Human Embryonal Carcinoma Stem Cells Expressing Green Fluorescent Protein Form Functioning Neurons In Vitro: A Research Tool for Co-culture Studies

Abstract

Neural differentiation is controlled by complex molecular mechanisms that determine cell fate and diversity within the nervous system. Interactions between developing tissues play an important role in regulating this process. In vitro co-culture experiments offer a method to study cell differentiation and function under controlled conditions, with the additional benefit of investigating how interactions between populations of cells influence cell growth and behavior. However, it can often be difficult to distinguish between populations of co-cultured cells. Here we report the development of a human embryonal carcinoma (EC) stem cell line (named TERA2.cl.SP12-GFP) that expresses the genetic marker, green fluorescent protein (GFP). Here, we demonstrate that TERA2.cl.SP12-GFP stem cells stably express GFP and that this remains detectable during retinoic acid-induced differentiation. Regulated expression of neural markers during cell development correlated with the formation of morphologically identifiable neurons. Populations of post-mitotic GFP-positive neurons were readily purified and electrophysiological characterization confirmed that such neurons were functionally active. Thus, cultured TERA2.cl.SP12-GFP cells can be readily distinguished from alternative cell types in vitro and provide an amenable system for live cell imaging to study the development and function of human neurons in isolation, and in co-culture with other tissue types.

An evaluation of a human stem cell line to identify risk of developmental neurotoxicity with antiepileptic drugs

“…In this study, we addressed the hypothesis that human stem cell technology used in vitro might be exploited to evaluate (or predict) the risk of neurodevelopmental toxicity of psychotropic drugs in vivo. Importantly, we used human EC stem cells because the neurons derived from them display key morphological, molecular and functional properties of mature neurons including voltage-and ligand-gated ion channels (Coyne et al, 2011;Stewart et al, 2004). Moreover, teratocarcinoma stem cells have been shown to have value in screening for developmental neurotoxicity with heavy metals such as lead, mercury and aluminum (Laurenza et al, 2013;Stern et al, 2014).…”

“…Stewart et al, 2004). Briefly, stem cells were maintained in 75 cm 2 tissue culture flasks at 37°C, 5% CO 2 , 100% relative humidity in Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich) supplemented with fetal bovine serum (FBS, 10%, v/v; Invitrogen), L-glutamine (2 mM; Sigma-Aldrich) and penicillin-streptomycin (100 U/ml, 100 lg/ml; Invitrogen).…”

“…The human EC stem cell line, TERA2.cl.SP12, is easy to grow in cell culture, does not require feeder layers and has enhanced propensity to differentiate into neurons and glia using a simple differentiation protocol (Przyborski, 2001). Specifically, when TERA2.cl.SP12 stem cells are exposed to retinoic acid the pattern of gene and protein expression, along with morphological changes in vitro, parallel the early stages of neural differentiation during embryonic development and, within 3-4 weeks, they develop into functional neurons expressing a range of voltage-and ligand-gated ion channels (Andrews, 2002;Coyne et al, 2011;Stewart et al, 2004).…”

On the Generation of Efficient Methodologies for Cell-Based Toxicity Screening by Comparative Protocol Analysis and Optimization

“…NT2 stem cells exhibit many aspects of human embryonic neural stem cells [20]. Upon treatment with retinoic acid (RA), NT2 cells differentiate into post-mitotic cells, showing properties of cells of the central nervous system, also referred to as NT2-N cells [21][22][23][24][25][26]. These cells are committed towards neurons, astrocytes, and oligodendrocytes, which are the three main cell lineages of the CNS, and have extensively been used for drug in vitro screening and for studying human neurogenesis, brain regeneration, and terminal differentiation [20].…”

“…There are numerous approaches reported in the literature on neuronal differentiation of NT2 cells in aggregate or suspension culture, as well as in monolayer cultures [21,[25][26][27][28][29][30][31][32]. These protocols Sepideh Abolpour Mofrad performed the present work in fulfillment of the requirements for obtaining the degree Dr. rer.…”