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

Abstract

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®, 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 characterizations 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 (≥ 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 analyzing 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. Furthermore, decellularized fibroblast-ECM could be a novel, attractive low-cost coating material.