Figure 3.

Multipotent Chick Crestospheres Differentiate into Multiple Neural Crest Derivatives

(A) qPCR shows a dramatic decrease in FOXD3 and SOX10 expression levels after 7 days of in vitro differentiation (n = 3).

(B–H) Seven day in vitro differentiation of either 2-week (B–E) or 7-week (F–H) cultured crestospheres results in production of melanoblasts (MELEM), neurons (TUJ-1, HUC/D), cells expressing P75, a common marker of many neural crest derived cells, glial cells (BCLP) as well as smooth muscle cells (SMA) and cartilage (RUNX2). Quantification of different derivatives shows similar results for 2- (B) and 7- (F) week spheres (n ranges: week 2, from 7 to 13; week 7, from 3 to 7). In contrast, few CNS-derived cells were observed as shown by oligodendrocyte marker 04.

(I) In vivo transplantation of 2-week cultured GFP crestospheres (CS) into the head mesenchyme of HH10 chick embryo (Day 0).

(J–L) GFP-positive cells were observed in the trigeminal ganglion (TGG) (J and K) and in a nerve bundle next to the left eye (L) 2 and 3 days after the transplantation, respectively. GFP⁺ cells express neuronal marker TUJ-1 in cell body and overlapping axons (white arrow); TUJ-negative presumptive Schwann cells are intertwined with the neuronal axons (blue arrow).

(M) GFP⁺ cells lining a blood vessel.

(N) Perinuclear HUC/D staining shows a GFP⁺ neuron in the mesenchyme below the lateral ventricle (lv). Arrowhead marks autofluorescent (AF) blood cells (bc) visible in all three channels. g, ganglia; mc, mouth cavity.

(O and P) Clonal GFP⁺ cells derived from individual premigratory neural crest cells grown as crestopheres give rise to neurons, melanocytes, and smooth muscle.

(Q) Summary of different derivatives formed. CSC, crestosphere culture. Scale bar represents 100 μm.