Restoration of Cardiac Progenitor Cells After Myocardial Infarction by Self-Proliferation and Selective Homing of Bone Marrow–Derived Stem Cells
Tissue-specific progenitor cells contribute to local cellular regeneration and maintain organ function. Recently, we have determined that cardiac side-population (CSP) cells represent a distinct cardiac progenitor cell population, capable of in vitro differentiation into functional cardiomyocytes. The response of endogenous CSP to myocardial injury, however, and the cellular mechanisms that maintain this cardiac progenitor cell pool in vivo remain unknown. In this report we demonstrate that local progenitor cell proliferation maintains CSP under physiologic conditions, with little contribution from extracardiac stem cell sources. Following myocardial infarction in adult mice, however, CSP cells are acutely depleted, both within the infarct and noninfarct areas. CSP pools are subsequently reconstituted to baseline levels within 7 days after myocardial infarction, through both proliferation of resident CSP cells, as well as through homing of bone marrow–derived stem cells (BMC) to specific areas of myocardial injury and immunophenotypic conversion of BMC to adopt a CSP phenotype. We, therefore, conclude that following myocardial injury, cardiac progenitor cell populations are acutely depleted and are reconstituted to normal levels by both self-proliferation and selective homing of BMC. Understanding and enhancing such processes hold enormous potential for therapeutic myocardial regeneration.
Tissue-specific progenitor cell populations maintain the regenerative capacity of terminally differentiated organs, both under basal conditions and following local tissue injury. Side population (SP) cells, characterized by their intrinsic capacity to efflux Hoechst dye through ATP-binding cassette transporters, contribute to the long-term regenerative potential of hematopoietic and extrahematopoietic tissues.1 Recently, we have demonstrated that cardiac SP (CSP) cells, immunophenotypically distinct from bone marrow (BM)–derived stem cells (BMC), are present in the adult heart and are capable of both biochemical and functional cardiomyogenic differentiation into mature cardiomyocytes, thereby identifying CSP as a distinct cardiac progenitor cell population.2 Supplementation of cardiac progenitor cell pools after myocardial infarction (MI) with exogenous cells has been shown to improve ventricular function by regenerating myocardium and cardiac vasculature.3–5 The response of endogenous CSP cells to myocardial injury, however, and the cellular mechanisms that maintain this endogenous cardiac progenitor cell pool under basal and after injury conditions remain unknown. We, therefore, serially assessed CSP pools in hearts following MI and determined the role of self-proliferation and BMC in reconstituting cardiac progenitor pools.
Methods and Materials
CSP were isolated from mouse hearts as described previously.2 MI was performed in mice via permanent coronary ligation.6 BM transplantation was performed in lethally irradiated mice using marrow isolated from C57bl/6-Tg(ACTbEGFP) mice.7 All animals were obtained from The Jackson Laboratory (Bar Harbor, Me), and animal studies were conducted under approved guidelines of the Institutional Animal Care and Use Committee. An expanded Methods and Materials section is available in the online data supplement at http://circres.ahajournals.org.
Depletion of Cardiac SP Cells With Myocardial Injury
To determine the response of endogenous CSP to myocardial injury, adult mice were subjected to sham operation or coronary occlusion (MI). Fluorescence-activated cell sorting (FACS) analysis of cardiomyocyte-depleted cardiac isolates from sham-operated mice revealed the presence of Hoechst-extruding, verapamil-sensitive CSP cells (supplemental Figure I). CSP cells expressed Sca-1, varied in their CD31 expression, and lacked hematopoietic stem cell markers such as CD34, CD45, and c-kit, similar to what we have reported previously (data not shown).2 With sham operation, CSP were unchanged over time. In contrast, MI animals exhibited an acute 40% depletion of CSP within one day following injury (Figure 1A). Importantly, CSP cells were progressively restored to baseline levels within 7 days after MI.
To further determine the response of CSP cell populations to tissue injury, MI hearts were subdivided into the infarcted left-ventricular free wall (infarct) and noninfarcted septum (noninfarct). Importantly, in nonoperated and in sham-operated animals, regional CSP levels were similar in the left-ventricular free wall and septum (data not shown). After MI, within the infarct region, CSP pools were significantly depleted by more than 60% within 1 day (Figure 1B). As seen with total SP cell counts, CSP within the infarct region were restored to baseline, sham values within 7 days (Figure 1B). Interestingly, within the noninfarct region a decrease in CSP levels, though to a lesser degree than in the infarct zone, was also observed, despite the lack of direct tissue injury (Figure 1C). Similar to what was seen within infarct region, this CSP pool was also returned to baseline values within 7 days.
In Vivo Proliferation of Cardiac SP Cells After MI
The cellular mechanisms responsible for reconstituting CSP populations following myocardial injury remain unknown. Prior work from our laboratory has demonstrated that CSP are capable of self-renewal, in vitro.2 We, therefore, quantified CSP in S–G2M phase from sham and MI animals, using the cell cycling marker, Ki67, and the DNA binding dye, propidium iodide. Approximately 10% of CSP were found to be actively cycling in sham-operated animals (Figure 2A and 2B), with no regional variation, suggesting a basal level of CSP cell turnover. After MI, CSP within the infarct region underwent self-proliferation and reentered the cell cycle at day 3, as marked by a 2-fold increase in Ki67, and remained cycling at day 7 after MI (Figure 2A). Within the noninfarct myocardium (Figure 2B), CSP proliferation was delayed, with increased Ki67 staining starting at 7 days following injury. Similar kinetics and fractions of S–G2M CSP were obtained by propidium iodide staining (data not shown). Taken together, these results suggest that CSP proliferation maintains cardiac progenitor pools under physiologic conditions and may contribute, at least in part, to the renewal of CSP cells in vivo following cardiac injury.
BMC Mobilization Contributes to CSP Cells After MI
The role of BMC in maintaining CSP pools during basal conditions and following cardiac injury also remains unknown. Thus, we first determined whether BMC contribute to CSP homeostasis during physiological postnatal heart growth. Newborn mice underwent transplantation with green fluorescence protein (GFP)-expressing BM cells following lethal irradiation. At 12 weeks of age, despite successful BMC reconstitution, FACS analysis of digested hearts showed no significant contribution of GFP-positive BMC to the CSP pool (<1%), suggesting a limited role of BMC in CSP homeostasis during basal conditions or postnatal heart growth (data not shown). Using a similar transplantation model in adult mice, we also assessed the contribution of BMC to CSP pools after cardiac injury. In sham-operated and MI animals before acute injury (day 0), <1% of total CSP were GFP positive (GFP+) (Figure 3A and 3B), suggesting that BMC contribute little to the long-term maintenance of CSP populations under physiologic conditions. After MI, however, within the infarct region (Figure 3A), BM-derived CSP increased significantly, with a marked rise in GFP+ CSP at 3 days, reaching approximately 25% of total CSP cells at 7 days. In the noninfarct region, BM-derived CSP remained minimal (<5%) (Figure 3B). Corresponding to the marked rise in BM-derived CSP within the infarct region, an increase in circulating SP cells and a decline in BM SP cells (BMSP) were observed (supplemental Figure II). These findings, therefore, suggest that following MI, BMC are mobilized into the peripheral circulation and selectively home to areas of myocardial injury, where they contribute significantly to reconstitution of cardiac progenitor cell pools.
To further elucidate the immunophenotype of BM-derived CSP, the expression of CD31 and the hematopoietic marker, CD45, were determined in GFP+ CSP cells. Early after MI, more than 50% of GFP+ CSP cells within the infarct zone were CD45+, confirming the hematopoietic origin and BM phenotype of these cells (Figure 3C). With time following myocardial injury, despite the increase in BM-derived (GFP+) CSP, the percentage of cells expressing CD45 decreased significantly. Within 7 days after MI, more than 90% of BM-derived CSP were negative for CD45, confirming their immunophenotypic conversion from BMC to CSP cells. Furthermore, CD31 expression was low among BM-derived (GFP+) CSP early after MI but increased significantly within 7 days (supplemental Figure III).
The response of resident cardiac progenitor cell populations to myocardial injury and the cellular mechanisms that maintain cardiac progenitor pools remain unknown. In this report we demonstrate that local progenitor cell proliferation likely constitutes the major mechanism by which CSP levels are maintained long term under physiologic conditions, with little contribution from extracardiac stem cells. During periods of acute myocardial injury, resident CSP populations are acutely depleted in the injured as well as noninjured regions, likely secondary to both cellular necrosis and apoptosis. CSP cell proliferation and self-renewal alone are inadequate to rapidly reconstitute cardiac progenitor cell populations. Under such period of acute injury, cardiac progenitor cell pools are reconstituted to baseline levels by both proliferation of existing cells as well as by selective homing of BMC and immunophenotypic conversion to adopt a CSP phenotype.
Intriguing studies in humans8 and animal models,5,9 have demonstrated that BMC contribute to cardiomyogenesis in vivo after cardiac injury. In contrast, however, we have found that BMC exhibit limited intrinsic potential for cardiomyogenic differentiation, in vitro.2 This report suggests that BMC, and specifically BMSP, may be mobilized into the peripheral circulation with MI, home to areas of tissue injury, and undergo tissue-specific immunophenotypic conversion to replenish CSP cells. These data are further supported by recent reports suggesting mobilization of labeled BMSP to the heart following MI.9 Thus, BMC, and specifically BMSP, may represent a reserve pool capable of contributing to tissue-specific progenitor cells under conditions of acute injury.
Although the biological signals that orchestrate the homing of BMC and transition into cardiac progenitor cells remain to be determined, understanding and enhancing such processes hold enormous potential for therapeutic myocardial regeneration.
This work was supported by NIH grants HL71775, HL67297, and HL73756 (to R.L.). F.M. and O.P. received funding support from the Federation Francaise de Cardiologie and Swiss National Science Foundation, respectively.
↵*Both authors contributed equally to this work.
Original received September 29, 2005; revision received October 18, 2005; accepted October 24, 2005.
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