Clonality of mouse and human cardiomyogenesis in vivo

Abstract

An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1-5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2-4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.F ate mapping protocols establish a lineage relationship between ancestors carrying the reporter gene and their descendents (1, 2), but do not provide information on the self-renewing property and clonogenicity of progenitor cells or clonal origin of daughter cells in vivo (3). Because of these limitations, viral gene-tagging remains the most accurate strategy for the analysis of stem cell growth (3-8). The semi-random insertion of retroviral and lentiviral vectors represents an effective tool for genetic marking, enabling the identification of the progeny generated by stem cell differentiation. Retroviruses and lentiviruses integrate permanently in the genome of the host cells; the insertion site of the viral genome is inherited by the population derived from the parental cell (6) and can be amplified by PCR. Thus, the detection of the sites of integration constitutes a unique approach for the documentation of self-renewal, clonogenicity, and multipotentiality of stem cells in vivo. So far, this methodology has been applied to the bone marrow (4-6) and the brain (3, 7, 8) and has not been used to characterize the mechanisms regulating cardiac homeostasis and pathology.The implementation of this technique in the adult heart is relevant for the incontrovertible demonstration of resident cardiac stem cells and the ability of the myocardium to undergo spontaneous regeneration. Moreover, the notion that cardiomyocytes have a long lifespan and ...

Regenerating new heart with stem cells

“…Among the different progenitor cell subsets, the c-kit-positive CSC has been well characterized in the mouse (42), rat (15), dog (16), and human (17,21) heart. The expression of the stem cell antigen c-kit is associated with a pool of undifferentiated cells that have essentially identical properties in vitro and in vivo and are indistinguishable among species.…”

Comparative study of immune regulatory properties of stem cells derived from different tissues

“…In other words, our data suggest that the immune modulatory properties of CSCs and LSCs here described in vitro for the first time may have a role in vivo in favoring their regenerative potential. Secondly, both CSCs and LSCs become structurally and functionally coupled with the cells of the recipient organ, creating new niches in which the SCs can divide asymmetrically [73,75,76], thus ensuring the preservation of the SC pool and the formation of parenchymal and vascular cells [50,53,77]. It is likely that the potential clinical implementation of these SC populations is significantly strengthened by their immunomodulatory function.…”

The effects of transplanted cells in stem cell therapy for myocardial ischemia

“…The discovery and recognition of the existence of cardiac stem cells led to a shift by researchers and physicians to explore the use of stem cells for new cardiovascular disease therapies (Bearzi et al, 2009;Hosoda et al, 2009;Urbanek et al, 2006). In the heart, cardiac stem cells (CSCs) have existed in their niches.…”

“…These factors induce interstitial CSCs to move through the myocardium to necrotic myocardium and scar areas. There, they divide and differentiate into heart cells and become involved in the process of new blood vessel formation (Bian et al, 2014;Hosoda et al, 2009;Tillmanns et al, 2008). CSC invasion to scar tissue is believed to be related to matrix metalloproteinase (MMP)-9 and -14 mediated regulation (Bax et al, 2012;Huang et al, 2011;Rota et al, 2008).…”