Rationale: Fibrosis is mediated partly by extracellular matrix–depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. Objective: We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. Methods and Results: We showed that Thy1 + CD45 − CD31 − CD11b − Ter119 − cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. Conclusions: The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

The mammalian heart has long been considered a postmitotic organ, implying that the total number of cardiomyocytes is set at birth. Analysis of cell division in the mammalian heart is complicated by cardiomyocyte binucleation shortly after birth, which makes it challenging to interpret traditional assays of cell turnover [Laflamme MA, Murray CE (2011) Nature 473(7347):326-335; Bergmann O, et al. (2009) Science 324(5923):98-102]. An elegant multi-isotope imaging-mass spectrometry technique recently calculated the low, discrete rate of cardiomyocyte generation in mice [Senyo SE, et al. (2013) Nature 493(7432):433-436], yet our cellularlevel understanding of postnatal cardiomyogenesis remains limited. Herein, we provide a new line of evidence for the differentiated α-myosin heavy chain-expressing cardiomyocyte as the cell of origin of postnatal cardiomyogenesis using the "mosaic analysis with double markers" mouse model. We show limited, life-long, symmetric division of cardiomyocytes as a rare event that is evident in utero but significantly diminishes after the first month of life in mice; daughter cardiomyocytes divide very seldom, which this study is the first to demonstrate, to our knowledge. Furthermore, ligation of the left anterior descending coronary artery, which causes a myocardial infarction in the mosaic analysis with double-marker mice, did not increase the rate of cardiomyocyte division above the basal level for up to 4 wk after the injury. The clonal analysis described here provides direct evidence of postnatal mammalian cardiomyogenesis.heart development | cardiovascular progenitors | aging | regeneration A lthough it was widely believed that the adult heart is a quiescent organ, in the past several years reports have argued in favor of the generation of new cardiomyocytes in the mouse and human hearts after birth. The strongest evidence to first incontrovertibly demonstrate this phenomenon "date-stamped" autopsied human hearts by correlating levels of 14 C in cardiomyocyte nuclei with atmospheric 14 C levels in different years, and revealed that a small percentage of cardiomyocytes is born during adulthood (1). Despite this significant finding, which indirectly correlated nuclear division with cell division, the parent cell of postnatal cardiomyogenesis, as well as the extent of division in the postnatal mammalian hearts, remains vigorously debated. Moreover, the effect of injury on the endogenous rate of mammalian cardiomyocyte generation is unresolved (2-5).After resection of the ventricular apex, both adult zebrafish and neonatal mice exhibit robust regeneration, which fate-mapping studies suggest occurs through a cardiomyocyte intermediate (6)(7)(8). However, the study of cardiomyocyte generation by division postnatally has been controversial (9, 10) in the mammalian heart because it often relies on indirect assays of cell division, which are challenging to interpret in the setting of cardiomyocyte polyploidy (11, 12) as well as potential DNA repair upon injury. Recently, it was shown using a ...

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 1864(1), the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival(2). In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2 + / CD13 + /KDR + /PDGFRα + cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2 + cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2 + /CD13 + /KDR + /PDGFRα + cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart. engraftment | surface markers | Stem cells | mature cardiomyocytes | clonal analysis