Differentiation of Human Embryonic Stem Cells to Sympathetic Neurons: A Potential Model for Understanding Neuroblastoma Pathogenesis

Figure 6

(a–c) Fluorescence-activated cell sorting (FACS) for p75 (neural crest stem cell marker). (a) FACS plots for H9 cells sorted on day 8 of differentiation. (a-i) 44.7% ± 6.1 p75+ viable cells in MACS® neuronal media. (a-ii) 32.5% ± 1.4 p75+ viable cells in neural BHK media. (b) Immunofluorescence of p75+ FACS-sorted H9 cells at different stages of differentiation. (b-i) PRPH and TH copositive H9 cells at 14 days post cell sort. (b-ii) PHOX2B and DBH copositive H9 cells at 14 days post cell sort. (c) mRNA expression by RT-PCR of FACS sorted H9 p75+ and p75− ve fractions showing high expression of TH, DBH, and PERIPHERIN in p75+ cells and undetectable expression in p75− cells and equivalent SNAIL expression in both populations. (d) RT-PCR of p75-enriched H9 cells isolated using anti-human p75 antibody-coated magnetic MicroBeads showing TH and p75 expression in p75-depleted population. +ve = p75 positive fraction, −ve = p75 negative fraction, and c = negative control.

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