SUMMARY Colony-forming units – fibroblast (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), are present in many organs, although the relationship between BM and organ-specific CFU-Fs in homeostasis and tissue repair is unknown. Here we describe a population of adult cardiac-resident CFU-Fs (cCFU-Fs) that occupy a perivascular, adventitial niche and show broad trans-germ layer potency in vitro and in vivo. CRE lineage tracing and embryo analysis demonstrated a proepicardial origin for cCFU-Fs. Furthermore, in BM transplantation chimeras, we found no interchange between BM and cCFU-Fs after aging, myocardial infarction, or BM stem cell mobilization. BM and cardiac and aortic CFU-Fs had distinct CRE lineage signatures, indicating that they arise from different progenitor beds during development. These diverse origins for CFU-Fs suggest an underlying basis for differentiation biases seen in different CFU-F populations, and could also influence their capacity for participating in tissue repair.

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Myocardial microenvironment plays a decisive role in guiding the function and fate of cardiomyocytes, and engineering this extracellular niche holds great promise for cardiac tissue regeneration. Platforms utilizing hybrid hydrogels containing various types of conductive nanoparticles have been a critical tool for constructing engineered cardiac tissues with outstanding mechanical integrity and improved electrophysiological properties. However, there has been no attempt to directly compare the efficacy of these hybrid hydrogels and decipher the mechanisms behind how these platforms differentially regulate cardiomyocyte behavior. Here, we employed gelatin methacryloyl (GelMA) hydrogels containing three different types of carbon-based nanoparticles: carbon nanotubes (CNTs), graphene oxide (GO), and reduced GO (rGO), to investigate the influence of these hybrid scaffolds on the structural organization and functionality of cardiomyocytes. Using immunofluorescent staining for assessing cellular organization and proliferation, we showed that electrically conductive scaffolds (CNT- and rGO-GelMA compared to relatively nonconductive GO-GelMA) played a significant role in promoting desirable morphology of cardiomyocytes and elevated the expression of functional cardiac markers, while maintaining their viability. Electrophysiological analysis revealed that these engineered cardiac tissues showed distinct cardiomyocyte phenotypes and different levels of maturity based on the substrate (CNT-GelMA: ventricular-like, GO-GelMA: atrial-like, and rGO-GelMA: ventricular/atrial mixed phenotypes). Through analysis of gene-expression patterns, we uncovered that the engineered cardiac tissues matured on CNT-GelMA and native cardiac tissues showed comparable expression levels of maturation markers. Furthermore, we demonstrated that engineered cardiac tissues matured on CNT-GelMA have increased functionality through integrin-mediated mechanotransduction (via YAP/TAZ) in contrast to cardiomyocytes cultured on rGO-GelMA.

Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.