Transcriptional Profiling of Neuronal Differentiation by Human Embryonal Carcinoma Stem Cells In Vitro

Abstract

Pluripotent stem cell lines can be induced to differentiate into a range of somatic cell types in response to various stimuli. Such cell-based systems provide powerful tools for the investigation of molecules that modulate cellular development. For instance, the formation of the nervous system is a highly regulated process, controlled by molecular pathways that determine the expression of specific proteins involved in cell differentiation. To begin to decipher this mechanism in humans, we used oligonucleotide microarrays to profile the complex patterns of gene expression during the differentiation of neurons from pluripotent human stem cells. Samples of mRNA were isolated from cultured NTERA2 human embryonal carcinoma stem cells and their retinoic-acid-induced derivatives and were prepared for hybridization on custom microarrays designed to detect the expression of genes primarily associated with the neural lineage. In response to retinoic acid, human NTERA2 cells coordinately regulate the expression of large numbers of neural transcripts simultaneously. Transcriptional profiles of many individual genes aligned closely with expression patterns previously recorded by developing neural cells in vitro and in vivo, demonstrating that cultured human pluripotent stem cells appear to form neurons in a conserved manner. These experiments have produced many new expression data concerning neuronal differentiation from human stem cells in vitro. Of particular interest was the regulated expression of Pax6 and Nkx6.1 mRNA and the absence of Pax7 transcription, indicating that neurons derived from NTERA2 pluripotent stem cells are characteristic of neuroectodermal cells of the ventral phenotype.

Evaluation of bias associated with high-multiplex, target-specific pre-amplification

“…We identified 34 regulated genes, which include well known stem cell biomarkers such as NANOG and neural cell biomarkers such as NEUROD1 ( Table 2 ). Most of these genes were previously characterized to be involved in NT2 differentiation [14] , [15] , [16] , [17] , which is confirmatory of our approach.…”

“…To determine if the PreAmp workflow can accurately quantify gene expression changes of natural targets, we analyzed RNA samples purified from NT2 cells, an established model system of human stem cell behavior. When treated with retinoic acid (RA), NT2 cells differentiate into neurons [12] , [13] ; during differentiation the expression of stem cell-specific biomarkers decrease while the expression of neuron-specific biomarkers increase [14] , [15] , [16] , [17] . To identify genes regulated during differentiation we applied the fold-change limits-of-detection criteria established in the previous section ( Table 1 ) to 267 genes involved in cell maintenance, cell fate and adaptation.…”

The knockdown of H19lncRNA reveals its regulatory role in pluripotency and tumorigenesis of human embryonic carcinoma cells

“…To further investigate the link between down-regulation of H19, pluripotency and early differentiation, transduced NCCIT cells were plated on laminin and poly-L-Lysine coated wells and then immunostained for Oct4 and Nestin, an early neural differentiation marker [ 23 ]. A significant decrease of Oct4 and a marked increase of Nestin intensities were observed in NCCIT/shH19 cells compared to control cells (Figure.…”

A robust and reproducible human pluripotent stem cell derived model of neurite outgrowth in a three-dimensional culture system and its application to study neurite inhibition

“…Characterisation of this model and assessment of markers associated with neural differentiation is consistent with previously published evidence (Przyborski, 2001; Przyborski et al., 2000, Przyborski et al., 2003, Przyborski et al., 2004; Stewart et al., 2004; Coyne et al., 2011), suggesting that the neurite outgrowth observed shares similarities with the early stages of nervous system development.…”

“…The embryonal carcinoma (EC) stem cell line, TERA2, readily differentiates into neuronal subtypes when exposed to ATRA. EC cells are the malignant counterpart of embryonic stem cells, and cells from the TERA2 lineage have provided the basis for many in vitro models of neural differentiation, function and neurite outgrowth (Tegengem et al., 2011, Satoh et al., 1997, Pewsey et al., 2010, Roloff et al., 2015, Przyborski, 2001, Przyborski et al., 2000, Przyborski et al., 2003, Przyborski et al., 2004, Stewart et al., 2004, Coyne et al., 2011). While such models are valuable, there is significant scope to enhance their reliability in terms of robustness and reproducibility.…”