Optimal technique for canine mesenchymal stem cells labeling with novel SPIO, MIRB™: for MRI detection of transplanted stem cells canine stroke model.

BACKGROUND: Cell-based therapy has emerged as a promising avenue for post-stroke recovery. A significant challenge lies in tracking the distribution and engraftment of transplanted cells within the target cerebral tissue. To address this, we turn to the potential of Brain MRI detection of mesenchymal stem cells (MSCs), achieved by labeling these cells with superparamagnetic iron oxide (SPIO). This is the first report of a technique to label canine MSCs using a commercially available SPIO, Molday ION Rhodamine B (MIRB), to optimize both viability and labeling efficacy for transplantation purposes." METHOD: Canine MSCs were incubated with addition of different MIRB concentration from 0, 10, 20, 30 µg Fe/ml. The cellular uptake of MIRB was confirmed through the analysis of fluorescent images and flow cytometry. The morphological characteristics of MSCs were assessed via microscopic visualization. Cellular viability was evaluated using both a cellometer and flow cytometry. RESULT: Fluorescent microscopic images of all MIRB incubated MSCs groups show >70% labeled cells with homogenous signal intensity. Notably, the morphology of MSCs remained unaltered in the 10 µg Fe/ml group compared to the control group. Furthermore, among the labeled groups, the 10 µg Fe/ml concentration exhibited the highest viability when assessed using two different flow cytometry methods (95.3%, p < 0.05). CONCLUSION: This study successfully labels canine MSCs with MIRB. The optimal concentration of 10 µg Fe/ml demonstrates optimal viability, labeling efficacy, and preserved cellular morphology.

Allogeneic Cell Combination Therapy Ameliorates Chronic Kidney Disease-Induced Heart Failure with Preserved Ejection Fraction.

BACKGROUND: Left ventricular hypertrophy and heart failure with preserved ejection fraction (HFpEF) are primary manifestations of the cardiorenal syndrome in patients with chronic kidney disease (CKD). Therapies that improve morbidity and mortality in HFpEF are lacking. Cell-based therapies promote cardiac repair in ischemic and non-ischemic cardiomyopathies. We hypothesized that cell-based therapy ameliorates CKD-induced HFpEF. METHODS AND RESULTS: Yorkshire pigs (n = 26) underwent 5/6 embolization-mediated nephrectomy. CKD was confirmed by increased creatinine and decreased glomerular filtration rate (GFR). Mean arterial pressure (MAP) was not different between groups from baseline to 4 weeks. HFpEF was evident at 4 weeks by increased LV mass, relative wall thickening, end-diastolic pressure, and end-diastolic pressure-volume relationship, with no change in ejection fraction (EF). Four weeks post-embolization, allogeneic (allo) bone marrow-derived mesenchymal stem cells (MSC; 1 × 107 cells), allo-kidney-derived stem cells (KSC; 1 × 107 cells), allo-cell combination therapy (ACCT; MSC + KSC; 1:1 ratio; total = 1 × 107 cells), or placebo (Plasma-Lyte) was delivered via intra-renal artery. Eight weeks post-treatment, there was a significant increase in MAP in the placebo group (21.89 ± 6.05 mmHg) compared to the ACCT group. GFR significantly improved in the ACCT group. EF, relative wall thickness, and LV mass did not differ between groups at 12 weeks. EDPVR improved in the ACCT group, indicating decreased ventricular stiffness. CONCLUSIONS: Intra-renal artery allogeneic cell therapy was safe in a CKD swine model manifesting the characteristics of HFpEF. The beneficial effect on renal function and ventricular compliance in the ACCT group supports further research of cell therapy for cardiorenal syndrome.

Comparative Effects of Bone Marrow-derived Versus Umbilical Cord Tissue Mesenchymal Stem Cells in an Experimental Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a life-threatening condition in preterm infants with few effective therapies. Mesenchymal stem or stromal cells (MSCs) are a promising therapeutic strategy for BPD. The ideal MSC source for BPD prevention is however unknown. The objective of this study was to compare the regenerative effects of MSC obtained from bone marrow (BM) and umbilical cord tissue (UCT) in an experimental BPD model. In vitro, UCT-MSC demonstrated greater proliferation and expression of anti-inflammatory cytokines as compared to BM-MSC. Lung epithelial cells incubated with UCT-MSC conditioned media (CM) had better-wound healing following scratch injury. UCT-MSC CM and BM-MSC CM had similar pro-angiogenic effects on hyperoxia-exposed pulmonary microvascular endothelial cells. In vivo, newborn rats exposed to normoxia or hyperoxia (85% O2) from postnatal day (P) 1 to 21 were given intra-tracheal (IT) BM or UCT-MSC (1 × 106 cells/50 µL), or placebo (PL) on P3. Hyperoxia PL-treated rats had marked alveolar simplification, reduced lung vascular density, pulmonary vascular remodeling, and lung inflammation. In contrast, administration of both BM-MSC and UCT-MSC significantly improved alveolar structure, lung angiogenesis, pulmonary vascular remodeling, and lung inflammation. UCT-MSC hyperoxia-exposed rats however had greater improvement in some morphometric measures of alveolarization and less lung macrophage infiltration as compared to the BM-MSC-treated group. Together, these findings suggest that BM-MSC and UCT-MSC have significant lung regenerative effects in experimental BPD but UCT-MSC suppresses lung macrophage infiltration and promotes lung epithelial cell healing to a greater degree.

The Interdisciplinary Stem Cell Institute's Use of Food and Drug Administration-Expanded Access Guidelines to Provide Experimental Cell Therapy to Patients With Rare Serious Diseases.

The U.S. Food and Drug Administration (FDA) provides guidance for expanded access to experimental therapies, which in turn plays an important role in the Twenty-first Century Cures Act mandate to advance cell-based therapy. In cases of incurable diseases where there is a lack of alternative treatment options, many patients seek access to cell-based therapies for the possibility of treatment responses demonstrated in clinical trials. Here, we describe the use of the FDA's expanded access to investigational new drug (IND) to address rare and emergency conditions that include stiff-person syndrome, spinal cord injury, traumatic brain stem injury, complex congenital heart disease, ischemic stroke, and peripheral nerve injury. We have administered both allogeneic bone marrow-derived mesenchymal stem cell (MSC) and autologous Schwann cell (SC) therapy to patients upon emergency request using Single Patient Expanded Access (SPEA) INDs approved by the FDA. In this report, we present our experience with 10 completed SPEA protocols.

A Phase II study of autologous mesenchymal stromal cells and c-kit positive cardiac cells, alone or in combination, in patients with ischaemic heart failure: the CCTRN CONCERT-HF trial.

AIMS: CONCERT-HF is an NHLBI-sponsored, double-blind, placebo-controlled, Phase II trial designed to determine whether treatment with autologous bone marrow-derived mesenchymal stromal cells (MSCs) and c-kit positive cardiac cells (CPCs), given alone or in combination, is feasible, safe, and beneficial in patients with heart failure (HF) caused by ischaemic cardiomyopathy. METHODS AND RESULTS: Patients were randomized (1:1:1:1) to transendocardial injection of MSCs combined with CPCs, MSCs alone, CPCs alone, or placebo, and followed for 12 months. Seven centres enrolled 125 participants with left ventricular ejection fraction of 28.6 ± 6.1% and scar size 19.4 ± 5.8%, in New York Heart Association class II or III. The proportion of major adverse cardiac events (MACE) was significantly decreased by CPCs alone (-22% vs. placebo, P = 0.043). Quality of life (Minnesota Living with Heart Failure Questionnaire score) was significantly improved by MSCs alone (P = 0.050) and MSCs + CPCs (P = 0.023) vs. placebo. Left ventricular ejection fraction, left ventricular volumes, scar size, 6-min walking distance, and peak oxygen consumption did not differ significantly among groups. CONCLUSIONS: This is the first multicentre trial assessing CPCs and a combination of two cell types from different tissues in HF patients. The results show that treatment is safe and feasible. Even with maximal guideline-directed therapy, both CPCs and MSCs were associated with improved clinical outcomes (MACE and quality of life, respectively) in ischaemic HF without affecting left ventricular function or structure, suggesting possible systemic or paracrine cellular mechanisms. Combining MSCs with CPCs was associated with improvement in both these outcomes. These results suggest potential important beneficial effects of CPCs and MSCs and support further investigation in HF patients.

A novel cardiomyogenic role for Isl1+ neural crest cells in the inflow tract.

The degree to which populations of cardiac progenitors (CPCs) persist in the postnatal heart remains a controversial issue in cardiobiology. To address this question, we conducted a spatiotemporally resolved analysis of CPC deployment dynamics, tracking cells expressing the pan-CPC gene Isl1 Most CPCs undergo programmed silencing during early cardiogenesis through proteasome-mediated and PRC2 (Polycomb group repressive complex 2)-mediated Isl1 repression, selectively in the outflow tract. A notable exception is a domain of cardiac neural crest cells (CNCs) in the inflow tract. These "dorsal CNCs" are regulated through a Wnt/ß-catenin/Isl1 feedback loop and generate a limited number of trabecular cardiomyocytes that undergo multiple clonal divisions during compaction, to eventually produce ~10% of the biventricular myocardium. After birth, CNCs continue to generate cardiomyocytes that, however, exhibit diminished clonal amplification dynamics. Thus, although the postnatal heart sustains cardiomyocyte-producing CNCs, their regenerative potential is likely diminished by the loss of trabeculation-like proliferative properties.

The impact of patient sex on the response to intramyocardial mesenchymal stem cell administration in patients with non-ischaemic dilated cardiomyopathy.

AIMS: Sex differences impact the occurrence, presentation, prognosis, and response to therapy in heart disease. Particularly, the phenotypic presentation of patients with non-ischaemic dilated cardiomyopathy (NIDCM) differs between men and women. However, whether the response to mesenchymal stem cell (MSC) therapy is influenced by sex remains unknown. We hypothesize that males and females with NIDCM respond similarly to MSC therapy. METHODS AND RESULTS: Male (n = 24) and female (n = 10) patients from the POSEIDON-DCM trial who received MSCs via transendocardial injections were evaluated over 12 months. Endothelial function was measured at baseline and 3 months post-transendocardial stem cell injection (TESI). At baseline, ejection fraction (EF) was lower (P = 0.004) and end-diastolic volume (EDV; P = 0.0002) and end-systolic volume (ESV; P = 0.0002) were higher in males vs. females. In contrast, baseline demographic characteristics, Minnesota Living with Heart Failure Questionnaire (MLHFQ), and 6-min walk test (6MWT) were similar between groups. EF improved in males by 6.2 units (P = 0.04) and in females by 8.6 units (P = 0.04; males vs. females, P = 0.57). EDV and ESV were unchanged over time. The MLHFQ score, New York Heart Association (NYHA) class, endothelial progenitor cell-colony forming units, and serum tumour necrosis factor alpha improved similarly in both groups. CONCLUSION: Despite major differences in phenotypic presentation of NIDCM in males and females, this study is the first of its kind to demonstrate that MSC therapy improves a variety of parameters in NIDCM irrespective of patient sex. These findings have important clinical and pathophysiologic implications regarding the impact of sex on responses to cell-based therapy for NIDCM.

Simulated Microgravity Impairs Cardiac Autonomic Neurogenesis from Neural Crest Cells.

Microgravity-induced alterations in the autonomic nervous system (ANS) contribute to derangements in both the mechanical and electrophysiological function of the cardiovascular system, leading to severe symptoms in humans following space travel. Because the ANS forms embryonically from neural crest (NC) progenitors, we hypothesized that microgravity can impair NC-derived cardiac structures. Accordingly, we conducted in vitro simulated microgravity experiments employing NC genetic lineage tracing in mice with cKitCreERT2/+, Isl1nLacZ, and Wnt1-Cre reporter alleles. Inducible fate mapping in adult mouse hearts and pluripotent stem cells (iPSCs) demonstrated reduced cKitCreERT2/+-mediated labeling of both NC-derived cardiomyocytes and autonomic neurons (P < 0.0005 vs. controls). Whole transcriptome analysis, suggested that this effect was associated with repressed cardiac NC- and upregulated mesoderm-related gene expression profiles, coupled with abnormal bone morphogenetic protein (BMP)/transforming growth factor beta (TGF-ß) and Wnt/ß-catenin signaling. To separate the manifestations of simulated microgravity on NC versus mesodermal-cardiac derivatives, we conducted Isl1nLacZ lineage analyses, which indicated an approximately 3-fold expansion (P < 0.05) in mesoderm-derived Isl-1+ pacemaker sinoatrial nodal cells; and an approximately 3-fold reduction (P < 0.05) in cardiac NC-derived ANS cells, including sympathetic nerves and Isl-1+ cardiac ganglia. Finally, NC-specific fate mapping with a Wnt1-Cre reporter iPSC model of murine NC development confirmed that simulated microgravity directly impacted the in vitro development of cardiac NC progenitors and their contribution to the sympathetic and parasympathetic innervation of the iPSC-derived myocardium. Altogether, these findings reveal an important role for gravity in the development of NCs and their postnatal derivatives, and have important therapeutic implications for human space exploration, providing insights into cellular and molecular mechanisms of microgravity-induced cardiomyopathies/channelopathies.

Allogeneic Mesenchymal Stem Cells Ameliorate Aging Frailty: A Phase II Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

BACKGROUND: Aging frailty, characterized by decreased physical and immunological functioning, is associated with stem cell depletion. Human allogeneic mesenchymal stem cells (allo-hMSCs) exert immunomodulatory effects and promote tissue repair. METHODS: This is a randomized, double-blinded, dose-finding study of intravenous allo-hMSCs (100 or 200-million [M]) vs placebo delivered to patients (n = 30, mean age 75.5 ± 7.3) with frailty. The primary endpoint was incidence of treatment-emergent serious adverse events (TE-SAEs) at 1-month postinfusion. Secondary endpoints included physical performance, patient-reported outcomes, and immune markers of frailty measured at 6 months postinfusion. RESULTS: No therapy-related TE-SAEs occurred at 1 month. Physical performance improved preferentially in the 100M-group; immunologic improvement occurred in both the 100M- and 200M-groups. The 6-minute walk test, short physical performance exam, and forced expiratory volume in 1 second improved in the 100M-group (p = .01), not in the 200M- or placebo groups. The female sexual quality of life questionnaire improved in the 100M-group (p = .03). Serum TNF-α levels decreased in the 100M-group (p = .03). B cell intracellular TNF-α improved in both the 100M- (p < .0001) and 200M-groups (p = .002) as well as between groups compared to placebo (p = .003 and p = .039, respectively). Early and late activated T-cells were also reduced by MSC therapy. CONCLUSION: Intravenous allo-hMSCs were safe in individuals with aging frailty. Treated groups had remarkable improvements in physical performance measures and inflammatory biomarkers, both of which characterize the frailty syndrome. Given the excellent safety and efficacy profiles demonstrated in this study, larger clinical trials are warranted to establish the efficacy of hMSCs in this multisystem disorder. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov: CRATUS (#NCT02065245).

Dose Comparison Study of Allogeneic Mesenchymal Stem Cells in Patients With Ischemic Cardiomyopathy (The TRIDENT Study).

RATIONALE: Cell dose and concentration play crucial roles in phenotypic responses to cell-based therapy for heart failure. OBJECTIVE: To compare the safety and efficacy of 2 doses of allogeneic bone marrow-derived human mesenchymal stem cells identically delivered in patients with ischemic cardiomyopathy. METHODS AND RESULTS: Thirty patients with ischemic cardiomyopathy received in a blinded manner either 20 million (n=15) or 100 million (n=15) allogeneic human mesenchymal stem cells via transendocardial injection (0.5 cc per injection × 10 injections per patient). Patients were followed for 12 months for safety and efficacy end points. There were no treatment-emergent serious adverse events at 30 days or treatment-related serious adverse events at 12 months. The Major Adverse Cardiac Event rate was 20.0% (95% confidence interval [CI], 6.9% to 50.0%) in 20 million and 13.3% (95% CI, 3.5% to 43.6%) in 100 million (P=0.58). Worsening heart failure rehospitalization was 20.0% (95% CI, 6.9% to 50.0%) in 20 million and 7.1% (95% CI, 1.0% to 40.9%) in 100 million (P=0.27). Whereas scar size reduced to a similar degree in both groups: 20 million by -6.4 g (interquartile range, -13.5 to -3.4 g; P=0.001) and 100 million by -6.1 g (interquartile range, -8.1 to -4.6 g; P=0.0002), the ejection fraction improved only with 100 million by 3.7 U (interquartile range, 1.1 to 6.1; P=0.04). New York Heart Association class improved at 12 months in 35.7% (95% CI, 12.7% to 64.9%) in 20 million and 42.9% (95% CI, 17.7% to 71.1%) in 100 million. Importantly, proBNP (pro-brain natriuretic peptide) increased at 12 months in 20 million by 0.32 log pg/mL (95% CI, 0.02 to 0.62; P=0.039), but not in 100 million (-0.07 log pg/mL; 95% CI, -0.36 to 0.23; P=0.65; between group P=0.07). CONCLUSIONS: Although both cell doses reduced scar size, only the 100 million dose increased ejection fraction. This study highlights the crucial role of cell dose in the responses to cell therapy. Determining optimal dose and delivery is essential to advance the field, decipher mechanism(s) of action and enhance planning of pivotal Phase III trials. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02013674.

Allogeneic Human Mesenchymal Stem Cell Infusions for Aging Frailty.

BACKGROUND: Impaired endogenous stem cell repair capacity is hypothesized to be a biologic basis of frailty. Therapies that restore regenerative capacity may therefore be beneficial. This Phase 1 study evaluated the safety and potential efficacy of intravenous, allogeneic, human mesenchymal stem cell (allo-hMSC)-based therapy in patients with aging frailty. METHODS: In this nonrandomized, dose-escalation study, patients received a single intravenous infusion of allo-hMSCs: 20-million (n = 5), 100-million (n = 5), or 200-million cells (n = 5). The primary endpoint was incidence of any treatment-emergent serious adverse events measured at 1 month postinfusion. The secondary endpoints were functional efficacy domains and inflammatory biomarkers, measured at 3 and 6 months, respectively. RESULTS: There were no treatment-emergent serious adverse events at 1-month postinfusion or significant donor-specific immune reactions during the first 6 months. There was one death at 258 days postinfusion in the 200-million group. In all treatment groups, 6-minute walk distance increased at 3 months (p = .02) and 6 months (p = .001) and TNF-α levels decreased at 6 months (p < .0001). Overall, the 100-million dose showed the best improvement in all parameters, with the exception of TNF-α, which showed an improvement in both the 100- and 200-million groups (p = .0001 and p = .0001, respectively). The 100-million cell-dose group also showed significant improvements in the physical component of the SF-36 quality of life assessment at all time points relative to baseline. CONCLUSIONS: Allo-hMSCs are safe and immunologically tolerated in aging frailty patients. Improvements in functional and immunologic status suggest that ongoing clinical development of cell-based therapy is warranted for frailty.

Randomized Comparison of Allogeneic Versus Autologous Mesenchymal Stem Cells for Nonischemic Dilated Cardiomyopathy: POSEIDON-DCM Trial.

BACKGROUND: Although human mesenchymal stem cells (hMSCs) have been tested in ischemic cardiomyopathy, few studies exist in chronic nonischemic dilated cardiomyopathy (NIDCM). OBJECTIVES: The authors conducted a randomized comparison of safety and efficacy of autologous (auto) versus allogeneic (allo) bone marrow-derived hMSCs in NIDCM. METHODS: Thirty-seven patients were randomized to either allo- or auto-hMSCs in a 1:1 ratio. Patients were recruited between December 2011 and July 2015 at the University of Miami Hospital. Patients received hMSCs (100 million) by transendocardial stem cell injection in 10 left ventricular sites. Treated patients were evaluated at baseline, 30 days, and 3-, 6-, and 12-months for safety (serious adverse events [SAE]), and efficacy endpoints: ejection fraction, Minnesota Living with Heart Failure Questionnaire, 6-min walk test, major adverse cardiac events, and immune biomarkers. RESULTS: There were no 30-day treatment-emergent SAEs. Twelve-month SAE incidence was 28.2% with allo-hMSCs versus 63.5% with auto-hMSCs (p = 0.1004 for the comparison). One allo-hMSC patient developed an elevated (>80) donor-specific calculated panel reactive antibody level. The ejection fraction increased in allo-hMSC patients by 8.0 percentage points (p = 0.004) compared with 5.4 with auto-hMSCs (p = 0.116; allo vs. auto p = 0.4887). The 6-min walk test increased with allo-hMSCs by 37.0 m (p = 0.04), but not auto-hMSCs at 7.3 m (p = 0.71; auto vs. allo p = 0.0168). MLHFQ score decreased in allo-hMSC (p = 0.0022) and auto-hMSC patients (p = 0.463; auto vs. allo p = 0.172). The major adverse cardiac event rate was lower, too, in the allo group (p = 0.0186 vs. auto). Tumor necrosis factor-α decreased (p = 0.0001 for each), to a greater extent with allo-hMSCs versus auto-hMSCs at 6 months (p = 0.05). CONCLUSIONS: These findings demonstrated safety and clinically meaningful efficacy of allo-hMSC versus auto-hMSC in NIDCM patients. Pivotal trials of allo-hMSCs are warranted based on these results. (Percutaneous Stem Cell Injection Delivery Effects on Neomyogenesis in Dilated Cardiomyopathy [PoseidonDCM]; NCT01392625).

The Effect of Gender on Mesenchymal Stem Cell (MSC) Efficacy in Neonatal Hyperoxia-Induced Lung Injury.

BACKGROUND: Mesenchymal stem cells (MSC) improve alveolar and vascular structures in experimental models of bronchopulmonary dysplasia (BPD). Female MSC secrete more anti-inflammatory and pro-angiogenic factors as compared to male MSC. Whether the therapeutic efficacy of MSC in attenuating lung injury in an experimental model of BPD is influenced by the sex of the donor MSC or recipient is unknown. Here we tested the hypothesis that female MSC would have greater lung regenerative properties than male MSC in experimental BPD and this benefit would be more evident in males. OBJECTIVE: To determine whether intra-tracheal (IT) administration of female MSC to neonatal rats with experimental BPD has more beneficial reparative effects as compared to IT male MSC. METHODS: Newborn Sprague-Dawley rats exposed to normoxia (RA) or hyperoxia (85% O2) from postnatal day (P) 2- P21 were randomly assigned to receive male or female IT bone marrow (BM)-derived green fluorescent protein (GFP+) MSC (1 x 106 cells/50 µl), or Placebo on P7. Pulmonary hypertension (PH), vascular remodeling, alveolarization, and angiogenesis were assessed at P21. PH was determined by measuring right ventricular systolic pressure (RVSP) and pulmonary vascular remodeling was evaluated by quantifying the percentage of muscularized peripheral pulmonary vessels. Alveolarization was evaluated by measuring mean linear intercept (MLI) and radial alveolar count (RAC). Angiogenesis was determined by measuring vascular density. Data are expressed as mean ± SD, and analyzed by ANOVA. RESULTS: There were no significant differences in the RA groups. Exposure to hyperoxia resulted in a decrease in vascular density and RAC, with a significant increase in MLI, RVSP, and the percentage of partially and fully muscularized pulmonary arterioles. Administration of both male and female MSC significantly improved vascular density, alveolarization, RVSP, percent of muscularized vessels and alveolarization. Interestingly, the improvement in PH and vascular remodeling was more robust in the hyperoxic rodents who received MSC from female donors. In keeping with our hypothesis, male animals receiving female MSC, had a greater improvement in vascular remodeling. This was accompanied by a more significant decrease in lung pro-inflammatory markers and a larger increase in anti-inflammatory and pro-angiogenic markers in male rodents that received female MSC. There were no significant differences in MSC engraftment among groups. CONCLUSIONS: Female BM-derived MSC have greater therapeutic efficacy than male MSC in reducing neonatal hyperoxia-induced lung inflammation and vascular remodeling. Furthermore, the beneficial effects of female MSC were more pronounced in male animals. Together, these findings suggest that female MSC maybe the most potent BM-derived MSC population for lung repair in severe BPD complicated by PH.

Physiological and hypoxic oxygen concentration differentially regulates human c-Kit+ cardiac stem cell proliferation and migration.

Cardiac stem cells (CSCs) are being evaluated for their efficacy in the treatment of heart failure. However, numerous factors impair the exogenously delivered cells' regenerative capabilities. Hypoxia is one stress that contributes to inadequate tissue repair. Here, we tested the hypothesis that hypoxia impairs cell proliferation, survival, and migration of human CSCs relative to physiological and room air oxygen concentrations. Human endomyocardial biopsy-derived CSCs were isolated, selected for c-Kit expression, and expanded in vitro at room air (21% O2). To assess the effect on proliferation, survival, and migration, CSCs were transferred to physiological (5%) or hypoxic (0.5%) O2 concentrations. Physiological O2 levels increased proliferation (P < 0.05) but did not affect survival of CSCs. Although similar growth rates were observed in room air and hypoxia, a significant reduction of ß-galactosidase activity (-4,203 fluorescent units, P < 0.05), p16 protein expression (0.58-fold, P < 0.001), and mitochondrial content (0.18-fold, P < 0.001) in hypoxia suggests that transition from high (21%) to low (0.5%) O2 reduces senescence and promotes quiescence. Furthermore, physiological O2 levels increased migration (P < 0.05) compared with room air and hypoxia, and treatment with mesenchymal stem cell-conditioned media rescued CSC migration under hypoxia to levels comparable to physiological O2 migration (2-fold, P < 0.05 relative to CSC media control). Our finding that physiological O2 concentration is optimal for in vitro parameters of CSC biology suggests that standard room air may diminish cell regenerative potential. This study provides novel insights into the modulatory effects of O2 concentration on CSC biology and has important implications for refining stem cell therapies.

Stimulatory Effects of Mesenchymal Stem Cells on cKit+ Cardiac Stem Cells Are Mediated by SDF1/CXCR4 and SCF/cKit Signaling Pathways.

RATIONALE: Culture-expanded cells originating from cardiac tissue that express the cell surface receptor cKit are undergoing clinical testing as a cell source for heart failure and congenital heart disease. Although accumulating data support that mesenchymal stem cells (MSCs) enhance the efficacy of cardiac cKit(+) cells (CSCs), the underlying mechanism for this synergistic effect remains incompletely understood. OBJECTIVE: To test the hypothesis that MSCs stimulate endogenous CSCs to proliferate, migrate, and differentiate via the SDF1/CXCR4 and stem cell factor/cKit pathways. METHODS AND RESULTS: Using genetic lineage-tracing approaches, we show that in the postnatal murine heart, cKit(+) cells proliferate, migrate, and form cardiomyocytes, but not endothelial cells. CSCs exhibit marked chemotactic and proliferative responses when cocultured with MSCs but not with cardiac stromal cells. Antagonism of the CXCR4 pathway with AMD3100 (an SDF1/CXCR4 antagonist) inhibited MSC-induced CSC chemotaxis but stimulated CSC cardiomyogenesis (P<0.0001). Furthermore, MSCs enhanced CSC proliferation via the stem cell factor/cKit and SDF1/CXCR4 pathways (P<0.0001). CONCLUSIONS: Together these findings show that MSCs exhibit profound, yet differential, effects on CSC migration, proliferation, and differentiation and suggest a mechanism underlying the improved cardiac regeneration associated with combination therapy using CSCs and MSCs. These findings have important therapeutic implications for cell-based therapy strategies that use mixtures of CSCs and MSCs.

Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus, from kefir with possible biotechnological interest.

Two thermophilic extracellular proteases, designated Lmm-protease-Lh ( approximately 29 kDa) and Hmm-protease-Lh ( approximately 62 kDa), were purified from the Lactobacillus helveticus from kefir, and found active in media containing dithiothreitol; the activity of Lmm-protease-Lh was increased significantly in media containing also EDTAK(2). Both novel proteases maintained full activity at 60 degrees C after 1-h incubation at 10 degrees C as well as at 80 degrees C, showing optimum k(cat)/K(m) values at pH 7.00 and 60 degrees C. Only irreversible inhibitors specific for cysteine proteinases strongly inhibited the activity of both novel enzymes, while they remained unaffected by irreversible inhibitors specific for serine proteinases. Both enzymes hydrolyzed the substrate Suc-FR-pNA via Michaelis-Menten kinetics; conversely, the substrate Cbz-FR-pNA was hydrolyzed by Lmm-protease-Lh via Michaelis-Menten kinetics and by Hmm-protease-Lh via substrate inhibition kinetics. Valuable rate constants and activation energies were estimated from the temperature-(k(cat)/K(m)) profiles of both enzymes, and useful results were obtained from the effect of different metallic ions on their Michaelis-Menten parameters.

Proton inventories constitute reliable tools in investigating enzymatic mechanisms: application on a novel thermo-stable extracellular protease from a halo-alkalophilic Bacillus sp.

A novel protease designated protease-A-17N-1, was purified from the halo-alkalophilic Bacillus sp. 17N-1, and found active in media containing dithiothreitol and EDTAK(2). This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum k(cat)/K(m) value at pH 7.50 and 33 degrees C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(k(cat)/K(m)) profile of protease-A-17N-1 was described by three pK(a)s in the acid limb, and one in the alkaline limb. Both are more likely due t3o the protonic dissociation of an acidic residue, and the development and subsequent deprotonation of an ion-pair, respectively, in its catalytic site, characteristic for cysteine proteinases. Moreover, both the obtained estimates of rate constant k(1) and the ratio k(2)/k(-1) at 25 degrees C, from the temperature-(k(cat)/K(m)) profile of protease-A-17N-1, were found similar to those estimated from the proton inventories of the same parameter, verifying the reliability of the latter methodology. Besides, the bowed-downward proton inventories of k(cat)/K(m), as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that k(cat)/K(m) = k(1). Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.