TISSUE-SPECIFIC STEM CELLS

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1 TISSUE-SPECIFIC STEM CELLS Alternative Generation of CNS Neural Stem Cells and PNS Derivatives from Neural Crest-Derived Peripheral Stem Cells MARLEN WEBER, a GALINA APOSTOLOVA, b DARIUS WIDERA, c MICHEL MITTELBRONN, d GEORG DECHANT, b BARBARA KALTSCHMIDT, c,e HERMANN ROHRER a Key Words. Neural crest Neural stem cell Peripheral nervous system Central nervous system Reprogramming a Max-Planck-Institute for Brain Research, Research Group Developmental Neurobiology, Frankfurt, Germany; b Innsbruck Medical University, Institute for Neuroscience, Innsbruck, Austria; c Institute of Cell Biology and e Molecular Neurobiology, University of Bielefeld, Bielefeld, Germany; d Edinger Institute (Neurological Institute), Frankfurt, Germany Correspondence: Hermann Rohrer, Dr. Max-Planck-Institute for Brain Research, Research Group Developmental Neurobiology, Max-von-Laue-Str. 4, Frankfurt, Germany. Telephone: ; Fax: ; Hermann.rohrer@ brain.mpg.de Received April 28, 2014; accepted for publication September 6, 2014; first published online in STEM CELLS EXPRESS October 21, VC AlphaMed Press /2014/$30.00/ /stem.1880 ABSTRACT Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rncscs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rncscs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pncscs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage. STEM CELLS 2015;33: INTRODUCTION The neural crest gives rise to a broad array of derivatives including neurons and glia of the peripheral nervous system (PNS), endocrine adrenal chromaffin cells, cartilage and bone of the face, heart vasculature, and melanocytes of the skin [1]. Neural crest cells are specified at the border between neural plate and nonneural ectoderm, delaminate during neurulation from the dorsal neural tube to subsequently migrate to their various target sites. Neural plate border, premigratory, and migratory neural crest are characterized by different compositions of transcription factors that form a neural crest gene regulatory network [2, 3]. The neural crest contains self-renewing and multipotent stem cell-like cells that are present in migratory neural crest and in postmigratory locations like gut and peripheral ganglia [4]. Much of the current interest in neural crest cells is due to their multipotent stem cell-like characteristics and their potential for regenerative medicine [5]. We are referring to multipotent, self-renewing cells derived from embryonic, postnatal, and adult PNS tissues as neural crest-derived stem cells (NCSCs) [6] although in the absence of melanocyte progeny these cells may also be considered as neural crest-derived progenitor cells [5]. NCSCs differ from neural stem cells in the central nervous system (CNS) by the expression of specific markers like Sox10, HNK1, p75, and by their potential to generate PNS progeny and mesenchymal derivatives. However, there is evidence that embryonic NCSCs can be reprogrammed to CNS neural stem cells in neurosphere (NS) cultures under the influence of culture conditions with heparin-enhanced fibroblast growth factor (FGF)-signaling, developed for the maintenance of CNS stem cells [6 8]. Reprogrammed NCSCs (rncscs) give rise to CNS progeny in vitro and in vivo, upon transplantation into embryonic and postnatal brain. The ability to produce myelinating oligodendrocytes that repair brain lesions in vivo is of considerable interest for regenerative medicine as self-renewing rncscs can be expanded in culture and are not genetically modified [6, 7]. STEM CELLS 2015;33: VC AlphaMed Press 2014

2 Weber, Apostolova, Widera et al. 575 The aim of this study was to characterize the generation of CNS neural stem cells from NCSCs in the PNS in more detail with respect to the underlying cellular and molecular mechanisms. In addition, NCSCs from the adult PNS were analyzed for their ability to acquire CNS properties in NS cultures. Using global gene expression analysis we demonstrate that rncscs are indistinguishable from neural stem cells derived from embryonic spinal cord (SCSCs) and show no traces of their previous PNS identity. Reprogramming proceeds rapidly by a simultaneous downregulation and upregulation of PNS and CNS markers, respectively, and leads to a homogenous population of Olig2 1, Sox3 1, and Lex 1 CNS stem cells. Reprogramming does not involve an epigenetic resetting to a pluripotent state, but rather direct transdifferentiation from PNS to CNS stem cells. PNS stem cells can be prevented from acquiring a CNS identity by the application of BMP4 to the culture medium. The PNS identity of BMP NCSCs is shown by the expression of neural crest marker genes and their potential to generate characteristic PNS progeny, that is peripheral neurons and Schwann cells. In addition, neural crest derivatives with the characteristic expression pattern of pericyte progenitors and mesenchymal stem cells (MSCs) are generated in the presence of BMP4. As cultured MSCs and pericytes show a very similar phenotype, ontogenetic relationship, marker gene expression, and differentiation potential we refer to these cells in the following as MSC-like cells [9 11]. Interestingly, NCSCs from adult mouse palate (pncscs) were also directed toward pericyte fates under conditions that allowed CNS reprogramming in embryonic NCSCs. These findings demonstrate that the potential of neural crest cells to generate MSC-like cells [12, 13] is maintained in postmigratory embryonic and adult NCSCs. MATERIALS AND METHODS Cell Culture NS cultures from embryonic DRG and spinal cord and from adult palate were generated with modifications of published procedures [6, 14], as detailed in Supporting Information. Immunocytochemistry Protocols of immunostainings for cell surface and intracellular antigens of short-term cultures and differentiated NS cultures are described in detail in Supporting Information. RT-PCR Total RNA was isolated from P3 rncscs, BMP NCSCs, SCSCs, pncscs, and mouse embryonic stem cells (ESCs) (kindly provided by A. Smith, University of Cambridge, U.K.) using the RNeasy Kit (Qiagen, Hilden, Germany). cdna from RNA was synthesized using the M-MLV Reverse Transcriptase Kit (Invitrogen, Karlsruhe, Germany). PCR protocol and primer list (Supporting Information Table S1) are available in Supporting Information. Microarray Analysis Total RNA was isolated using TRIzol reagent (Life Technologies, Darmstadt, Germany), purified by RNeasy MinElute kit (Qiagen, Hilden, Germany), and analyzed for RNA integrity (2100 Bioanalyzer). RNA samples from three independent cell cultures of rncscs, BMP NCSCs, SCSCs, and pncscs were labeled and hybridized to Affymetrix Mouse Genome Arrays. Microarray hybridization was performed at the Expression Profiling Unit of the Medical University Innsbruck. Preprocessing and differential expression analysis were performed in R ( using packages from the Bioconductor project [15]. Raw expression values were normalized and summarized using the GeneChip robust multiarray analysis (GCRMA) method [16]. The moderated t test [17] was applied to assess the significance of differential expression between the compared groups; the resulting p-values were adjusted for multiple hypothesis testing following the method of Benjamini and Hochberg [18] for a strong control of false discovery rate. A gene was considered as differentially expressed when it was upregulated or downregulated at least 1.5-fold and its adjusted p-value was lower than.05. Gene ontology (GO) analysis was carried out to study the biological function of genes differentially expressed between rncscs, BMP NCSCs, and pncscs using DAVID [19]. Principal component analysis (PCA) and unsupervised hierarchical clustering (HC) were performed on samples based on normalized expression of all genes. To correct for batch effects either Com- Bat package was used [20] or SCAN a single-sample normalization approach which allows concurrent use of independent gene expression datasets [21]. Profiling results have been deposited at the gene expression omnibus (GEO) under accession GSE The following publicly available expression data downloaded from GEO were used for comparison with the microarray data generated in this study: GSE50824 (GSM , GSM , GSM , and GSM ); GSE8034 (GSM200043, GSM200044, and GSM200045). RESULTS Olig2 Is Induced in DRG-Derived NSs Under Proliferation Conditions (EGF/FGF) DRG-derived NCSCs are reprogrammed in NS cultures to rncscs that give rise under differentiation conditions to CNS progeny including Olig2 1 cells with oligodendrocyte morphology and markers O4, CNPase, and Sox10 [6] (Supporting Information Fig. S1A). To analyze the reprogramming efficiency, we use short-term cultures of third passage (P3) NSs in proliferation medium and characterize the properties of rncscs. Virtually all cells in P3 rncsc NSs express the CNS-specific gene Olig2 (99% 6 0.4%; n 5 3; mean 6 SEM; Fig. 1A; Supporting Information Fig. S1B), whereas the PNS marker p75 and the Schwann cell/oligodendrocyte marker O4 are not detectable. Sox10 1 cells, Tuj1 1 neurons, and glial fibrillary acidic protein (GFAP 1 ) glia are present in very low numbers (Fig. 1A, a f). Comparable results are obtained for P3 NSs derived from E12.5 mouse spinal cord (SCSCs) analyzed for comparison (Fig. 1A, g l). These data indicate that reprogramming of embryonic DRG-derived NCSCs occurs under conditions of NS cell proliferation and results in a homogenous Olig2 1 CNS stem cell population that does not maintain p75/ Sox10 PNS markers. As these conclusions are based on the analysis of a small number of marker genes, genome-wide expression profiles of P3 rncscs and SCSCs were analyzed by Affymetrix microarrays. Notably, the gene expression profiles of SCSCs and rncscs are virtually identical (Fig. 1B). Among the VC AlphaMed Press 2014

3 576 Generation of CNS Stem Cells from PNS Progenitors Figure 1. VC AlphaMed Press 2014 STEM CELLS

4 Weber, Apostolova, Widera et al. 577 probe sets on the array, only one (Efcab7) was differentially expressed following the established criteria for differential expression (adjusted p-value <.05; fold change 1.5). In both SCSCs and rncscs, genes expressed during normal development in spinal cord neuroepithelial cells and ventricular zone cells like Fabp7 [22, 23], Ptprz1, Tnc [24], Olig1, Olig2 [25], Sox1, Sox2, and Sox3 are highly abundant, whereas neural crest markers such as Tcfap2a, Tcfap2b, Dlx1, Dlx2, Erbb3, and Sox10 [26] are expressed at background levels (Fig. 1B). The selective expression of CNS markers Olig1, Olig2, Pax6, and Sox1 and the absence of marker genes for neural crest and neural crest derivatives (Tcfap2a, Tcfap2b, Dlx1, Dlx2, Erbb3, Mgp, Lgals3, and Bgn) were confirmed by RT-PCR (Fig. 1C). HC (Fig. 1E) and PCA (Fig. 1F) of the expression profiles from rncscs, SCSCs, and NSCs from cortex (radial glia progenitors (RGPs)) [27] and cerebellum (granule neuron progenitors (GNPs)) [28] demonstrate that rncscs and SCSCs cluster together and are distinct from NSCs in developing brain. The anteroposterior identity is maintained by DRG-derived NSs as shown by the continued expression of Hox genes (Hoxb6 and Hoxb10) and the absence of forebrain markers (Foxg1, Emx1, and Nr2e1) (Supporting Information Fig. S2 and [6]). DRG-Derived rncscs Acquire a Spinal Cord Neural Progenitor Identity Analyzing the gene expression of the total NS cell population leaves the question open whether rncscs NSs are composed of neural stem/progenitor cells with different identities that may correspond to neural plate and/or different dorsoventral locations in the spinal cord. An alternative would be that NS culture conditions induce or select a common neural progenitor identity. To address this issue, rncscs and SCSCs were analyzed by immunostaining for marker genes with either broad or restricted expression in neural plate and spinal cord. Sox3, the LewisX (LeX) glycan, detected by the mabs 5750 LeX and 487 LeX (data not shown) and the chondroitin sulfate proteoglycan (CH-PG) recognized by the monoclonal antibody (mab) 473HD are widely expressed by nestin 1, NS-forming neural precursor cells during early stages of CNS development, including spinal cord [29 34]. Pax6, Fabp7, Olig1/2, and Sox1 are observed during development in spinal cord rather than neural plate and Fabp7, Olig1/2, and Nkx2.2 are ventral spinal cord markers [23, 35]. The uniform expression of all markers analyzed in NSs derived from both embryonic DRG and spinal cord (Fig. 1D) argues in favor of a common ventral spinal progenitor identity, which is induced by culturing in FGF/epidermal growth factor (EGF). Kinetics and Mechanism of Reprogramming To define the kinetics of reprogramming, Olig2 and p75 as CNS- and PNS-markers, respectively, were analyzed in the E12.5 DRG starting population and at different passages. Already after P1, which corresponds to approximately 2 weeks, approximately 70% of the cells express Olig2 and the proportion of p75 1 cells is decreased to below 10%. After P2 a homogenous Olig2 1 /p75 2 population is present, which is maintained at least up to P10, the latest passage analyzed (Fig. 2A). Sox10 expression is lost within a similar time frame [6]. Cells coexpressing p75 and Olig2 were not detected (Fig. 2B), suggesting that the loss of p75 expression precedes Olig2 induction. This observation also raises the issue of potential transitory states between neural crest and rncscs/scscs. Generation of rncscs may involve a resetting of the epigenetic state from neural crest to a pluripotent embryonic state followed by the acquisition of CNS identity. Pluripotency genes are indeed expressed in NCSCs derived from various tissues, including craniofacial and trunk skin, palate, olfactory, and respiratory mucosa [36, 37]. However, the expression levels of Oct4, Lin28, Nanog, Klf4, and other pluripotency markers were much lower in NCSCs as compared to ESCs [37, 38]. Also rncscs express much lower levels of Oct4, Nanog, and Klf4 than ESCs and from the core pluripotency genes that are induced by different combinations of transcription factors during direct reprogramming only Sox2 is expressed (Fig. 2C). The low Oct4, Nanog, and Klf4 expression, together with the lack of teratoma formation upon implantation of rncscs into embryonic brains [6] argue against pluripotent properties of rncscs and against a transient pluripotent state during reprogramming [6, 7]. This is supported by undetectably low expression of Oct4 and Nanog also during the first passage, that is, during the switch from PNS to CNS identity (data not shown). NCSC NSs Cultured in the Presence of BMP4 Generate Neural Crest Progenitors NS culture conditions have been developed and optimized for the growth of CNS neural stem cells and may not be Figure 1. rncscs show uniform expression of CNS. Third passage rncscs and SCSCs neurospheres were analyzed via immunocytochemistry (A, D), affymetrix gene array (B, E, F), and RT-PCR (C). (A): rncscs and SCSCs neurosphere cells under proliferation conditions express Olig2 (a, g) and are devoid of p75 (b, h). Only very few cells express Sox10 (c, i), Tuj1 (d, j) and GFAP (e, k). O4 1 oligodendrocytes were never detected (f, l) under proliferation conditions. (B): Scatter plot demonstrates a virtual identical gene expression pattern of rncscs and SCSCs. Genes characteristic for CNS stem cells, neural crest, and neural crest derivatives and ESCs genes are indicated (red). Differential expression represents log twofold change. (C): Differential expression of marker genes is confirmed by RT-PCR (CNS marker: Olig1/2, Pax6, Sox1; NC/mesenchymal NC marker: Tcfap2a/2b, Dlx1/2, Erbb3, Mgp, Lgals3, Bgn). Trunk AP identity is shown by Hoxb6 expression. PCRs of all marker genes were run in parallel and were analyzed on the same gel. (D): Virtually all cells express LeX epitopes 473HD (a, g) and Lex5750 (b, h), Sox3 (c, i), Fabp7 (d, j) and are devoid of the peripheral nervous system markers Tcfap2a (e, k) and Phox2b (f, l). Arrows in Ac and Ai point to Sox10 1 cells. (E): Hierarchical clustering of rncscs, SCSCs samples, and NSCs from cortex (GFP low /prominin 1 radial glia [RGPs]) [27] and cerebellum (GNPs) [28] (average linkage algorithm) based on normalized expression of all genes. In the dendrogram, Pearson correlation is used as a distance metric. Please note that the biological replicates cluster together, that rncscs and SCSCs form a cluster that strongly differs from RGPs and GNPs. (F): Unsupervised principal component analysis comparing gene expression profiles of rncscs, SCSCs, RGP [27], and GNP [28]. Each dot represents the global gene expression of a single microarray sample. Distances between dots indicate the difference between the samples. Scale bars in (A) and (D) 5 30 mm. Abbreviations: GFAP, glial fibrillary acidic protein; GNP, granule neuron progenitor; NCSC, neural crest-derived stem cell; RGP, radial glial progenitor; SCSC, stem cells derived from embryonic spinal cord. VC AlphaMed Press 2014

5 578 Generation of CNS Stem Cells from PNS Progenitors Figure 2. Reprogramming of rncscs to cells with CNS identity is complete at passage 2 and maintained at least up to passage 10. (A): NCSC neurospheres at passages 1 3 (P1 3) and P10 were analyzed in short-term cultures by immunocytochemistry and quantification of marker-positive cells and compared to dissociated E12.5 DRG cells. The peripheral nervous system marker p75 is downregulated (DRG: 96.5% 6 0.5%, P1: 7.8% 6 5.9%, P2: 0.1% 6 0.0% [n 5 3 4; mean 6 SEM]) and is not detected from P3 onwards. CNS marker Olig2 is upregulated (P1: 73.7% %, P2: 90.7% 6 1.8%, P3: 94.2% 6 1.6%, and P10: 89.7% 6 2.0% [n 5 3 4; mean 6 SEM]). Olig2 is not detected in DRG cultures (n 5 5). (B): p75 1 /Olig2 1 cells were not identified in P1 rncscs. (C): From the pluripotency genes analyzed by RT PCR in P3 rncscs and SCSCs Oct4 was not detectable. Klf4 and Nanog were expressed at very low levels as compared to ESCs. Sox2 is expressed at similar levels in rncscs, SCSCs, and ESCs. PCRs of all marker genes were run in parallel and were analyzed on the same gel. Arrows in (B) point to p75 1 /Olig2 2 cells. Scale bars 5 30 mm. Abbreviations: DRG, dorsal root ganglion; ESCs, embryonic stem cells; rncscs, reprogrammed neural crest-derived stem cells; SCSCs, stem cells derived from embryonic spinal cord. appropriate for culturing of PNS neural stem cells. We have previously shown that PNS identity of NCSCs is maintained in presence of chick embryo extract and BMP4 [6]. Here, we demonstrate that the addition of BMP4 to the NS culture medium is sufficient to antagonize the reprogramming effect of the NS culture, which is reflected by the complete absence of Olig2 in P3 BMP4-treated NSs (BMP NCSCs) (Fig. 3D). The neural crest and glia marker Sox10, which is downregulated in rncscs (Fig. 1A, 1B), remained expressed in approximately 45% of BMP NCSCs at P3 (Fig. 3A, 3D). The majority of Sox10 1 cells may represent glia progenitors that downregulate p75 [39, 40] as p75 is retained only in a subpopulation of 5% 10% of BMP NCSCs from P1 onwards (Fig. 3A, 3D; Supporting Information Fig. S3). The neural crest and glia marker Tcfap2a and the autonomic progenitor marker Phox2b are expressed in 79% 6 4% (n 5 3) and 5% 6 3% (n 5 3) of BMP NCSCs (Fig. 3D, g, h). The neural crest progenitor identity of BMP NCSCs is confirmed by comparative gene expression profiling of BMP NCSCs and rncscs. The expression profile of BMP NCSCs differs massively from the profile of rncscs/scscs as observed by scatter plot (Fig. 3B), HC (Fig. 3C; Supporting Information Fig. S4), and PCA (Supporting Information Fig. S4). Twentyeight percent of all probe-sets are significantly differentially expressed (1.5-fold-change, adjusted p-value <.05). BMP NCSCs show strong and selective expression of established neural crest markers like Tcfap2a, Tcfap2b, Dlx1, Dlx2, Erbb3, Sox10 and of the pan-autonomic marker Phox2b and undetectably low expression of CNS stem cell marker genes. Expression of pluripotency genes was restricted to Sox2 with low Klf4 expression and undetectable expression of Oct4 and Nanog. Selective expression was confirmed by immunostaining VC AlphaMed Press 2014 and RT-PCR (Fig. 3D, 3E, Table 1). GO analysis of microarray data showed that genes characteristic for developing nonneural tissues, including vasculature, cartilage, and bone were enriched in BMP NCSCs (Supporting Information Table S2). The strong expression of Pdgfrb, Rgs5, S1pr3, Acta2, Spp1, Timp2, CD248, and Ly6a (Table 1) which are associated with MSCs/progenitors for perivascular pericytes and vascular smooth muscle cells [41, 42] explains the enrichment of the GO term vasculature. Differential expression was also observed for the MSC/pericyte markers Kcnj8 and Abcc9 (Fig. 3E). The expression of genes associated with chondrocyte and osteoblast development (Acan, Bgn, Mgp, Calcylin, galectin, Dlx2, Col2a1, Col9a1, Fmod, Acan, and osteoglycin [Table 1, Fig. 3E]) indicates the generation of mesenchymal neural crest derivatives induced by BMP and FGF [43 52]. Besides endoneurial fibroblasts [53], non-neural progeny is restricted to cranial neural crest during normal development, but trunk neural crest cells also possess the potential for mesenchymal differentiation and MSC generation [12, 13, 54 57]. Our present findings support and extend these observations by the demonstration that MSC-like cells can be generated from postmigratory NCSCs in peripheral ganglia. The expression of many trunk-specific Hox-genes observed in the gene expression profiling and confirmed by RT-PCR for Hoxb6 (Fig. 3E) argues against a shift in the anteroposterior identity in the presence of BMP4 [6, 58]. However, approximately 45% of P3 BMP NCSCs maintain Sox10, which marks progenitors of peripheral neurons and glia [59, 60] but is downregulated in neural crest cells during mesenchymal progenitor generation [12]. PNS progenitors in BMP NCSCs were identified by their potential to produce Phox2b 1 /Tuj1 1 immature autonomic neurons (Fig. 3D, g), p75 1 /Tuj1 1 (Fig. 3F, e), and peripherin 1 / STEM CELLS

6 Weber, Apostolova, Widera et al. 579 Tuj1 1 (Fig. 3G) PNS neurons and the potential to differentiate to O4 1 /p75 1 peripheral glial cells in the presence of neuregulin and forskolin (Fig. 3H). Notably, this treatment was unable to elicit the generation of p75 1 PNS glia in rncsc cultures [6]. Conversely, GFAP 1 /p75 2 astrocytes and Olig2 1 /O4 1 oligodendrocyte-like cells are generated from rncscs (Supporting Information Fig. S1) but not from BMP NCSCs (Fig. 3F, a, b). Taken together these results demonstrate that the Figure 3. VC AlphaMed Press 2014

7 580 Generation of CNS Stem Cells from PNS Progenitors addition of BMP4 prevents CNS reprogramming, supports progenitors of PNS neurons and glia, and induces MSC development in DRG-derived trunk NCSCs. BMP NCSCs and rncscs Are Stably Restricted to PNS and CNS Fates, Respectively Our finding that reprogramming can be prevented by BMP signaling raised the question whether rncscs and BMP NCSCs can be switched back to the identity of neural crest and CNS stem cells, respectively [61]. Omission of BMP4 in P3 BMP NCSCs does not result in the generation of CNS progeny, as shown by the absence of Olig2 1 and GFAP 1 cells and the continued expression of p75 and Sox10 at P5 (Fig. 4A). Similar proportions of Sox10 1 and p75 1 cells are observed in BMP NCSC cultures with or without BMP4 (Supporting Information Fig. S3). Conversely, delayed addition of BMP4 to P3 rncscs did not reduce the proportion of GFAP 1 astrocytes and of Olig2 1 cells and also did not elicit p75 expression at P5 (Fig. 4B). However, Sox3 expression was inhibited and the differentiation to O4 1 /Olig2 1 oligodendrocytes was completely blocked (Fig. 4B, Supporting Information Fig. S5). Although Sox3 expression is used as early neural plate marker, which is antagonized by BMP signaling, it has recently been shown to be essential for oligodendrocyte differentiation [62 64]. Together with the established block of oligodendrocyte differentiation by BMP [65, 66] these findings imply selective effects of BMPs on rncsc oligodendrocyte differentiation rather than a loss of CNS identity upon delayed BMP treatment. Together, these data imply a stable CNS and PNS identity for rncscs and BMP NCSCs, respectively. Adult NCSCs from Mouse Palate Acquire Cranial Neural Crest Rather than CNS Fates in NS Cultures The characterization of rncscs as cells with complete CNS identity, together with the previously demonstrated ability of these cells to differentiate in vivo to myelinating oligodendrocytes, suggests the use of rncscs for cell replacement therapies. Therapeutical application of rncscs requires, however, an easily accessible source of adult NCSCs and efficient methods for reprogramming. As DRGs are not readily accessible we have chosen the sensory innervation of adult mouse palate as source of adult NCSCs. NS-generating cells in rat and human palate represent a convenient source for adult NCSCs that display multipotent properties [14]. It was unclear, however, whether palate NCSCs (pncscs) have the potential to generate CNS progeny. In NS cultures with heparin-enhanced FGF signaling, pncscs from adult mouse palate downregulate Sox10 and p75 with increasing passage number, but in contrast to embryonic and postnatal NCSCs, do not induce any of the CNS progenitor markers expressed by rncscs (Fig. 5A, 5B). Also under differentiation conditions neither Olig2 1 nor GFAP 1 cells were detected (Fig. 5A). The comparison of the gene expression profile of pncsc NSs and rncsc NSs revealed strong differences, that is, 31% of probesets are differentially expressed (1.5-fold-change, adjusted p-value <.05) and the expression of CNS markers is restricted to rncscs (e.g., Fabp7, Olig1/2, Pax6, Sox1, Slc1a3, and Prprz1) (Figs. 3C, 5C, 5D; Table 2). pncscs express genes characteristic for neural crest and neural crest derivatives, including the transcription factors Tfap2a, Snai2, Erbb3, Twist1, and Dlx1/2 (Fig. 5C, 5D; Table 2). However, GO analysis (Supporting Information Table S3) revealed the strongest enrichment for the GO terms vasculature development, blood vessel development and morphogenesis, and angiogenesis, suggesting that pncscs resemble mesenchymal cranial neural crest. This notion is supported by the lack of Sox10, which is downregulated in mesenchymal progenitors and the strongly enriched expression of MSC/pericyte marker genes Pdgfrb, Rgs5, Abcc9, S1pr3, Kcnj8, Enpep, Spp1, Ly6a, Timp2, and CD248 (Fig. 5C, 5D; Table 2). Notably, pericytes and vascular smooth muscle cells (vsmcs) in head, meninges, brain, retina, optic nerve, and thymus are derived from cranial neural crest [57, 67 70]. Thus, the induction of MSCs/pericyte development in pncscs NS cultures reflects a normal fate of cranial NCSCs [41, 42]. The role of MSCs in immune homeostasis and tissue repair may account for the enrichment of the annotation cluster with the response to wounding, defense response and inflammatory response terms [9, 71] (Supporting Information Table S3). As MSCs isolated from various tissues are thought to originate from perivascular Figure 3. BMP NCSCs maintain their PNS identity and show strong differences in gene expression pattern compared to rncscs. (A): BMP NCSCs neurospheres were analyzed in short-term cultures at P1 3 by immunocytochemistry and quantification of marker-positive cells and compared to dissociated E12.5 DRG cells. A high proportion of BMP4 NCSCs express Sox10 (DRG: 22.1% 6 1.7%, P1: 65.6% 6 6.0%, P2: 61.3% 6 3.5%, P3: 45.5% 6 1.8% [n 5 3; mean 6 SEM]). p75 is downregulated but remains expressed al low levels (DRG: 96.5% 6 0.5%, P1: 5.4% 6 1.3%, P2: 6.4% 6 2.8%, P3: 5.3% 6 2.3% [n 5 3; mean 6 SEM]). (B): Scatter plot demonstrates strong differences in gene expression profile of BMP NCSCs and rncscs. Central nervous system (CNS), ESC, and neural crest marker genes are indicated (red). (C): Hierarchical clustering of rncscs, SCSCs, BMP NCSCs, and adult pncscs samples (average linkage algorithm) based on normalized expression of all genes. In the dendrogram, Pearson correlation is used as a distance metric. Please note that the biological replicates cluster together, that rncscs and SCSCs form a cluster that strongly differs from BMP NCSCs and pncscs. (D): P3 BMP NCSCs generate p75 1 and Tuj1 1 cells with neuronal morphology (b, d), Tuj1 1 /Phox2b 1 autonomic neurons (g; arrows). O4 1 and GFAP 1 glial cells were not observed (e, f). BMP NCSCs are devoid of CNS markers Olig2 (a) 473HD (i), Lex5750 (j), Sox3 (k), and Fabp7 (l) and express NC markers Tcfap2a (h) and Sox10 (c). (E): Differential expression of marker genes is confirmed by RT-PCR (CNS marker: Olig1/2, Pax6, Sox1; NC/NC derivative marker Tcfap2a/2b, Dlx1/2, Erbb3, Mgp, Lgals3, Bgn. Mesenchymal stem cell marker: Rgs5, Pdgfrb, S1pr3, Abcc9, Kcnj8). Sox2 is strongly expressed in BMP NCSCs, rncscs, and ESCs, whereas Oct4, Nanog, and Klf4 are only expressed at high levels in ESCs. Trunk AP identity is shown by Hoxb6 expression. PCRs of all marker genes were run in parallel and were analyzed on the same gel. (F H): BMP NCSCs differentiate into PNS cell types. (F): In differentiated cultures of P3 BMP NCSCs Olig2 is not observed (a). PNS identity of BMP NCSC derivatives is shown by p75 1 /Sox10 1 cells (c), p75 1 /GFAP 2 cells (d), and Tuj1 1 /p75 1 PNS neurons (e). (G): PNS identity of neuronal progeny of BMP NCSCs is shown by peripherin expression. (H): BMP NCSCs differentiate into O4 1 /p75 1 PNS glial cells when treated with neuregulin and forskolin. Scale bar 5 30 mm. Abbreviations: DRG, dorsal root ganglion; ESCs, embryonic stem cells; GFAP, glial fibrillary acidic protein; pncsc, palate neural crest-derived stem cell; rncsc, reprogrammed neural crest-derived stem cell; SCSC, stem cells derived from embryonic spinal cord. VC AlphaMed Press 2014 STEM CELLS

8 Weber, Apostolova, Widera et al. 581 Table 1. Comparison of gene expression of reprogrammed neural crest-derived stem cells (rncscs) and BMP NCSCs Gene symbol Gene name Fold change p-value Early neural genes (neural plate) Zic1 Zinc finger protein of the cerebellum E-10 Sox3 SRY-box containing gene E-08 Gbx2 Gastrulation brain homeobox Sox11 SRY-box containing gene E-08 Pou3f1 (Oct6) POU domain, class 3, transcr. factor E-05 Pou3f4 (Brn4) POU domain, class 3, transcr. factor E-10 Spinal cord ventricular zone Fabp7 Fatty acid binding protein 7, brain E-11 Olig1 oligodendrocyte Transcription factor E-11 Olig2 Oligodendrocyte transcription factor E-11 Pax6 Paired box gene E-08 Sox1 SRY-box containing gene E-06 Slc1a3 Glial high affinity glut. transp. (Glast) E-05 Ptprz1 Protein tyrosine phosphatase, receptor z E-11 Tnc Tenascin C E-08 Neural crest Snai2 Snail homolog 2 (Drosophila) E-10 Erbb3 v-erb-b2 homolog E-10 Sox10 SRY-box containing gene E-10 Tcfap2a Transcription factor AP-2, alpha E-07 Tcfap2b Transcription factor AP-2, beta E-05 Dlx2 Distal-less homeobox E-09 Chondrocyte/osteoblast Bgn Biglycan E-11 Mgp Matrix gla protein E-09 Lgals3 Galectin E-09 Col2a1 Collagen, type II, alpha E-11 Col9a1 Collagen, type IX, alpha E-08 Fmod Fibromodulin E-10 Acan Aggrecan E-08 Ogn Osteoglycin E-08 MSC/pericyte Pdgfrb Pdgf receptor, beta E-08 Rgs5 Regulator of G-protein signaling E-09 S1pr3 Endothelial diff. spingolipid G-pr. coupl. rec E-09 Acta2 Smooth muscle actin, alpha E-05 Spp1 Secreted phosphoprotein E-06 Timp2 Tissue inhibitor of metalloprotease E-06 CD248 Endosialin E-05 Ly6a Lymphocyte antigen 6 comp.; Sca Analysis of gene expression in rncsc and BMP NCSC neurospheres by Affymetrix microarrays. We selected the following genes for display: (a) genes expressed in neural plate epithelial cells, (b) in the spinal cord ventricular zone, (c) in neural crest, (d) in mesenchymal neural crest derivatives, and (e) in MSC/perivascular cells. The average signal fold change is shown (log twofold values). p-values adjusted for multiple hypothesis testing (Benjamin and Hochberg [18]). Abbreviation: MSC, mesenchymal stem cell. pericytes, the MSC-like cells in pncscs cultures could either be derived from p75 1, nestin 1 glial stem/progenitor cells [14], or by the selective propagation of neural crest-derived perivascular pericytes. To address this issue, the generation of NSs was followed in pncsc cultures from a Sox10-eGFP mouse line [72]. The vast majority of initial NSs in pncsc cultures is composed of green fluorescent protein (GFP 1 ) cells (Fig. 5E). After 5 days in culture 94.5% 6 2% (n 5 2) of NSs contained Sox10 1 cells whereas Sox10 2 NSs represented only 5.5% 6 2% (n 5 2). The strongly decreased proportion of Sox10 1 NSs after 7 days in culture (52% 6 2%; n 5 2) is compatible with the notion that palate NSs are derived from Sox10 1 cells in the sensory innervation that rapidly lose Sox10 expression. Although MSC-like cells are generated in both pncsc and BMP NCSC cultures, there are pncsc-specific traits, including the absence of Hox gene expression and of BMP-controlled genes like Phox2b, Msx2, and Tbx20. Different gene expression profiles, demonstrated by HC and PCA analysis (Fig. 3C, Supporting Information Fig. S4), are expected due to the difference in rostrocaudal origin and culture conditions. In addition, the comparison of markers associated with bone and cartilage development (cf. Tables 1 and 2) suggests a different extent or type of mesenchymal differentiation. DISCUSSION NCSCs from the embryonic PNS can be reprogrammed in NS cultures to CNS stem cells that generate oligodendrocytes, astrocytes, and CNS neurons. We now demonstrate that rncscs display the full identity of spinal cord-derived neural stem cells and are completely devoid of PNS characteristics. In the presence of BMP4, CNS reprogramming is prevented which allows to propagate either PNS or CNS neural stem cells in NS cultures from embryonic DRG. Mesenchymal fates VC AlphaMed Press 2014

9 582 Generation of CNS Stem Cells from PNS Progenitors Figure 4. The CNS/PNS identity of reprogrammed neural crest-derived stem cells (rncscs) and BMP NCSCs stays stable. (A): BMP NCSCs kept without BMP4 (wobmp) between P3 and P5 were analyzed after differentiation and compared with BMP NCSCs cultured in continuous presence of BMP4. BMP NCSCs without BMP4 are devoid Olig2 1 CNS cells (a), differentiate into p75/sox10 coexpressing cells (e), and lack GFAP 1 (g) and O4 1 (a, c) glial cells similar to the P5 BMP NCSCs control (b, d, f, h). (B): rncscs were supplemented between P3 and P5 with BMP4 (delayed BMP) and analyzed after differentiation for 7 days. rncscs with delayed BMP treatment express Olig2 (a), are devoid of p75 (c, g), and differentiate into GFAP 1 astrocytes (e, g), as P5 rncscs controls. However, O4 1 /Olig2 1 oligodendrocyte-like cells were not detected after delayed BMP4 application (cf. a and c). Scale bars 5 30 mm. Abbreviation: GFAP, glial fibrillary acidic protein. are induced in embryonic BMP NCSCs and in adult NCSCs from mouse palate demonstrating that the potential of NCSCs for MSC generation is maintained up to the adult stage. VC AlphaMed Press 2014 Complete Reprogramming of PNS-Derived NCSCs to CNS Stem Cells with Spinal Cord Identity (SCSCs) From previous investigations on CNS reprogramming of embryonic NCSCs it was unclear whether reprogramming takes place in proliferating NSs, whether the change in gene expression is complete and if all NS cells acquire CNS properties [6, 7]. Considering the identical expression profiles of rncsc and SCSC NSs, that are distinct from those of NSCs from cortex and cerebellum, we conclude that NCSCs completely change their gene expression profile and acquire an identity, which is indistinguishable from SCSCs. rncscs/scscs display high level expression of genes characteristic for neuroepithelial and ventricular zone cells, like Fabp7, Olig1, Olig2, Ptprz1, TnC, Pax6, Sox1, and only background levels of genes expressed by neural crest and neural crest derivatives. Only for one gene differential expression reached statistical significance but as it is not expressed in a CNS or PNS-specific manner, it seems to be unrelated to CNS reprogramming. The results of global expression analysis could reflect the properties of mixed cell populations at different developmental stages or of a homogenous stem cell population. Immunostaining revealed that rncscs/scscs expressed Olig2, Sox3, Fabp7, the CH-PG recognized by the mab 473HD, and the LewisX (LeX) glycan recognized by the mabs 5750 Lex and 487 Lex. CH-PG and LeX glycan are carried by Tenascin C and Receptor Protein Tyrosine Phosphatase b/f that are highly enriched in rncscs and SCSCs, and are important components of the CNS stem cell niche that control cell STEM CELLS

10 Figure 5. pncscs are not able to acquire a CNS phenotype but express genes characteristic for pericyte progenitors/mesenchymal stem cells (MSCs). Under proliferation conditions (A, a f) and after differentiation for 7 days (A, g l), P3 pncscs are devoid of Olig2 1 CNS cells (a, g), lack GFAP (e, k) and O4 (I, f). Small populations of Sox10 1 cells (c, i) and Tuj1 1 /p75 1 cells (d, j) are present. (B): pncscs were analyzed in short-term cultures at P1 and P3 by immunocytochemistry and quantification of marker-positive cells. p75 (P1: 63.3% 6 0.2%, P3: 11.3% 6 7.3% [n 5 3; mean 6 SEM]) and Sox10 (P1: 34.5% 6 1.9%, P3: 1.2% 6 0.7% [n 5 3 5; mean 6 SEM]) are downregulated in culture. (C): Scatter plot demonstrates strong differences in gene expression pattern of pncscs and rncscs. Genes characteristic for CNS stem cells, ESCs, neural crest, chondrocyte/osteoblasts, and MSC/pericyte progenitors are indicated (red). (D): Differential expression of marker genes is confirmed by RT-PCR. PCRs of all marker genes were run in parallel and were analyzed on the same gel. (E): Freshly dissociated palate cells from Sox10 GFP mice show GFP expression in cells with Schwann cell morphology (a, b). GFP-expressing initial pncsc neurosphere at 5 days in culture. Scale bars 5 30 mm. Abbreviations: ESCs, embryonic stem cells; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; pncsc, palate neural crest-derived stem cell; rncsc, reprogrammed neural crestderived stem cell.

11 584 Generation of CNS Stem Cells from PNS Progenitors Table 2. Comparison of gene expression of reprogrammed neural crest-derived stem cells (rncscs) and palate NCSCs Gene symbol Gene name Fold change p-value Early neural genes (neural plate) Zic1 Zinc finger protein of the cerebellum E-09 Sox3 SRY-box containing gene E-08 Gbx2 Gastrulation brain homeobox Sox11 SRY-box containing gene E-08 Pou3f1 (Oct6) POU domain, class 3, transcr. factor Pou3f4 (Brn4) POU domain, class 3, transcr. factor E-08 Spinal cord ventricular zone Fabp7 Fatty acid binding protein 7, brain E-08 Olig1 Oligodendrocyte transcription factor E-11 Olig2 Oligodendrocyte transcription factor E-11 Pax6 Paired box gene E-09 Sox1 SRY-box containing gene E-07 Slc1a3 Glial high affinity glut. transp. (Glast) E-08 Ptprz1 Protein tyrosine phosphatase, receptor z E-11 Neural crest Snai2 Snail homolog 2 (Drosophila) E-10 Erbb3 v-erb-b2 homolog E-10 Tcfap2a Transcription factor AP-2, alpha E-07 Twist1 Twist1 transcription factor Dlx1 Distal-less homeobox Dlx2 Distal-less homeobox Chondrocyte/osteoblast Bgn Biglycan E-11 Mgp Matrix gla protein E-10 Lgals3 Galectin E-10 Col2a1 Collagen, type II, alpha ns Col9a1 Collagen, type IX, alpha ns Fmod Fibromodulin ns Acan Aggrecan ns Ogn Osteoglycin ns MSC/pericyte Pdgfrb Pdgf receptor beta E-10 Rgs5 Regulator of G-protein signaling E-12 Abcc9 ATP-binding cassette, subfamily C, E-11 Ly6a Lymphocyte antigen 6 comp.; Sca E-08 S1pr3 Endothelial diff. spingolipid G-pr. coupl. rec E-11 Kcnj8 Potassium inwardly rect. Channel J E-08 Enpep Glutamylaminopeptidase E-05 Spp1 Secreted phosphoprotein E-08 Timp2 Tissue inhibitor of metalloprotease E-10 CD248 Endosialin E-06 Analysis of gene expression in rncsc and BMP NCSC neurospheres by Affymetrix microarrays. We selected the following genes for display: (a) genes expressed in neural plate epithelial cells, (b) in the spinal cord ventricular zone, (c) in neural crest, (d) in mesenchymal neural crest derivatives, and (e) in MSC/perivascular cells. The average signal fold change is shown (log twofold values). p-values adjusted for multiple hypothesis testing (Benjamin and Hochberg [18]). Abbreviation: MSC, mesenchymal stem cell. proliferation and differentiation [73 75]. Since all neural stem cell markers analyzed are uniformly expressed by virtually all rncscs and SCSCs we conclude that a common CNS stem cell identity is induced in NS cultures. As many early neural genes are expressed both at the neural plate stage and by progenitor cells in the spinal cord ventricular zone it is difficult to decide whether rncscs correspond to early neural plate or spinal cord [76 78]. However, the expression of Pax6, Fabp7, Olig2, and Sox1, which are undetectable at the neural plate stage, and the absence of Pou5f1 (Oct4), which is transiently expressed at the neural plate stage [79 82], imply a spinal cord rather than neural plate identity. In addition, a ventral spinal cord identity is suggested as Fabp7, Olig2, and Nkx2.2 are expressed in the ventral neural tube during normal development [6, 25, 83]. The anterior-posterior identity of the NPCs is maintained as trunkspecific Hox genes but not forebrain-specific markers are expressed [6, 84]. VC AlphaMed Press 2014 Kinetics and Mechanism of Reprogramming The majority of NCSCs has acquired the CNS marker Olig2 and lost p75 after the first passage. Cells with mixed phenotype were not detected. This raised the question whether reprogramming involves a transient pluripotent state. rncscs/scscs express, however, much lower levels of pluripotency genes as compared to ESCs. From the core pluripotency genes only Sox2 is expressed at high levels, Nanog and Klf4 expression are very low and Oct4 was not detected. Also during the first passage, that is, the switch from PNS to CNS identity we did not observe increased Oct4 and Nanog expression. As Sox2 expression in the developing spinal cord is not restricted to ventricular zone stem cells but maintained in the glial cell lineage, Sox2 expression cannot be taken as evidence for pluripotency [64]. It should also be noted that SCSCs and rncscs display identical pluripotency gene expression although SCSC generation does not involve a change of identity. Taken together these results argue against a transient pluripotent state during CNS STEM CELLS

12 Weber, Apostolova, Widera et al. 585 reprogramming. A more likely possibility is that NCSCs transdifferentiate to spinal cord neural stem cells. Notably, the transcriptional circuitry that controls the generation of the neural plate during development, in particular the class III Pou transcription factors Pou3f1 (Oct6) and Pou3f4 (Brn4), is highly expressed in rncscs [85]. Class III Pou transcription factors elicit Sox2 expression, which leads to induction of nestin expression by the synergistic action of Pou and Sox factors [86]. Neural Crest Fate Is Maintained by BMP4 in NCSC NS Cultures The neural crest is a transient population of stem and progenitor cells that in culture and in vivo rapidly differentiate to various derivatives. Thus, there are only very few cell culture models that allow to propagate embryonic neural crest stem cells [87 89]. We now demonstrate that the presence of BMP4 is sufficient to prevent CNS reprogramming and allows longterm culture of DRG-derived NCSCs. PNS progenitor identity of BMP NCSCs is shown by the expression of the neural crest markers Snai2, Erbb3, Sox10, Dlx1/2, Tcfap2a/b and the ability to generate O4 1 /p75 1 PNS glia as well as Phox2b 1 /Tuj1 1 and peripherin 1 /Tuj1 1 PNS neurons. The generation of Phox2b 1 autonomic progenitors is expected in the presence of BMPs that are essential and sufficient for autonomic neuron development [90 92]. BMPs are also involved in mesenchymal differentiation including cartilage, bone, and tooth development and may induce MSC-like cells and mesenchymal differentiation in NCSC cultures. MSC-like cells are included in the Sox10 2 subpopulation of BMP NCSC cultures as the generation of mesenchymal neural crest progeny involves Sox10 downregulation [12]. Removal of BMP4 from the NS culture medium did not induce a loss of PNS or the induction of CNS markers. This may be explained by a continued endogenous BMP expression, which would interfere with FGF/EGF signaling. Indeed, expression levels of BMP2 and BMP5 are highly enriched in BMP NCSCs (data not shown). BMP4 application to rncscs did also not result in a loss of CNS identity, as shown by maintained Olig2 and lack of p75 expression and the generation of GFAP 1 astrocytes under differentiation conditions. The generation of O4 1 /Olig2 1 oligodendrocytes from rncscs is prevented by pretreatment with BMP4, which is explained, however, by interfering with oligodendrocyte differentiation[65,66].impairedproductionofo4 1 /Olig2 1 oligodendrocytes may be linked to the loss of Sox3, which plays an important role in oligodendrocyte differentiation [64]. The observation that the CNS identity of rncscs is a stable property implies that the developmental plasticity of NCSCs is restricted to the initial, DRG-derived NCSC population. This conclusion is also supported by the restriction to CNS fates upon injection of rncscs into embryonic and postnatal brains [6, 7]. Adult NCSCs from Mouse Palate Acquire an MSC-Like Fate in NS Culture NCSCs can be isolated from a number of adult tissues from the head region like hair follicles, dermis, dental pulp, olfactory and respiratory epithelium, and palate [14, 38, 93, 94]. They can be propagated in NS culture and generate neural, neuroectodermal, and mesenchymal progeny. Here, we have used the mouse palate as easily accessible source of adult NCSCs to investigate whether CNS reprogramming can be induced as observed for NCSCs from embryonic DRG. Although pncscs lose the PNS marker Sox10 and p75 with time in culture, this is not paralleled by the induction of Olig2 and Sox3. Comparing the gene expression profile of adult pncscs and embryonic rncscs revealed a selective enrichment of CNS-specific gene expression in rncscs and neural crest marker expression in pncscs. Interestingly, the set of marker genes most highly expressed in pncscs is associated with pericyte progenitors/mscs. As pericytes and vsmcs in head, brain, and thymus are neural crest-derived, pncscs are directed to a normal cranial neural crest fate [57, 67 70]. Previous studies suggested that pncscs are derived from p75 1 nestin 1 cells associated with the innervation of Meissner corpuscules and Merkel cells [95]. The present analysis of pncsc NS from Sox10-GFP mice supports the notion that pncscs are derived from peripheral glial cells that lose Sox10 and acquire MSC characteristics. MSCs are operationally defined as cells that give rise in vitro to osteoblasts, chondrocytes, and adipocytes. MSCs have a perivascular origin and recent lineage tracing studies showed that MSCs are derived, at least in part, from migrating neural crest cells [9, 12, 13, 42]. Our results demonstrate that not only NCSCs in migrating neural crest but also developing and adult PNS have the potential for MSC development. Why is CNS reprogramming efficiently induced in embryonic NCSCs but not observed in pncscs? The most straightforward explanation, that is, that NCSCs lose plasticity with age is supported by our finding that NCSCs from postnatal DRG are only partly reprogrammed and maintain PNS properties [6]. The demonstration that CNS phenotypes can be obtained from adult NCSCs isolated from hair bulge (hepi- NCSC) [96], skin [97], and inferior turbinate [38] (M uller and Kaltschmidt, unpublished observation) argues against a general loss of plasticity but rather suggests that NCSCs isolated from various stem cell niches differ in their ability to transdifferentiate to CNS neural stem cells. In addition, specific culture conditions may be required for the induction of CNS identity in adult NCSCs, avoiding the generation of MSCs. However, the complete switch from neural crest to CNS identity demonstrated for embryonic NCSCs represents the important proof of principle that CNS cells can be generated from PNS progenitors in the absence of genetic manipulation. CONCLUSION In summary, our data demonstrate that NCSCs from embryonic DRG can be reprogrammed in NS cultures to acquire a full CNS identity and completely lose neural crest characteristics. In the presence of BMP4 neural crest progenitor identity is maintained, which allows to either propagate PNS or CNS neural stem cells from embryonic DRG. The presence of MSC-like cells in cultures of embryonic BMP NCSCs and adult pncscs reveals that postmigratory NCSCs are a source for MSC-like cells throughout development. The present molecular analysis of NCSC reprogramming to CNS fates complements our previous demonstration that rncscs produce myelinating oligodendrocytes and repair brain lesions [6]. Together, these results suggest that CNS stem cells generated without genetic modification from PNS progenitors could be a potential source for CNS cell therapy. ACKNOWLEDGMENTS We gratefully acknowledge the help in microarray normalization by Lin Gan and Bernd Denecke, RWTH Aachen University. VC AlphaMed Press 2014