Of Mice and Men: Cell Therapy for the Aging Heart

Background.

Heart failure (HF) remains a major cause of mortality and morbidity¹ accounting for one in three deaths. Nearly 800,000 Americans experience a myocardial infarction (MI) each year. While acute interventions including angioplasty have saved many of these lives, about half of those experiencing an MI develop HF within 5 years. This highlights the urgent need for novel therapeutic approaches to repair the injured heart muscle, improve cardiac function, quality of life and long-term survival.

Aging and Heart Function.

Age is a major risk factor for developing HF.² Other chronic, age related co-morbidities including obesity, diabetes and chronic kidney disease are contributors to HF progression¹. Therefore, understanding the potential of novel therapies for heart failure in the aging population remains an unmet socio-economic challenge. At the functional level, studies have demonstrated that in the absence of co-morbidities, aging is associated with reduced cardiac output and stroke volume under resting conditions as well as with lower cardiac reserve.³ At the structural level, epicardial fat deposition, calcification of the aortic valve, atrial hypertrophy and dilation, left ventricular hypertrophy and fibrosis occur with aging. Ventricular myocyte loss with increases in cardiac fibroblast populations represent the dynamic changes in cellular populations throughout the aging heart.⁴ Expression profiling in aged murine hearts suggest that changes occur in inflammation and immunity related genes as well as elevated proapoptotic signaling but no actual increases in apoptosis.³ Thus, understanding processes of myocardial aging are complex and precise molecular signaling, cell population dynamics and resulting changes to heart muscle structure and function remain unmet aims required for developing novel therapeutic strategies for this HF patient segment.

Cell Therapy for Failing Hearts: Interspecies Differences.

In this Issue, Zhao et al.⁵ have evaluated the potential of Cardiosphere-Derived Cells (CDCs) to improve cardiac structure and function in aged mice. CDCs were directly injected into the hearts of 21-month old mice and outcomes were compared to young, 2-month old mice. Direct injections of 2 × 10⁴ murine CDCs (mCDCs) derived from neonatal mice were made into each of four locations throughout the left ventricles of the recipient mice with reduced function relative to the young mouse hearts. This study did not test the hypothesis that CDCs derived from young or neonatal donors have greater potential than CDCs from older donors. Note that CDCs derived from patients with advanced HF were shown to have greater therapeutic potential in a murine MI model.⁶ Zhao et al.⁵ evaluated cardiac structure and function assessed by echocardiographic, histological and molecular analyses of cardiac tissues. They further evaluated peripheral responses of key inflammation markers in blood and rejuvenation by hair regrowth. In this case there were no statistical differences in LVEF, as might be expected from a clinical perspective as many cases of age related heart failure are characterized as heart failure with preserved ejection fraction. Left ventricular diastolic wall thickness increased after CDC injection, resulting in reduced diastolic LV volume. Flow velocities were unaffected. Furthermore, both CDC treated and age matched control animals had similar pressure-volume parameters. At the molecular level, cardiomyocyte telomere length was also not impacted by mCDCs in aged mice. Taken together, there was no detectable improvement due to mCDC delivery to aging hearts in this study. Noticeably absent is a longitudinal study of murine cardiac structure and function data to show the development of age related heart failure in the mouse to define efficacious therapeutic timing. In spite of nearly identical methodologies, these results are in sharp contrast to those from a rat study by Grigorian-Shamagian et el.⁷ They delivered cell neonatal rat CDCs (rCDCs) and young human CDCs either via cross-clamping, or by direct injection into the aging rat heart. The results revealed significant improvements in cardiac function, reduced fibrosis, increased cardiomyocyte rejuvenation in cardiomyocyte telomer length and improved systemic markers of inflammation and hair regrowth and improved kidney function. Taken together, these results would suggest that the benefit of cell therapies to aging patients with reduced cardiac function is uncertain and sourcing of therapeutic stem cell preparations should be a component of further investigations.

The contrasting results between these studies highlight current challenges in translating pre-clinical cell delivery outcomes. The controversies that were recently highlighted by Keith and Bolli⁸ continue with challenges in characterizing and stabilizing molecular and functional cardiac derived progenitors.⁹

Cell Source Preparation and Characterization:

While the preparation of CDCs in by Zhao et al.⁵ and by Grigorian-Shamagian et al.⁷ appear to be strikingly similar, standardized endpoints representing functional and molecular phenotypes are not available to ensure that the resulting populations are comparable. As an example of potential, but unknown differences between the CDC preparations, especially interspecies differences. Zhao et al.⁵ report that 6% of CDCs are cKit positive while Smith et al¹⁰report nearly 3 times that value in human CDC isolation. It is notable that at least 97% of cardiac progenitor cells isolated from human LVAD samples were shown to be express cKit in culture though this receptor was internalized in tissue culture¹¹. In this elegant study by Monsanto et al., cardiac CPCs, endothelial progenitors and cardiac mesenchymal stem cells, all potential components of some CDC preparations, were isolated in parallel from individual human cardiac samples, and had significantly different cKit, CD90/105, CD31 and CD34 expression profiles and functional activities. With the internalization of several of these surface markers, there is uncertainty regarding the value of classical of flow cytometric quantitation of these cell preparations in understanding the nature of population, interspecies differences in CDC populations and functional dynamics. CDC soluble and exosomal secretomes are by consensus, the presumed active cell products responsible for therapeutic efficacy. The interspecies variance, in secretome compositions is not yet fully characterized and could be partially responsible for the lack of improvement in aged murine hearts in contrast to aged rat hearts. Therefore a scientific rationale for rodent interspecies differences in outcomes cannot be determined without more detailed characterization of CDCs.

It should also be recognized that in both this murine study by Zhao et al.⁵ as well as the rat study by Grigorian-Shamagian⁷ used CDCs isolated from neonatal animals and their clinical relevance has not been made clear. Indeed, infarcted heart CDCs from chronic HF patients have been reported to be more therapeutic via cardiomyogenesis in the infarcted heart than from non-infarcted heart.⁶ Thus there is potential for CDCs from infarcted donor hearts to positively impact aging hearts via paracrine and nonparacrine effects.

Challenges in Characterizing CDCs and Cardiac Progenitor Cells (CPCs).

With the internalization of surface markers in tissue culture and the extremely low surface expression of some markers here by Zhao et al.⁵ and in hCDCs by Smith et al.,¹² the relative contribution of cKit+ cardiac progenitors EPCs and MSCs in CDC preparations is expected to be dynamic. Furthermore, Smith et al.¹² suggest that at MSCs may play a role in supporting the secretory activity of other CPCs in culture and thus CPC population dynamics could impact therapeutic efficacy of mixed populations. The cKit expressing cells in CDC preparations have been reported to be less important contributors efficacy suggesting that they might play indirect or minor roles in the therapeutic secretome of CDCs.¹³ However, that report employed magnetically activated cell sorting, which relies on cell surface cKit expression which would not capture internalized cKit. In addition, use of secretome signatures should become a component in the suite of assays used to characterize and compare both cell efficacies and cell product efficacies in the future Until this unmet need is met, the efficacies of delivered cells and cell secreted products will remain controversial.

In summary, there is the potential for many uncontrolled variables to be responsible for the lack of efficacy of mCDCs in aging mice compared to rCDCs or hCDCs in aging rats and the controversies continue. Cell therapies remain an emerging technology. Cardiac derived progenitors are proving to be highly dynamic, producing secretomes with complex activities when delivered to the aging heart. It is likely that controversies will continue until methods are adopted for routine standardized characterization of cells being delivered as well as controlling their functional phenotypes.