Rationale

Autologous stem cell therapy using human c-Kit⁺ cardiac progenitor cells (hCPCs) is a promising therapeutic approach for treatment of heart failure (HF). However, hCPCs derived from aged patients with HF with genetic predispositions and comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impair overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with proregenerative molecules ex vivo is crucial for improving the therapeutic outcome in patients with HF.

Objective

To improve hCPC proliferation and migration responses that are critical for regeneration by targeting proregenerative P2Y₂ nucleotide receptor (P2Y₂R) activated by extracellular ATP and UTP molecules released following injury/stress.

Methods and results

c-Kit⁺ hCPCs were isolated from cardiac tissue of patients with HF undergoing left ventricular assist device implantation surgery. Correlations between P2 nucleotide receptor expression and hCPC growth kinetics revealed downregulation of select P2 receptors, including P2Y₂R, in slow-growing hCPCs compared with fast growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y₂R. Mechanistically, P2Y₂R-induced proliferation and migration were dependent on activation of YAP (yes-associated protein)-the downstream effector of Hippo signaling pathway.

Conclusions

Proliferation and migration of functionally impaired hCPCs are enhanced by P2Y₂R-mediated YAP activation, revealing a novel link between extracellular nucleotides released during injury/stress and Hippo signaling-a central regulator of cardiac regeneration. Functional correlations exist between hCPC phenotypic properties and P2 purinergic receptor expression. Lack of P2Y₂R and other crucial purinergic stress detectors could compromise hCPC responsiveness to presence of extracellular stress signals. These findings set the stage for subsequent studies to assess purinergic signaling modulation as a potential strategy to improve therapeutic outcome for use of hCPCs in patients with HF.

Senescence-associated dysfunction deleteriously affects biological activities of human c-Kit⁺ cardiac progenitor cells (hCPCs), particularly under conditions of in vitro culture. In comparison, preservation of self-renewal and decreases in mitochondrial reactive oxygen species (ROS) are characteristics of murine CPCs in vivo that reside within hypoxic niches. Recapitulating hypoxic niche oxygen tension conditions of ∼1% O₂ in vitro for expansion of hCPCs rather than typical normoxic cell culture conditions (21% O₂ ) could provide significant improvement of functional and biological activities of hCPCs. hCPCs were isolated and expanded under permanent hypoxic (hCPC-1%) or normoxic (hCPC-21%) conditions from left ventricular tissue explants collected during left ventricular assist device implantation. hCPC-1% exhibit increased self-renewal and suppression of senescence characteristics relative to hCPC-21%. Oxidative stress contributed to higher susceptibility to apoptosis, as well as decreased mitochondrial function in hCPC-21%. Hypoxia prevented accumulation of dysfunctional mitochondria, supporting higher oxygen consumption rates and mitochondrial membrane potential. Mitochondrial ROS was an upstream mediator of senescence since treatment of hCPC-1% with mitochondrial inhibitor antimycin A recapitulated mitochondrial dysfunction and senescence observed in hCPC-21%. NAD⁺ /NADH ratio and autophagic flux, which are key factors for mitochondrial function, were higher in hCPC-1%, but hCPC-21% were highly dependent on BNIP3/NIX-mediated mitophagy to maintain mitochondrial function. Overall, results demonstrate that supraphysiological oxygen tension during in vitro expansion initiates a downward spiral of oxidative stress, mitochondrial dysfunction, and cellular energy imbalance culminating in early proliferation arrest of hCPCs. Senescence is inhibited by preventing ROS through hypoxic culture of hCPCs. Stem Cells 2019;37:555-567.

Rationale

The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration.

Objective

Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy.

Methods and results

Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm³ pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit⁺ cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit^- mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit⁺ population is further enriched by selection for a CD133⁺ endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry.

Conclusions

Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients.

Rationale

Biological significance of c-Kit as a cardiac stem cell marker and role(s) of c-Kit+ cells in myocardial development or response to pathological injury remain unresolved because of varied and discrepant findings. Alternative experimental models are required to contextualize and reconcile discordant published observations of cardiac c-Kit myocardial biology and provide meaningful insights regarding clinical relevance of c-Kit signaling for translational cell therapy.

Objective

The main objectives of this study are as follows: demonstrating c-Kit myocardial biology through combined studies of both human and murine cardiac cells; advancing understanding of c-Kit myocardial biology through creation and characterization of a novel, inducible transgenic c-Kit reporter mouse model that overcomes limitations inherent to knock-in reporter models; and providing perspective to reconcile disparate viewpoints on c-Kit biology in the myocardium.

Methods and results

In vitro studies confirm a critical role for c-Kit signaling in both cardiomyocytes and cardiac stem cells. Activation of c-Kit receptor promotes cell survival and proliferation in stem cells and cardiomyocytes of either human or murine origin. For creation of the mouse model, the cloned mouse c-Kit promoter drives Histone2B-EGFP (enhanced green fluorescent protein; H2BEGFP) expression in a doxycycline-inducible transgenic reporter line. The combination of c-Kit transgenesis coupled to H2BEGFP readout provides sensitive, specific, inducible, and persistent tracking of c-Kit promoter activation. Tagging efficiency for EGFP+/c-Kit+ cells is similar between our transgenic versus a c-Kit knock-in mouse line, but frequency of c-Kit+ cells in cardiac tissue from the knock-in model is 55% lower than that from our transgenic line. The c-Kit transgenic reporter model reveals intimate association of c-Kit expression with adult myocardial biology. Both cardiac stem cells and a subpopulation of cardiomyocytes express c-Kit in uninjured adult heart, upregulating c-Kit expression in response to pathological stress.

Conclusions

c-Kit myocardial biology is more complex and varied than previously appreciated or documented, demonstrating validity in multiple points of coexisting yet heretofore seemingly irreconcilable published findings.

Cardiomyocyte ploidy has been described but remains obscure in cardiac interstitial cells. Ploidy of c-kit+ cardiac interstitial cells was assessed using confocal, karyotypic, and flow cytometric technique. Notable differences were found between rodent (rat, mouse) c-kit+ cardiac interstitial cells possessing mononuclear tetraploid (4n) content, compared to large mammals (human, swine) with mononuclear diploid (2n) content. In-situ analysis, confirmed with fresh isolates, revealed diploid content in human c-kit+ cardiac interstitial cells and a mixture of diploid and tetraploid content in mouse. Downregulation of the p53 signaling pathway provides evidence why rodent, but not human, c-kit+ cardiac interstitial cells escape replicative senescence. Single cell transcriptional profiling reveals distinctions between diploid versus tetraploid populations in mouse c-kit+ cardiac interstitial cells, alluding to functional divergences. Collectively, these data reveal notable species-specific biological differences in c-kit+ cardiac interstitial cells, which could account for challenges in extrapolation of myocardial from preclinical studies to clinical trials.