Human neural crest cells display molecular and phenotypic hallmarks of stem cells

Abstract

The fields of both developmental and stem cell biology explore how functionally distinct cell types arise from a self-renewing founder population. Multipotent, proliferative human neural crest cells (hNCC) develop toward the end of the first month of pregnancy. It is assumed that most differentiate after migrating throughout the organism, although in animal models neural crest stem cells reportedly persist in postnatal tissues. Molecular pathways leading over time from an invasive mesenchyme to differentiated progeny such as the dorsal root ganglion, the maxillary bone or the adrenal medulla are altered in many congenital diseases. To identify additional components of such pathways, we derived and maintained self-renewing hNCC lines from pharyngulas. We show that, unlike their animal counterparts, hNCC are able to self-renew ex vivo under feeder-free conditions. While cross species comparisons showed extensive overlap between human, mouse and avian NCC transcriptomes, some molecular cascades are only active in the human cells, correlating with phenotypic differences. Furthermore, we found that the global hNCC molecular profile is highly similar to that of pluripotent embryonic stem cells when compared with other stem cell populations or hNCC derivatives. The pluripotency markers NANOG, POU5F1 and SOX2 are also expressed by hNCC, and a small subset of transcripts can unambiguously identify hNCC among other cell types. The hNCC molecular profile is thus both unique and globally characteristic of uncommitted stem cells.

Figure 1.

Primary neural crest cells can be isolated from human embryos. (A) Human neural tube (NT) from an embryo at Carnegie stage (C)13. (B) After 16 h, most hNCC have migrated away from the dorsal NT. (C) The intact NT is detached from the culture dish. (D) An enriched hNCC population remains, phenotypically similar to murine or avian NCC. (E) Empirical evaluation of hNCC migration (none to few, some and many) from 31 explanted neural tubes. Third-order polynomial regressions reflect the rostral-to-caudal temporal maturation gradient of the human NT and maturation of NCC. Peaks occur during C11 at cephalic levels, and during late C12 at rostral trunk levels (segments extending from somites 5 through the last-formed somite pair).

Figure 2.

Protein markers of hNCC indicate a heterogeneously uncommitted phenotype. In vitro, hNCC synthesized (A) α-smooth muscle actin and/or NCAM, separately and sometimes in the same cells; (B) neuron-specific beta III tubulin; (C) nuclear SOX9; (D) nuclear and cytoplasmic SOX2; and unpolymerized GFAP (not shown). Pigment or immunoreactivity to calcitonin or tyrosine hydroxylase was never observed in these culture conditions. Inset: negative control with non-specific primary antibody.

Figure 3.

Expression validation of the hNCC SAGE bank. (A) Presence in five distinct hNCC lines (N1–N5) of typical animal NCC gene transcripts, as shown by RT–PCR. Most of these genes are also expressed in the C12 and C13 human neural tube (T12 and T13) and in the C13 liver bud (L), as well as in the adult human liver, below. (B) GJA1 (CX43) and PAX3 are expressed more in hNCC than embryonic liver. ACTB expression was used to normalize the data before calculating the expression ratio of each gene in the five hNCC lines compared to the C13 liver bud. (C) Choline acetyltransferase (CHAT) and paired-like homeobox 2b (PHOX2B) are expressed by sympathetic neurons and tyrosinase-related protein 1 (TYRP1) by melanocytes, both hNCC derivatives: none of the five hNCC lines express all three markers while T12/T13/L samples do. (D) Expression in the five distinct hNCC lines of genes reported in the literature as highly characteristic of hES cells.

Figure 4.

In situ expression of SOX11, MAZ and GJA1 in the human embryo. (A) Hematoxylin-eosin (HE) stain of caudal trunk-level transverse section at stage Carnegie (C)13. (B) Adjacent section. Presumptive dermatome, neural tube excluding floorplate, pronephros and splanchnopleural mesoderm and neural crest cells (arrowheads) express SOX11. (C) The same tissues express GJA1 in a simultaneously hybridized adjacent section. Expression is higher in the dermatome, dorsal neural tube and migratory neural crest cells (arrowheads). (D) MAZ is expressed in an adjacent section in most of the neural tube aside from the floorplate (indicated as extension of dotted lines), and lightly in neural crest (arrowheads), dermamyotome and pronephros. (E) Forelimb-level HE transverse section in separate C13 embryo. (F) GJA1 antisense probe-hybridized adjacent section. Limb bud, dorsal neural tube and dorsal roots (arrowheads) hybridize more strongly than other tissues, nearly all of which have some basal GJA1 expression. (G and J) GJA1 sense probe-hybridized adjacent section to previous frame, demonstrating specificity of antisense hybridizations. (H) Rostral trunk-level HE section of same embryo as in (A–D). (I) GJA1 is highly expressed in the dorsal neural tube, the dorsal roots and ganglia, the dermatome and the splanchnopleural mesoderm, with basal expression in all other tissues of the section as compared with (J). Abbreviations: aer, apical ectodermal ridge; am, amnion; cœ, cœlom; cv, cardinal vein; da, dorsal aorta; dC, duct of Cuvier; dr/g, dorsal root more or less in plane of ganglion; dt, dermatome; g, gut; la, left atrium; lb, limb bud; lv, liver bud; my, myotome; ncc, neural crest cells; nd, notochord; nt, neural tube; pn, pronephros; scl, sclerotome; spl, splanchnopleura; sv, sinus venosus. Bar = 250 µm.

Figure 5.

Human and mouse neural crest transcriptomes have much in common but are also species-specific. (A) Venn diagram with common and specific genes to hNCC, mouse (m)NCSC (early cultures) and mNCP (later cultures) as described by Hu et al. (22). (B and C) Functional annotation of those hNCC genes differentially expressed (P < 0.001; Fisher's exact t-test with Benjamini–Hochberg correction for multiple testing) with respect to the combined set of m(NCSC+NCP). (B) Statistically over-represented functional groups in hNCC with number of molecules assigned to a given group over each bar. (C) Under- and over-expression of genes assigned to individual pathways in hNCC relative to the mouse represented in green and red, respectively. White represents those members of a category absent from one or the other dataset. (D) Schematic view of individual components of part of the Notch pathway from (C) with the same color convention, as expressed in hNCC. In contrast to mNCSC/mNCP, hNCC express many Notch ligands, receptors, co-activators, effectors and transcriptional targets. Abbreviations: a, cell signaling; b, cell death; c, gene expression; d, growth and proliferation; e, cell cycle; f; cytokinesis; g, nervous system development and function; h, cell morphology; i, cell–cell interaction; j, embryonic development; k, hematological system development and function; AA, eicosanoids; EGF, epidermal growth factor; ERK, Microtubule-associated protein kinases; hNCC, human neural crest cells; IGF1, insulin-related growth factor 1; IR, insulin receptor; ITG, integrins; mNCP, mouse neural crest progenitors; mNCSC, mouse neural crest stem cells; NRG, neuregulins; NT, neurotrophins; PI3K, Phosphoinositide 3-kinases; SHH, sonic hedgehog; TGFb, transforming growth factor beta family; TLR, Toll-like receptors; VEGF, vascular endothelial growth factors.

Figure 6.

Pluripotent stem cell markers are expressed by uncommitted hNCC. (A) Hierarchical cluster dendrogram of hNCC-expressed transcript list compared to 14 publicly available, normal human tissue SAGE banks (uncentered correlation, average linkage). The global transcriptome of hNCC is most similar to two hES cell lines (hES3 and hES4) relative to the transcriptomes of the substantia nigra (subst nig); mesenchymal stem cells from the umbilical cord (umb MSC) or bone marrow (BM-MSC), highly similar to each other by this analysis; pulmonary epithelium (lung ep), Schwann cells in vitro, prostate, sciatic nerve, cerebellum, brain white matter (wh matt), kidney, liver or skeletal (sk) muscle. Correlation coefficients are indicated in red. (B) Expression of NANOG mRNA in transverse section of human Carnegie stage 13 (C13) embryo (cf. Fig. 4H) is discrete but present in neural tube, neural crest cells in the dorsal root (arrowhead) and dermatome, when compared with a sense probe hybridized adjacent section (C). (D) Expression of POU5F1 in simultaneously hybridized adjacent section is more visible in equivalent structures, and seems to have a widespread basal expression in all tissues. (E) NANOG expression at C15 (cf. Fig. 9F and G). After a few days’ growth, expression is more distinct in the proliferating neuroepithelium, dorsal root ganglia, the motor horns, stomach, sympathetic ganglia, liver bud and migrating myotome cells. (F) Adjacent section hybridized with sense probe. (G) POU5F1 is expressed in a similar pattern. Both NANOG and POU5F1 transcripts appear to be excluded from the roofplate of the neural tube at this stage (extension of dotted lines). (H) Heat map of target genes whose promoters can be co-occupied by SOX2, NANOG and POU5F1 in hNCC, hES3, hES4, BM-MSC, UC-MSC, adult liver and lung cells, respectively. White stands for a tags-per-million (tpm) value equal to 0, light blue for 1 ≤ tpm ≤ 49, dark blue for 50 ≤ tpm ≤ 100 and magenta for tpm value >100. Global modulation is similar in hNCC and hES3/4 compared to the more distantly clustered cell types; for example, SOX2 itself, although other genes are expressed differentially, such as ZIC1 or ZFHX1B. Abbreviations: da, dorsal aorta; dm, dermamyotome; drg, dorsal root ganglion; es, stomach; lv, liver; mn, mesonephros; nt, neural tube; sg, sympathetic ganglion.

Figure 7.

Growth factor pathways are similarly activated in hNCC and hES cells, relative to other cell types. Heat map representing expression levels in hNCC, hES3, hES4, UC-MSC, BM-MSC, adult liver and adult lung cells of lists of genes attributed by IP software to various signaling pathways. These include but are not exclusive to the epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), NOTCH, Sonic hedgehog (SHH), transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF) and WNT pathways. Gene symbols for the more abundant hNCC tags are listed on the left, with high concomitant hES expression in red text. White, tags-per-million (tpm) value equal to 0; light blue, 1 ≤ tpm ≤ 49; dark blue, 50 ≤ tpm ≤ 100; magenta, tpm value >100.

Figure 8.

Schematic view of part of the IGF-1 and integrin signaling pathway genes transcribed by hNCC. Pink-tinted symbols are genes expressed by hNCC, and documented physical interactions exist for each edge depicted.

Figure 9.

Prediction of mRNA subset specific to hNCC. (A) RT–PCR for four genes with TPE score=1 shows that it is possible to amplify AMIGO3 and ZNF417 from an adult human liver, although below the level of detection by a publicly available SAGE bank from another sample. HOXC5 and C2ORF63 were specific to most or all hNCC lines. (B) In situ hybridization to HOXC5 demonstrates expression in the human embryo at Carnegie stage 13 (C13; cf. Fig. 4E) in most cells at the level of the limb bud, with higher levels in the neural tube, the neural crest (arrowhead), the limb mesenchyme and the rest of the somatopleural mesoderm. (C and E) Sense probe-hybridized adjacent transverse sections to previous frames demonstrate specificity of signal. (D) HOXC5 in a different C13 embryo section taken at a more rostral transverse level than in (B); expression appears reduced in the floorplate relative to the rest of the neural tube. (F) At C15 (cf. Fig. 6E–G), HOXC5 is expressed by the developing vertebra, muscle precursors, ventricular zone of the neural tube and motor horns; it is also transcribed by all other tissues in this section when compared with the sense-hybridized adjacent section (not shown). (G) Adjacent HE section. Thus, markers are only specific to a particular cell type within a given spatiotemporal context. Abbreviations: cœ, cœlom; da, dorsal aortae; h, heart; hm, hypaxial muscle precursors; lb, limb bud; lv, liver; m, muscle; nd, notochord; nt, neural tube; vt, vertebra.