Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells

Abstract

Recent reports on ES cell differentiation have suggested the possibility that information on in vivo neurogenesis might be systematically linked to stem cell technology (3, 4). However, it remains to be known whether ES cell-derived neural precursors generated in vitro can produce the full dorsal-ventral range of neuroectodermal derivatives in response to embryonic positional information. To address this question, we have tested in this study whether SDIA-treated ES cells have the competence of differentiating into the dorsal-(neural crest) and ventralmost (floor plate) cells under embryologically relevant conditions. Materials and MethodsCell Culture and Treatment with Patterning Factors. Mouse ES cells (EB5), primate ES cells (cynomolgus monkey-derived; purchased from Asahi Technoglass, Funabashi, Japan), and PA6 cells were maintained and used for induction as described (1, 2, 5). Human bone morphogenetic protein (BMP)4 and mouse were purchased from R&D Systems and freshly added at each medium change. The day on which ES cells are seeded on PA6 is defined as day 0.Immunocytochemistry, Statistics, and RT-PCR. Cells were fixed with 4% paraformaldehyde, and immunostaining was performed with secondary antibodies conjugated with FITC, cy3, or cy5. For statistics, Ϸ100 colonies were observed in each experiment, and three or more experiments were performed. P values for statistical significance (t test) are described in the corresponding figure legends. The values shown in graphs represent the mean Ϯ SD. RT-PCR was performed with ES cell colonies detached from feeder cells as described (1). The primary antibodies and primers used are described in Supporting Materials and Methods, which are published as supporting information on the PNAS web site, www.pnas.org.Colony Isolation and Axon Guidance Assays. The 3D collagen gel assay for axon guidance was performed by using isolated ES cell colonies (day 8; ref. 1) and the cerebellar plate region excised from embryonic day 13 Wistar rats as a responder (6). Results Positional Identity of Neural Tissues Induced from ES Cells by SDIA.We first examined the expression of rostral-caudal CNS markers by RT-PCR (Fig. 1A). SDIA-treated mouse ES cells express the forebrain markers Otx2 and Six3, the ventral diencephalon marker Rx, the ventral forebrain marker Nkx2.1, the midbrainhindbrain border marker En2, and the hindbrain marker Gbx2, but not the spinal cord markers Hoxb4 and b9 (lane 2). These results show that a majority of neural cells induced by SDIA express rostral neural markers. This idea is consistent with our previous report that dopaminergic neurons generated by the SDIA method are those of the midbrain type (1).We next attempted to alter the rostral-caudal identity of SDIA-induced neural cells by the caudalizing factor retinoic acid (RA; ref. 7). RA treatment (0.2 M all-trans RA; Fig. 1 A, lane 3) suppressed the forebrain markers Otx2, Six3, Rx, and Nkx2.1, whereas it induced the hindbrain marker Gbx2 and the spinal cord markers Hoxb4 and b9. RA treatment did not sign...

Roles of TGF-β family signaling in stem cell renewal and differentiation

“…While BMP signals inhibit neural differentiation at early gastrula stages, the same signals induce differentiation of neural crest cells at later stages in embryos. In accordance with in vivo status, late exposure of mouse and primate ES cells to BMP-4 results in the generation of neural crest cells [67]. Furthermore, low and high concentrations of BMP-4 induced sensory and autonomic neurons from ES cells, respectively, which is consistent with the results of ex vivo experiments using neural tube explants of rats [68].…”

BMP-4 induces a Smad-dependent apoptotic cell death of mouse embryonic stem cell-derived neural precursors

“…6 Late BMP-4 exposure causes differentiation of neural crest cells and dorsalmost CNS cells, while Sonic hedgehog suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons. 30 In these studies 6 and in our hands, only the early exposure to BMP-4 (between day 3 and day 5) led to the inhibition of neural cell differentiation. Here we demonstrate that, even though cells expressing markers of differentiated neural cells (tubulin or NeuN), or even the undifferentiated neural progenitors Nestinpositive cells, were not the target of BMP-4 induced apoptosis, we systematically observed apoptosis in clusters where these markers were expressed.…”

Neural crest cells retain their capability for multipotential differentiation even after lineage‐restricted stages

“…Sox10þ/Kitþ cells isolated from the E10.5 embryonic neural tube generated TuJ-1-positive N under those conditions, as did Sox10þ/KitÀ cells (Fig. 7A, B Mizuseki et al (2003), and found that these Sox10þ/Kitþ cells also generated TuJ-1-positive N and GFAP-positive G (Fig. 7C-E …”

“…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.…”