Self-renewal capacity is a widespread property of various types of neural crest precursor cells

Abstract

In vertebrates, trunk neural crest (NC) generates glia, neurons, and melanocytes. In addition, it yields mesectodermal derivatives (connective tissues, chondrocytes, and myofibroblasts lining the blood vessels) in the head. Previous in vitro clonal analyses of avian NC cells unraveled a hierarchical succession of highly pluripotent, followed by various intermediate, progenitors, suggesting a model of progressive restrictions in the multiple potentialities of a totipotent stem cell, as prevails in the hematopoietic system. However, which progenitors are able to self-renew within the hierarchy of the NC lineages is still undetermined. Here, we explored further the stem cell properties of quail NC cells by means of in vitro serial subcloning. We identified types of multipotent and oligopotent NC progenitors that differ in their developmental repertoire, ability to self-maintain, and response to exogenous endothelin 3 according to their truncal or cephalic origin. The most striking result is that bipotent progenitors are endowed with self-renewal properties. Thus glia-melanocyte and glia-myofibroblast progenitors behave like stem cells in that they are able both to self-renew and generate a restricted progeny. In our culture conditions, glia-myofibroblast precursors display a modest capacity to self-renew, whereas glia-melanocyte precursors respond to endothelin 3 by extensive self-renewal. These findings may explain the etiology of certain multiphenotypic NC-derived tumors in humans. Moreover, the presence of multiple stem cell phenotypes along the NC-derived lineages may account for the rarity of the "totipotent NC stem cell" and may be related to the large variety and widespread dispersion of NC derivatives throughout the body.

Fig. 1.

Phenotypic analysis of NCC in primary clones on 3T3 cells (A–L) and in subclones on collagen (M–P). (A–H) Cephalic NCC in control medium. (A–D) GNMF clone with cells expressing SMP (A), neurofilament (B, arrow), MelEM (C), and α-SMA (D) antigens. (E–H) GMFC clone showing cartilage islets (arrows in E, phase–contrast), a pigment cell (F, bright field), and cells positive for SMP (G) and α-SMA (H). (I–L) Trunk NCC in presence of ET3. GNMF clone with cells expressing SMP (I), MelEM (J), tyrosine hydroxylase (K), and α-SMA (L) is shown. (M and N) Cephalic NCC secondary cloning in the presence of ET3. GF subclone with cells immunoreactive for SMP (M) and α-SMA (N) is shown. (O and P) Trunk NCC tertiary cloning in the presence of ET3. GM subclone with cells positive for SMP (O) and MelEM (P) is shown. (Scale bar in P represents 50 μm; scale bar in D is 15 μm for D only.)

Fig. 2.

Self-renewal of cephalic NCC. Primary clones (I) from GF and GM founders were subcloned serially in control (A) and ET3-supplemented (B) media. At each generation, the different types of clones (and mean percentage of total clones), as well as the fold increase in the number of GF or GM produced by self-renewing founder cells, are indicated. The subcloned colonies were two GF I and GF II (control); and five GF I, two GF II, and one GM at each subcloning I–III (ET3) (see Tables 4 and 5 and Supporting Text).

Fig. 3.

Self-renewal of trunk NC precursors in presence of ET3. The progeny of identified GF, GM, and GMF precursors was analyzed during serial subcloning. The different types of subclones (and mean percentage of total clones), as well as the fold increase in the number of parental-like progeny produced by individual founders, are indicated. The increase in GM progeny is given for GMF founders (*). The subcloned colonies were as follows: three GF I; six GM I, five GM II, two GM III, and one GM IV; and one GMF (I) and two GM at cloning II and III (see Tables 6–8 and Supporting Text).

Fig. 4.

Model for cell-lineage segregation in cephalic (A) and trunk (B) NC. Progenitors are classified according to the number of developmental potentials, and those identified for the first time in the present study are shown in shaded circles. Data from in vitro clonal analysis (refs. – and and this study) indicate that differentiation of neurons, glia, melanocytes, myofibroblasts, and cartilage from totipotent quail NCC involves progressive developmental restrictions yielding several intermediate oligopotent precursors. Here, we identified a highly pluripotent GNMF precursor that could be a totipotent trunk NCC. The existence of a totipotent cephalic progenitor (GNMFC) and the filiations (dashed arrows) are hypothetical. The GM and GF precursors behave as stem cells. GF precursors self-renew independently of ET3 (curved arrows), whereas GM (and possibly GMF) progenitors display ET3-induced high self-renewal activity (bold curved arrows). ET3 also promotes GM differentiation into glial and melanocytic cells and biased GF stem cell progeny toward a glial fate (bold arrows).