Cardiac interstitial tetraploid cells can escape replicative senescence in rodents but not large mammals

Abstract

Cardiomyocyte ploidy has been described but remains obscure in cardiac interstitial cells. Ploidy of c-kit+ cardiac interstitial cells was assessed using confocal, karyotypic, and flow cytometric technique. Notable differences were found between rodent (rat, mouse) c-kit+ cardiac interstitial cells possessing mononuclear tetraploid (4n) content, compared to large mammals (human, swine) with mononuclear diploid (2n) content. In-situ analysis, confirmed with fresh isolates, revealed diploid content in human c-kit+ cardiac interstitial cells and a mixture of diploid and tetraploid content in mouse. Downregulation of the p53 signaling pathway provides evidence why rodent, but not human, c-kit+ cardiac interstitial cells escape replicative senescence. Single cell transcriptional profiling reveals distinctions between diploid versus tetraploid populations in mouse c-kit+ cardiac interstitial cells, alluding to functional divergences. Collectively, these data reveal notable species-specific biological differences in c-kit+ cardiac interstitial cells, which could account for challenges in extrapolation of myocardial from preclinical studies to clinical trials.

Adaptation within embryonic and neonatal heart environment reveals alternative fates for adult c-kit+ cardiac interstitial cells

“…Developing embryos are comprised exclusively of diploid cells, whereas tetraploid cells are depleted from the epiblast lineage by mid‐gestation stage, excluded from the ICM, and instead reside among trophoblast layer contributing to extra‐embryonic membranes . The extra‐embryonic membrane localization of blastocyst‐injected cCICs (Figure ) is consistent with tetraploid DNA content of in vitro‐expanded cCIC . Tetraploid (4n) content of cCICs used for this study was confirmed by nuclear DNA content and larger nuclear size compared with sperm (haploid, 1n) or bone marrow cells (BMC, diploid, 2n) by flow cytometry and microscopy‐based nuclear intensity quantification (Figure A‐C).…”

“…Taken further, our group found that relatively short‐term 2D cell culture for five serial passages results in loss of cell‐specific identity markers and increased homogeneity in a CIC subpopulation enriched for tyrosine‐protein kinase kit or CD117 (c‐Kit + ) cardiac interstitial cell (cCIC) compared with correspondingly selected freshly isolated cells by single‐cell RNA‐Seq transcriptional profiling . Findings such as these support the contention that CIC isolation and propagation conditions exert profound influences upon biological and functional properties, consistent with our recent reports of hypoxic culture conditions antagonizing mitochondrial dysfunction and senescence in human cCICs as well as tetraploid conversion of murine cCICs . Surprisingly, despite irrefutable evidence of alterations following in vitro expansion of primary CIC isolates, there are essentially no studies to document the extent of such changes as permanent or transient and whether CICs undergo another round of phenotypic and functional adaptation following reintroduction to their native environment of in vivo myocardium.…”

“…Even for the extensively characterized cCIC subpopulation, phenotypic properties and changes experienced by culture expanded cells upon reintroduction to a myocardial environment remain largely unknown. Heightened awareness of profound biological changes exerted by limited ex vivo culture expansion upon cCICs including transcriptional reprogramming and ploidy alteration emphasized the need to evaluate responsiveness of cCICs to myocardial exposure.…”

“…Similarly, chimeric placental tissue forms following injection of tetraploid hybrid cells into blastocysts . Cultured murine cCICs acquire tetraploid DNA content with serial passaging and override cellular senescence . Indeed, the tetraploid nature of cCICs (Figure ) likely accounts for the mechanism behind engraftment into TE and amniotic membrane integration (Figure and Figure F‐G), since embryo chimerism by blastocyst injection requires karyotypic normalcy of donor stem cells .…”

Ex uno, plures–From One Tissue to Many Cells: A Review of Single-Cell Transcriptomics in Cardiovascular Biology

“…Both studies combined bulk transcriptomic data on laser capture microdissected samples, with single-cell Taqman ® sc-qPCR through Fluidigm Dynamic Arrays, to show the beneficial factors secreted by the cells of interest, post-injection in the infarcted hearts. More recently, three studies have focused on the controversial population of c-kit + progenitor cells [ 83 , 84 , 85 ]. In all cases, scRNAseq analysis showed that freshly isolated c-kit + cells are heterogeneous and include cells with mesenchymal or endothelial features; thus, they are unlikely to differentiate in CMs.…”