Graduate Candidate University of California, Santa Barbara, California, United States
Abstract: The power of stem cells and gene editing is the ability to introduce mutations into isogenic lines; especially for heart disease-linked mutations which contribute to 1 in 5 deaths worldwide. These mutations have measurable effects on the contractility and morphology of hiPSC-CMs. Yet, heterogeneity from line to line and batch to batch remains a challenge and can be exacerbated by gene editing, different media, handling by different researchers, differentiation, etc. We sought to analyze this heterogeneity using mixed effects models. We included known clusters (e.g. researcher or batch) and allowed random slopes and intercepts of those clusters within the model hierarchy. Clustering improves estimates for repeat sampling, unbalanced sampling, and variation. We modeled this heterogeneity in multiple isogenic hiPSC-CMs cell lines from one donor (over 58 batches) and multiple cell lines from donors of different sexes (over 35 batches). We analyzed published Traction Force Microscopy (TFM) data from the diseased cell line collection, generated from cell line GM25256 by the Allen Institute for Cell Science, to model heterogeneity associated with gene editing and researcher (Lee, 2023 and Pardon, 2024). We found robustly similar morphological and contractile phenotypes across users and differentiation batches with small but significant differences from the gene editing process. However, differences between multiple isogenic control lines, undergoing the same editing processes, were dwarfed by the differences caused by the deleterious mutations edited into the disease lines. Thus, we conclude a single isogenic control line can serve to compare the effects of these isogenic mutation lines. We also found mTeSR1 media used on pre-differentiated hiPSCs resulted in larger spread area in hiPSC-CMs when compared to Essential 8 (E8). Additionally, we analyzed published TFM data from multiple male and female donor lines, obtained from the Stanford Cardiovascular Institute, to measure sex differences (Chirikian, 2022). Male cells had 42% higher twitch force (p = 8.8E-6) with 25% higher contraction (p = 1.77E-5) and relaxation (p = 0.0006) velocities. Knowing that deleterious mutations and cellular sex cause larger changes than other sources of heterogeneity of hiPSC-CMs will improve hiPSC-CMs as a cardiac cell model.
Funding Source: This project was supported by the NSF BRITE Fellow award #2227509, NSF Data Driven Biology NRT award #2125644, Connie Frank Biomedical Fellowship, CIRM EDUC4-12821, and the NIH 1RM1GM131981-01.
