Survival and retention of c-kit+ CSCs following transplantation
A number of studies have reported that stem cells of various sources suffer low viability, and only few persist several weeks following transplantation into the injured myocardium [
60‐
67]. Similarly, one of the problems with the current therapy with c-kit+ CSCs is poor survival and retention of the injected cells in the heart [
41•,
42,
60]. It is thought that myocardial ischemia/reperfusion and infarction create a hostile environment for stem cells, because of the presence of inflammatory cells and cytokines/mediators, lack of extracellular matrix and supporting cells, and poor supply of oxygen and nutrients, all of which conspire to promote death of the grafted cells [
68,
69]. For this reason, increasing survival and retention of the transplanted CSCs in the heart currently constitutes one of the major challenges in the field of CSC therapy.
Troubleshooting this issue would require close and accurate monitoring of the number, distribution, and fate of the transplanted donor cells, and correlation of these variables with changes in functional parameters. Our research group has recently developed a novel quantitative PCR (qPCR)-based method to quantify the
absolute numbers of male mouse CSCs remaining in the female recipient heart at a given time point [
60]. The method involves adding cells of another genotype (e.g., human cells) to each tissue sample as an internal standard prior to genomic DNA isolation. Since the ratio of the internal standard DNA (i.e., human DNA) to the male mouse donor DNA should remain constant regardless of the efficiency of DNA isolation, the amount of human DNA in the sample can be used to calculate the total, original amount of male donor DNA present in the entire tissue. The amount of DNA of a particular genotype is acquired by qPCR and converted to the number of cells by dividing it by the amount of genomic DNA present per cell (e.g., 6.26 pg DNA per diploid human cell). Another unique feature of the assay is that it targets a novel, male-specific, multiple-copy gene named
Rbmy [
70,
71], which increases the sensitivity of detection of male DNA by several folds over a traditional male marker,
Sry [
60].
In our previous study [
60], we measured the absolute numbers of male CSCs remaining in the recipient heart following intramyocardial or intracoronary delivery into the female mouse heart. When intramyocardial injection of 10
5 CSCs were made (at 2 days after ischemia-reperfusion), only 43 % of the injected cells were found at 5 min after injection. This immediate and massive cell loss is likely due to leakage of cells through transepicardial puncture holes. Greater than 75 % of CSCs present at 5 min were lost in the ensuing 24 h, and only 7.6 % of the CSCs present at 5 min were found at 7 days. By 35 days, only 2.8 % of the cells present at 5 min (i.e., 1,224 ± 257 cells/heart; 1.2 ± 0.2 % of total cells injected) were detected in the recipient heart. Following intracoronary infusion of the same number of CSCs (i.e., 10
5), > 60 % of the injected CSCs were already lost by 5 min, suggesting that the majority of cells failed to be retained and were washed off almost immediately due to the coronary blood flow. Moreover, more than 85 % of the cells at 5 min were lost in the next 24 h. Only 3.5 % and 2.4 % (i.e., 987 ± 211 cells/heart; 1.0 ± 0.2 % of total cells injected) of the cells present at 5 min were found at 7 days and 35 days, respectively. In addition to the heart, presence of the intracoronarily delivered donor cells was detected in lungs and kidneys of the recipient mice, yet they were absent in livers and spleens (Hong et al., manuscript submitted). Although the number of donor cells in the heart declined slightly faster following intracoronary infusion (versus intramyocardial injection), the percentage of transplanted cells remaining after 35 days was similar between the two different delivery methods, reaching approximately 1 % (i.e., ~1,000 cells out of 100,000). These quantitative studies clearly demonstrate that the transplanted CSCs suffer low survival and/or retention in the recipient heart, regardless of the delivery methods, and strongly support the notion that the poor survival and/or retention of the donor CSCs is indeed a major factor limiting the efficacy of the current CSC therapy.
Several studies have tested different strategies to overcome the problem of poor survival or retention of the cells in the hostile environment of the infarcted heart. For example, Mohsin and colleagues tested the effect of ex vivo gene delivery of a pro-survival gene, Pim-1 kinase on survival/engraftment and reparative potential of human CSCs using a mouse model of ischemic cardiomyopathy [
46]. The same group had previously identified Pim-1 as a kinase responsible for cardioprotection downstream of pro-survival Akt signaling [
72,
73]. Human CSCs engineered to overexpress Pim-1 were superior over the control cells in terms of cellular engraftment and differentiation. Bioluminescence imaging analysis of luciferase-expressing donor cells [
74] showed that the presence of Pim-1 CSCs persisted up to 8 weeks post-transplantation, whereas control cells became undetectable by 1 week. Moreover, the persistence of cells in the recipient heart was accompanied by improvement in vasculature and reduction in infarct size, which were coupled with increased hemodynamic performance at 20 weeks post-transplantation [
46]. Interestingly, a subsequent report by the same group showed that the increases in proliferation and telomere lengths observed in Pim-1-expressing CSCs are only transient, suggesting that Pim-1 overexpression does not lead to immortalization or oncogenic transformation of CSCs [
75]. The results are promising and indicate that ex vivo delivery of a pro-survival gene prior to transplantation may serve a viable option in overcoming current limitations in the field.
Such strategy also has proven fruitful in terms of promoting survival and therapeutic efficacy of other stem cell sources, including MSCs [
61,
76,
77]. One of the first attempts to improve post-transplantation donor MSC survival was reported by Mangi and colleagues [
78]. They genetically engineered rat MSCs ex vivo using retroviral transduction to overexpress the pro-survival gene
Akt1 [
79,
80]. At 24 h following intramyocardial injection of MSCs into the rat MI heart, they observed a greater number of donor cells and a lower rate of apoptosis with MSCs overexpressing Akt (Akt-MSC) compared to the control GFP-expressing MSCs [
78]. Subsequently, transplantation of MSCs into the ischemic heart significantly reduced intramyocardial inflammation, fibrosis and myocyte hypertrophy, and normalized cardiac function. The study reported that Akt-MSCs restored fourfold greater myocardial volume compared to the control MSCs. Another study tested the utility of stably introducing heme oxygenase-1 (HO-1) expression in MSCs [
61]. HO-1 is known to exert a potent anti-apoptotic, anti-oxidant and cytoprotective activity in an ischemic environment [
81,
82]. When they injected HO-1 MSCs into acute MI heart, their survival was fivefold greater than that of the control MSC group (expressing lacZ) at 7 days of implantation, and this was consistent with a significantly reduced rate of apoptosis among HO-1 MSCs [
61]. Importantly, HO-1 MSCs were also superior over the control cells in attenuating LV remodeling and enhancing the functional recovery of injured hearts [
61]. Similarly, the pro-survival or cardio-protective effects of various candidate genes, including GATA4 [
76], heat shock protein 27 [
83], miRNA-1 [
84], and protein kinase G1α [
85], have been recently exploited to promote survival and efficacy of MSCs in the context of hypoxia-reoxygenation injury.
Pharmacologic activation of innate cytoprotective mechanisms seems to be another attractive option to enhance the survival and engraftment of CSCs. In a recent study, Cai et al. showed that treatment of human c-kit+ CSCs with cobalt protoporphyrin (CoPP), a well known HO-1 inducer [
86], promoted cell survival after increased oxidative stress in vitro [
87]. The cytoprotective effects of CoPP were dependent on the upregulation of HO-1, cyclooxygenase-2 (COX-2), and nuclear factor-like 2 (NRF2). Interestingly, preconditioning CSCs with CoPP also led to a global increase in release of a variety of cytokines, and the conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells also play a major role in the pro-survival effects of CoPP [
87]. Another study by Zafir and colleagues examined the role of protein O-GlcNAcylation (β-O-linkage of N-acetylglucosamine, O-GlcNAc), a universal cytoprotective pathway [
88] in the protection of mouse CSCs against hypoxia-reoxygenation injury [
89]. Protein O-GlcNAcylation was significantly elevated in post-hypoxic CSCs upon reoxygenation. Reduction in the O-GlcNAc signal via pharmacological inhibition sensitized CSCs to post-hypoxic injury, whereas increasing O-GlcNAc levels enhanced cell survival. The study has an important implication that pharmacologic augmentation of protein O-GlcNAc levels can be effective in priming CSCs for survival within the unfavorable environment of the infarcted myocardium. Likewise, preconditioning of cells prior to transplantation or co-treatment with different agents has been shown to be a successful strategy for reinforcing viability and therapeutic efficacy of the donor MSCs [
90‐
94]. To this end, our group has recently discovered that activation of c-kit-mediated signaling enhances viability of CSCs under stress conditions in vitro. The preliminary data indicate that activation of c-kit by its ligand, stem cell factor, not only prolongs the survival of c-kit+ CSCs under serum deprivation, but also stimulates their migration robustly in vitro (Vajravelu and Hong, unpublished observations). It would be of interest to see if activation of c-kit receptor on CSCs can enhance viability of the donor cells, as well as their ability to home into the infarcted myocardium.
Preconditioning or priming the stem cells with pharmacological agents prior to autologous transfer is likely to be clinically viable, for it is relatively easy to implement and does not involve direct genetic manipulation of the cells. Undoubtedly, identification of important cytoprotective mechanisms within CSCs and potential pro-survival candidate genes will be critical in developing novel strategies for augmenting post-transplantation survival and engraftment of donor CSCs.
Lack of robust, direct differentiation of transplanted CSCs
Although a variety of stem cell populations, including c-kit+ CSCs, have been shown to play regenerative and/or protective functions following transplantation in animal models of acute and chronic myocardial infarction, it has been widely accepted that therapeutic benefits of stem cell therapy are mainly contributed by paracrine factors secreted by the donor cells, rather than by direct differentiation of transplanted stem cells into cardiac cell types [
69,
95‐
98]. Consistent with this notion, our group has also observed that most of the transplanted c-kit+ CSCs do not give rise to mature cardiomyocytes, despite that a number of the transplanted cells begin to express markers of cardiomyocytes [
41•,
42]. Even after 35 days post-transplantation, nearly all of the cardiomyogenic progenies (based on α-sarcomeric actin expression) of the donor cells were small and failed to exhibit striations. The reason for this is not clear, but may be due to either alteration of the cellular properties during ex vivo expansion or lack of proper molecular and cellular cues at the site of transplantation. Regardless of the cause, there is a disconnection between functional benefits of CSC therapy and the lack of robust differentiation of transplanted cells. Based on this, one can argue that the contribution of direct cardiovascular differentiation of the donor stem cells to the salutary effects is negligible.
However, previous studies on transplantation of non-cardiac stem cells suggests that direct cardiac differentiation of the donor stem cells is essential for the functional benefits they provide. Yoon and colleagues investigated the contribution of cardiovascular differentiation of BM-derived mononuclear cells (BMMNCs) to the renewal of myocardium following acute MI by using a clever system in which the differentiated progenies of the donor cells can be selectively ablated in a temporally controlled manner [
99]. They engineered the donor cells to express a vector that encoded a prodrug-activated suicide gene (
herpes simplex virus thymidine kinase;
HSVtk) under the control of endothelium (eNOS)-, smooth muscle (SM22α)-, or cardiomyocyte (α-MHC)-specific promoters, which permitted selective depletion of the individual cardiac lineage acquired by the donor cells via administration of a prodrug, ganciclovir. When each donor cell-derived lineage was ablated two weeks after transplantation of the engineered BMMNCs, they found that depletion of endothelium-committed or smooth-muscle–committed cells, but not cardiomyocyte-committed cells, induced a significant decline in EF [
99]. This demonstrated that vascular differentiation is one of the essential mechanisms by which BMMNCs contribute to the functional recovery of the injured heart.
Another study by Behfar et al. also suggested that cardiogenic potential of the donor BM-derived MSC population dictates the functional outcome of stem cell therapy [
100•]. While the majority of patient-derived MSC failed to elicit significant improvements in EF, stem cells from few individuals harbored a spontaneous capacity to improve cardiac performance in an animal model of chronic ischemic cardiomyopathy. Interestingly, the stem cells exhibiting the reparative activity expressed relatively high basal levels of early (e.g., NKX-2.5, TBX5, and MESP1) and late (e.g., MEF2C) cardiac transcription factors, compared to the non-effective MSC counterparts. Based on this observation, the authors formulated a “cardiogenic cocktail” (containing TGF-β1, BMP-4, activin A, retinoic acid, IGF-1, FGF-2, α-thrombin, and IL-6) to stimulate cardiogenic potential/differentiation of naïve MSCs prior to transplantation. This strategy not only potentiated expression of cardiac transcription factors across different patient MSC populations, but also enhanced therapeutic efficacy of MSCs against chronic ischemic cardiomyopathy in mice [
100•]. The study provides an interesting implication that enhancing differentiation potential or properties of donor stem cells is feasible, and is an effective strategy to further improve the efficacy of stem cell therapy. Along the same lines, our research group has been exploring the idea of “priming” CSCs prior to transplantation to facilitate their differentiation. We have been experimenting with the approach of “forward reprogramming” human CSCs by introducing individual or combination of different cardiac transcription factors, including GATA4, NKX-2.5, MEF2C, TBX5, and BAF60C. Our results in vitro are promising that overexpression of GATA4 potentiates expression of marker genes associated with multiple cardiovascular lineages in human CSCs (Hong and Al-Maqtari et al., manuscript in revision). It remains to be seen whether or not such strategy can lead to robust and full differentiation of human CSCs following transplantation, and thereby enhance their regenerative activity in the infarcted heart.