Background The use of non-autologous stem cells isolated from healthy donors for stem cell therapy is an attractive approach since the stem cells can be culture expanded in advance, thoroughly tested, and formulated into off-the-shelf medicine. However, HLA compatibility and related immunosuppressive protocols can compromise therapeutic efficacy and cause unwanted side effects. Methods Mesenchymal stem cells (MSCs) have been postulated to possess unique immune regulatory function. We explored the immunomodulatory property of human and porcine MSCs for the treatment of δ-sarcoglycan-deficient dystrophic hamster muscle without immunosuppression. Circulating as well as tissue markers of inflammation were analyzed. Muscle regeneration and stem cell fate were characterized. Results Total white blood cell counts and leukocyte distribution profiles were similar among the saline- and MSC-injected dystrophic hamsters one month post treatment. Circulating levels of immunoglobulin A, vascular cell adhesion molecule-1, myeloperoxidase, and major cytokines involved in inflammatory response were not elevated by MSCs, nor were expression of the leukocyte common antigen CD45 and the cytokine transcriptional activator NF-κB in the injected muscle. Treated muscles exhibited increased cell cycle activity and attenuated oxidative stress. Injected MSCs were found to be trapped in the musculature, and contribute to both preexisting and new muscle fibers, and mediates capillary formation. Conclusions Intramuscular injection of non-autologous MSCs can be safely used for the treatment of dystrophic muscle in immunocompetent hosts without inflaming the host immune system.

Therapeutic implantation of mesenchymal stem cells (MSCs) is entering the realm of clinical trials for several human diseases, and yet much remains uncertain regarding their dynamic distribution and cell fate after in vivo application. Discrepancies in the literature can be attributed in part to the use of different cell labeling/tracking methods and cell administration protocols. To identify a stem cell detection method suitable for myocardial implantation in a large animal model, we experimented on three different MSC labeling methods: adenovirus-mediated expression of enhanced green fluorescence protein (EGFP) and β-galactosidase (LacZ), and nuclear staining with DAPI. Intramuscular and intracoronary administrations of labeled porcine MSCs identified the nuclear affinity dye to be a reliable stem cell tracking marker. Stem cell identification is facilitated by an optimized live cell labeling condition generating bright blue fluorescence sharply confined to the nucleus. DAPI-labeled MSCs retained full viability, ceased proliferation, and exhibited an increased differentiation potential. The labeled MSCs remained fully active in expressing key growth factor and cytokine genes, and notably exhibited enhanced expression of the chemokine receptor CXCR4 and its ligand SDF1, indicating their competency in response to tissue injury. Histological analysis revealed that approximately half a million MSCs or ϳ2% of the administered MSCs remained localized in the normal pig heart 2 weeks after coronary infusion. That the vast majority of these identified MSCs were interstitial indicated the ability of MSCs to migrate across the coronary endothelium. No evidence was obtained indicating MSC differentiation to cardiomyocyte.Key words: Mesenchymal stem cells; Heart; Tracking; Implantation INTRODUCTIONfusion, and intercellular communication through GAP junctions or nanotubes have been proposed to account for the seeming pluripotency of adult stem cells in variStem cell therapy has in recent years received much attention in the field of regenerative medicine. Theraous implantation studies (26,32,50,53,62). Technically, these discrepancies may be caused in part by the use of peutic implantation of mesenchymal stem cells (MSCs), in particular, has been attempted in several clinical trials different cell labeling/tracking methods, cell administration protocols, timing of cell transplantation after tissue for hematological disorders, cardiovascular diseases, osteogenesis imperfecta, neurological pathologies, and even injury, and possibly misinterpretation of microscopy results (4). cancer therapy (9,14,23). The ease of isolation, capacity for large-scale expansion, and alleged immunoprivileged Many cell labeling techniques have been used for tracking implanted stem cells in vivo. Fluorescent quanstatus of MSCs (3,21,29) are likely to generate more clinical applications in the future. Central to these efforts tum dots and iridium nanoparticles are randomly internalized by cells during culturing (13,41). Membrane is a thorough unders...

BackgroundCardiosphere-derived cells (CDCs) improve ventricular function and reduce fibrotic volume when administered via an infarct-related artery using the “stop-flow” technique. Unfortunately, myocyte loss and dysfunction occur globally in many patients with ischemic and non-ischemic cardiomyopathy, necessitating an approach to distribute CDCs throughout the entire heart. We therefore determined whether global intracoronary infusion of CDCs under continuous flow improves contractile function and stimulates new myocyte formation.Methods and ResultsSwine with hibernating myocardium from a chronic LAD occlusion were studied 3-months after instrumentation (n = 25). CDCs isolated from myocardial biopsies were infused into each major coronary artery (∼33×106 icCDCs). Global icCDC infusion was safe and while ∼3% of injected CDCs were retained, they did not affect ventricular function or myocyte proliferation in normal animals. In contrast, four-weeks after icCDCs were administered to animals with hibernating myocardium, %LADWT increased from 23±6 to 51±5% (p<0.01). In diseased hearts, myocyte proliferation (phospho-histone-H3) increased in hibernating and remote regions with a concomitant increase in myocyte nuclear density. These effects were accompanied by reductions in myocyte diameter consistent with new myocyte formation. Only rare myocytes arose from sex-mismatched donor CDCs.ConclusionsGlobal icCDC infusion under continuous flow is feasible and improves contractile function, regresses myocyte cellular hypertrophy and increases myocyte proliferation in diseased but not normal hearts. New myocytes arising via differentiation of injected cells are rare, implicating stimulation of endogenous myocyte regeneration as the primary mechanism of repair.

Rationale Allogeneic bone marrow-derived mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs) have each entered clinical trials but a direct comparison of these cell types has not been performed in a large animal model of hibernating myocardium. Objective Using completely blinded methodology, compare the efficacy of global intracoronary allogeneic MSCs (icMSCs, ~35×106) and CDCs (icCDCs, ~35×106) vs. vehicle in cyclosporine-immunosuppressed swine with a chronic LAD stenosis (n=26). Methods and Results Studies began 3-months after instrumentation when wall-thickening (%WT) was reduced (LAD%WT 38±11% (mean ± SD) vs. 83±26% in remote, p<0.01) and similar among groups. Four-weeks after treatment, LAD%WT increased similarly following icCDCs and icMSCs, while it remained depressed in vehicle-treated controls (icMSCs: 51±13%; icCDCs: 51±17%; vehicle: 34±3%, treatments p<0.05 vs. vehicle). There was no change in myocardial perfusion. Both icMSCs and icCDCs increased LAD myocyte nuclear density (icMSCs: 1601±279 nuclei/mm2, icCDCs: 1569±294 nuclei/mm2, vehicle: 973±181 nuclei/mm2, treatments p<0.05 vs. vehicle) and reduced myocyte diameter (icMSCs: 16.4±1.5 μm, icCDCs: 16.8±1.2 μm, vehicle: 20.2±3.7 μm, treatments p<0.05 vs. vehicle) to the same extent. Similar changes in myocyte nuclear density and diameter were observed in the remote region of cell-treated animals. Cell fate analysis using Y-FISH demonstrated rare cells from sex-mismatched donors. Conclusions Allogeneic icMSCs and icCDCs exhibit comparable therapeutic efficacy in a large animal model of hibernating myocardium. Both cell types produced equivalent increases in regional function and stimulated myocyte regeneration in ischemic and remote myocardium. The activation of endogenous myocyte proliferation and regression of myocyte cellular hypertrophy support a common mechanism of cardiac repair.

Sarcopenia and frailty are highly prevalent in older individuals, increasing the risk of disability and loss of independence. High intensity interval training (HIIT) may provide a robust intervention for both sarcopenia and frailty by achieving both strength and endurance benefits with lower time commitments than other exercise regimens. To better understand the impacts of HIIT during aging, we compared 24-month-old C57BL/6J sedentary mice with those that were administered 10-minute uphill treadmill HIIT sessions three times per week over 16 weeks. Baseline and end point assessments included body composition, physical performance, and frailty based on criteria from the Fried physical frailty scale. HIIT-trained mice demonstrated dramatic improvement in grip strength (HIIT 10.9% vs -3.9% in sedentary mice), treadmill endurance (32.6% vs -2.0%), and gait speed (107.0% vs 39.0%). Muscles from HIIT mice also exhibited greater mass, larger fiber size, and an increase in mitochondrial biomass. Furthermore, HIIT exercise led to a dramatic reduction in frailty scores in five of six mice that were frail or prefrail at baseline, with four ultimately becoming nonfrail. The uphill treadmill HIIT exercise sessions were well tolerated by aged mice and led to performance gains, improvement in underlying muscle physiology, and reduction in frailty.

Vitamin D insufficiency (serum 25-OH vitamin D < 30 ng/ml) affects 70-80% of the general population, yet the long-term impacts on physical performance and the progression of sarcopenia are poorly understood. We therefore followed 6-month-old male C57BL/6J mice (n=6) consuming either sufficient (STD, 1000 IU) or insufficient (LOW, 125 IU) vitamin D3/kg chow for 12 months (equivalent to 20-30 human years). LOW supplemented mice exhibited a rapid decline of serum 25-OH vitamin D levels by two weeks that remained between 11-15 ng/mL for all time points thereafter. After 12 months LOW mice displayed worse grip endurance (34.6 ± 14.1 versus 147.5 ± 50.6 seconds, p=0.001), uphill sprint speed (16.0 ± 1.0 versus 21.8 ± 2.4 meters/min, p=0.0007), and stride length (4.4 ± 0.3 versus 5.1 ± 0.3, p=0.002). LOW mice also showed less lean body mass after 8 months (57.5% ± 5.1% versus 64.5% ± 4.0%, p=0.023), but not after 12 months of supplementation, as well as greater protein expression of atrophy pathway gene atrogin‑1. Additionally, microRNA sequencing revealed differential expression of mIR‑26a in muscle tissue of LOW mice. These data suggest chronic vitamin D insufficiency may be an important factor contributing to functional decline and sarcopenia.

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A major problem in translating stem cell therapeutics is the difficulty of producing stable, long-term severe left ventricular (LV) dysfunction in a large animal model. For that purpose, extensive infarction was created in sinclair miniswine by injecting microspheres (1.5 × 106 microspheres, 45 μm diameter) in LAD. At 2 months after embolization, animals (n = 11) were randomized to receive allogeneic cardiosphere-derived cells derived from atrium (CDCs: 20 × 106, n = 5) or saline (untreated, n = 6). Four weeks after therapy myocardial function, myocyte proliferation (Ki67), mitosis (phosphor-Histone H3; pHH3), apoptosis, infarct size (TTC), myocyte nuclear density, and cell size were evaluated. CDCs injected into infarcted and remodeled remote myocardium (global infusion) increased regional function and global function contrasting no change in untreated animals. CDCs reduced infarct volume and stimulated Ki67 and pHH3 positive myocytes in infarct and remote regions. As a result, myocyte number (nuclear density) increased and myocyte cell diameter decreased in both infarct and remote regions. Coronary microembolization produces stable long-term ischemic cardiomyopathy. Global infusion of CDCs stimulates myocyte regeneration and improves left ventricular ejection fraction. Thus, global infusion of CDCs could become a new therapy to reverse LV dysfunction in patients with asymptomatic heart failure.