses J. Topf N. Harrison N.L. Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2004; 101: 12543-12548Crossref PubMed Scopus (857) Google Scholar, 17Rhee Y.H. Ko J.Y. Chang M.Y. Yi S.H. Kim D. Kim C.H. Shim J.W. Jo A.Y. Kim B.W. Lee H. et al.Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.J. Clin. Invest. 2011; 121: 2326-2335Crossref PubMed Scopus (197) Google Scholar The NPCs derived from hESCs (hES-NPCs) proliferate in the presence of the mitogen basic fibroblast growth factor (bFGF), while maintaining NPC marker expression (Figures 1A and 1B ), and efficiently differentiate into neurons under terminal differentiation culture conditions (Figure 1C).1Brederlau A. Correia A.S. Anisimov S.V. Elmi M. Paul G. Roybon L. Morizane A. Bergquist F. Riebe I. Nannmark U. et al.Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma formation.Stem Cells. 2006; 24: 1433-1440Crossref PubMed Scopus (366) Google Scholar, 7Perrier A.L. Tabar V. Barberi T. Rubio M.E. Bruses J. Topf N. Harrison N.L. Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2004; 101: 12543-12548Crossref PubMed Scopus (857) Google Scholar, 17Rhee Y.H. Ko J.Y. Chang M.Y. Yi S.H. Kim D. Kim C.H. Shim J.W. Jo A.Y. Kim B.W. Lee H. et al.Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.J. Clin. Invest. 2011; 121: 2326-2335Crossref PubMed Scopus (197) Google Scholar Cells positive for the undiff. hPSC marker OCT3/4 (up to 5% of total cells) occasionally remain in NPC cultures after neural induction of hESCs (Figure 1D),10Ko J.Y. Lee H.S. Park C.H. Koh H.C. Lee Y.S. Lee S.H. Conditions for tumor-free and dopamine neuron-enriched grafts after transplanting human ES cell-derived neural precursor cells.Mol. Ther. 2009; 17: 1761-1770Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar and cells expressing the undiff. hPSC marker were consistently found during NPC expansion with the mitogen bFGF (Figure 1F). However, the OCT3/4+ cells in hES-NPC cultures completely disappeared within 4 days of terminal differentiation induced by concurrent withdrawal of bFGF and supplementation of the culture medium with the neurogenic factors brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and cyclic AMP (cAMP; Figure 1E). None of the neurogenic factors added to the differentiation medium resulted in the elimination of the OCT3/4+ cells (Figure 1F and data not shown), but bFGF withdrawal alone was sufficient to eliminate these cells (Figure 1F). The mitogens epidermal growth factor (EGF), sonic hedgehog (SHH), and fibroblast growth factor 8 (FGF8) also induced hES-NPC expansion, but unlike bFGF did not maintain the OCT3/4+ cells (Figure 1G). When bFGF was added after disappearance of the OCT3/4+ cells, the undiff. marker-positive cells did not reappear (data not shown). We further verified the efficient disappearance of OCT3/4 expression by withdrawal of bFGF using real-time PCR analysis (Figure 1H). These findings indicate that hES-NPC donor cells free from residual PSC-marker+ cells can be obtained simply by pre-conditioning hES-NPCs in bFGF-free culture media for several days prior to grafting. When OCT3/4 expression levels were estimated by mean fluorescent intensity (MFI) in the individual immunofluorescently stained cells, the MFIs of the residual OCT3/4+ cells in the hES-NPC cultures were significantly weaker than those in undiff. hESC cultures (Figures S1A–S1C). Furthermore, other pluripotent cell markers (NANOG and TRA-1-60) were not expressed in the OCT3/4+ cells detected in hES-NPC cultures (Figures S1D–S1G). Primitive neuroepithelial cells at early neural development are also reported to express OCT3/4.18Akamatsu W. DeVeale B. Okano H. Cooney A.J. van der Kooy D. Suppression of Oct4 by germ cell nuclear factor restricts pluripotency and promotes neural stem cell development in the early neural lineage.J. Neurosci. 2009; 29: 2113-2124Crossref PubMed Scopus (63) Google Scholar, 19Shimozaki K. Nakashima K. Niwa H. Taga T. Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures.Development. 2003; 130: 2505-2512Crossref PubMed Scopus (113) Google Scholar Notably, Nestin, a marker specific for neuroepithelial cell (and NPC), was co-localized in OCT3/4+ cells in hES-NPC cultures (Figure S1I), but not in undiff. hESC cultures (Figure S1H), suggesting the OCT3/4+ cells in hES-NPC cultures are not true hESCs, but rather neuroepithelial cells (early NPCs), which are not associated with teratoma formation. Because the OCT3/4+ cells in the hES-NPC cultures were a minority sub-population (<5% of total cells) and appeared not to be true undiff. hESCs, it may be questionable whether pre-conditioning in bFGF-free medium is an effective method to eliminate the OCT3/4+ cells when true undiff. hESCs represent a larger proportion of the cells present in NPC cultures. To address this question, we intentionally mixed single-cell dissociates of hES-NPCs (70%) with those of undiff. hESCs labeled with GFP (pEF1α-GFP-hESCs, 30%) and cultured them together in bFGF-free differentiation medium (Figure 2A). Because bFGF is required for survival of undiff. hESC cultures, we suspected cell death to be the mechanism underlying the disappearance of OCT3/4+ cells shown in Figure 1H. However, unexpectedly, the GFP+ cells did not die off, but rather survived and expanded to generate growing cell clusters during the bFGF-free culture period (Figure 2B). Apoptotic cells detected by cleaved (activated) caspase-3 immunostaining were much fewer among the GFP+-hESC-origin cells than among the co-cultured GFP(−)-NPCs, and the low level of apoptotic cell death in the hESC-origin cell population was maintained during the co-culture period (Figure 2E), supporting that cell death was not the mechanism for the disappearance of undiff. PSC marker expressions in the co-culture. On the other hand, the undiff. hESC markers OCT3/4 and NANOG were gradually lost from the GFP-labeled hESC-origin cells during culture, and none of the GFP+ cells were positive for the undiff. markers after 8–9 days in culture (Figures 2C and 2D). Collectively, these findings indicate that hESCs co-cultured with NPCs do not undergo cell death, but rapidly lose their undiff. hESC phenotypes under bFGF-free differentiation culture conditions. Interestingly, along with the decline of OCT3/4 expression in the GFP+ cell clusters, expression of promyelocytic leukaemia zinc finger protein (PLZF), a primitive neuroepithelial cell marker,20Elkabetz Y. Panagiotakos G. Al Shamy G. Socci N.D. Tabar V. Studer L. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage.Genes Dev. 2008; 22: 152-165Crossref PubMed Scopus (516) Google Scholar gradually increased over the first 7 days of culture and then decreased at day 9 (Figure 3A). A similar time-dependent expression pattern of PAX6, an early neuroectodermal determinant, was also evident (Figure 3B). At day 9, the cells assembled in GFP+ cell clusters were those of young neuron morphology with bipolar processes, indicating conversion of hESCs toward the neural lineage. Neural conversion of hESCs by co-culturing with NPCs was further confirmed by using hESC lines expressing GFP under the control of the Hes5 (pHes5-GFP-hESC) and delta-like canonical Notch ligand 1 (Dll1; pDll1-GFP-hESC)21Placantonakis D.G. Tomishima M.J. Lafaille F. Desbordes S.C. Jia F. Socci N.D. Viale A. Lee H. Harrison N. Tabar V. Studer L. BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.Stem Cells. 2009; 27: 521-532Crossref PubMed Scopus (69) Google Scholar promoters specific for NPCs and differentiated neurons, respectively.22Campos L.S. Duarte A.J. Branco T. Henrique D. mDll1 and mDll3 expression in the developing mouse brain: role in the establishment of the early cortex.J. Neurosci. Res. 2001; 64: 590-598Crossref PubMed Scopus (49) Google Scholar, 23Castro D.S. Skowronska-Krawczyk D. Armant O. Donaldson I.J. Parras C. Hunt C. Critchley J.A. Nguyen L. Gossler A. Göttgens B. et al.Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif.Dev. Cell. 2006; 11: 831-844Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 24Henrique D. Adam J. Myat A. Chitnis A. Lewis J. Ish-Horowicz D. Expression of a Delta homologue in prospective neurons in the chick.Nature. 1995; 375: 787-790Crossref PubMed Scopus (920) Google Scholar, 25Kawaguchi A. Ikawa T. Kasukawa T. Ueda H.R. Kurimoto K. Saitou M. Matsuzaki F. Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis.Development. 2008; 135: 3113-3124Crossref PubMed Scopus (161) Google Scholar, 26Kawaguchi D. Yoshimatsu T. Hozumi K. Gotoh Y. Selection of differentiating cells by different levels of delta-like 1 among neural precursor cells in the developing mouse telencephalon.Development. 2008; 135: 3849-3858Crossref PubMed Scopus (68) Google Scholar, 27Park C.-H. Kang J.S. Yoon E.-H. Shim J.-W. Suh-Kim H. Lee S.-H. Proneural bHLH neurogenin 2 differentially regulates Nurr1-induced dopamine neuron differentiation in rat and mouse neural precursor cells in vitro.FEBS Lett. 2008; 582: 537-542Crossref PubMed Scopus (38) Google Scholar, 28Shimojo H. Ohtsuka T. Kageyama R. Oscillations in notch signaling regulate maintenance of neural progenitors.Neuron. 2008; 58: 52-64Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar None of the pHes5-GFP-hESCs or pDll1-GFP-hESCs cultured under ESC culture conditions expressed GFP (data not shown). The percentage of cells expressing GFP under the control of the Hes5 or Dll1 promoter gradually increased during co-culture with NPCs (Figures 3C, 3D, and S2). The GFP+ cells derived from pDll-GFP-hESCs at days 8–9 exhibited neuronal shapes characterized by a small cell body (5–15 μm in diameter), neurite outgrowths (Figures 3D–3G), and expression of the neuronal marker TUJ1 (Figures 3F and 3G). Collectively, these findings indicate that neural conversion of hESCs underlies the disappearance of undiff. ESC markers in the co-cultures. Further, notably, given derivation of neurons from hESC or iPSCs is a lengthy process requiring several weeks,7Perrier A.L. Tabar V. Barberi T. Rubio M.E. Bruses J. Topf N. Harrison N.L. Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2004; 101: 12543-12548Crossref PubMed Scopus (857) Google Scholar, 9Chambers S.M.S.S.M. Fasano C.A. Papapetrou E.P. Tomishima M. Sadelain M. Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.Nat. Biotechnol. 2009; 27: 275-280Crossref PubMed Scopus (2431) Google Scholar, 14Ko J.-Y. Park C.-H. Koh H.-C. Cho Y.-H. Kyhm J.-H. Kim Y.-S. Lee I. Lee Y.S. Lee S.H. Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons.J. Neurochem. 2007; 103: 1417-1429Crossref PubMed Scopus (62) Google Scholar neural induction from hESCs within 8 days is unprecedented. We investigated further in order to learn how neural differentiation of hESCs is induced under co-culture conditions. Culturing pDll1-GFP-hESCs alone in terminal differentiation medium (insulin-transferrin-selenite [ITS] medium supplemented with 0.2 mM ascorbic acid [ITSA]) did not produce significant neural induction, as evidenced by no or only a few GFP+ cells at day 7 of culture (Figures 4A and 4E ). In contrast, when pDll1-GFP-hESCs were cultured in medium conditioned by differentiating hES-NPCs, about 50% of the cells expressed GFP and PLZF 7 days after culture (Figures 4B and 4E). In order to examine the contribution of cell-to-cell contact-mediated signals, the hESC lines were cultured on a feeder layer of hES-NPCs fixed with paraformaldehyde.29Kawasaki H. Mizuseki K. Nishikawa S. Kaneko S. Kuwana Y. Nakanishi S. Nishikawa S.I. Sasai Y. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.Neuron. 2000; 28: 31-40Abstract Full Text Full Text PDF PubMed Scopus (1117) Google Scholar, 30Wurmser A.E. Nakashima K. Summers R.G. Toni N. D’Amour K.A. Lie D.C. Gage F.H. Cell fusion-independent differentiation of neural stem cells to the endothelial lineage.Nature. 2004; 430: 350-356Crossref PubMed Scopus (310) Google Scholar Robust neural conversion, as confirmed by GFP/PLZF expression, was attained in the hESCs cultured on the fixed NPC feeder layers (Figures 4C and 4E), and the effects were indistinguishable from those in cultures plated on live NPC feeder layers (Figures 4D and 4E). These findings suggest that both diffusible soluble factors and cell-to-cell contact-mediated signals from differentiating NPCs are responsible for neural conversion of hESCs, but cell-to-cell contact signals are the major contributor. To gain insight into candidate molecules responsible for neural induction of hESCs, we carried out RNA-sequencing (RNA-seq) analyses for H9 hES-NPCs and undiff. hESCs (H9), and analyzed the data with three different ways. First, we created intersections between the gene ontology (GO) of “neural development” and GOs for [“cell-to-cell” or “cell-matrix communication”] from QUICK GO (https://www.ebi.ac.uk/QuickGO). In addition, we also created interactions between the GO of “neural development” and GO for “secreted molecules and growth factors.” After unrelated genes were manually removed from the gene list, the genes in the intersections were defined as “neurogenic adhesion molecules” and “neurogenic soluble molecules,” respectively, and their expressions (log10[fold changes of hES-NPC/undiff. hESC]) are shown in Figure 4F. Second, we attempted to identify candidate cell adhesion or extracellular matrix (ECM) molecules from a published gene set for “Neuron-ECM interaction,” which were upregulated in 3D-neural culture versus 2D-neural culture.31Simão D. Silva M.M. Terrasso A.P. Arez F. Sousa M.F.Q. Mehrjardi N.Z. Šarić T. Gomes-Alves P. Raimundo N. Alves P.M. Brito C. Recapitulation of Human Neural Microenvironment Signatures in iPSC-Derived NPC 3D Differentiation.Stem Cell Reports. 2018; 11: 552-564Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar We selected this gene set because the ECMs enriched in the 3D culture contribute to establishing a more physiologic environment similar to the developing brain,32Baker B.M. Chen C.S. Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues.J. Cell Sci. 2012; 125: 3015-3024Crossref PubMed Scopus (1193) Google Scholar and thus are regarded as neurogenic molecules. The candidate gene expressions are shown in Figure 4G. Lastly, to define candidate genes in another way, we applied a conventional GO analysis for the genes upregulated in hES-NPCs versus undiff. hESCs (Figure 4I). Out of top eight GOs (upregulated fold change > 4, NPCs versus hESCs) from DAVID, four were GOs correlated with neural development and cell adhesion (Figure 4I, left). Candidate genes are listed in the intersections between the GOs of [“CNS development” or “nervous system development”] and [“hemophilic cell adhesion” or “cell adhesion”] (Figure 4I, right), and their expressions are shown in Figure 4H. Out of the 66 genes identified in Figures 4F–4H, 58 genes were upregulated in hES-NPC culture and thus suggested as candidate molecules to induce neural differentiation. Among the candidate genes, CDH2 (Cadherin-2; N-cadherin) has been shown to induce the differentiation of embryonic carcinoma P19 cells toward a neural lineage.33Gao X. Bian W. Yang J. Tang K. Kitani H. Atsumi T. Jing N. A role of N-cadherin in neuronal differentiation of embryonic carcinoma P19 cells.Biochem. Biophys. Res. Commun. 2001; 284: 1098-1103Crossref PubMed Scopus (84) Google Scholar Furthermore, it has also been reported that an N-cadherin-based biomimetic substrate promotes ESC or iPSC differentiation into NPCs without neuro-inducing chemical treatment.34Haque A. Adnan N. Motazedian A. Akter F. Hossain S. Kutsuzawa K. Nag K. Kobatake E. Akaike T. An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors.PLoS ONE. 2015; 10: e0135170Crossref PubMed Scopus (19) Google Scholar Thus, the cell adhesion molecule N-cadherin is regarded as one of the most viable candidates responsible for the neural induction of hPSCs. The undiff. state of hPSCs is maintained by the actions of pluripotent factors, and hPSC differentiation toward certain somatic cell lineages is attained by repressing pluripotency.35Niwa H. How is pluripotency determined and maintained?.Development. 2007; 134: 635-646Crossref PubMed Scopus (661) Google Scholar Out of the pluripotent factors expressed in hESCs or hiPSCs, KLF4 has been shown to specifically block hESCs neural differentiation by upregulating miR-371-3, a microRNA highly expressed in hESC lines with low neurogenic propensities.15Kim H. Lee G. Ganat Y. Papapetrou E.P. Lipchina I. Socci N.D. Sadelain M. Studer L. miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells.Cell Stem Cell. 2011; 8: 695-706Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar Based on the role of Notch signaling to downregulate KLF4 expression,36Ai X. Jia Z. Liu S. Wang J. Zhang X. Notch-1 regulates proliferation and differentiation of human bladder cancer cell l" @default.
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• W2938006872 title "Efficient Neural Differentiation of hPSCs by Extrinsic Signals Derived from Co-cultured Neural Stem or Precursor Cells" @default.
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• W2938006872 doi "https://doi.org/10.1016/j.ymthe.2019.04.011" @default.
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