Enhancing the Potential of Cardiac Progenitor Cells: Pushing Forward with Pim-1

There is no cure for ischemic heart disease; therefore, limiting damage and salvaging viable myocardium are the current gold standard for patient care. In recent years, much interest has surrounded the idea of cell-based myocardial regeneration as a therapeutic alternative to conventional treatment regimens. However, success in human trials using a variety of stem cells or progenitor-like cells has been limited¹. This has left the field to ponder how this approach can be improved. It seems as though the ideal candidate cell type should effectively maintain the stem cell/progenitor population and efficiently differentiate into functional cardiomyocytes. Admittedly, this is no easy task. The reprogramming of somatic cells to iPS cells², or even directly to cardiomyocyte-like cells³, is remarkable and is expected to have lasting effects on stem cell biology and medicine in general. Yet this reprogramming approach to organ regeneration remains limited in its current state due, at least in part, to the 1:1 ratio between starting material and finished product.

It was reported nearly 10 years ago that a population of cells exists in the adult heart that harbors both the ability to self-renew and the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells⁴. This population was termed cardiac progenitor cells (CPCs) and remains a viable target for manipulation and myocardial regeneration. CPC treatment has been demonstrated to reduce infarct size and fibrosis⁵, improve left ventricular (LV) function^{6, 7}, attenuate LV dilation⁷ and increase coronary artery formation⁸ in experimental models. Although somewhat effective in their basal state, if CPCs could be engineered to further promote either their ability to self-renew or differentiate into a desired cell type, their regenerative capacity might be further enhanced. This then begs the question, what regulates these crucial characteristics of stem cells in vivo?

Asymmetric cell division is generally defined as the generation of distinctly destined daughter cells from a single mother cell and is a hallmark of stem and progenitor cells⁹. There are two described mechanisms by which this phenomenon occurs. Intrinsically mediated asymmetrical cell division occurs when the mother cell segregates cell fate determinants non-randomly⁹. This ensures that one daughter cell receives a disproportionate amount of “stem cell” factors compared to the other daughter cell, which is more likely to differentiate and lose its stemness. Niche-mediated asymmetrical cell division utilizes the surrounding extracellular environment, which is critical to maintaining the stem cell population, to “favor” one daughter cell over the other by placing it in close contact with the niche, thereby prolonging its stemness¹⁰.

Asymmetry can also occur at the DNA level through the non-random segregation of sister chromosomes¹¹. The mechanism by which this occurs, however, is still controversial^{12, 13}. One theory, known as the “Immortal Strand” hypothesis, was put forth by Cairns in 1975 and proposes that template DNA strands co-segregate (and are retained in stem cell populations) as a way to limit replication-associated DNA damage, thereby preserving DNA integrity for long durations and many cell divisions¹⁴. Findings in support of this hypothesis have been reported in various cell types by several independent groups^{15, 16}, yet there is still no empirical evidence linking this observation to mutation rates or cancer. An alternative theory is the “Silent Sister” hypothesis which proposes that asymmetric cell division and cell fate are codirected by epigenetic differences between sister chromatids¹³. It argues that distinct marks at centrosomal DNA and perhaps other regions direct non-random segregation of chromatids during mitosis. It also predicts differences in post-mitotic gene expression as a mechanism to influence cell fate. However, for both theories, the mechanism by which cells distinguish between sister chromatids remains to be established.

Pim-1 is a highly conserved serine/threonine kinase and an established downstream target of the cardioprotective kinase Akt that promotes survival in a variety of cell types including cardiomyocytes¹⁷. Pim-1 is constitutively active in its nascent state and is regulated through transcription, translation and degradation¹⁸. Pim-1 also regulates cell cycle progression and chromatin dynamics, thereby modulating proliferation¹⁸. In the heart, Pim-1 expression is increased in response to injury and protects against myocardial infarction (MI)^{17, 19}. Myocardial Pim-1 transgenic mice have increased levels of Bcl-2, Bcl-xL and p-Bad, healthier mitochondria and are protected against cardiac injury²⁰.

Pim-1 expression also regulates CPCs. Increasing Pim-1 levels via lentiviral transduction were shown to promote proliferation, differentiation and survival of CPCs²¹. Following MI, Pim-1-transduced CPCs caused reduced infarct, stimulated increased differentiation into cardiomyocytes and increased neovascularization²². The ability of Pim-1 to promote increased CPC self-renewal and differentiation, while avoiding unwanted off-target effects resulting from gene manipulation, makes it an extremely attractive target for optimizing cell-based therapies for heart disease.

The current study by Sundararaman et al.²³ provides mechanistic insight into how Pim-1 can accomplish these beneficial effects in CPCs by focusing on asymmetric chromosome segregation (ACS). First, the authors are able to quantify the rate of ACS in CPCs cultured from adult mouse hearts. Second, they demonstrate that CPCs transduced with Pim-1 are nearly twice (4.95% vs. 9.19%) as likely to undergo ACS compared to eGFP-transduced CPC controls. As a corollary, if increased Pim-1 expression promotes ACS, does depletion of Pim-1 reduce it, i.e., is endogenous Pim-1 required for ACS? This pertinent question remains unanswered.

The observation that less than 5% of the CPC population practices ACS raises the question of heterogeneity among CPCs and stem cells in general. If differences exist between subpopulations of CPCs, how then can we identify and isolate those subpopulations better suited for regeneration? Perhaps novel markers, in addition to those already employed, will help to identify progenitors with an increased propensity for proliferation, self-renewal and differentiation. Recent work has demonstrated that CPCs positive for the IGF-1 receptor have increased regenerative capacity, suggesting that subpopulations of CPCs can be isolated and may prove to be more effective for cell-based therapy²⁴.

Two complimentary methods are used by Sundararaman et al. to quantify ACS in CPCs: the label release assay and the label retention assay. For the label release assay, cells are mitotically synchronized and incubated (pulsed) with BrdU for a short period (6hrs, one expected mitotic event) so that the BrdU label is uniformly distributed/incorporated. Cells are then chased in medium lacking BrdU, arrested during cytokinesis, and BrdU intensity is measured in the binucleated cells. If ACS has occurred, then one daughter cell should contain the majority (>70%) of BrdU signal compared to the sister. An absolute ratio (100:0) is not used, as previous work has demonstrated that not all chromosomes segregate nonrandomly²⁵.

The label retention assay also requires synchronization of cells prior to incubation with BrdU label; however, cells are grown in BrdU medium for an extended period (18-25 days) to ensure that all DNA is labeled in all cells. CPCs are then chased in BrdU-free medium for two rounds of mitosis. If ACS occurs then the stem/progenitor cell population will retain the BrdU label (70:30). Conversely, symmetric chromosome segregation will give rise to an evenly diluted BrdU signal intensity. While this method allows for the effective identification and quantification (via signal intensity) of asymmetric versus symmetric chromosome segregation, it is not without limitations. Importantly, the fixation and labeling requirements interfere with the ability to study the functional significance of ACS in living cells.

Insulin-like growth factor 1 (IGF-1) has been used to improve the efficacy of cell-based therapies for treatment of myocardial ischemia. Delivery of CPCs in the presence of IGF-1 was shown to increase myocardial regeneration and improve outcomes²⁶. Mechanistically, IGF-1 has been shown to increase survival, selfrenewal and proliferation of pluripotent cell types²⁷. It can also promote the differentiation of progenitor cells to cardiomyocytes, smooth muscle cells and endothelial cells^{28, 29}. The multifaceted benefits of IGF-1 are rather unique and make it an attractive candidate for improving cell-based therapy. IGF-1 is a potent activator of Akt and has been shown to increase Akt localization in the nucleus. Since nuclear Akt can activate Pim-1 to elicit cardioprotection¹⁷, it is possible that IGF-1-mediated activation of Akt causes increased Pim-1 signaling in CPCs and promotes ACS.

The manuscript by Sundararaman et al. provides exciting new insight into the dynamics of chromosome segregation and the ability of Pim-1 to enhance ACS in CPCs. It also gives rise to many still unanswered questions. It will be extremely interesting to next determine the functional significance of this asymmetrical enhancement. Does this translate to a larger pool of stem cell-like progenitors? Will this, in turn, lead to more differentiated cardiomyocytes? Does ACS serve to limit mutagenesis of DNA in stem and progenitor cells, thereby prolonging their stemness and delaying aging? Similarly, do CPCs undergoing ACS have an increased regenerative potential versus symmetrical CPCs, and, if so, could this difference be exploited therapeutically (see Figure)?

Figure.

A schematic summary of the current findings by Sundararaman et al. Briefly, increased Pim-1 expression in CPCs promotes asymmetrical chromosome segregation, which may explain why Pim-1 can enhance CPC proliferation, selfrenewal and differentiation.

Recent work continues to highlight the attractiveness of Pim-1 as a target to enhance the efficacy of CPCs and perhaps the regenerative capacity of the injured myocardium. What, then, is the most promising approach to translating these findings into something more therapeutically relevant? The current manuscript provides ample evidence that Pim-1 should be pursued and warrants further investigation into the mechanism responsible for the enhanced proliferation, differentiation and self-renewal of CPCs elicited by this cardioprotective kinase. Specifically, what are the downstream targets necessary to mediate these beneficial outcomes? Is it possible to directly stimulate Pim-1 expression? Could Pim-1 therapy be combined with additional interventions, such as IGF-1, to further enhance the regenerative potential of CPCs? It is certainly possible to imagine a course of treatment involving 1) the isolation of CPCs from a patient, 2) providing these CPCs with a “regenerative boost” through modulation of Pim-1 or other additional factors, and 3) the subsequent re-administration of these CPCs to the injured myocardium. A more complete understanding of the mechanism underlying the benefits of enhanced ACS, the identification of novel markers of CPCs with increased regenerative capacity, and more effective methods of isolating these true stem-like progenitor cell populations should further improve cell-based therapy for ischemic heart disease.