Determining Whether Clonal Lineage Identity Impacts hiPSC Cardiac Differentiation with Functional Guide-RNA Cell Barcodes

Introduction: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have great potential in tissue engineering applications, but their clinical relevance is hindered by the heterogeneity of cardiac differentiation, which yields varying abundances of CMs and CM subtypes, even within a single hiPSC line. A potential source of this heterogeneity is differential responses to cardiac differentiation cues that arise from epigenetic differences between subpopulations of hiPSC clonal lineages. This may make lineages “primed” to differentiate into different cell fates in the cardiac niche. A potential solution to address this challenge is to employ cell barcoding to analyze lineage dynamics during cardiac differentiation. Utilizing the functional cell barcoding platform ClonMapper from Dr. Amy Brock's lab, it is possible to live-isolate specific lineages throughout differentiation. This approach can facilitate evaluation of differential expression in lineages that are "primed" for different cell fates within the cardiac niche. Characterizing the cardiac differentiation properties of different hiPSC lineages may elucidate methods into improving homogeneity of cardiac differentiation. This will be accomplished with the following objectives: i) establish a ClonMapper barcoded hiPSCs to assess if barcoding negatively impacts differentiation capacity, and ii) determine if any hiPSC lineages preferentially differentiate into hiPSC-CMs.

Materials and

Methods: ClonMapper guide-RNA barcodes were integrated into hiPSC genomes via lentivirus transduction. Successfully transduced hiPSCs were sorted via fluorescence activated cell sorting, as the barcodes are conjugated to a gene encoding blue fluorescent protein. Barcoded (BC), sorted hiPSCs were seeded on Geltrex coated plates for cardiac differentiation via Wnt modulation. Pluripotency was assessed via immunostaining for pluripotent markers in BC hiPSCs, and via immunostaining and qPCR for cardiac markers in BC hiPSC-CMs. hiPSC-CM yield was measured via flow cytometry and hiPSC-CM functional metrics were measured based on excitation-contraction coupling analyzed via a MATLAB pipeline. Abundance sequencing of lineages was performed by sequencing gDNA extracted from BC hiPSCs and BC hiPSC-CMs and non-CMs isolated after differentiation to track changes in lineage abundance through differentiation.

Results, Conclusions, and Discussions: Immunostaining showed BC hiPSCs retain pluripotent marker expression and can differentiate into cTnT-expressing hiPSC-CMs, suggesting barcoding does not negatively impact pluripotency. Flow cytometry after SIRPα staining revealed no significant difference in CM yield (Fig. 1A) and our MATLAB analytical pipeline revealed no significant differences in functionality (Fig. 1B) between control and BC hiPSC-CMs. Abundance sequencing revealed lineages that are enriched in either hiPSC-CM or non-CM populations. Our data show BC hiPSC-CMs maintain pluripotency and cardiac differentiation capacity, and there is no difference in the yield or functionality of BC and control CMs. Our data also reveal that there are certain hiPSC lineages that are enriched in CM or non-CM populations, suggesting that these lineages preferentially differentiate into either CMs or non-CMs (Fig. 2). While additional analysis is needed to characterize these hiPSC lineages to determine whether the observed differences in abundance result in meaningful differences in cardiac differentiation outcomes, the data we present indicate that hiPSC cardiac differentiation may be influenced by clonal lineage identity.

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