In this study, we have shown that: 1) there is a significant increase in the proliferative capacity of CS-forming cells isolated from the “middle aged” heart following acute MI resulting in a significant rise in the number of CSs in vitro; 2) this increase is time-dependent and is most pronounced within the first week post-MI in the animal model studied; 3) transplanted CSs from infarcted myocardium engraft in ischemic myocardium, improve cardiac function, and promote endogenous angiogenesis; 4) adult heart contains Isl1+ cells and Isl1 expression in CSs is 17-fold higher than in total adult cardiac tissue; 5) Isl1 expression in the Sca-1+CD45− subpopulation within CSs is 3-fold higher than in total CSs; 6) Sca-1+CD45− cells in CSs can be cloned, expanded, and have the characteristics of multipotent cardiac progenitor cells in vitro; and 7) after injection into ischemic myocardium, cloned Sca-1+CD45− cells not only survive long-term, but also differentiate into endothelial and smooth muscle cells, promote endogenous angiogenesis, reduce cardiomyocyte apoptosis, reduce infarct size, and improve cardiac function. Of special note, these experiments have been performed in middle-aged mice, rather than in young adults, to simulate a more clinically relevant disease model.
Previous studies 
have shown that the number of Sca-1+
cardiac progenitor cells increases post-MI. Our data agrees with these reports and further shows that the potential for CSs generation in vitro
is highly time dependent post-MI. The number of CSs from 1- and 2-week post-MI hearts greatly increases compared to uninjured hearts, and this increase is attenuated by 4 weeks post-MI. This suggests that acute MI induces the proliferation of cardiac progenitor cells, and this increase in proliferation gradually dissipates over a 4-week period post-MI. Therefore early acquisition of tissue from post-MI hearts would facilitate higher yields of CSs in vitro
. However, the optimal timing in humans may be different than the ones reported in our study and future research is necessary to better define the ideal timing of tissue acquisition in patients post-MI.
A previous report 
showed that stem cell niches distribute preferentially to the apex and atria of the heart, where the wall stress is relatively low. MI may affect their distribution. Our results show that various regions of 1-week post-MI hearts have similar abilities to produce CSs. This suggests that although MI mainly affects the LV, the CS-forming cells throughout the heart are activated post-MI. As such, taking tissue from any region of the heart appears to yield similar numbers of CSs per unit of tissue. The mechanism by which MI increases CSs production is worthy of further investigation. Importantly, the septum and right ventricle yield the same numbers of CSs. Thus, percutaneous right ventricular endomyocardial biopsy, as is performed routinely for other clinical indications, could potentially be used to generate CSs in the post-MI setting.
It has been previously reported that CSs from non-infarcted hearts could differentiate into cardiac cells and preserve cardiac function
. Whether CSs derived from early stage post-MI hearts have the same abilities has not been demonstrated until now. Our results show for the first time that the CS cells obtained from 1-week post-MI hearts engraft in ischemic myocardium and restore cardiac function at 25 days post-injection in vivo
. However, we did not find evidence for differentiation of these cells into mature cardiomyocytes or new vessels. Our data demonstrate that the injected CSs promoted angiogenesis in vivo
, suggesting that engrafted CSs cells likely have a paracrine, pro-angiogenic effect in the ischemic myocardium. Secreted VEGF from engrafted CSs may contribute to angiogenesis in the infarcted hearts 
. This paracrine effect may play an important role in attenuating adverse LV remodeling and preserving cardiac function.
Recent reports have found Isl1+
cells in adult mouse, rat and human hearts 
. Our results not only confirm the presence of Isl1+
cells in adult murine hearts, but we show that these can be efficiently isolated and expanded by culturing Sca-1+
cells from CSs. Since Isl1 is not expressed on the cell surface, it has been difficult to isolate and purify these cells by immune selection. However, we now demonstrate that isolating Sca-1+
cells from CSs results in an enriched population of Isl1+
cardiac progenitors for autologous cardiac cell-therapy.
While cloned Sca-1+
cells improved cardiac function post-MI in transplanted mice, we did not find evidence for differentiation of these cells into cardiomyocytes in vivo
. One possible explanation is that the cloned Sca-1+
cells may need a longer time to differentiate into cardiomyocytes in situ
. This is supported by the observation that there was no differentiation seen from cloned cells in the first 25 days, while endothelial and smooth muscle cell differentiation occurred only after 75 days. Another possible explanation is that there may be subpopulations of Sca-1+
CS cells with distinct differentiation capacities. Whether resident cardiac progenitor cells in adult hearts are capable of cardiomyogenic differentiation in vivo
remains controversial 
. The therapeutic effects of CS-derived Sca-1+
cells in vivo
do suggest, however, that these cells might be responsible for the overall effects of CSs. Since the Sca-1+
cells can be clonally expanded in vitro
, they provide a feasible approach to rapidly generating therapeutic quantities of cardiac progenitor cells.
A recent report has suggested that CSs are composed of fibroblasts, and not cardiac progenitor cells 
. Although many fibroblasts grow out from the cardiac explant during the first stage of culture, we demonstrate here that our CSs contain cardiac progenitor cells that are capable of clonal expansion and multi-lineage cardiac differentiation. Furthermore, our demonstration that cloned Sca-1+
cells have a beneficial therapeutic effect, similar to heterogeneous CSs, argues strongly against the hypothesis that fibroblasts are the major contributors to cardiac repair in CSs.
There are several limitations to this study. First, under the experimental conditions used, we did not find evidence for differentiation of cloned Sca-1+
cells into cardiomyocytes in vivo
. It is possible that a longer follow-up period might be required for this differentiation to be observed in vivo
. Despite this, we have demonstrated that these cells do indeed have “progenitor” characteristics given their ability to differentiate into other cell types both in vivo
and in vitro
. Second, we show increased angiogenesis and reduced cardiomyocyte apoptosis after injection of cloned Sca-1+
cells. However, given our experimental conditions, we are unable to address whether the injected cells recruit and influence endogenous cardiac progenitors in vivo
. Recently, using a genetic lineage mapping approach, Loffredo et al 
have reported new cardiomyocyte formation in infarcted hearts derived from endogenous cardiac stem cells 8 weeks after injecting with murine bone marrow c-kit+
cells. There are several possible explanations for the consistent improvements in ventricular function including: reduction in cardiomyocyte apoptosis 
, prevention of infarct scar expansion by mechanically stiffening the infarct zone, facilitation of hypertrophy of the border zone cardiomyocytes by enhanced angiogenesis 
. Other groups have reported stimulation of the endogenous adult cardiomyocytes to re-enter cell cycle and divide 
and recruitment and/or activation of resident cardiac progenitors 
as possible additional mechanisms. Each of these mechanisms has been implicated in improved cardiac function seen with cell therapy. However, the exact contribution of each of these mechanisms to the overall benefit of therapy remains the focus of intense research.
In summary, our data suggest that the cloned Sca-1+CD45− cells derived from CSs from post-MI hearts are enriched in Isl1+ progenitors, have the characteristics of progenitor cells, and are an attractive source of autologous cells for myocardial therapy post-MI.