Cardiac Cell Therapy:Tumorigenicity Evaluation And Paracrine Effect Exploration

Background:Ever since stem cell science began, scientists have been trying to harness stem cells to reverse advanced heart failure which caused by kinds of cardiovascular diseases. Recently, the field of cardiac cell therapy has witnessed two major progresses. A novel candidate cell:induced pluripotent stem (iPS) cell have been on the horizon; And a different working mechanism:paracrine effect has been acknowledged. This dissertation, composed of three parts, is to study the application of iPS cell and paracrine effect in cardiac cell therapy.iPS cell is intrinsically tumorigenic due to its reprogramming process. So, it requires further investigation prior to use in a clinical setting. In part one of this dissertation, we investigated whether undifferentiated iPS cells are tumorigenic after intramyocardial transplantation into immunocompetent allogeneic recipients.Mounting evidence demonstrates that the benefits of cardiac stem cell therapy are not derived from in situ regeneration, but from the transplanted cell’s paracrine effects on cardiac host cells. Cardiomyocytes, occupying the most volume of the heart, are the performers of cardiac function; Cardiac fibroblasts (CF), the most numerous cells in the heart, play critical roles in cardiac structural remodeling as well as electrical remodeling. In part two and part three of this dissertation, we explored stem cell’s paracrine effects on these two kinds of cardiac host cells.The dogma that the adult mammalian heart is a terminally differentiated organ has been challenged by the concept that the heart is capable of cardiomyocyte turnover. Accordingly, cellular senescence may have an important role in the pathology of heart aging. Therefore, in part two, we tested our hypothesize that iPS cell could inhibit cardiac cellular senescence via paracrine mechanism.Animal research shows that bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation could reduce cardiac fibrosis. And in vitro study shows that rat MSC could, via paracrine effects, inhibit rat cardiac fibroblast proliferation and collagen synthesis. Besides, animal researches and clinical trials suggest that MSC transplantation could maintain cardiac electrical stability. Therefore, in order to explore the mechanism via which MSC could prevent cardiac structural and electrical remodeling, we studied MSC’s paracrine effects on CF’s phenotype, function and electrophysiology.Methodology:In part one, we transplanted2×104,2×105, or2×106cells from the established rat iPS cell line M13intramyocardially into intact or infarcted hearts of immunocompetent allogeneic rats. Transplant duration was2,4, or6weeks. Histological examination with hematoxylin-eosin staining is used to analyze tumor formation and differentiation.In part two, we collected iPS cell culture supernatant, with or without oxidative stress, as conditioned medium (CM) for rat cardiomyocyte-derived cell line H9c2. H9c2cells were underwent hypoxia/reoxygenation to induce cellular senescence, with or without CM.In part three, we confirmed that serum starved human cardiac fibroblasts-adult ventricle (HCF-av) could be used as an in vitro model of cardiac fibroblast in injured heart, which suffered mainly from ischemia/hypoxia. Then, we co-cultured human BM-MSC (hBM-MSC) with HCF-av in a non-contact manner. By real-time PCR, we analyzed hBM-MSC’s effects on the mRNA expression of fibroblast markers, extracellular matrixes and connexins in HCF-av. Apart from that, we analyzed HCF-av’s expression patterns of sodium channels and potassium channels in normal culture (baseline) group, serum starvation group and co-culture group.Results:1. Undifferentiated rat iPS cells could generate heterogeneous tumors in both intracardiac and extracardiac sites. Furthermore, tumor incidence was independent of cell dose, transplant duration, and the presence or absence of myocardial infarction.2. iPS derived CM reduced senescence-associated-β-galactosidase activity, attenuated G1cell cycle arrest, and reduced the expressions of senescent markers p16INK4a, p21Wafl/CiP1and p53in H9c2cells. Furthermore, the CM contained more trophic factors:tissue inhibitor of metalloproteinase-1(TIMP1), and vascular endothelial growth factor (VEGF) than H9c2derived CM.3. hBM-MSC could enhance the expression of alpha smooth muscle actin (a-SMA), thymocyte differentiation antigen1(THY-1, CD90); reduce the expression of major extracellular matrixes, including collagen I, fibronectin, tenascin-C, laminin and tissue-inhibitor of metalloproteinase1; and could also reduce the expression of connexins, CX40, CX43and CX45included. Our data showed that Navl.5and Navl.7are expressed in HCF-av. Navl.5expression was significantly increased in serum starvation group, which could reduce by hBM-MSC co-culture; while the expression of Navl.7showed no significant difference among the three groups. Besides, we found that potassium channels expressed in HCF-av includes:Kvl.3, Kv1.5, Kv4.2, Kv4.3, Kca1.1, Kca3.1, Kir2.1, Kir2.3, Kir6.1and Kir6.2. All of the potassium channels increased their expression when suffered from serum starvation, and the increased expression could be significantly down-regulated by hBM-MSC co-culture. Kv7.1, Kv11.1and Kir4.1were undetectable in HCF-av.Conclusions:1. Allogeneic iPS cell transplantation in the heart will likely result in in situ tumorigenesis, while the cells leaked from the beating heart are a potential source of tumor spread.2. Paracrine factors released from iPS cells, even under oxidative stress, prevent stress induced senescence of H9c2cells via inhibiting both p53-p21and p16-pRb pathways.3. The benefits of MSC transplantation on cardiac structural and electrical remodeling could exert via paracrine effects. The mechanisms involved may include:1) reducing extracellular matrix deposit;2) enhancing cardiac fibroblasts’ ability and efficiency of mending the injured tissue, preventing their excess proliferation, thus inhibiting fibrosis;3) down-regulating the pathological couple between fibroblasts and cardiomyocytes, so to diminish the electrophysiological alteration of cardiomyocytes; and4) reverting the membrane currents of fibroblasts to their normal pattern.Significance:1. This is the first report demonstrating that allogeneic iPS cell transplantation into the heart can cause in situ tumorigenesis in immunocompetent recipients, underscoring the importance of evaluating the safety of future iPS cell therapy for cardiac disease.2. This is the first report demonstrating that antisenescence effects of stem cell therapy may be a novel therapeutic strategy for age-related cardiovascular disease.3. We present the first proof of hBM-MSC’s paracrine effects on HCF. Furthermore, we proposed a novel idea that MSC could prevent cardiac electrical remodeling via its paracrine effects on CF.