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 (hiPSC) 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 hiPSC as well as the differentiation of hiPSC into target cells that are used for toxicity testing, mainly depend on the presence of extracellular matrix (ECM) coatings. The most widely used ECM coating is Matrigel, an animal product that is derived from mice sarcomas. Drawbacks of Matrigel are widely recognized and include batch-to batch variations, usage of animal rather than human material and ethical concerns for its production. While alternative coatings exist, factors such as higher cost and limited characterizations may hinder a broader uptake by the scientific community. Here, we report an extensive comparison of three commercial 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 by analyzing copy number variations by shallow DNA sequencing. Furthermore, a low-cost alternative, decellularized ECM produced by human neonatal dermal fibroblasts was analyzed. In addition, all alternative coatings were tested for hiPSC differentiation into renal podocyte-like cells in a genome-wide transcriptomics screen. Our results showed that all tested coatings were highly comparable to the 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