Comparison of Human Recombinant Protein Coatings and Fibroblast-ECM to Matrigel for Induced Pluripotent Stem Cell Culture and Renal Podocyte Differentiation

Human induced pluripotent stem cells (hiPSCs) offer great opportunities within the 3R framework. In the field of toxicology, they may contribute greatly to the reduction and eventually replacement of animal models. However, culturing hiPSCs as well as differentiation of hiPSCs into target cells that are used for toxicity testing depend on the presence of extracellular matrix (ECM) coating the growth surface. The most widely used ECM is Matrigel & REG;, an animal product that is derived from mouse sarcoma. Drawbacks of Matrigel are widely recognized and include batch-to batch variations, use of animal rather than human material, and ethical concerns about its production. While alternative coatings exist, higher cost and limited charac-terizations may hinder their broader uptake by the scientific community. Here, we report an extensive comparison of three commercially available human ECM coatings, vitronectin, laminin-511, and laminin-521, to Matrigel in three different hiPSC lines in long-term culture (& GE; 9 passages). Characterization included expression of pluripotent markers in a genome-wide transcriptomics study (TempO-Seq), capacity to differentiate into embryoid bodies, and karyotype stability assessed by ana-lyzing copy number variations by shallow DNA sequencing. Furthermore, a low-cost, decellularized ECM produced by human neonatal dermal fibroblasts was tested. In addition, all alternative coatings were tested for hiPSC differentiation into renal podocyte-like cells in a genome-wide transcriptomics screen. Our results show that all tested coatings were highly comparable to animal-derived Matrigel for both hiPSC maintenance and differentiation into renal podocyte-like cells. Fur-thermore, decellularized fibroblast-ECM could be a novel, attractive low-cost coating material.

Automated summary

Human induced pluripotent stem cells (hiPSCs) show potential for replacing animal models in toxicology, but their culture and differentiation depend on extracellular matrix (ECM) coating. Matrigel, the most widely used ECM, has drawbacks such as batch variations and ethical concerns. This study compares three commercially available human ECM coatings (vitronectin, laminin-511, and laminin-521) to Matrigel for hiPSC maintenance and differentiation into renal podocyte-like cells. Results show that all tested coatings are comparable to Matrigel, and decellularized fibroblast-ECM is a promising low-cost alternative.