Fibroblast feeder layers inhibit differentiation of retinoic acid-treated embryonal carcinoma cells by increasing the probability of stem cell renewal

Murine embryonal carcinoma cells can differentiate into a varied spectrum of cell types. We observed the abundant and precocious development of neuronlike cells when embryonal carcinoma cells of various pluripotent lines were aggregated and cultured in the presence of nontoxic concentrations of retinoic acid. Neuronlike cells were also formed in retinoic acid-treated cultures of the embryonal carcinoma line, P19, which does not differentiate into neurons in the absence of the drug. The neuronal nature of these cells was confirmed by their staining with antiserum directed against neurofilament protein in indirect immunofluorescence experiments. Retinoic acid-treated cultures also contained elevated acetylcholinesterase activity. Glial cells, identified by immunofluorescence analysis of their intermediate filaments, and a population of fibroblastlike cells were also present in retinoic acid-treated cultures of P19 cells. We did not observe embryonal carcinoma, muscle, or epithelial cells in these cultures. Neurons and glial cells appeared in cultures exposed to retinoic acid for as little as 48 h. We found no evidence for retinoic acid toxicity, suggesting that the effect of the drug was to induce the development of neurons and glia rather than to select against cells differentiating along other developmental pathways.

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Maintenance of active chromatin states by HMGN2 is required for stem cell identity in a pluripotent stem cell model

Epigenetics & Chromatin ◽

10.1186/s13072-019-0320-7 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 3

Author(s):

Sylvia Garza-Manero ◽

Abdulmajeed Abdulghani A. Sindi ◽

Gokula Mohan ◽

Ohoud Rehbini ◽

Valentine H. M. Jeantet ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Neuronal Differentiation ◽

Embryonal Carcinoma ◽

Cell Model ◽

Carcinoma Cells ◽

Adult Brain ◽

Embryonal Carcinoma Cells ◽

Stem Cell Model ◽

Neural Stem Progenitor Cells

Abstract Background Members of the HMGN protein family modulate chromatin structure and influence epigenetic modifications. HMGN1 and HMGN2 are highly expressed during early development and in the neural stem/progenitor cells of the developing and adult brain. Here, we investigate whether HMGN proteins contribute to the chromatin plasticity and epigenetic regulation that is essential for maintaining pluripotency in stem cells. Results We show that loss of Hmgn1 or Hmgn2 in pluripotent embryonal carcinoma cells leads to increased levels of spontaneous neuronal differentiation. This is accompanied by the loss of pluripotency markers Nanog and Ssea1, and increased expression of the pro-neural transcription factors Neurog1 and Ascl1. Neural stem cells derived from these Hmgn-knockout lines also show increased spontaneous neuronal differentiation and Neurog1 expression. The loss of HMGN2 leads to a global reduction in H3K9 acetylation, and disrupts the profile of H3K4me3, H3K9ac, H3K27ac and H3K122ac at the Nanog and Oct4 loci. At endodermal/mesodermal genes, Hmgn2-knockout cells show a switch from a bivalent to a repressive chromatin configuration. However, at neuronal lineage genes whose expression is increased, no epigenetic changes are observed and their bivalent states are retained following the loss of HMGN2. Conclusions We conclude that HMGN1 and HMGN2 maintain the identity of pluripotent embryonal carcinoma cells by optimising the pluripotency transcription factor network and protecting the cells from precocious differentiation. Our evidence suggests that HMGN2 regulates active and bivalent genes by promoting an epigenetic landscape of active histone modifications at promoters and enhancers.

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