Biomedicines, Vol. 10, Pages 3143: Cardiac Mesenchymal Stem Cell-like Cells Derived from a Young Patient with Bicuspid Aortic Valve Disease Have a Prematurely Aged Phenotype

1. IntroductionA number of resident cardiac stem cell populations have been identified, several of which are present in the human heart [1,2,3,4,5]. However, clinical trials utilizing bone marrow or cardiac-derived stem cells have delivered only modestly successful results, with only a few patients showing meaningful improvements in cardiac function [6,7].It is now accepted that most therapeutic benefits of MSC transplantation for cardiac repair are driven by paracrine secretions of numerous growth factors, cytokines and extracellular vesicles that aid in promoting cardiomyocyte survival, angiogenesis, and steering remodeling towards a less fibrotic phenotype [8]. Despite this, their mechanisms of action within the heart remain controversial and there has also been consideration of their possible role in tumor formation [9,10]. Further challenges are presented in the production of clinically relevant cell therapy products, including the influence of in vitro ageing during culture expansion [11], and limited retention and survival of donor cells at the target site, which requires combination of cells with supporting biocompatible materials [12,13,14,15,16]. It is well-known that not all donor cells are created equal [17]. For example, a previous study has shown that myocardial infarction adversely influences the therapeutic potential of bone marrow-derived cells from the same donor [18]. Furthermore, the secretome varies dynamically according to phenotype of the originating cells. Extracellular vesicles derived from post-MI mouse hearts were shown to aggravate inflammation and worsened heart function in other animals [19]. Thus, the source of donor therapeutic cells might also impact on the therapeutic outcome. By their very nature, patients requiring cardiac surgical intervention are rarely healthy or absent of cardiovascular disease. Therefore, if cardiac-derived cells are to be used for therapy, better understanding of the effects of cardiac disease and/or aging on those cells is needed.Coronary artery disease (CAD) is a common disease caused by the buildup of atheroma in the coronary arteries that causes narrowing of the arteries and reduced blood flow to the heart [20]. This atherosclerotic process is, however, not restricted to the coronary arteries or even to the heart and can affect a number of different organs in the body and can therefore be considered a systemic disease [21]. The loss and/or dysfunction of MSCs has been associated with many systemic diseases (reviewed in Vizoso et al., 2019 [22]), while circulating endothelial progenitor cells (CPCs) expressing osteocalcin have been shown to be present in higher numbers in patients with coronary atherosclerosis than those without [23]. These studies support a role for progenitors/stem cells as either contributing to, or as being targets of, disease progression in some systemic diseases, including CAD. Contrary to this, bicuspid aortic valve defects are a form of congenital heart defect, often associated with several other cardiac complications, the symptoms of which frequently become apparent with increasing age [24,25].One cell fate known to impede stem cell function is cellular senescence, controlled by the p16-pRb and p53-p21 pathways and defined as a cell cycle arrest, alterations in gene and protein expression and the production of the senescence-associated secretory phenotype (SASP) [26], a cocktail of pro-inflammatory cytokines, chemokines, matrix proteases and growth factors, which in the heart can impact tissue function, attenuate regeneration, induce fibrosis, extracellular matrix degeneration and drive inflammation [27]. Cardiac regenerative potential declines with age [28] and populations of human cardiac progenitor cells (CPCs) accumulate the senescence phenotype with age, express p16 and are unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart [29]. Our recent studies demonstrate that in the heart, cardiomyocyte, fibroblast and endothelial senescence can be induced not only by ageing but also as a result of cellular stress and disease [30,31]. The effect of disease on progenitor cells independent of ageing has not yet been investigated.We have previously reported on the identification of human cardiac mesenchymal stem cell-like cells (CMSCLC), which have stem cell-like characteristics and an immunophenotype typical of MSC. These cells were capable of low levels of adipogenic differentiation but failed to differentiate into osteoblasts or chondrocytes. However, these CMSCLC did, under cardiac differentiation conditions, have the phenotype of both mature and immature cardiac cells, expressing troponin C and Nkx2.5, respectively [5]. CMSCLC express many of the bioactive molecules that make up the cardio-beneficial paracrine secretome, including, interleukin-10 (IL10), fibroblast growth factor-2 (FGF2), vascular endothelial growth factor (VEGF), transforming growth factor (TGF) and hepatocyte growth factor (HGF) [5], and their potential for therapeutic application demonstrated in vivo by improved cell retention, survival, extracellular vesicle production, and promotion of functional cardiac repair when encapsulated and delivered to a murine model of myocardial infarction ischemic injury [32].

Clinical presentation of a 21-year-old female patient with relatively rare bicuspid aortic valve disease (BAVD) provided an opportunity to present a case-based study that contributes to advancing the understanding of the impact of both age and different cardiomyopathies on the health of CMSCLC, and thus the potential implications for application of cardiac-derived stem cell-based therapies in clinic. We have isolated and compared CMSCLC from the atrial appendage of both the BAVD patient and from a 78-year-old female patient with coronary artery disease (CAD). We provide evidence that CMSCLC from the 21-year-old BAVD patient have a prematurely aged phenotype when compared with CMSCLC from the 78-year-old CAD patient. This suggests that premature aging of resident cardiac stem cells may contribute to additional cardiac complications observed in some patients with BAVD.

4. DiscussionBicuspid aortic valve defects are a common form of congenital heart defect, often associated with a number of other cardiac complications, the symptoms of which frequently become apparent with increasing age [24,25]. The causes of BAVD, however, remain unclear. We report for the first time on differences between CMSCLC isolated from of a young female patient with BAVD disease and an elderly female patient with CAD disease. We observed that both the CAD-CMSCLC and BAVD-CMSCLC expressed cell surface makers normally expressed by MSCs and lack expression of hematopoietic lineage makers. Moreover, the CMSCLCs adhere to plastic under standard MSC culture conditions [41,42]. However, morphologically, while the CAD-CMSCLC displayed an MSC-like morphology [41], the BAVD-CMSCLC did not; they displayed characteristics associated with aged MSCs [43].One well-recognized stem cell characteristic is the ability to form colonies in culture; we observed that CAD-CMSCLC formed more CFU-Fs than the BAVD-CMSCLC. They also formed fewer CFU-Fs than for other patient-derived CMSCLC reported in our previous study [5]. Moreover, the number of cells that could be derived from the CAD-CFU-Fs was higher than those that could be derived from the BAVD-CFU-Fs. BAVD-CMSCLC also had a slower doubling rate and a reduced capacity for cell division compared with CAD-CMSCLC, both of which are characteristics of aging [43]. The BAVD-CMSCLC also had a shorter telomere length than the CAD-CMSCLC. The replicative potential of hematopoietic stem cells is related to telomere length and as such this provides an indication of stem cell function and there is increasing evidence that telomerase activity and telomere length are important for the function of bone marrow derived MSCs. Mouse MSCs that lack telomerase activity show an inability to differentiate into adipocytes or chondrocytes [44,45] while human MSCs, forced to overexpress telomerase, have increased proliferative potential [45]. It has been suggested that in human MSCs telomerase activity is required to bring about regenerative capacity and differentiation potential [46]. The shorter telomeres in the BAVD-CMSCLC may therefore be an indicator of the inferior quality of these cells compared with the CAD-CMSCLC and suggest that like bone marrow MSCs telomerase activity and telomere length is required to maintain the CMSCLC proliferative potential.NANOG is associated with biological processes important for stem cell function including proliferation and differentiation potential [47,48,49]. In aging murine bone marrow MSCs, the over-expression of NANOG reversed ageing-associated loss of proliferation and myogenic potential [50]. We observed NANOG expression in both CMSCLC populations; however, there was decreased expression in terms of cell numbers in BAVD-CMSCLC. Furthermore, p16 an inhibitor of cell cycle progression most commonly associated with cell senescence including, tissue resident MSC and cardiomyocytes, was upregulated in BAVD-CMSCLC compared to CAD-CMSCLC [30,51,52]. While p16 is a robust marker of human senescence in vivo [40], the senescent phenotype can be heterogeneric and dependent on cell type and stimuli as such a deeper analysis of senescence-associated gene expression would provide insight into the signature of senescent CMSCLCs [53]. Cell cycle analysis revealed difference in cell cycle kinetics between the CAD and BAVD CMSCLC, with a subpopulation of BAVD cells being in the G2/M phase of the cell cycle which can be indicative of a stressed or senescent cell phenotype [50]. Recent studies have demonstrated that epigenetic biomarkers of ageing are prognostic of disease across multiple tissues including the cardiovascular system. [54,55,56]. While it is beyond the scope of the current study, a future investigation of epigenetic changes within CMSCLC populations could shed light on if epigenetic changes are associated with CMSCLC age and disease-related dysfunction.Finally, we investigated the health of both populations of cells. Quantification of metabolic activity by alamarBlue® assay is an established indicator of cell health whereby measurement of the reducing power of the intracellular environment includes contribution from mitochondrial and cytoplasmic reductases, and is therefore a function of both aerobic glycolysis and oxidative phosphorylation, respectively [34]. Our observation that the BAVD-CMSCLC were less metabolically active than the CAD-CMSCLC suggests that these cells have an impaired cellular metabolism, consistent with the established phenotype of senescent cells [34,57,58]. Metabolic balance is known to couple bioenergetic state with broader physiological pathways that regulate MSCs phenotype and function, including the metabolic switch of aerobic glycolysis to oxidative phosphorylation that drives differentiation [59]. Whilst aerobic glycolysis might be expected to be the predominant metabolic pathway in rapidly proliferating CMSCLC, the involvement of oxidative phosphorylation in the production of metabolite intermediates that form the precursors of biosynthetic pathways, as well as the metabolism of glutamine to glutathione for the regulation of REDOX signaling should also be considered [60,61,62]. Dysregulation of these integrated metabolic pathways is linked to elevated production of reactive oxygen species (ROS) and cellular oxidative stress that drives the senescent phenotype, and we propose this mechanism as one that warrants further interrogation in determining the health status of patient-derived CMSCLC [60,61].The premature senescence evidenced in the BAVD-CMSCLCs is intriguing when considering that BAVD has no anatomical or embryological link with the right atrium (the aortic valve originates from neural crest and mutations tend not to affect right heart), whereas CAD associated with significant atherosclerosis is a systemic illness as opposed to a very organ-specific abnormality [63,64]. We suggest that in atherosclerotic cardiovascular disease, vascular remodeling, whilst being initiated by dysfunctional endothelial cells, is contributed to by stem cells originating from several sources. However, these cells have been shown to differentiate primarily to adipocytes, chondrocytes and osteocytes [65]. In our previously published study, CMSCLC from RAA of patients with CAD also had a very poor ability to undergo this tri-lineage differentiation [5], suggesting that CMSCLC from RAA are distinct from the stem cells contributing to cardiovascular disease. Resident cardiac stem cells known as CASCs, have also been isolated from atrial appendages based on aldehyde dehydrogenase activity and have been shown to be present in both human and pig heart. In the pig examination of different regions of the heart for the presence of CASCs revealed the highest numbers to be present in RAA and it is therefore a good source of stem cells that are relevant to cardiac-specific therapeutic application [66]. Interestingly, it has been proposed that individuals with BAV are predisposed to senescence [67]. Individuals with BAV display an increased prevalence of thoracic aortic aneurysm (TAA) and have a significantly increased risk of aortic dissection compared to those with a typical tricuspid valve (TAV), while senescence is associated with TAA in both TAV and BAV patients, only aortas of individuals with BAV contained senescence cells in the absence of TAA [68]. Observations that suggest BAV aortas have an increased predisposition to senescence even in the absence of symptomatic disease. Although the cause of this predisposition is unknown, however the genetics contributing to the disease may play a role, therefore it is possible that other cardiac resident cells including CMSCLCs share this susceptibility to senescence. The ultimate goal for stem cell research is cellular therapy; therefore, it is imperative that we fully understand how donor disease and genetics effects stem cell biology. Whilst our study is limited by access to cardiac tissue of one donor with relatively rare BAVD, our data suggests that genetic valvular disease can promote a senescence phenotype, and raises important questions regarding the appropriateness of using stem cells from diseased individuals, which may be required for autologous transplantation. Studies investigating age-related senescence have demonstrated that c-kit expressing cardiac progenitor cells have impaired stem cell function and express a proinflammatory SASP that promotes senescence in healthy CPC populations [29]. As such, cellular therapies that use cell populations, which include senescent cells may not only be less effective but may in fact be detrimental to both the transplanted population as a whole and the organ into which they are transplanted. Indeed, in animal models, transplantation of small numbers of senescent cells induces age-related disease, increases frailty, and increases mortality [6,7,8]. It is possible that in the future some of these challenges could be overcome isolated senescent CPCs can be “rejuvenated” via the treatment with senolytics which induce senescent but not proliferative cells to apoptosis [29].

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