Generation of Murine Sympathoadrenergic Progenitor-Like Cells from Embryonic Stem Cells and Postnatal Adrenal Glands
Figure 2
ESC-derived NCSC-like cells are enriched by sorting for expression of CD57.
ESC-derived NPCs were cultured in medium containing chicken embryonic extract, retinoic acid, bFGF and IGF-1 under hypoxic conditions to generate low-enriched NCSC-like cells. (A) A subset of low-enriched NCSC-like cells expresses CD57 and can be isolated by FACS. Low-enriched NCSC-like cells were used. Left upper panel shows isotype control, right upper panel unsorted CD57-stained cells and lower panels post-sort plots of the CD57− and CD57+ fractions. (B) Stronger expression of some NCSC and SAP markers in CD57+ cells. RNA isolated from unsorted, CD57− and CD57+ cells was analyzed by RT-PCR to detect markers of CD57 biosynthesis, pluripotent cells, NCSCs, SAPs and sympathetic neurons. As positive control for TrkA and peripherin RT-PCR, cDNA from Neuro2a (a mouse NB) was used, while for TrkB mouse brain cDNA was employed. (C) CD57+ cells have more trilineage NCSC potential as compared to the CD57− fraction. CD57−sorted cells were cultured at clonal density in NCSC medium and resultant clones were simultaneously stained for NF160, GFAP, SMA and DAPI. A trilineage clone is shown. Scale bar equals 100 µm. The percentages of clones with cells of all three lineages, two lineages, one lineage or none of the lineage (“other”) were quantified. The means of three independent experiments (with a total of 183 clones) are depicted. Statistical analysis was performed using the t-test. *, p = 0.019. (D) Clones from CD57+ cells do not contain cells expressing peripherin protein. Clones (left panel) and neuro2a NB cells used as positive control (right panel) were stained for peripherin. Nuclei were counterstained with DAPI. Scale bar equals 100 µm.