Markers of Pluripotency and Differentiation in Human Neural Precursor Cells Derived from Embryonic Stem Cells and CNS Tissue

Abstract

Cell transplantation therapies for central nervous system (CNS) deficits such as spinal cord injury (SCI) have been shown to be effective in several animal models. One cell type that has been transplanted is neural precursor cells (NPCs), for which there are several possible sources. We have studied NPCs derived from human embryonic stem cells (hESCs) and human fetal CNS tissue (hfNPCs), cultured as neurospheres, and the expression of pluripotency and neural genes during neural induction and in vitro differentiation. mRNA for the pluripotency markers Nanog, Oct-4, Gdf3, and DNMT3b were downregulated during neural differentiation of hESCs. mRNA for these markers was found in nonpluripotent hfNPC at higher levels compared to hESC-NPCs. However, Oct-4 protein was found in hESC-NPCs after 8 weeks of culture, but not in hfNPCs. Similarly, SSEA-4 and CD326 were only found in hESC-NPCs. NPCs from both sources differentiated as expected to cells with typical features of neurons and astrocytes. The expressions of neuronal markers in hESC-NPCs were affected by the composition of cell culture medium, while this did not affect hfNPCs. Transplantation of hESC-NPC or hfNPC neurospheres into immunodeficient mouse testis or subcutaneous tissue did not result in tumor formation. In contrast, typical teratomas appeared in all animals after transplantation of hESC-NPCs to injured or noninjured spinal cords of immunodeficient rats. Our data show that transplantation to the subcutaneous tissue or the testes of immunodeficient mice is not a reliable method for evaluation of the tumor risk of remaining pluripotent cells in grafts.

Involvement of DNMT3B in the pathogenesis of Hirschsprung disease and its possible role as a regulator of neurogenesis in the human enteric nervous system

“…15,22,23 We observed reduced levels of DNMT3B mRNA and protein levels in neural precursor cells of HSCR-NLBs. We also detected a high number of enteric precursors with different DNA methylation patterns in HSCR-NLBs, which may lead to variations in the transcriptional regulation and the pluripotency stage during the cell differentiation process of these precursors.…”

“…15 This de novo DNA methylation of the genome suggests the existence of conserved mechanisms during mammalian development. [22][23][24] Nowadays, the isolation of ENS progenitor cells from the human postnatal gut represents a powerful tool for the study of ENS development. The ENS progenitor cells are cultured in order to form cell clusters, which subsequently grow as aggregates or neurosphere-like bodies (NLBs) in suspension and include stem cells and their progeny derived from the neural crest.…”

BMP-4 increasesactivin Agene expression during osteogenic differentiation of mouse embryonic stem cells

“…This was not the same with Oct-4 gene expression, which diminished independently of the BMP-4 stimulus. Furthermore, discovery of gene expression of these markers is not unique to ES cells as these pluripotent markers have been described in non pluripotent cells as central nervous system and endometrium (Allickson et al, 2011;Sundberg et al, 2011).…”

Antiproliferative and cytotoxic effects of different type cytostatics on mouse pluripotent stem and teratocarcinoma cells

“…However, even when their differentiation induced by different factors some cells remain undif ferentiated, i.e. in most cases in vitro differentiation of pluripotent cells is asynchronous and incomplete (Brederlau et al, 2006;Boyd et al, 2008;Sundberg et al, 2011). The causes of this phenomenon are unclear, but it largely limits the development of pluri potent stem cell based therapy, because the residual undifferentiated cells in transplants initiate the ter atoma development.…”