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Keywords:

  • neural crest;
  • embryonic stem cells;
  • Kit;
  • Sox10;
  • mouse

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Multipotency of neural crest cells (NC cells) is thought to be a transient phase at the early stage of their generation; after NC cells emerge from the neural tube, they are specified into the lineage-restricted precursors. We analyzed the differentiation of early-stage NC-like cells derived from Sox10-IRES-Venus ES cells, where the expression of Sox10 can be visualized with a fluorescent protein. Unexpectedly, both the Sox10+/Kit− cells and the Sox10+/Kit+ cells, which were restricted in vivo to the neuron (N)-glial cell (G) lineage and melanocyte (M) lineage, respectively, generated N, G, and M, showing that they retain multipotency. We generated mice from the Sox10-IRES-Venus ES cells and analyzed the differentiation of their NC cells. Both the Sox10+/Kit− cells and Sox10+/Kit+ cells isolated from these mice formed colonies containing N, G, and M, showing that they are also multipotent. These findings suggest that NC cells retain multipotency even after the initial lineage-restricted stages. Developmental Dynamics 240:1681–1693, 2011. © 2011 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Neural crest cells (NC cells) emerge from the dorsal region of the neural tube and then migrate throughout the embryo. During their migration or at the target tissues, they respond to various environment factors, such as BMPs, EGFs, and Wnt proteins (Shah and Anderson, 1997; Lee et al., 2004), and differentiate into many cell types, including neurons (N) and glial cells (G) of the peripheral sensory and autonomic ganglia, Schwann cells, melanocytes (M), endocrine cells, smooth muscle cells, and skeletal and connective tissue cells of the craniofacial complex (Le Douarin and Kalcheim, 1999). With these important characteristics of high mobility and capability for multi-potent differentiation, NC cells play critical roles in embryogenesis.

In the trunk, the NC cells migrate along 2 major routes: the first is the ventral pathway between the neural tube and somites, where cells give rise to the autonomic ganglia (sympathetic ganglia and parasympathetic ganglia), to dorsal root ganglia, to glial cells, and to chromaffin cells. The second is the dorsolateral pathway between the overlying ectoderm and the somites, where cells give rise to Melanocytes (Le Douarin and Teillet, 1974; Le Douarin and Kalcheim, 1999). The NC cells are thought to lose their multipotency before or soon after they emerge from the neural tube becoming restricted in their differentiation abilities depending on the direction of their migration. The ventrally migrating NC cells become restricted to N and G fates and the dorsolaterally migrating ones, to the M fate. For example, NC cells isolated from the ventral pathway do not give rise to melanocytes when explanted in culture (Reedy et al., 1998). Also, a naturally occurring chick mutant whose pigment cells migrate ventrally has no ectopic neurogenesis (Faraco et al., 2001).

A large number of studies have addressed when and where the NC cells lose their multipotency and become restricted in their fate. Clonal analysis of NC cells shows that almost half of the initial NC cell population is a lineage-restricted population, which generates clones of only a single cell type (Henion and Weston, 1997), or is a population comprising bipotent and unipotent precursors (Lahav et al., 1998). In particular, a subpopulation of early NC cells that express the receptor tyrosine kinase Kit exclusively express M-lineage markers in rodents (Wilson et al., 2004), and avian Kit-expressing NC cells invariably give rise to clones containing only melanocytes (Henion and Weston, 1997; Luo et al., 2003). Furthermore, none of the ventrally migrating NC cells express Kit (Wilson et al., 2004), and they do not differentiate into M even under conditions that favor melanocyte development (Reedy et al., 1998). These studies suggest that migratory NC cells are fate-restricted precursors that give rise to 1 or 2 cell types. On the other hand, multipotent NC stem cells are maintained even in adult rodent tissues, being found in adult sciatic nerve, gut, skin, and carotid bodies, where NC cells have migrated and are able to differentiate into components such as N or G or M (Stemple and Anderson, 1992; Morrison et al., 1999; Kruger et al., 2002; Fernandes et al., 2004; Pardal et al., 2007). The relevance of NC stem cells among migrating NC cells, as well as the origin of terminally maintained NC stem cells, is still obscure.

Several transcriptional factors have been identified that regulate the formation and differentiation of NC cells. For example, FoxD3, which belongs to the forkhead family of transcription factors, is sufficient to induce NC cell formation from neural tube cells (Kos et al., 2001); Pax3, which is a member of the paired box family of transcription factors, is required for NC cell migration (Conway et al., 1997); Snail, which is a zinc-finger transcriptional factor, is involved in NC cell formation (del Barrio and Nieto, 2002); and the group E of Sox proteins, which are in the SRY-like HMG box family of transcriptional factors, are essential for formation and differentiation of NC cells (Wegner and Stolt, 2005). Among the group E of Sox proteins, Sox10 is particularly important for NC cell development. Sox10 expression starts in premigratory NC cells and continues in migrating NC cells and NC cell-derivatives (Mollaaghababa and Pavan, 2003); however, loss of Sox10 function does not affect NC formation and migration. Mice lacking Sox10 have NC cells that undergo apoptosis before reaching their maturation stage (Herbarth et al., 1998; Southard-Smith et al., 1998; Britsch et al., 2001; Paratore et al., 2001; Sonnenberg-Riethmacher et al., 2001). Dominant megacolon mice (Dom), which have a spontaneous mutation in their Sox10 locus, have pigmentation defects and intestinal aganglionosis, which are symptoms of a neurocristopathy (Southard-Smith et al., 1998, 1999). In humans, mutations in Sox10 cause Waardenburg syndrome type IV, which combines the characteristics of Waardenburg's syndrome and Hirschsprung's disease, i.e., enteric ganglionosis in the distal region of the colon, cochlear deafness, and pigmentary defects (Pingault et al., 1998). On the other hand, an in vitro Sox10 gain-of-function study suggested that Sox10 inhibits neuronal differentiation and maintains the multipotency of NC cells (Kim et al., 2003).

We previously reported that NC-like cells could be generated from mouse ES cells in in vitro culture (Motohashi et al., 2007). When ES cells are co-cultured with ST2 stromal cells, NC-like cells can be discriminated as Kit-expressing cells in the ES cell cultures. These NC-like cells differentiate into autonomic N, G, smooth muscle cells, and M, just like NC cells in the trunk do in vivo (Motohashi et al., 2007). In this present study, we further analyzed the early developmental commitment of the NC-like cells by using mouse ES cells designed to express green fluorescent protein under the control of the Sox10 promoter. By analyzing the expression of Sox10 and Kit in the ES cell cultures, we reconfirmed that Kit-expressing NC-like cells are able to differentiate into N, G, and M. In addition, we show that Sox10-positive but Kit-negative NC-like cells, which are thought to be the ventral path-migrating NC cells, were able to differentiate into M as well as N and G. Furthermore, we generated Sox10-GFP mice from the ES cells and showed that Kit-positive NC cells and Sox10-positive NC cells both had the potential to differentiate into N, G, and M. These results thus suggest that NC cells maintain their multipotency even after lineage restriction.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Generation of Sox10-IRES-Venus ES Cells

Previously, we reported the generation of NC-like cells from mouse ES cells (Motohashi et al., 2007). NC-like cells were isolated as Kit-expressing cells by flow cytometry, and they differentiated into N, G, and M, suggesting that these Kit-expressing cells have multipotency. However, in vivo Kit-expressing NC-derived cells are reported to migrate dorsolaterally in the skin and differentiate only into M (Henion and Weston, 1997; Luo et al., 2003; Wilson et al., 2004). To further investigate the commitment potential of these early NC-like cells, we took advantage of the Sox10 transcription factor as a well-characterized NC marker. To detect Sox10-expressing cells, we placed the Venus GFP variant gene (Nagai et al., 2002) under the control of the internal ribosomal entry site (IRES) downstream of the Sox10 stop codon (Fig. 1A). As shown in Figure 1A, homologous recombination would be expected to insert IRES-Venus into exon 5 without disrupting the expression of Sox10, therefore resulting in Sox10 and Venus co-expression. The targeting vector was used to transfect D3 ES cell lines, and homologous recombination events were identified by Southern blot and PCR screening. Of 700 ES cell clones analyzed, 3 of them were positive for homologous recombination (Sox10-IRES-Venus ES cells, Fig. 1B, C). To examine whether the expression of Venus synchronized with the expression of Sox10 in the ES cells, we cultured the Sox10-IRES-Venus ES cells by the previously reported culture method in which NC-like cells are generated, and analyzed the expression of Sox10 and Venus. As shown in Figure 1D–G, Sox10-IRES-Venus ES cells expressed Sox10 protein together with Venus after 21 days of culture.

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Figure 1. Targeting strategy for production of Sox10-IRES-Venus ES cells and expression of Venus in Sox10-IRES-Venus ES cells. A: Schematic representation of the targeting strategy employed to insert the IRES sequence and Venus gene. Shown are the endogenous Sox10 locus, including the exon structure and the relative location of the Hind III (H), Nhe I (N), and Xba I (X) restriction sites (top), the targeting construct used for homologous recombination (middle), and the targeted locus after homologous recombination (bottom). The location of the 3′ probe used to hybridize to Southern blots of Nhe I–digested genomic DNA is also shown. Arrows indicate the PCR primers. Neo, neomycin resistance gene cassette; DT-A, diphtheria toxin coding sequences; IRES, internal ribosomal entry site. B: PCR analysis of wild-type (WT) and targeted (KI) ES cells. PCR primers are shown in “A.” C: Southern blot analysis after Nhe I-digestion. The expected sizes of the fragments detected by the probe used for hybridization are shown. D–G: Immunocytochemical analysis of cultured Sox10-IRES-Venus ES cells. Sox10-IRES-Venus ES cells were cultured by the previously reported method (Motohashi et al., 2007) and immunostained with anti-Sox10. All photos are of the same field. Scale bar = 200 μm.

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NC-like Sox10+ Cells Derived From Sox10-IRES-Venus ES Cells Differentiate Into N, G, and M, Irrespective of Their Kit Expression

Next, we analyzed when the Sox10-IRES-Venus ES cells started to express Kit or Sox10 during the culture period. To exclude hematopoietic cells, which also express Kit, we used the hematopoietic cell-specific CD45 marker. During days 0 to 5 of the cultures, no Sox10-positive cells emerged. At day 6 of the culture period, a small number of Sox10-positive cells were identified; but they disappeared by day 7 or 8 (Fig. 2A). At day 9, 1.0% of the cells were Sox10 single-positive ones (Sox10+/Kit− cells), and 0.5% of the cells were Sox10 and Kit double-positive cells (Sox10+/Kit+ cells). After day 9, these populations continued to expand day by day (Fig. 2A). RT-PCR analysis showed that the Sox10+/Kit− and Sox10+/Kit+ cells expressed the NC cell marker genes Pax3 and Snail, together with Sox10 (Fig. 2B). The M lineage–specific marker Mitf-M was more highly expressed in Sox10+/Kit+ cells than in the Sox10+/Kit− cells (Fig. 2B), implying that the Sox10+/Kit+ cells might have been the cells whose fate is inclined toward the M lineage.

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Figure 2. Analysis and isolation of ES cell–derived NC-like cells. A: Expression of Sox10 (Venus) and Kit in Sox10-IRES-Venus ES cell cultures. B: Sox10-/Kit-, Sox10-/Kit+, Sox10+/Kit−, and Sox10+/Kit+ cells were sorted at each day of culture and analyzed by RT-PCR for expression of the indicated genes. C–F: Typical multipotent colonies generated from Sox10+/Kit+ cells. All pictures were taken of a single typical colony. The same visual field is shown in C and D, and in E and F. Scale bar = 200 μm. G: Sox10+/Kit− and Sox10+/Kit+ cells were inoculated at 100 cells/well onto ST2 monolayers. After 21 days in culture, the colonies were immunostained with TuJ-1 and anti-GFAP. M were detected as pigmented cells. Frequencies of the production of the different types of colonies from Sox10+/Kit− and Sox10+/Kit+ cells were calculated. M/N/G indicates that the colonies contained M, N, and G. M/N, M/G, and N/G refer to colonies containing 2 types of cell, and M, N, and G, to those containing a single cell type. C indicates the colonies not containing M, N, or G. The percentages were calculated as follows: (number of colonies containing the indicated types of cells)/(total number of colonies) × 100. The experiment was performed 2 times.

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Next, we analyzed the differentiation potential of the Sox10+/Kit− cells and Sox10+/Kit+ cells. One hundred Sox10+/Kit− and Sox10+/Kit+ cells were isolated from day-9, -10, and -12 cultures and re-cultured on ST2 monolayers. After 21 days, colonies containing pigmented M were generated from Sox10+/Kit+ cells isolated at days 9, 10, and 12 of the culture period, as previously reported (Motohashi et al., 2007). Surprisingly, Sox10+/Kit− cells also formed colonies containing pigmented M. Sox10-/Kit- cells and Sox10-/Kit+ cells from day-9, -10, and -12 cultures did not form colonies containing M (data not shown). Immunostaining showed that the M colonies that emerged from Sox10+/Kit+ and Sox10+/Kit− cells contained TuJ-1-positive N and GFAP-positive G as well (Fig. 2C–F). Some colonies also contained α smooth muscle actin-positive cells (see Supp. Fig. S1, which is available online). We classified the colonies generated, as shown in Figure 2G. Both Sox10+/Kit−cells and Sox10+/Kit+ cells formed colonies composed of 2 cell types (M/N and M/G) or of 1 cell type (M or N or G). We also detected some colonies composed of cells other than N, G, and M (Fig. 2G, indicated as “C”). The Sox10+/Kit−cells tended to form more C colonies than did the Sox10+/Kit+ cells. This difference might indicate that Sox10+/Kit−cells are a different type of NC-like cell, and one that differentiates into cell types other than N, G, and M. In addition, we found that the Sox10-positive cells differentiated into TuJ-1-positive N on PA6 stromal cells (Supp. Fig. S2), on which mouse ES cells differentiate into NC cell descendants (Mizuseki et al., 2003). Some of these cells also expressed tyrosine hydroxylase (TH, see Supp. Fig. S2), which is expressed in the peripheral autonomic N. These results suggest that both Sox10+/Kit− and Sox10+/Kit+ cells are multipotent NC-like cells that can differentiate into N, G, and M.

Sox10+/Kit− cells and Sox10+/Kit+ cells isolated from day 9 to 12 of the culture period were inoculated 1 cell per well onto ST2 monolayers in 96-well plates, and the colonies that appeared were identified immunocytochemically. As shown in Figure 3, the Sox10+/Kit+ cells showed higher plating efficiency than the Sox10+/Kit− cells from day 9 to 12 of the culture period. All types of colonies, N or G or M, were equally formed from Sox10+/Kit− cells and Sox10+/Kit+ cells, indicating that both cell-types have a similar capability for differentiation. However, the colonies composed of the cells other than N, G, and M (Fig. 3, indicated as “C”) more frequently differentiated from Sox10+/Kit− cells, similar to the experimental results shown in Figure 2G, again indicating that Sox10+/Kit− cells might be more immature NC-like cells when compared to Sox10+/Kit+ cells. Although Sox10-/Kit- cells and Sox10-/Kit+ cells formed a few colonies, no N or G or M were generated (data not shown).

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Figure 3. Frequencies of the production of different types of colonies from Sox10+/Kit− or Sox10+/Kit+ cells based on the cell types present in colonies derived from a single cell. “Plating” indicates the plating efficiency of the inoculated cells that actually formed colonies. M/N/G, M/N, M/G, N/G, M, N, G, and C are defined in the legend of Figure 2G. The experiment was performed 3 times, and each result is indicated as the average for all 3 experiments.

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Inhibition of BMP Signaling Suppresses the Generation of Sox10+ Cells

To further confirm that Sox10+/Kit+ cells and Sox10+/Kit− cells are NC-like cells similar to in vivo NC cells, we cultured Sox10-IRES-Venus ES cells in the presence or absence of 500 ng/ml Noggin. Noggin inhibits BMP signaling, which has crucial roles in NC cell generation during embryogenesis (Selleck et al., 1998; Sela-Donenfeld and Kalcheim, 1999). From day 9 to day 12 of the culture period, the generation of Sox10-expressing cells was reduced about two- or threefold by the addition of Noggin (Fig. 4). The Sox10-positive cells that were generated in a very low number in the presence of Noggin were sorted and re-cultured on ST2 stromal cells. Although a few colonies were formed, no N, G, and M were generated from these Sox10-expressing cells (data not shown). The results further confirm that the Sox10-expressing cells derived from Sox10-IRES-Venus ES cells are NC-like cells similar to in vivo NC cells.

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Figure 4. Noggin inhibited the generation of Sox10+ cells in Sox10-IRES-Venus ES cell cultures. The Sox10-IRES-Venus ES cells were cultured in the presence or absence of Noggin and analyzed by Flow-cytometry. “Vehicle” indicates the solvent in which Noggin was dissolved.

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Multipotential Differentiation Ability of Sox10+/Kit− and Sox10+/Kit+ Cells Isolated From Developing Embryos

During embryogenesis, Sox10+/Kit− NC-like cells are thought to be ventrally migrating immature NC cells and Sox10+/Kit+ NC-like cells are thought to be the dorsolaterally migrating NC cells. As mentioned earlier, most of the Kit+ NC cells are believed to migrate into the ectoderm and differentiate into M only, whereas Kit- NC cells migrate ventrally to finally differentiate into N or G, and not into M (Luo et al., 2003; Wilson et al., 2004). To examine the discrepancy between the differentiation potential of ES cell–derived Sox10+ NC-like cells tested in vitro and that of their embryonic counterpart, we generated mice from the Sox10-IRES-Venus ES cells. Since we could not obtain mice from the Sox10-IRES-Venus ES cells derived from D3 ES cell lines, we generated such cells from V6.5 ES cell lines and obtained mice from them. The Sox10-IRES-Venus ES cells generated from these V6.5 ES cells were shown to express Venus protein and Venus+/Kit- cells or Venus+/Kit+ cells induced from these cells differentiated into N, G, and M on ST2 cells (Supp. Fig. S3). These mouse embryos expressed Venus in their migrating NC cells or in the tissues containing NC-derived cells (Fig. 5A–D). The pattern of Venus expression was similar to the β-galactosidase staining patterns of the Sox10lacZ/+ mouse embryos previously reported (Britsch et al., 2001). Briefly, at embryonic day 8.5 (E8.5), Venus was detected mainly in the cephalic fold (Fig. 5A). At E9.5, strong Venus expression was detected in the otic vesicles, in migrating NC cells of branchial arches 1 and 2, and in NC cells during the formation of the dorsal root ganglia (Fig. 5B). At E10.5, the cranial ganglia, dorsal root ganglia, and spinal nerves strongly expressed Venus (Fig. 5C). At E12.5, the expression continued to progress toward the periphery and expanded to the embryonic skin region (Fig. 5D). The Sox10-IRES-Venus neonates were born at the expected Mendelian ratio, were healthy, grew into adults, and did not exhibit the megacolon and pigmentation defects observed in Sox10-mutant mice (data not shown).

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Figure 5. Sox10 expression pattern in Sox10-IRES-Venus mice. The heterozygous mice derived from Sox10-IRES-Venus ES cells expressed Venus along the NC cell migration pathways at embryonic day 8.5 (E8.5, A), 9.5 (E9.5, B), 10.5 (E10.5, C), and 12.5 (E12.5, D).

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Using these established Sox10-IRES-Venus mice, we first investigated the expression of Sox10 and Kit in their embryonic NC cells. To compare the expression pattern with that of ES cell–derived NC-like cells, we prepared the cells from dissected trunk neural tubes and the adjacent tissues. At E8.5, 0.45% of the trunk neural tube and the adjacent cells expressed Sox10 only (Fig. 6A). By E9.5, 3.15% of the cells were Sox10+/Kit− and 0.03% of the cells were Sox10+/Kit+ (Fig. 6A). At E10.5 and E12.5, 3.44 and 4.32% of the cells were Sox10+/Kit−cells, respectively, and 0.06 and 0.17% of them were Sox10+/Kit+ cells, respectively (Fig. 6A). RT-PCR analysis showed that both Sox10+/Kit− and Sox10+/Kit+ cells expressed the NC cell marker genes Pax3 and Snail, together with Sox10 (Fig. 6B). FoxD3, which is an N- or G-lineage NC cell marker gene, was expressed only in the Sox10+/Kit−cells (Fig. 6B). The M lineage–specific marker Mitf-M was expressed more strongly in Sox10+/Kit+ cells than in Sox10+/Kit−cells (Fig. 6B). We isolated Sox10+/Kit− and Sox10+/Kit+ cells, and cultured them clonally on ST2 stromal cells, and analyzed the progeny by immunostaining. At E8.5, only Sox10+/Kit−cells were isolated; and the cells did not form colonies containing N or G or M (data not shown). Sox10+/Kit− cells taken from E9.5 embryos formed colonies containing M/N/G in addition to those containing N/G, N, or G after 21 days in culture (Fig. 6C), in accordance with Sox10+/Kit−cells derived from ES cells. In contrast, Sox10+/Kit+ cells generated a few colonies containing N/G or G and colonies containing M were not detected (Fig. 6C). Sox10+/Kit−cells taken from E10.5 embryos formed colonies containing M (M/N/G, M/N, M) in addition to N/G and N colonies (Fig. 6C). Similarly, Sox10+/Kit+ cells from E10.5 embryos formed colonies comprising M/N/G, N/G, M/N, N or G (Fig. 6C–G). In the case of E12.5 embryos, the Sox10+/Kit−cells predominantly formed colonies consisting of N/G, N or G, although the cells still could form colonies containing M (M/N/G and M/N, Fig. 6C). The E12.5 Sox10+/Kit+ cells also differentiated into colonies containing M/N/G, N/G, M/N, N or G (Fig. 6C). N generated from Sox10+/Kit−cells and Sox10+/Kit+ cells also expressed other neuronal markers, i.e., nestin and neurofilaments (Supp. Fig. S4A, B). Neither Sox10-/Kit- nor Sox10-/Kit+ cells from E9.5, E10.5 or E12.5 embryos formed colonies containing N or G or M (data not shown). Thus, under these permissive culture conditions, Sox10+ NC cells isolated from the embryo exerted their multipotential differentiation abilities irrespective of the Kit expression.

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Figure 6. Isolation and analysis of Sox10+/Kit− and Sox10+/Kit+ cells from Sox10-IRES-Venus mice.

A: Expression of Sox10 (Venus) and Kit in heterozygous mice. B: Sox10+/Kit− (S) and Sox10+/Kit+ (S/K) cells were sorted at each day of culture and analyzed for the indicated gene expression by RT-PCR. C: Sox10+/Kit− and Sox10+/Kit+ cells were cultured on ST2 monolayers. Sox10+/Kit+ cells from E9.5, E10.5, and E12.5 embryos were inoculated at 30, 70, 150 cells/well, respectively, and the Sox10+/Kit−cells at 200 cells/well. After 21 days in culture, the colonies were immunostained with TuJ-1 and anti-GFAP. M was detected as pigmented cells. The numbers of the different types of colonies produced from Sox10+/Kit− and Sox10+/Kit+ cells were counted. M/N/G, M/N, M/G, N/G, M, N, and G are defined in the legend of Figure 2G. The experiment was performed 3 times. D–G: Typical multipotent colonies generated from Sox10+/Kit+ cells from an E10.5 embryo. All pictures were taken of a single typical colony. The same visual field is shown in each panel. Scale bar = 200 μm.

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In summary, Sox10+/Kit−cells originating from the neural tube, whose differentiation is normally restricted to sensory N or G in vivo, have the potential to differentiate into M. Similarly, Sox10+/Kit+ cells, normally restricted to the M lineage in vivo, have the potential to differentiate not only into M, but also into N or G.

Embryonic Sox10+/Kit+ Cells Differentiate Into N, G Either Under NC Stem Cell Culture Conditions or in Co-Culture With PA6 Stromal Cells

To test their multipotential cell fate under other in vitro conditions, we cultured Sox10+/Kit+ and Sox10+/Kit− cells purified from embryonic neural tubes under conditions previously reported (Stemple and Anderson, 1992; Morrison et al., 1999). Sox10+/Kit+ cells isolated from the E10.5 embryonic neural tube generated TuJ-1-positive N under those conditions, as did Sox10+/Kit− cells (Fig. 7A, B, Table 1), indicating that Sox10+/Kit+ cells have the potential to differentiate into N similar to the Sox10+/Kit− cells, which correspond to ventrally migrating NC cells. Furthermore, we cultured Sox10+/Kit+ cells on PA6 stromal cells according to Mizuseki et al. (2003), and found that these Sox10+/Kit+ cells also generated TuJ-1-positive N and GFAP-positive G (Fig. 7C–E, Table 1). These results suggest that embryonic Sox10+/Kit+ NC cells have the capability to differentiate not only into M, but also into N or G, under various in vitro conditions supporting neural differentiation.

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Figure 7. In vivo–derived Sox10+/Kit+ cells cultured as NC cell cultures or on PA6 cells also differentiated into N or G. Sox10+/Kit+ and Sox10+/Kit−cells were isolated by flow cytometry from E10.5 embryonic neural tubes and cultured as NC cell cultures for 14 days (A, B) or on PA6 monolayers for 21 days (C–E). A, B: The cell clusters that emerged from Sox10+/Kit+ cells (A) and Sox10+/Kit− cells (B) were immunostained with TuJ-1 (Red). The nuclei were stained with Hoechst 33258 (Blue). C–E: A typical N/G colony generated from the Sox10+/Kit+ cells cultured on PA6 cells. The colonies were immunostained with TuJ-1 (Red) and anti-GFAP (Green). The same visual field is shown in C–E. Scale bar = 200 μm.

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Table 1. Sox10+/Kit+, Sox10+/Kit−, and Sox10−/Kit− Cells Were Cultured as NC Cell Cultures or on PA6 Stromal Cellsa
 NC culturePA6 culture
 N/GNGN/GNG
  • a

    Sox10+/Kit+, Sox10+/Kit−, and Sox10−/Kit− cells were isolated from E10.5 embryonic neural tubes, cultured as NC cell cultures (NC culture) or on PA6 cells (PA6 culture), and immunostained with TuJ-1 and anti-GFAP. The Sox10+/Kit+ cells for NC cultures were inoculated at 25 cells/well; and those for PA6 cultures, at 40 cells/well. Others were inoculated at 200 cells/well. The numbers of the different types of clusters produced by the cells were counted. N/G indicates clusters that contained N and G; and N or G, those containing a single cell type. The experiment was performed three times.

Sox10+/Kit+04.6 ± 0.500.3 ± 0.63.5 ± 1.80.2 ± 0.3
Sox10+/Kit−026.0 ± 5.309.8 ± 3.68.6 ± 2.31.4 ± 1.9
Sox10−/Kit−00.2 ± 0.4004.4 ± 2.60.2 ± 0.4

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

In the previous study, we generated NC-like cells from mouse ES cells by culturing them on monolayers of ST2 stromal cells. The cells were isolated as Kit-expressing cells and had characteristics of NC cells in vivo, i.e., high motility and multipotency (Motohashi et al., 2007). Furthermore, we have also previously shown that Kit-positive melanoblasts, even after they have already migrated throughout the skin, are multipotent and able to generate N, G, and smooth muscle cells in addition to M (Motohashi et al., 2009). These studies suggest that the Kit+ NC cells maintain their multipotency even after they have migrated into skin and differentiated into melanoblasts. However, the potential of NC cells in their early developmental stage, especially that of the Kit-NC population, remained unclear.

A construct in which the complete open reading frame of Sox10 was replaced by lacZ sequences was previously reported (Britsch et al., 2001). Although the NC cells in the resultant mice in the heterozygous state are precisely marked by the lacZ expression, a significant population of these mice exhibit megacolon and pigmentation defects and die during the first postnatal weeks, as is the case for Sox10Dom mutant mice (Southard-Smith et al., 1999). In this study, we generated Sox10-IRES-Venus ES cells, which expressed the fluorescent protein “Venus” under the control of Sox10 expression (Fig. 1A), which did not impede Sox10 gene expression. In this case, the Sox10-Venus-marked cells in Sox10-IRES-Venus mice should accurately reflect the behavior of the wildtype NC cells. Recently, chimeric mice generated from ES cells in which exon I of the Sox10 had been replaced by the GFP sequence were also reported to mark NC cells in vivo (Kawaguchi et al., 2010).

In both ES cell cultures and the developing neural tube, Sox10+/Kit−cells were generated prior to the emergence of Sox10+/Kit+ cells (Fig. 2A, Day 9; Fig. 6A, E8.5–9.5). In avian trunk neural tube explant cultures, NC cells that differentiate exclusively into N and G are the first to migrate from the neural tube, whereas NC cells that differentiate into M do not migrate until 6–12 hr later (Henion and Weston, 1997; Reedy et al., 1998). Also in mouse embryos, the migratory population of P75+ NC cells, which likely represents ventrally migrating NC cells, were reported to precede P75+/Kit+ NC cells representing NC cells migrating to the ectoderm (Wilson et al., 2004). The Sox10+/Kit− cells in our study might correspond to the cells migrating first, i.e., the ventrally migrating NC cells, and the Sox10+/Kit+ cells, to the later dorsolaterally migrating NC cells that differentiate into M. This notion is supported by the fact that Sox10+/Kit+ cells generated more colonies containing M than Sox10+/Kit−cells and also by the fact that Sox10+/Kit−cells differentiated more frequently to N or G than did the Sox10+/Kit+ cells (Figs. 2G, 6C).

Our present results clearly indicate that the Kit+ NC cells have multipotency just after they arise in the ES cell culture or in the developing neural tube. Our previous report (Motohashi et al., 2009) indicated that Kit+ melanoblasts maintain their multipotency during migration in the embryonic skin and after settling in the hair follicles. By culturing the purified Sox10+/Kit+ cells under established NC culture conditions or co-culturing them with PA6 stromal cells, we further showed that they are capable of differentiating into N or G (Fig. 7). In contrast to our studies, an in vitro culture study using quail embryo NC cells showed that the Kit+ NC cells invariably give rise to clones containing only M (Luo et al., 2003). Additionally, an in vivo immunohistochemistry study on mouse embryos showed that the migrating Kit+ NC cells give rise to only M (Wilson et al., 2004). All these in vivo observations suggest the unipotent feature of Sox10+/Kit+ NC cells. Our study, however, revealed the persistent multipotency of NC lineage cells, which had been thought to be a characteristic restricted to NC stem cells. It should be emphasized that we collected all of the Kit+ NC lineage cells as the Sox10+/Kit+ cell population and that no N, G, or M cells were derived from the Sox10-/Kit+ population (Fig. 6, data not shown), indicating that the Kit+ NC cells contain multipotential cells. Clearly, environmental cues are restricting the multipotential capacity of Sox10+/Kit+ to differentiate into M in the developing mouse skin.

As we noticed a broad range of variation for the amount of Sox10 and Kit expression, the populations with higher Sox10 or Kit expression (Sox10-high, Kit-high) and lower Sox10 or Kit expression (Sox10-low, Kit-low) were separated from day 11 of the ES cells culture, and then replated on ST2 stromal cells (Supp. Fig. S5A). The percentages of the distinct colony types generated were similar between Sox10-high and Sox10-low or Kit-high and Kit-low populations (Supp. Fig. S5B). This finding indicates that the migrating NC cells have the ability to differentiate into M, N, and G, irrespective of the expression of Sox10 and Kit. We then cultured Sox10-positive cells prepared from the day-11 ES cells on ST2 stromal cells for 14 days and then separated these cultured cells into Sox10-high, Sox10-low, Kit-high, and Kit-low populations for a third culture on ST2 stromal cells (Supp. Fig. S5C). Sox10-low, Kit-high, and Kit-low populations generated similar types of colonies including M; however, the Sox10-high population generated no M/N/G colonies, and instead G or M colonies increased in number (Supp. Fig. S5D). Thus, Sox10 expression in forming colonies might be related to the cell-fate restriction.

Unexpectedly, Sox10+/Kit− cells derived from ES cells or the embryonic neural tube also showed multipotency as in the case of the Sox10+/Kit+ cells (Figs. 2G, 6C). Our result was preceded by the finding that a small population of NC cells that have already migrated into the epidermis have both melanogenic and neurogenic potential in vitro (Richardson and Sieber-Blum, 1993), although their identity was not precisely determined by the use of cell- surface markers. As recently shown, Schwann cell precursors that have migrated via the ventral pathway and myelinating Schwann cells retain their competence to differentiate into M (Adameyko et al., 2009). In contrast, Krispin et al. (2010) reported that avian neural fate-restricted NC cells misexpressing the endothelin receptor B2, a gene that directs lateral migration, fail to differentiate into M, albeit they migrate along the dorso-lateral pathway. This discrepancy might be based on the difference between mammals and avians; however, manifestation of maintained multipotency of NC cells might be difficult in in vivo environments in contrast to the in vitro culture. In any case, we show in this present study that Kit- NC cells have multipotential cell fate including that for the M lineage as indicated for Kit+ NC cells, at least under our in vitro culture conditions.

Why did all these NC cell populations show their multipotency only in vitro, whereas their fates are more or less restricted in vivo? In vivo cell lineage analysis shows deterministic restriction of premigratory NC cells for differentiation into sensory neurons by GDF7 (Lo et al., 2005). A study using purified rat enteric neural precursors transplanted into chick embryo also suggested that the cell fate of NC-derived precursor cells is more restricted in the in vivo situation (White and Anderson, 1999). Also, spatially distinct NC stem cells were suggested to have differences in their fate determination in an in vivo transplantation study (Mosher et al., 2007). But, on the other hand, the sciatic NC stem cells that preferentially generate parasympathetic N and not noradrenergic N in vivo can differentiate into noradrenergic neurons in a BMP-2 dose-dependent manner in vitro (White et al., 2001). We report here that only the in vitro cultured Sox10+/Kit− or Sox10+/Kit+ cells showed the unexpected multipotency. In vivo Sox10+/Kit+ NC cells differentiate into M precursors in the epidermal area, which contains many keratinocytes. In the culture of neonatal skin containing keratinocytes and melanoblasts, no N or G are generated except for dendritic M or Dct-positive cells (Hirobe, 1992). The in vivo microenvironments, i.e., keratinocytes, might restrict the multipotency of NC cells to the uni-potency for M. We showed that the embryonic skin-derived Sox10+/Kit+ cells did not express FoxD3, known to restrict NC cells to the N- and G-lineage NC cells, implying that in vivo microenvironments might have a role in this lineage restriction. Apparently, further studies are necessary to clarify the in vivo mechanisms restricting the multipotential capability of Sox10+ NC cells. Also, in this context, multipotential stem cells established from dissociated skin (Fernandes et al., 2004; Amoh et al., 2005) could possibly be generated from a differentiated subpopulation of NC cells such as Sox10+/Kit+ melanoblast precursors.

In this study, we show that ES cell-derived NC-like cells share their developmental potentials, including an unexpected multipotency exerted in vitro, with their in vivo counterparts. A recent finding that NC-derived Schwann cells are developmentally progeny for follicular melanocytes (Adameyko et al., 2009) clearly demonstrates the multipotential capability of seemingly differentiated cells. It should be emphasized that distinctive NC stem cell populations are also present even in the later developmental stages (Stemple and Anderson, 1992; Morrison et al., 1999; Kruger et al., 2002; Fernandes et al., 2004; Pardal et al., 2007), probably indicating the importance of these stem cell populations for the homeostatic maintenance of NC-derived cells in each part of the body. Currently, we do not have any evidence showing the significance of the multipotential capacity of NC cells after lineage restriction. The participation of “multipotential” differentiated NC cells in scheduled developmental processes or unscheduled regenerative processes is an important question to be addressed.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Sox10-IRES-Venus ES Cells and Sox10-IRES-Venus Mice

The Sox10 locus was isolated from a 129/Sv genomic λ library. The target vector was constructed by standard techniques (Fig. 1A). The linearized target vector was electroporated into mouse D3 ES cells or V6.5 ES cells (provided by Dr. R. Jaenish), which were then selected with G418. The selected ES cells were screened for homologous recombination by PCR and Southern hybridization using probes located at the 3′ end of the targeting vector (Fig. 1B, C). For the generation of Sox10-IRES-Venus mice, targeted ES cell clones were injected into C57Bl/6J blastocysts, generating chimeras and mutant mouse strains. Genotyping of the mutant mice was performed by PCR (Fig. 1B). The primers used for PCR were 5′-TTTGACTATTCTGACCATCAGCC-3′ (Primer A) and 5′-TTC TGCTGGTAGTGGTCGGCGA-3′ (Primer B). The Sox10-IRES-Venus mice were maintained in our animal faculty. Noon of the day the vaginal plug was detected was designated as day 0.5 of gestation (E0.5). The developmental stages of embryos were judged by their morphological appearance as described in “The Mouse” (Rugh, 1990). All animal experiments were performed in accordance with the Regulations of Animal Experiments in Gifu University.

ES Cell Culture and Generation of NC-Like Cells

Mouse D3 ES cells, V6.5 ES cells, and ST2 cells were maintained as previously described (Motohashi et al., 2007). The Sox10-IRES-Venus ES cells generated from D3 ES cells and V6.5 ES cells were also maintained as previously described (Motohashi et al., 2007). For the induction of NC-like cells, 500–1,500 ES cells were inoculated into 6-well plates (Nunc, Rochester, NY) previously seeded with ST2 stromal cells and containing α-MEM (Invitrogen, La Jolla, CA) supplemented with 10% FCS, 10−7 M dexamethasone (Dex; Sigma, St. Louis, MO), 20 pM fibroblast growth factor-2 (bFGF; R&D Systems, Minneapoliis, MN), 10 pM cholera toxin (CT; Sigma), and 100 ng/ml human recombinant endothelin-3 (EDN3; Peptide Institute, Inc., Osaka, Japan) in 5% CO2 at 37°C. Usually, 100 nM all-trans retinoic acid (RA; Sigma) was also present in the medium from day 0 to day 9 of the culture period. The medium was changed every 2 days, and the day when ES cells were seeded onto ST2 monolayers was defined as day 0. For FACS analysis, cultured ES cells were dissociated by incubating them for 6 min at 37°C in Dispase II (2.4 U/ml in Ca, Mg-free PBS; Sanko Jyunyaku, Tokyo, Japan). The digestion was quenched with 2 volumes of staining medium (SM: PBS containing 3% FSC). For inhibition of BMP signaling, the Sox10-IRES-Venus ES cells were cultured in the presence of 500 ng/ml Noggin/Fc chimera (R&D Systems).

Preparation of Cell Suspensions From Mouse Embryos

E9.5, E10.5, and E12.5 embryos were incubated in 0.75 mg/ml collagenase (Wako Jyunyaku) at room temperature for 20 min. After having been washed with PBS, each embryo was dissected in the region corresponding to the end of the branchial arches during observation through a binocular microscope (Carl Zeiss, Thornwood, NY; Stereomicroscope DV4); and the cranial region was discarded. In the case of the E12.5 embryo, the embryo was dissected rostral to the forefeet with fine scissors. The neural tube and the adjacent areas were removed from the trunk region of the embryos, incubated for 15 min at room temperature in 1× Dispase II, and then gently dissociated by passage through a 21-gauge needle. The digestion was quenched with 2 volumes of SM.

Flow-Cytometric Analysis and Cell Sorting

Flow-cytometric analysis and cell sorting were performed as previously described (Motohashi et al., 2007). Briefly, the cells were washed with SM, and blocked with rat anti-mouse Fc gamma receptor (2.4-G2; BD Biosciences, San Jose, CA) on ice for 30–40 min. After another wash with SM, the cells were stained with phycoerythrin-conjugated rat anti-mouse CD45 (30-F11; BD Bioscience) and allophycocyanin-conjugated rat-anti mouse Kit (2B8; BD Bioscience). All cell sorting and analysis were performed with a FACS Vantage (Becton-Dickinson, Franklin Lakes, NJ). To exclude hematopoietic cells, we analyzed Kit expression except in the CD45-positive population. The sorted cells were directly inoculated into 6- or 96-well plates previously seeded with ST2 stromal cells by using the CloneCyt Plus v 3.1 System (Beckton-Dickinson).

NC Cell Cultures

For differentiation of NC cells into neuronal cells, isolated Sox10+/Kit+, Sox10+/Kit−, and Sox10-/Kit-cells were directly inoculated into 6-well plates previously coated with poly-D-Lysine (Biomedical Technologies Inc., Stoughton, MA) and human plasma fibronectin (Gibco, Gaithersburg, MD) and containing a 5:3 mixture of DMEM-low:neurobasal medium (Invitrogen) supplemented with 15% CEE, 1% N2 supplement (Gibco), 2% B27 supplement (Gibco), 50 μM 2-mercaptoethanol (Sigma), 35 ng/ml all-trans retinoic acid (RA; Sigma), 20 ng/ml IGF-1 (R&D Systems), and 20 ng/ml bFGF (R&D Systems). The culture system based on Morrison et al. ( 1999) was used, and the cultures were continued for 9 days. For culturing on PA6 stromal cells, isolated cells were directly inoculated into 6-well plates seeded with PA6 stromal cells and containing GMEM (Invitrogen) supplemented with 10% KSR, 2 mM glutamine (Gibco), 1 mM pyruvate (Sigma), 0.1 mM non-essential amino acid (Gibco), and 0.1 mM 2-mercaptoethanol (Sigma). The medium was changed every 2 days, and the cultures were continued for 21 days.

RT-PCR Analysis

Target cells were isolated by using the FACS Vantage. Total RNA was purified by using Isogen (Nippon Gene, Tokyo, Japan), and first-strand cDNA synthesis was carried out with Superscript III reverse transcriptase (Invitrogen) primed with random hexamer. PCR reactions were performed with the following parameters: 94°C, 2 min; 35–40 cycles of 94°C, 30 s- gene-specific annealing temperature for 30 s to 72°C 60 s. The primers used for PCR are described in Supp. Table S1.

Immunohistochemical Analysis

The colonies were sequentially fixed in 4% PFA for 15 min, made permeable by immersion in 0.1% Triton-X100 in 5 % BSA PBS for 30 min, washed in PBS, and blocked in a mixture of 2% goat serum and 5% BSA in PBS or 5% BSA in PBS for 30 min. Primary antibodies were then added and allowed to react at room temperature. The following primary antibodies were used: anti-mouse neuronal class III β-tubulin (1:500; TuJ-1, BABCO), anti-mouse glial fibrillary acidic protein (GFAP, 1:500; Z0334, Dakocytomation), anti-mouse Sox10 (1:50; N-20, Santa Cruz Biotechnology, Santa Cruz, CA), and anti-Dct (Provided by Dr. Vincent. J. Hearing). After having been washed in PBS, the cells were stained with the secondary antibodies in the same manner. The following secondary antibodies were used: Texas-Red-conjugated anti-mouse IgG (1:1,000; Molecular Probes, Eugene, OR), Texas-Red-conjugated anti-rabbit IgG (1:1,000; Molecular Probes), Alexa-Fluor 546 anti-goat IgG (1:1,000; Molecular Probes), and Alexa-Fluor 488-conjugated-anti-rabbit IgG (1:1,000; Molecular Probes). Nuclei were stained with Hoechst 33258 (Sigma). Colonies were examined by using an Olympus IX-71 fluorescence-microscope.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Dr. Hitoshi Niwa (RIKEN CDB) for constructing the IRES-Venus, Dr. Rudolf Jaenisch (Whitehead Institute) for V6.5 ES cells, Dr. Yasuhiro Yamada (Kyoto University) for technical advice, Dr. Vincent J. Hearing (NIH) for anti-Dct, and other members in our laboratory for discussion and critical reading of the manuscript. This study was supported by a grant from the program Grants-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (20592086).

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  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_22658_sm_suppinfofig1.tif1396KSupporting Information Figure 1. Some Sox10+/Kit+ cells differentiated into α smooth muscle actin-positive cells. Sox10+/Kit+ cells derived from Sox10-IRES-Venus ES cells were inoculated at 100 cells/well onto ST2 monolayers in a 6-well dish. After 21 days in culture, the colonies were immunostained with antibody against α smooth muscle actin (1:500; 1A4, Sigma). The colonies contained some α smooth muscle actin-positive cells (αSM) together with melanocytes. The same colony is shown in both photos. Scale bars = 200 μm.
DVDY_22658_sm_suppinfofig2.tif2631KSupporting Information Figure 2. Some Sox10-expressing cells differentiated into peripheral autonomic neurons. Sox10-expressing cells derived from Sox10-IRES-Venus ES cells were inoculated at 100 cells/well onto PA6 monolayers in a 6-well dish. After 21 days in culture, the colonies were immunostained for neuronal marker β-tubulin (TuJ-1 antibody) and tyrosine hydroxylase (TH; anti-mouse tyrosine hydroxylase, 1:500; AB152, Chemicon, Temecula, CA). The colonies contained TuJ-1-positive N and some TuJ-1-positive N cells also expressed tyrosine hydroxylase (TH), which is known to be expressed in peripheral autonomic neurons. All photos show the same field. Scale bar = 100 μm.
DVDY_22658_sm_suppinfofig3.tif2088KSupporting Information Figure 3. Analysis of NC-like cells derived from Sox10-IRES-Venus V6.5 ES cells. A: Flow-cytometry analysis of expression of Sox10 and Kit in Sox10-IRES-Venus V6.5 ES cells in a day-12 culture. The cultured Sox10-IRES-Venus ES cells were stained with anti-Kit-APC and anti-CD45-PE antibodies. To eliminate the hematopoietic cells, we analyzed Sox10 and Kit expression except in the CD45-positive population. B: Sox10+/Kit- and Sox10+/Kit+ cells were inoculated separately at 100 cells/well onto ST2 monolayers in 6-well dishes at day 12 of culture. After 21 days in culture, the colonies were immunostained for neuronal marker (TuJ-1) and glial marker (GFAP). Melanocytes were detected as pigmented cells. Frequencies of the different types of colonies produced from Sox10+/Kit- and Sox10+/Kit+ cells were calculated. M/N/G indicates that the colonies contained M, N, and G. M/N, M/G, and N/G refer to colonies containing 2 types of cells. M, N, and G refer to those containing a single cell type. The percentages were calculated as in Figure 2. The experiment was performed 3 times. C: Typical multipotent colonies generated from Sox10+/Kit+ cells. All photos show the same field. Scale bar = 200 μm.
DVDY_22658_sm_suppinfofig4.tif2161KSupporting Information Figure 4. In vivo-derived Sox10+/Kit+ cells differentiated into neuronal precursors. The colonies generated from Sox10+/Kit+ cells from E10.5 embryonic neural tubes were immunostained for neuronal markers, nestin (anti-mouse Nestin, 1:500; Rat 401, Chemicon) and neurofilaments (anti-mouse Neurofilament, 1:500; AB1981, Chemicon). A: The colonies derived from Sox10+/Kit+ cells contained nestin-positive neurons. B: A few TuJ-1-positive (not shown) cells also expressed neurofilament (NF). Photos in A and B show the same field. Scale bar = 200 μm.
DVDY_22658_sm_suppinfofig5.tif778KSupporting Information Figure 5. Sox10 or Kit expression status and differentiation potential. A,B: The Sox10 low-expressing (Sox10 low, gate a), high-expressing (Sox10 high, gate b), Kit low-expressing (Kit low, gate c), and high-expressing (Kit high, gate d) cells were isolated from day 11 of ES cell culture by flow-cytometry and cultured on stromal cells for 21 days. Colonies emerged from Sox10-high, Sox10-low, Kit-high and Kit-low cells were stained TuJ-1 and anti-GFAP. Melanocytes were detected as pigmented cells. Frequencies of the production of different types of colonies from each type of cell were calculated. The percentages were calculated as in Supplementary Figure 3B. The experiment was performed 2 times. C,D: The Sox10-expressing cells were isolated from day 11 of ES cell culture by flow-cytometry and cultured on stromal cells for 14 days. The colonies that emerged were dissociated and the Sox10 low-expressing (Sox10 low, gate a), high-expressing (Sox10 high, gate b), Kit low-expressing (Kit low, gate c) and high-expressing (Kit high, gate d) cells were isolated and again cultured on stromal cells for 21 days. Colonies emerged from Sox10-high, Sox10-low, Kit-high, and Kit-low cells were stained and frequencies of the production of different types of colonies from each type of cell were calculated as B. The experiment was performed 2 times.
DVDY_22658_sm_suppinfotable.doc52KSupporting Information

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