Abstract
BackgroundHuman induced pluripotent stem cells (hiPSC) hold great promise for use in cell therapy applications and for improved in vitro models of human disease. So far, most hiPSC differentiation protocols to astroglia use undefined, animal-containing culture matrices. Laminins, which play an essential role in the regulation of cell behavior, offer a source of defined, animal-free culture matrix.MethodsIn order to understand how laminins affect astroglia differentiation, recombinant human laminin-521 (LN521), was compared to a murine Engelbreth-Holm-Swarm sarcoma derived laminin (L2020). Astroglia expression of protein and mRNA together with glutamate uptake and protein secretion function, were evaluated. Finally, these astroglia were evaluated in a coculture model of the blood–brain barrier (BBB).ResultsAstroglia of good quality were generated from hiPSC on both LN521 and L2020. However, astroglia differentiated on human LN521 showed higher expression of several astroglia specific mRNAs and proteins such as GFAP, S100B, Angiopoietin-1, and EAAT1, compared to astroglia differentiated on murine L2020. In addition, glutamate uptake and ability to induce expression of junction proteins in endothelial cells were affected by the culture matrix for differentiation.ConclusionOur results suggest that astroglia differentiated on LN521 display an improved phenotype and are suitable for coculture in a hiPSC-derived BBB model. This provides a starting point for a more defined and robust derivation of astroglia for use in BBB coculture models.
Highlights
Human induced pluripotent stem cells hold great promise for use in cell therapy applications and for improved in vitro models of human disease
Astroglia specific protein and mRNA expression Astroglia differentiated on human LN521 or murine laminin from murine Engelbreth-Holm-Swarm sarcoma (L2020) were characterized based on morphology and astroglia marker expression after 28 days of differentiation
Analysis of the expression of astroglia marker genes (Fig. 2b–i) revealed higher expression of several mRNAs in astroglia differentiated on LN521 when compared to L2020
Summary
Human induced pluripotent stem cells (hiPSC) hold great promise for use in cell therapy applications and for improved in vitro models of human disease. Human induced pluripotent stem cells (hiPSC) can be used to generate an unlimited supply of specialized cell types from a patient’s own tissue and hold great promise to overcome issues with donor variability and low availability of human tissue. Glia cells play a key role in the formation and maintenance of the blood–brain barrier (BBB) through physical interaction with the brain microvascular endothelial cells and secretion of modulating factors [12,13,14,15,16]. The tight cellular interactions between the BMEC in the BBB act as a physical barrier for pathogens, cells, proteins and watersoluble agents. The BM contains specific, highly conserved proteins, and consists mostly of laminin, type IV collagen, agrin, perlecan, fibronectin and nidogen [17]
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