Enhanced xeno-free differentiation of hiPSC-derived astroglia applied in a blood–brain barrier model

Abstract

Background: Human 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. Methods: In 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). Results: Astroglia 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. Conclusion: Our 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.

Recent advances in human iPSC-derived models of the blood–brain barrier

“…More recent work has focused on differentiating iPSCs into the additional cells of the NVU and combining these with iBMECs for fully iPSC-derived BBB models. Similar to results with primary cells, co-culture of iBMECs with iPSC-derived cells of the NVU can also raise TEER values and improve barrier function [26][27][28][29][30][31], however these additional cells are not required for iBMECs to achieve physiological TEER levels and may only improve barrier properties under suboptimal starting conditions or stress [32]. These studies have initiated a personalized approach to BBB modeling, which will likely provide new insights into genetic-based neurological diseases that may involve cell-cell interactions of the NVU.…”

Modeling Human Brain Tumors and the Microenvironment Using Induced Pluripotent Stem Cells

“…Coculture of iBMECs with pericytes, astrocytes, and neurons is an attractive approach to enhance BBB properties in models, including transendothelial electrical resistance (TEER) and low passive permeability [97][98][99]. This has led to the generation of iPSC-derived NVU supporting cells and combining them with iBMECs for an isogenic model of human BBB [95,[100][101][102]. Such models should allow a deeper understanding of physiological processes in both healthy and diseased states.…”

Challenges and opportunities in the use of transcriptomic characterization of human iPSC-derived BBB models

“…The extensive variety of cells used for co-culture together with the different in vitro platforms with diverse levels of cell-cell contact and spatial orientations, leads to a wide diversity in the iPSC-derived BBB models (Appelt-Menzel et al, 2017;Jamieson et al, 2019). Even the slightest change, like another extracellular matrix, in the differentiation of the co-cultured cells, can influence the expression levels of BBB markers (Delsing et al, 2019).…”