Plasmatic Membrane Expression of Adhesion Molecules in Human Cardiac Progenitor/Stem Cells Might Explain Their Superior Cell Engraftment after Cell Transplantation.
ABSTRACT: Human bone marrow mesenchymal stem cells (BM-MSCs) and cardiac progenitor/stem cells (CPCs) have been extensively studied as a potential therapeutic treatment for myocardial infarction (MI). Previous reports suggest that lower doses of CPCs are needed to improve cardiac function relative to their bone marrow counterparts. Here, we confirmed this observations and investigated the surface protein expression profile that might explain this effect. Myocardial infarction was performed in nude rats by permanent ligation of the left coronary artery. Cardiac function and infarct size before and after cell transplantation were evaluated by echocardiography and morphometry, respectively. The CPC and BM-MSC receptome were analyzed by proteomic analysis of biotin-labeled surface proteins. Rats transplanted with CPCs showed a greater improvement in cardiac function after MI than those transplanted with BM-MSCs, and this was associated with a smaller infarct size. Analysis of the receptome of CPCs and BM-MSCs showed that gene ontology biological processes and KEGG pathways associated with adhesion mechanisms were upregulated in CPCs compared with BM-MSCs. Moreover, the membrane protein interactome in CPCs showed a strong relationship with biological processes related to cell adhesion whereas the BM-MSCs interactome was more related to immune regulation processes. We conclude that the stronger capacity of CPCs over BM-MSCs to engraft in the infarcted area is likely linked to a more pronounced cell adhesion expression program.
Project description:Cell-based cardiac therapy is a promising therapeutic strategy to restore heart function after myocardial infarction (MI). However, the cell type selection and ensuing effects remain controversial. Here, we intramyocardially injected Isl1+ cardiac progenitor cells (CPCs) derived from EGFP/luciferase double-tagged mouse embryonic stem (dt-mES) cells with vehicle (fibrin gel) or phosphate-buffered saline (PBS) into the infarcted area in nude mice to assess the contribution of CPCs to the recovery of cardiac function post-MI. Our results showed that Isl1+ CPCs differentiated normally into three cardiac lineages (cardiomyocytes (CMs), endothelial cells and smooth muscle cells) both on cell culture plates and in fibrin gel. Cell retention was significantly increased when the transplanted cells were injected with vehicle. Importantly, 28 days after injection, CPCs were observed to differentiate into CMs within the infarcted area. Moreover, numerous CD31+ endothelial cells derived from endogenous revascularization and differentiation of the injected CPCs were detected. SMMHC-, Ki67- and CX-43-positive cells were identified in the injected CPC population, further demonstrating the proliferation, differentiation and integration of the transplanted CPCs in host cells. Furthermore, animal hearts injected with CPCs showed increased angiogenesis, decreased infarct size, and improved heart function. In conclusion, our studies showed that Isl1+ CPCs, when combined with a suitable vehicle, can produce notable therapeutic effects in the infarcted heart, suggesting that CPCs might be an ideal cell source for cardiac therapy.
Project description:<h4>Aims</h4>Myocardial infarction (MI) is associated with irreversible loss of viable cardiomyocytes. Cell therapy is a potential option to replace the lost cardiomyocytes and restore cardiac function. However, cell therapy is faced with a number of challenges, including survival of the transplanted cells in the infarct region, which is characterized by abundant levels of oxidants and lack of a pro-survival support mechanism. The goal of the present study was to evaluate the effect of supplemental oxygenation on cell engraftment and functional recovery in a rat model.<h4>Methods and results</h4>MI was induced in rats by a 60-min occlusion of the coronary artery, followed by restoration of flow. Mesenchymal stem cells (MSCs), isolated from adult rat bone marrow, were transplanted in the MI region. Rats with transplanted MSCs were exposed to hyperbaric oxygen (HBO: 100% O(2), 2 atmospheres absolute) for 90 min, 5 days/week for 4 weeks. The experimental groups were: MI (control), Ox (MI + HBO), MSC (MI + MSC), and MSC + Ox (MI + MSC + HBO). HBO exposure (oxygenation) was started 3 days after induction of MI. MSCs were transplanted 1 week after induction of MI. Echocardiography showed a significant recovery of cardiac function in the MSC + Ox group, when compared with the MI or MSC group. Oxygenation increased the engraftment of MSCs and vascular density in the infarct region. Molecular analysis of infarct tissue showed a four-fold increase in NOS3 expression in the MSC + Ox group compared with the MI group.<h4>Conclusions</h4>The results showed that post-MI exposure of rats to daily cycles of hyperoxygenation (oxygen cycling) improved stem cell engraftment, cardiac function, and increased NOS3 expression.
Project description:Bone marrow-mesenchymal stem cell (BM-MSC) therapy improves the recovery of cardiac function after myocardial infarction (MI); however, the underlying molecular mechanisms are not completely understood. Recent studies have shown that microRNAs (miRNAs) modulate the pathophysiology of cardiovascular diseases. Here, we investigated the mechanisms underlying the effects of BM-MSC-derived paracrine factors and cardiac miRNAs on myocardial regeneration after MI. In our study, MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery. BM-MSCs transplanted in infarcted rats significantly downregulated the expression of miRNA-23a and miRNA-92a and inhibited apoptosis in the myocardium. An in vitro experiment showed that supernatant from BM-MSCs cultured under hypoxia contained higher levels of vascular endothelial growth factor (VEGF) than that from BM-MSCs under normoxia. In addition, inhibition of miRNA-23a and miRNA-92a reduced cardiac apoptosis. Moreover, the VEGF-containing BM-MSC supernatant inhibited miRNA-23a and miRNA-92a expression and reduced apoptotic signaling in cardiomyocytes under hypoxia. These effects were inhibited when the supernatant was treated with neutralizing antibodies against VEGF. Our results indicate that the paracrine factor, VEGF, derived from transplanted BM-MSCs, regulated the expression of miRNAs such as miRNA-23a and miRNA-92a and exerted anti-apoptotic effects in cardiomyocytes after MI.
Project description:<h4>Aims</h4>Cardiovascular diseases caused by loss of functional cardiomyocytes (CMs) are a major cause of mortality and morbidity worldwide due in part to the low regenerative capacity of the adult human heart. Human pluripotent stem cell (hPSC)-derived cardiovascular progenitor cells (CPCs) are a potential cell source for cardiac repair. The aim of this study was to examine the impact of extensive remuscularization and coincident revascularization on cardiac remodelling and function in a mouse model of myocardial infarction (MI) by transplanting doxycycline (DOX)-inducible (Tet-On-MYC) hPSC-derived CPCs in vivo and inducing proliferation and cardiovascular differentiation in a drug-regulated manner.<h4>Methods and results</h4>CPCs were injected firstly at a non-cardiac site in Matrigel suspension under the skin of immunocompromised mice to assess their commitment to the cardiovascular lineage and ability to self-renew or differentiate in vivo when instructed by systemically delivered factors including DOX and basic fibroblast growth factor (bFGF). CPCs in Matrigel were then injected intra-myocardially in mice subjected to MI to assess whether expandable CPCs could mediate cardiac repair. Transplanted CPCs expanded robustly both subcutis and in the myocardium using the same DOX/growth factor inducing regime. Upon withdrawal of these cell-renewal factors, CPCs differentiated with high efficiency at both sites into the major cardiac lineages including CMs, endothelial cells, and smooth muscle cells. After MI, engraftment of CPCs in the heart significantly reduced fibrosis in the infarcted area and prevented left ventricular remodelling, although cardiac function determined by magnetic resonance imaging was unaltered.<h4>Conclusion</h4>Replacement of large areas of muscle may be required to regenerate the heart of patients following MI. Our human/mouse model demonstrated that proliferating hPSC-CPCs could reduce infarct size and fibrosis resulting in formation of large grafts. Importantly, the results suggested that expanding transplanted cells in situ at the progenitor stage maybe be an effective alternative causing less tissue damage than injection of very large numbers of CMs.
Project description:Biomaterials in conjunction with stem cell therapy have recently attracted attention as a new therapeutic approach for myocardial infarction (MI), with the aim to solve the delivery challenges that exist with transplanted cells. Self-assembling peptide (SAP) hydrogels comprise a promising class of synthetic biomaterials with cardiac-compatible properties such as mild gelation, injectability, rehealing ability, and potential for sequence modification. Herein, we developed an SAP hydrogel composed of a self-assembling gel-forming core sequence (RADA) modified with SDKP motif with pro-angiogenic and anti-fibrotic activity to be used as a cardioprotective scaffold. The RADA-SDKP hydrogel was intramyocardially injected into the infarct border zone of a rat model of MI induced by left anterior descending artery (LAD) ligation as a cell-free or a cell-delivering scaffold for bone marrow mesenchymal stem cells (BM-MSCs). The left ventricular ejection fraction (LVEF) was markedly improved after transplantation of either free hydrogel or cell-laden hydrogel. This cardiac functional repair coincided very well with substantially lower fibrotic tissue formation, expanded microvasculature, and lower inflammatory response in the infarct area. Interestingly, BM-MSCs alone or in combination with hydrogel could not surpass the cardiac repair effects of the SDKP-modified SAP hydrogel. Taken together, we suggest that the RADA-SDKP hydrogel can be a promising cell-free construct that has the capability for functional restoration in the instances of acute myocardial infarction (AMI) that might minimize the safety concerns of cardiac cell therapy and facilitate clinical extrapolation.
Project description:INTRODUCTION: Among the plethora of cells under investigation to restore a functional myocardium, mesenchymal stromal cells (MSCs) have been granted considerable interest. However, whereas the beneficial effects of bone marrow MSCs (BM-MSCs) in the context of the diseased heart are widely reported, data are still scarce on MSCs from the umbilical cord matrix (UCM-MSCs). Herein we report on the effect of UCM-MSC transplantation to the infarcted murine heart, seconded by the dissection of the molecular mechanisms at play. METHODS: Human umbilical cord tissue-derived MSCs (UCX®), obtained by using a proprietary technology developed by ECBio, were delivered via intramyocardial injection to C57BL/6 females subjected to permanent ligation of the left descending coronary artery. Moreover, medium produced by cultured UCX® preconditioned under normoxia (CM) or hypoxia (CMH) was collected for subsequent in vitro assays. RESULTS: Evaluation of the effects upon intramyocardial transplantation shows that UCX® preserved cardiac function and attenuated cardiac remodeling subsequent to myocardial infarction (MI). UCX® further led to increased capillary density and decreased apoptosis in the injured tissue. In vitro, UCX®-conditioned medium displayed (a) proangiogenic activity by promoting the formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs), and (b) antiapoptotic activity in HL-1 cardiomyocytes subjected to hypoxia. Moreover, in adult murine cardiac Sca-1+ progenitor cells (CPCs), conditioned medium enhanced mitogenic activity while activating a gene program characteristic of cardiomyogenic differentiation. CONCLUSIONS: UCX® preserve cardiac function after intramyocardial transplantation in a MI murine model. The cardioprotective effects of UCX® were attributed to paracrine mechanisms that appear to enhance angiogenesis, limit the extent of the apoptosis, augment proliferation, and activate a pool of resident CPCs. Overall, these results suggest that UCX® should be considered an alternative cell source when designing new therapeutic approaches to treat MI.
Project description:Stem cells are promising for the treatment of myocardial infarction (MI) and large animal models should be used to better understand the full spectrum of stem cell actions and preclinical evidences. In this study, bone marrow mesenchymal stem cells (BM-MSCs) were transplanted into swine heart ischemia model. To detect glucose metabolism in global left ventricular myocardium and regional myocardium, combined with assessment of cardiac function, positron emission tomography-computer tomography (PET-CT) and magnetic resonance imaging (MRI) were performed. To study the changes of glucose transporters and glucose metabolism-related enzymes and the signal transduction pathway, RT-PCR, Western-blot, and immunohistochemistry were carried out. Myocardium metabolic evaluation by PET-CT showed that mean signal intensity (MSI) increased in these segments at week 4 compared with that at week 1 after BM-MSCs transplantation. Moreover, MRI demonstrated significant function enhancement in BM-MSCs group. The gene expressions of glucose transporters (GLUT1, GLUT4), glucose metabolism-related enzymes phosphofructokinase (PFK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) and 70-kDa ribosomal protein S6 kinase (p70s6k) in BM-MSCs injected areas were up-regulated at week 4 after BM-MSCs transplantation and this was confirmed by Western-blot and immunohistochemistry. In conclusions, BM-MSCs transplantation could improve cardiac function in swine MI model by activation of mTOR signal transduction pathway.
Project description:To develop an effective therapeutic strategy for cardiac regeneration using bone marrow mesenchymal stem cells (BM-MSCs), the primary mouse BM-MSCs (1(st) BM-MSCs) and 5(th) passage BM-MSCs from ?-galactosidase transgenic mice were respectively intramyocardially transplanted into the acute myocardial infarction (AMI) model of wild type mice. At the 6(th) week, animals/tissues from the 1(st) BM-MSCs group, the 5(th) passage BM-MSCs group, control group were examined. Our results revealed that, compared to the 5(th) passage BM-MSCs, the 1(st) BM-MSCs had better therapeutic effects in the mouse MI model. The 1(st) BM-MSCs maintained greater differentiation potentials towards cardiomocytes or vascular endothelial cells in vitro. This is indicated by higher expressions of cardiomyocyte and vascular endothelial cell mature markers in vitro. Furthermore, we identified that 24 proteins were down-regulated and 3 proteins were up-regulated in the 5(th) BM-MSCs in comparison to the 1(st) BM-MSCs, using mass spectrometry following two-dimensional electrophoresis. Our data suggest that transplantation of the 1(st) BM-MSCs may be an effective therapeutic strategy for cardiac tissue regeneration following AMI, and altered protein expression profiles between the 1(st) BM-MSCs and 5(th) passage BM-MSCs may account for the difference in their maintenance of stemness and their therapeutic effects following AMI.
Project description:<h4>Background</h4>Stem cell therapy has emerged as a promising therapeutic strategy for myocardial infarction (MI). However, the poor viability of transplanted stem cells hampers their therapeutic efficacy. Hypoxic preconditioning (HPC) can effectively promote the survival of stem cells. The aim of this study was to investigate whether HPC improved the functional survival of bone marrow mesenchymal stem cells (BM-MSCs) and increased their cardiac protective effect.<h4>Methods</h4>BM-MSCs, isolated from Tg(Fluc-egfp) mice which constitutively express both firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP), were preconditioned with HPC (1% O<sub>2</sub>) for 12 h, 24 h, 36 h, and 48 h, respectively, followed by 24 h of hypoxia and serum deprivation (H/SD) injury.<h4>Results</h4>HPC dose-dependently increased the autophagy in BM-MSCs. However, the protective effects of HPC for 24 h are most pronounced. Moreover, hypoxic preconditioned BM-MSCs (<sup>HPC</sup>MSCs) and nonhypoxic preconditioned BM-MSCs (<sup>NPC</sup>MSCs) were transplanted into infarcted hearts. Longitudinal in vivo bioluminescence imaging (BLI) and immunofluorescent staining revealed that HPC enhanced the survival of engrafted BM-MSCs. Furthermore, <sup>HPC</sup>MSCs significantly reduced fibrosis, decreased apoptotic cardiomyocytes, and preserved heart function. However, the beneficial effect of HPC was abolished by autophagy inhibition with 3-methyladenine (3-MA) and Atg7siRNA.<h4>Conclusion</h4>This study demonstrates that HPC may improve the functional survival and the therapeutic efficiencies of engrafted BM-MSCs, at least in part through autophagy regulation. Hypoxic preconditioning may serve as a promising strategy for optimizing cell-based cardiac regenerative therapy.
Project description:<h4>Background</h4>The beneficial functions of bone marrow mesenchymal stem cells (BM-MSCs) decline with decreased cell survival, limiting their therapeutic efficacy for myocardial infarction (MI). Irisin, a novel myokine which is cleaved from its precursor fibronectin type III domain-containing protein 5 (FNDC5), is believed to be involved in a cardioprotective effect, but little was known on injured BM-MSCs and MI repair yet. Here, we investigated whether FNDC5 or irisin could improve the low viability of transplanted BM-MSCs and increase their therapeutic efficacy after MI.<h4>Methods</h4>BM-MSCs, isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein positive (Fluc<sup>+</sup>-eGFP<sup>+</sup>) transgenic mice, were exposed to normoxic condition and hypoxic stress for 12?h, 24?h, and 48?h, respectively. In addition, BM-MSCs were treated with irisin (20?nmol/L) and overexpression of FNDC5 (FNDC5-OV) in serum deprivation (H/SD) injury. Furthermore, BM-MSCs were engrafted into infarcted hearts with or without FNDC5-OV.<h4>Results</h4>Hypoxic stress contributed to increased apoptosis, decreased cell viability, and paracrine effects of BM-MSCs while irisin or FNDC5-OV alleviated these injuries. Longitudinal in vivo bioluminescence imaging and immunofluorescence results illustrated that BM-MSCs with overexpression of FNDC5 treatment (FNDC5-MSCs) improved the survival of transplanted BM-MSCs, which ameliorated the increased apoptosis and decreased angiogenesis of BM-MSCs in vivo. Interestingly, FNDC5-OV elevated the secretion of exosomes in BM-MSCs. Furthermore, FNDC5-MSC therapy significantly reduced fibrosis and alleviated injured heart function.<h4>Conclusions</h4>The present study indicated that irisin or FNDC5 improved BM-MSC engraftment and paracrine effects in infarcted hearts, which might provide a potential therapeutic target for MI.