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 batchto 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. *

Differentiation and Subculturing of Renal Proximal Tubular‐like Cells Derived from Human iPSC

“…This protocol outlines the procedure to differentiate iPSC into PTL within 14 days. PTL can be differentiated on either growth factor reduced Geltrex or de-cellularized extracellular matrix (ECM) derived from cultured RPTEC/TERT1 cells, or from cultured human dermal fibroblasts fHDF/TERT166, as previously described (Chandrasekaran et al, 2021;Murphy et al, 2023).…”

“…RPTEC/TERT1 cells were cultured as previously described (Aschauer et al, 2013), and ECM was harvested 4 days after reaching confluency as previously described (Chandrasekaran et al, 2021). ECM from fHDF/TERT166 cells was harvested immediately after confluency was reached as previously described (Murphy et al, 2023). Depending on the cell line, additional time after reaching confluency to produce sufficient ECM may be necessary.…”

“…Nevertheless, for smaller formats such as a 96-well plate, or smaller, a coating is recommended. In such cases, we recommend one of the following coatings: RPTEC/TERT1 or fHDF/TERT166-ECM coating (see Support Protocol 2) (Chandrasekaran et al, 2021;Murphy et al, 2023), or human collagen IV coating (see Support Protocol 3).…”

“…We tested the cell lines RPTEC/TERT1 and fHDF/TERT166. Both models are immortalized with hTERT and were obtained from Evercyte GmbH (Murphy et al, 2023;Wieser et al, 2008). RPTEC/TERT1 cells were cultured as previously described (Aschauer et al, 2013), and ECM was harvested 4 days after reaching confluency as previously described (Chandrasekaran et al, 2021).…”

Engagement of scientists with the public and policymakers to promote alternative methods

“…Science should also be communicated and disseminated to policymakers independently of the political and legislative agenda. The science of alternative methods is a complex topic with its own dilemmas, e.g., cell culture techniques commonly require the use of animal-derived materials that may be associated with animal suffering such as fetal bovine/calf serum, basement membrane extracts (e.g., Matrigel), antibodies obtained from ascites fluid, primary cells from animals, and other animal-derived products obtained by invasive sampling (van der Valk et al, 2018;Murphy et al, 2022;Gruber and Hartung, 2004;Cassotta et al, 2022). Communication activities build trust in the topic, make policymakers familiar with technical terms used by the scientific community, and build a relationship, but are challenged by recurring emergencies as policymakers (elected or non-elected) are increasingly crisis-driven.…”