Developmental regulation of neurogenesis in the pluripotent human embryonal carcinoma cell line NTERA‐2

Abstract

Embryonal carcinoma (EC) cells provide a caricature of pluripotent embryonic stem (ES) cells and may be used as surrogates for investigating the mechanisms that regulate cell differentiation during embryonic development. NTERA-2 is a human EC cell line that differentiates in response to retinoic acid yielding cells that include terminally differentiated neurons. The expression of genes known to be involved in the formation of the vertebrate nervous system was examined during retinoic acid-induced NTERA-2 differentiation. Differentiation of these human EC cells into neurons could be divided into three sequential phases. During phase 1, in the first week of differentiation, hath1 mRNA showed a small transient increase that correlated with the rapid accumulation of nestin message, a marker of neuroprogenitors. Transcripts of nestin were quickly downregulated during phase 2 as expression of neuroD1, characteristic of neuroprogenitors exiting the cell cycle, was induced. A neural cell surface antigen, detected by the monoclonal antibody A2B5, was expressed by cells exiting the cell cycle, correlating with the expression of neuroD1 as the cells became post-mitotic. Markers of mature neural cells (e.g. synaptophysin and neuron-specific enolase) were subsequently increased during phase 3 and were maintained. This regulated pattern of gene expression and commitment to the neural lineage indicates that differentiation of NTERA-2 neurons in vitro follows a similar pathway to that observed by neural ectodermal precursors during vertebrate neurogenesis in vivo.

Hybrids of pluripotent and nullipotent human embryonal carcinoma cells: Partial retention of a pluripotent phenotype

“…Few, if any, morphologically identifiable neurons were seen in retinoic acid-induced C10 cultures, in contrast to TG11.nR, and the rare neurofilament-positive cells observed did not exhibit a neuronal morphology; such cells are also seen in NTERA2 and TG11.nR cultures but their identity is obscure. 15 Genes associated with neural differentiation, neuroD1 39 and Mal 38 were not induced. Of these, neuroD1 is particularly striking as a marker for committed post-mitotic neuroblasts, the precursors of the mature neurons during both embryogenesis 52 and the differentiation of NTERA2 EC cells in culture.…”

“…Of these, neuroD1 is particularly striking as a marker for committed post-mitotic neuroblasts, the precursors of the mature neurons during both embryogenesis 52 and the differentiation of NTERA2 EC cells in culture. 39 Thus, such neuroblasts are apparently absent from differentiating C10 cultures, and the block to neural differentiation in these cells must be at a relatively early stage. These results suggest that the 2102Ep parental cells contribute an inhibitory mutation, specifically active during neural differentiation, to the hybrids.…”

Establishment and initial characterization of SOX2-overexpressing NT2/D1 cell clones

“…In response to RA, NT2 cells differentiate into neurons expressing transcription factors typically observed in the neurons found in dorsal and ventral forebrain, hindbrain and spinal cord (Coyle et al, 2011). Importantly, post-mitotic NT2 neurons obtained by treatment with RA are able to generate action potentials and calcium spikes, express, release and respond to neurotransmitters (Houldsworth et al, 2001;Przyborski et al, 2000Przyborski et al, , 2003Coyle et al, 2011).…”

“…Differentiation of NT2/D1 cells into neurons could be divided into three sequential phases (Przyborski et al, 2000). Phase 1 is characterized by the accumulation of nestin mRNA, while expression of neuroD1 and synaptophysin is up-regulated during phases 2 and 3, respectively (Przyborski et al, 2000).…”

“…Phase 1 is characterized by the accumulation of nestin mRNA, while expression of neuroD1 and synaptophysin is up-regulated during phases 2 and 3, respectively (Przyborski et al, 2000).…”