Human Embryonal Carcinoma Cells in Serum-free Conditions as an In Vitro Model System of Neural Differentiation

Abstract

Serum is generally regarded as an essential component of many eukaryotic cell culture media, despite the fact that serum composition varies greatly and may be the source of a wide range of artefacts. The objective of this study was to assess serum-free growth conditions for the human embryonal carcinoma cell line, NT2/D1. These cells greatly resemble embryonic stem cells. In the presence of retinoic acid (RA), NT2/D1 cells irreversibly differentiate along the neuronal lineage. We have previously shown that the early phases of neural induction of these cells by RA involve the up-regulation of SOX3 gene expression. Our goal was to compare RA-induced differentiation of NT2/D1 cells in serum-containing and serum-free media, by using SOX3 protein levels as a marker of differentiation. We found that NT2/D1 cells can be successfully grown under serum-free conditions, and that the presence or absence of serum does not affect the level of SOX3 protein after a 48-hour RA induction. However, six days of RA treatment resulted in a marked increase in SOX3 protein levels in serum-free media compared to serum-containing media, indicating that serum might have an inhibitory effect on the expression of this neural differentiation marker. This finding is important for both basic and translational studies that hope to exploit cell culture conditions that are free of animal-derived products.

Reactive and Senescent Astroglial Phenotypes as Hallmarks of Brain Pathologies

“…However, the availability of human primary astrocytes is limited, underlining the need for alternative, more accessible sources of human astrocytes for in vitro studies. Human pluripotent embryonal teratocarcinoma NT2/D1 cell line has been used as neural model system in different studies [252][253][254][255]. This is an immortalized cell line that has the ability to differentiate along the neural lineage when treated with retinoic acid, yielding both neuronal and glial cell populations [236,256].…”

Advanced materials and biofabrication technologies to design in vitro functional central nervous system models

“…However, it is important to take into account, for the in vitro expansion of mESCs, the relative difficulties in controlling the culture environment (possibly due to excessive cell aggregation problems), the requirement of intensive manipulation and media supplements, the needed constant (and time consuming) characterization of newly derived ESC lines before germline testing or in vitro differentiation (e.g., by assays for mycoplasma, bacteria, fungi or mouse pathogens, SNP paneling analysis and periodic Karyotipic assessments), not disregarding mice employment issues (related to the production or renewal of mESCs from specific strains) (Rodrigues et al, 2011;Czechanski et al, 2014;Martin-Lemaitre et al, 2020). Among the pluripotent stem cells, it is also possible to mention the embryonal carcinoma cells (ECCs), derived from teratomas, which generally, upon culture with retinoic acid (RA), could differentiate into well-developed, morphologically and immunophenotypically CNS-like neurons, providing a more efficient and more reproducible system in terms of growth and manipulation, compared to ESCs (Jasnic-Savovic et al, 2015). However, ECCs are rarely employed in regenerative therapies due to their tumorigenic properties, but can be used for in vitro disease modeling.…”