Comparative Single Cell Transcriptomics Reveals Distinct Cell Fate Transition Statuses during Human Cardiac Reprogramming

Direct lineage conversion among various somatic cell types revolutionized the field of stem cell and regenerative medicine. In addition, the platform of cellular reprogramming offered a powerful system to gain new knowledge about cell plasticity and cell fate determination and ultimately challenged previous notions of cell identity. Previously, we successfully utilized single cell transcriptomics to reconstruct the molecular routes of how a murine fibroblast adopts cardiomyocyte fate following a continuum of states. In this study, we employed a comparative single cell transcriptomics approach to study human cardiac reprogramming. In comparison with murine fibroblasts and other human cell types, we identified unexpected heterogeneity in human cardiac fibroblasts that was mainly due to variance in cell cycle status. Trajectories inferred by SLICER suggest molecular routes and pathways taken by human fibroblasts when transiting into CM fate. Importantly, by assigning a “cell fate index” to each single cell on the iCM trajectories resolved from both mouse and human fibroblasts, we discovered species differences in fibroblast plasticity and intermediate cell fate statuses when reprogrammed towards a CM fate. Collectively, our comparative single cell transcriptomics study of human cardiac reprogramming revealed previously unrecognized molecular features of human cardiac fibroblasts and regulatory mechanisms in human cardiomyocyte cell fate control. Primary human cardiac fibroblasts were isolated and reprogrammed with polycistronic human MEF2C, GATA4, TBX5 and miR-133. Single cells were captured using the Fluidigm C1 system with the capacity of up to 96 single cells per experiment. A total of eight individual single-cell experiments were performed. One plate (D0hCF) contained uninfected d0 human primary fibroblasts. Two plates (D3M1 and D3M2) contained d3 MGT+miR-133-transduced cells. The fourth plate (D3UN) contained d3 DsRed-transduced cells (DsRed+ by FACS) mixed with d3 uninfected cells at 3:1 ratio. The rest four plates contained MGT+miR-133-transduced cells mixed with Tdtomato-transduced cells (Tdtomato+ by FACS) at 3:1 ratio, which were collected on d3 (D3M3), d5 (D5M1), d7 (D7M1), and d9 (D9M1), respectively. Control RNA spike-ins were added to the plate before cell lysis and processed in parallel to cellular RNA. Lysate from 704 wells containing single cells were selected for mRNA sequencing (raw data files) and a total of 652 high-quality single-cell transcriptomes were analyzed after QC (processed data).