Preclinical studies have suggested that transplanted human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) grafts expand due to proliferation.1 This knowledge came from cell cycle activity measurements that cannot discriminate between cytokinesis or DNA synthesis associated with hypertrophy. To refine our understanding of hPSC-CM cell therapy, we genetically engineered a cardiomyocyte-specific fluorescent barcoding system into an hPSC line. Since cellular progeny have the same color as parental hPSC-CMs, we could identify subsets of engrafted hPSC-CMs with greater clonal expansion.
HPSC lines were generated by knocking four copies of the Cre-dependent Brainbow 3.2 lineage reporter2 into WTC11 cells (Figure A). These rainbow hPSCs were transduced with cardiac troponin T (cTnT)-driven Cre, which restricts expression of the rainbow barcoding system to committed cardiomyocytes (Figure A). Rainbow-labeling was observed after 7 days of differentiation, and immunostaining confirmed that labeled cells express cTnT (Figure B). A sparse labeling strategy was used to avoid expression of the same color code in neighboring cells, hence permitting single cell tracking over time (Figure C). Cre-mediated recombination elicited all eighteen of the possible hues (Figure D).
By day 14, hPSC-CMs had clonally expanded (Figure E). On average, the number of cardiomyocytes per clone went from 1.03 to 1.71 (day 7 versus 14, p<0.03, Figure F). While most rainbow hPSC-CMs had not proliferated, some were highly proliferative and a subset of hPSC-CMs continued to proliferate after replating at day 14 (Figure F), including hPSC-CMs that were lactate selected. Repeat imaging of replated hPSC-CMs over days 15–28 confirmed that neighboring cells had unique hues and daughter cardiomyocytes inherited the parental fluorescent-barcode (Figure G), definitively demonstrating these clusters arise from clonal expansion. We observed that hPSC-CM displayed limited cell migration (Figure G) and underwent hypertrophic growth as measured by cell area (Figure H). At day 28, clonally expanded hPSC-CMs were 4.07-fold smaller than non-dividing hPSC-CMs (p<0.0001, Figure H). Staining confirmed rainbow labeling demarcated cardiomyocytes and showed multinucleation in non-dividing, hypertrophied hPSC-CMs (Figures I and J). Consistent with the subset of clonally expanded hPSC-CMs, we conducted unbiased graph-based clustering on raw single cell RNA sequencing data3 from isogenic day 15 and day 30 WTC11 hPSC-CMs and found a unique subset (686 of 18073 cells) with increased mitosis and cytokinesis gene expression (AURKB, CDK1, CCNB1, 5.54 to 6.18-fold increase versus all cells, p<0.0001). These results suggest the heterogeneity in hPSC-CM proliferation may be regulated transcriptionally.
For the transplantation studies, sparsely labeled day 14 hPSC-CMs were dissociated, resuspended in Matrigel with prosurvival cocktail, and transplanted into the hearts of immune-compromised athymic rats.1 All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health and was approved by the University of Washington institutional animal care and use committee (IACUC protocol # 4376–01). TUNEL staining demonstrated 17.8 ± 2.6% of hPSC-CMs underwent apoptosis 24-hours post injection. Two weeks after engraftment, most hPSC-CMs hypertrophied (1.75-fold increase versus injectate, p<0.002) while some subsets clonally expanded (Figures K and L). To ensure that the results were not driven by false positives, the cell injectate was imaged to confirm that neighboring hPSC-CMs did not express the same barcode at the time of transplantation (Figure K). Differences in proliferative potential among engrafted hPSC-CMs was not due to differences in sarcomere content, as the proportion of α-actinin+ area was the same in both clonally expanding and non-expanding groups (p>0.77, Figure M). After 6 weeks of engraftment, cumulative clonal expansion increased further, demonstrating hPSC-CMs continue to proliferate in vivo at later timepoints, with the average number of cells per clone being 1.05 (injectate), 1.77 (2 weeks), and 5.41 (6 weeks, p < 0.001 versus 2 weeks, p < 0.0001 versus injectate, Figures N and O). Notably, the heterogenous amount of clonal expansion among engrafted hPSC-CMs would not have been observed without the rainbow single-cell reporter.
As hPSC-CM therapy is rapidly approaching clinical use, it is critical to understand how these cells behave in vivo. Single cell transcriptomics assays3 and DNA content analysis4 have revealed profound molecular heterogeneity among hPSC-CMs. By generating a cTnT lineage rainbow reporter longitudinal tracking of the hypertrophic and proliferative growth of individual hPSC-CMs is now possible. This approach demonstrated that hPSC-CMs have heterogeneous levels of proliferation in vitro and after engraftment in host myocardium. By examining the generation of newly formed cardiomyocytes, rather than utilizing proxies for cell proliferation, this study distinguished bona fide cardiomyocyte division versus incomplete cell cycle activation. The heterogenous proliferative capacity among hPSC-CMs is consistent with findings that demonstrated only a few clonally dominant cardiomyocytes generate most of the adult zebrafish heart5, suggesting a similar mechanism may underlie hPSC-CM graft expansion. Proliferative hPSC-CM express normal levels of sarcomere contractile elements, suggesting increased hPSC-CM clonal expansion could efficiently repopulate myocardium lost to injury. This is in line with cardiac cell therapy optimizations, such as co-transplantation of hPSC-CMs with epicardial cells1, demonstrating improved outcomes from stimulating grafted hPSC-CM proliferation, though graft cell function and clinical relevance remains uncertain. Thus, controlling engrafted hPSC-CM clonal expansion holds promise for improving cardiac regenerative therapies. With respect to data sharing, all data and materials will be available from the corresponding author by request.
Sources of Funding
This work was supported by NIH HL141187 & HL142624 (J.D.), NSF CMMI-1661730 (N.J.S.), NIH F32HL143851 (D.E.), NIH T32AG066574 (D.B.), Gree Family Gift (J.D./N.J.S./C.E.M.). C.E.M. was also supported by NIH grants R01HL128362, U54DK107979, R01HL128368, R01HL141570, R01HL146868, and a grant from the Foundation Leducq Transatlantic Network of Excellence.
Footnotes
Disclosures
C.E.M. is a founder and equity holder in Sana Biotechnology. C.E.M. and D.E. hold patents related to heart regeneration technology. The other authors report no conflicts of interest.
References
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