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Tracking fusion of human mesenchymal stem cells after transplantation to the heart.

ABSTRACT:

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Evidence suggests that transplanted mesenchymal stem cells (MSCs) can aid recovery of damaged myocardium caused by myocardial infarction. One possible mechanism for MSC-mediated recovery is reprogramming after cell fusion between transplanted MSCs and recipient cardiac cells. We used a Cre/LoxP-based luciferase reporter system coupled to biophotonic imaging to detect fusion of transplanted human pluripotent stem cell-derived MSCs to cells of organs of living mice. Human MSCs, with transient expression of a viral fusogen, were delivered to the murine heart via a collagen patch. At 2 days and 1 week later, living mice were probed for bioluminescence indicative of cell fusion. Cell fusion was detected at the site of delivery (heart) and in distal tissues (i.e., stomach, small intestine, liver). Fusion was confirmed at the cellular scale via fluorescence in situ hybridization for human-specific and mouse-specific centromeres. Human cells in organs distal to the heart were typically located near the vasculature, suggesting MSCs and perhaps MSC fusion products have the ability to migrate via the circulatory system to distal organs and engraft with local cells. The present study reveals previously unknown migratory patterns of delivered human MSCs and associated fusion products in the healthy murine heart. The study also sets the stage for follow-on studies to determine the functional effects of cell fusion in a model of myocardial damage or disease.

Significance

Mesenchymal stem cells (MSCs) are transplanted to the heart, cartilage, and other tissues to recover lost function or at least limit overactive immune responses. Analysis of tissues after MSC transplantation shows evidence of fusion between MSCs and the cells of the recipient. To date, the biologic implications of cell fusion remain unclear. A newly developed in vivo tracking system was used to identify MSC fusion products in living mice. The migratory patterns of fusion products were determined both in the target organ (i.e., the heart) and in distal organs. This study shows, for the first time, evidence of fusion products at sites distal from the target organ and data to suggest that migration occurs via the vasculature. These results will inform and improve future, MSC-based therapeutics.

SUBMITTER: Freeman BT

PROVIDER: S-EPMC4449091 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Human mesenchymal stem cell (hMSC) delivery has demonstrated promise in preclinical and clinical trials for myocardial infarction therapy; however, broad acceptance is hindered by limited understanding of hMSC-human cardiomyocyte (hCM) interactions. To better understand the electrophysiological consequences of direct heterocellular connections between hMSCs and hCMs, three original mathematical models were developed, representing an experimentally verified triad of hMSC families with distinct functional ion channel currents. The arrhythmogenic risk of such direct electrical interactions in the setting of healthy adult myocardium was predicted by coupling and fusing these hMSC models to the published ten Tusscher midcardial hCM model. Substantial variations in action potential waveform-such as decreased action potential duration (APD) and plateau height-were found when hCMs were coupled to the two hMSC models expressing functional delayed rectifier-like human ether à-go-go K+ channel 1 (hEAG1); the effects were exacerbated for fused hMSC-hCM hybrid cells. The third family of hMSCs (Type C), absent of hEAG1 activity, led to smaller single-cell action potential alterations during coupling and fusion, translating to longer tissue-level mean action potential wavelength. In a simulated 2-D monolayer of cardiac tissue, re-entry vulnerability with low (5%) hMSC insertion was approximately eight-fold lower with Type C hMSCs compared to hEAG1-functional hMSCs. A 20% decrease in APD dispersion by Type C hMSCs compared to hEAG1-active hMSCs supports the claim of reduced arrhythmogenic potential of this cell type with low hMSC insertion. However, at moderate (15%) and high (25%) hMSC insertion, the vulnerable window increased independent of hMSC type. In summary, this study provides novel electrophysiological models of hMSCs, predicts possible arrhythmogenic effects of hMSCs when directly coupled to healthy hCMs, and proposes that isolating a subset of hMSCs absent of hEAG1 activity may offer increased safety as a cell delivery cardiotherapy at low levels of hMSC-hCM coupling.

Project description:Mesenchymal stem cells (MSCs) are promising cells for cardiovascular regenerative medicine. However, their potential may be limited, because of their restricted cardiovascular differentiation potential and decline in their number and functional characteristics with increasing donor age. We have previously shown that rat fetus heart harbors primitive MSCs and administration of these cells improved left ventricular (LV) function after ischemia/reperfusion injury in rats. To evaluate their potential as a new cell type for clinical cardiovascular cell therapy, we have undertaken this study on the isolation and characterization of human fetal cardiac MSCs (hfC-MSCs).MSCs were isolated from the heart of five 14-16-week-old aborted human fetuses and studied for their growth characteristics, karyotypic stability and senescence over successive passages, expression of mesenchymal and embryonal markers by flow cytometry and immunocytochemistry, constitutive expression of cardiovascular genes by RT-PCR, differentiation into cells of the cardiovascular lineage and their immunomodulatory properties.The hfC-MSCs grew as adherent monolayer with spindle shaped morphology and exhibited rapid proliferation with an average population doubling time of 34 hours and expansion to up to more than 80 population doublings with maintenance of a normal karyotype and without senescence. Immunophenotyping showed that they had similar phenotype as human bone marrow mesenchymal stem cells (hBM-MSCs) expressing CD73, CD90, CD105 and lacking expression of CD31, CD34, CD45, HLA-DR. However, hfC-MSCs expressed significantly higher levels of CD117 and SSEA-4 compared to hBM-MSCs. In addition, hfC-MSCs expressed the embryonal markers Oct-4, Nanog and Sox-2 as compared to hBM-MSCs. Further, hfC-MSCs had significantly higher expression of the cardiovascular genes viz. ISL-1, flk-1, GATA-4, NKX2.5 and MDR-1 as compared to hBM-MSCs, and could be differentiated into major cardiovascular cells (cardiomyocytes, endothelial cells, smooth muscle cells). Interestingly, hfC-MSCs markedly reduced T-lymphocyte proliferation with an increased secretion of TGF-? and IL-10.Our results show that human fetus heart is a novel source of primitive MSCs with cardiovascular commitment which may have a potential therapeutic application in cardiovascular regenerative medicine.

Project description:Bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promise in in vitro neuronal differentiation and in cellular therapy for neurodegenerative disorders, including Parkinson' disease. However, the effects of intracerebral transplantation are not well defined, and studies do not agreed on the optimal neuronal differentiation method. Here, we investigated three growth factor-based neuronal differentiation procedures (using FGF-2/EGF/PDGF/SHH/FGF-8/GDNF), and found all to be capable of eliciting an immature neural phenotype, in terms of cell morphology and gene/protein expression. The neuronal-priming (FGF-2/EGF) method induced neurosphere-like formation and the highest NES and NR4A2 expression by hMSCs. Transplantation of undifferentiated and neuronal-primed hMSCs into the striatum and substantia nigra of 6-OHDA-lesioned hemiparkinsonian rats revealed transient graft survival of 7 days, despite the reported immunosuppressive properties of MSCs and cyclosporine-immunosuppression of rats. Neither differentiation of hMSCs nor induction of host neurogenesis was observed at injection sites, and hMSCs continued producing mesodermal fibronectin. Strategies for improving engraftment and differentiation post-transplantation, such as prior in vitro neuronal-priming, nigral and striatal grafting, and co-transplantation of olfactory ensheathing cells that promote neural regeneration, were unable to provide advantages. Innate inflammatory responses (Iba-1-positive microglia/macrophage and GFAP-positive astrocyte activation and accumulation) were detected around grafts within 7 days. Our findings indicate that growth factor-based methods allow hMSC differentiation toward immature neuronal-like cells, and contrary to previous reports, only transient survival and engraftment of hMSCs occurs following transplantation in immunosuppressed hemiparkinsonian rats. In addition, suppression of host innate inflammatory responses may be a key factor for improving hMSC survival and engraftment.

Project description:BACKGROUND:Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. METHODS:A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n?=?10), MSCs (MSCT, n =?10), a combination of SSCs and MSCs (MS-SSCT, n?=?10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n?=?10). RESULTS:The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P?<?0.001). TFI after MSi-SSCT (83.3?±?19.5%) was significantly higher compared to MS-SSCT (71.5?±?21.7%, P?=?0.036) but did not differ statistically compared to SSCT (78.2?±?12.5%). In contrast, TFI after MSCT (50.2?±?22.5%) was significantly lower compared to SSCT (P?<?0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8?±?8.0%) compared to SSCT (1.9?±?1.1%; P?<?0.001), MSCT (0.0?±?0.0%; P?<?0.001), and MS-SSCT (3.4?±?1.9%; P?<?0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs. CONCLUSION:Co-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.

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Tracking fusion of human mesenchymal stem cells after transplantation to the heart.

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