Bone Marrow-derived Mononuclear Cells Treatment Research Articles

Long-Term Outcomes of BMMSC Compared with BMMNC for Treatment of Critical Limb Ischemia and Foot Ulcer in Patients with Diabetes

We first compared long-term clinical outcomes in treating critical limb ischemia (CLI) and foot ulcer in patients with diabetes between autologous bone marrow mesenchymal stem cell (BMMSC) and bone-marrow-derived mononuclear cell (BMMNC) transplants. Forty-one patients were enrolled and followed up for 3 years. They received an 18-day standard treatment before stem cell transplantation. Patients with bilateral CLI and foot ulcer were injected intramuscularly or basally with BMMSC, BMMNC, or normal saline (NS). Cox model analysis showed significant differences in the hazard ratio (HR) for amputation with treatment by BMMSC (HR 0.21 [95% CI (0.05, 0.95)], P = 0.043), infection of foot (HR 5.30 [95% CI (1.89, 14.92)], P = 0.002), and age ≥64 (HR 3.01 [95% CI (1.11, 8.15)], P = 0.030), but no significant differences by BMMNC at 9 months after transplantation. Regarding ulcer healing and recurrence rate, the BMMSC group demonstrated a significant difference from the NS group during the 3–6 months after transplantation or healing, but the BMMNC group did not. This trial suggests that, compared with BMMNC treatment, BMMSC treatment leads to a longer time of limb salvage and blood flow improvement, and, when compared with conventional therapy, it can promote limb blood flow and ulcerative healing, and reduce ulcer recurrence and amputation within 9 months.

GW28-e0590 Effect of bone marrow-derived mononuclear cell treatment in myocardial infarction

GW28-e0590 Effect of bone marrow-derived mononuclear cell treatment in myocardial infarction

Effect of Bone Marrow-Derived Mononuclear Cell Treatment, Early or Late After Acute Myocardial Infarction: Twelve Months CMR and Long-Term Clinical Results.

Intracoronary delivery of autologous bone marrow-derived mononuclear cells (BM-MNC) may improve remodeling of the left ventricle (LV) after acute myocardial infarction (AMI). To demonstrate long-term efficacy of BM-MNC treatment after AMI. In a multicenter study, we randomized 200 patients with large AMI in a 1:1:1 pattern into an open-labeled control and 2 BM-MNC treatment groups. In the BM-MNC groups, cells were either administered 5 to 7 days (early) or 3 to 4 weeks (late) after AMI. Cardiac magnetic resonance imaging was performed at baseline and after 12 months. The current analysis investigates the change from baseline to 12 months in global LV ejection fraction, LV volumes, scar size, and N-terminal pro-brain natriuretic peptide values comparing the 2 treatment groups with control in a linear regression model. Besides the complete case analysis, multiple imputation analysis was performed to address for missing data. Furthermore, the long-term clinical event rate was computed. The absolute change in LV ejection fraction from baseline to 12 months was -1.9±9.8% for control (mean±SD), -0.9±10.5% for the early treatment group, and -0.7±10.1% for the late treatment group. The difference between the groups was not significant, both for complete case analysis and multiple imputation analysis. A combined clinical end point occurred equally in all the groups. Overall, 1-year mortality was low (2.25%). Among patients with AMI and LV dysfunction, treatment with BM-MNC either 5 to 7 days or 3 to 4 weeks after AMI did not improve LV function at 12 months, compared with control. The results are limited by an important drop out rate. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00355186.

Systematic Review and Meta-Analysis of Bone Marrow-Derived Mononuclear Cells in Animal Models of Ischemic Stroke.

Bone marrow-derived mononuclear cells (BMMNCs) offer the promise of augmenting poststroke recovery. There is mounting evidence of safety and efficacy of BMMNCs from preclinical studies of ischemic stroke; however, their pooled effects have not been described. Using Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines, we conducted a systematic review of preclinical literature for intravenous use of BMMNCs followed by meta-analyses of histological and behavioral outcomes. Studies were selected based on predefined criteria. Data were abstracted by 2 independent investigators. After quality assessment, the pooled effects were generated using mixed-effect models. Impact of possible biases on estimated effect size was evaluated. Standardized mean difference and 95% confidence interval for reduction in lesion volume was significantly beneficial for BMMNC treatment (standardized mean difference: -3.3; 95% confidence interval, -4.3 to -2.3). n=113 each for BMMNC and controls. BMMNC-treated animals (n=161) also had improved function measured by cylinder test (standardized mean difference: -2.4; 95% confidence interval, -3.1 to -1.6), as compared with controls (n=205). A trend for benefit was observed for adhesive removal test and neurological deficit score. Study quality score (median: 6; Q1-Q3: 5-7) was correlated with year of publication. There was funnel plot asymmetry; however, the pooled effects were robust to the correction of this bias and remained significant in favor of BMMNC treatment. BMMNCs demonstrate beneficial effects across histological and behavioral outcomes in animal ischemic stroke models. Although study quality has improved over time, considerable degree of heterogeneity calls for standardization in the conduct and reporting of experimentation.

Abstract 17510: The First Prediction Model for Response to Autologous Bone Marrow-derived Mononuclear Cells After Myocardial Infarction Based on the REPAIR-AMI Trial

Background: Administration of autologous bone marrow-derived mononuclear cells (BM-MNC) resulted in favorable outcomes after myocardial infarction (MI) compared to placebo in the REPAIR-AMI trial. Pre-clinical studies have shown that individual risk factors negatively influence cell function. To date, it is not known how these risk factors modify treatment effect in clinical trials and if potential (non-)responders can be identified. Aim: To investigate the effect of individual risk factors on functional outcome after BM-MNC therapy and to establish a prediction model for treatment response in patients with MI. Methods: Data from the REPAIR-AMI trial were used, consisting of 186 patients who had complete baseline and follow-up measurements. We performed univariable and multivariable linear regression with 18 predefined baseline characteristics using the difference in baseline EF and after 4 months (ΔEF4) as primary outcome. Our main goal was to identify interaction terms of predictors with BM-MNC treatment; i.e. effect modifiers of treatment response. An individual estimate of treatment effect over placebo (ΔΔEF4) was created by extrapolating an ‘untreated’ reference value for ΔEF4, based on the placebo-arm and comparing it to the observed value after treatment. Treatment response was defined as a ΔΔEF4 of >5% (so 5% over a predicted placebo value). Discrimination was quantified by the area under the ROC-curve (AUC). Results: The predictors age, weight, baseline EF and ESV showed an interaction with cell treatment in multivariable analysis with backward selection. Subsequently, the prediction model for individual ΔΔEF4 included age (-0.18/year, p=0.008), weight (+0.16/kg, p=0.01), baseline EF (-0.46/%, p=0.0001) and ESV (-0.09/ml, p=0.06). The predictive capacity for response to therapy showed an AUC of 79.2% (95% CI 69.7-88.6), which retained 74.8% (95% CI 65.0-84.5) after shrinkage. Conclusion: Response to BM-MNC administration after MI is influenced by age, weight, and baseline cardiac parameters. Using these continuous predictors, potential treatment responders can be accurately identified. Interestingly, an adverse risk-factor profile was associated with greater response, with potential implications for future patient selection.

Granulocyte colony-stimulating factor (G-CSF) treatment in combination with transplantation of bone marrow cells is not superior to G-CSF treatment alone after cortical stroke in spontaneously hypertensive rats.

Granulocyte-colony stimulating factor (G-CSF) and bone marrow derived mononuclear cells (BM-MNCs) have both been shown to improve functional outcome following experimental stroke. These effects are associated with increased angiogenesis and neurogenesis. In the present study, we aimed to determine synergistic effects of G-CSF and BM-NMC treatment on long-term structural and functional recovery after photothrombotic stroke. To model the etiology of stroke more closely, we used spontaneously hypertensive (SH) rats in our experiment. Bone marrow derived mononuclear cells transplantation was initiated 1 h after the onset of photothrombotic stroke. Repeated G-CSF treatment commenced immediately after BM-MNC treatment followed by daily injections for five consecutive days. The primary endpoint was functional outcome after ischemia. Secondary endpoints included analysis of neurogenesis and angiogenesis as well as determination of infarct size. Granulocyte-colony stimulating factor treated rats, either in combination with BM-MNC or alone showed improved somatosensory but not gross motor function following ischemia. No beneficial effect of BM-MNC monotherapy was found. Infarct volumes were comparable in all groups. In contrast to previous studies, which used healthy animals, post-stroke neurogenesis and angiogenesis were not enhanced by G-CSF. In conclusion, the combination of G-CSF and BM-MNC was not more effective than G-CSF alone. The reduced efficacy of G-CSF treatment and the absence of any beneficial effect of BM-MNC transplantation might be attributed to hypertension-related morbidity.

Bone marrow-derived mononuclear cells do not exert acute neuroprotection after stroke in spontaneously hypertensive rats

Bone marrow-derived mononuclear cells (BM-MNCs) were shown to improve the outcome in animal stroke models and clinical pilot studies on BM-MNCs for stroke patients were already conducted. However, relevant aspects of pre-clinical evaluation, such as the use of animals with comorbidities and dose-response studies, were not thoroughly addressed so far. We therefore investigated different BM-MNC doses in the clinical meaningful stroke model of spontaneously hypertensive (SH) rats. Three hours after the onset of transient middle cerebral artery occlusion (MCAO) animals received either one of three syngeneic BM-MNC doses or placebo intravenously. The primary endpoint was the infarct size. Secondary endpoints included functional outcome, mortality, inflammatory processes, and the dose-response relationship. In contrast to previous studies which used healthy animals no beneficial effect of BM-MNCs was found. Infarct volumes, mortality, behavioral outcomes, and the extent of the inflammatory response to cerebral ischemia were comparable in all groups. In conclusion, we could not demonstrate that early BM-MNC treatment improves the outcome after stroke in SH rats. Whether BM-MNCs improve neurological recovery after delayed treatment initiation was not investigated in the present study, but our data indicates that this should be determined in co-morbid animal stroke models before moving to large-scale clinical studies. Future preclinical stroke studies on co-morbid animals should also include groups of healthy animals in order to determine whether negative results can be attributed to the comorbid condition.

Combined Delivery of Bone Marrow‐Derived Mononuclear Cells in Chronic Ischemic Heart Disease: Rationale and Study Design

Treatment with bone marrow-derived mononuclear cells (BM-MNC) may improve left ventricular (LV) function in patients with chronic ischemic heart disease (IHD). Delivery method of the cell product may be crucial for efficacy. We aimed to demonstrate that the combination of intramyocardial and intracoronary injection of BM-MNC is safe and improves LV function in patients with chronic IHD. After a safety/feasibility phase of 10 patients, 54 patients will be randomly assigned in a 1:1:1 pattern to 1 control and 2 BM-MNC treatment groups. The control group will be treated with state-of-the-art medical management. The treatment groups will receive either exclusively intramyocardial injection or a combination of intramyocardial and intracoronary injection of autologous BM-MNC. Left ventricular function as well as scar size, transmural extension, and regional wall-motion score will be assessed by cardiac magnetic resonance imaging studies at baseline and after 6 months. The primary endpoint is the change in global LV ejection fraction by cardiac magnetic resonance from 6 months to baseline. The results, it is hoped, will have important clinical impact and provide essential information to improve the design of future regenerative-medicine protocols in cardiology. As cell delivery may play an important role in chronic IHD, we aim to demonstrate feasibility and efficacy of a combined cell-delivery approach in patients with decreased LV function.

Intracoronary Injection of Bone Marrow–Derived Mononuclear Cells Early or Late After Acute Myocardial Infarction

Background— Intracoronary administration of autologous bone marrow–derived mononuclear cells (BM-MNC) may improve remodeling of the left ventricle (LV) after acute myocardial infarction. The optimal time point of administration of BM-MNC is still uncertain and has rarely been addressed prospectively in randomized clinical trials. Methods and Results— In a multicenter study, we randomized 200 patients with large, successfully reperfused ST-segment elevation myocardial infarction in a 1:1:1 pattern into an open-labeled control and 2 BM-MNC treatment groups. In the BM-MNC groups, cells were administered either early (ie, 5 to 7 days) or late (ie, 3 to 4 weeks) after acute myocardial infarction. Cardiac magnetic resonance imaging was performed at baseline and after 4 months. The primary end point was the change from baseline to 4 months in global LV ejection fraction between the 2 treatment groups and the control group. The absolute change in LV ejection fraction from baseline to 4 months was −0.4±8.8% (mean±SD; P =0.74 versus baseline) in the control group, 1.8±8.4% ( P =0.12 versus baseline) in the early group, and 0.8±7.6% ( P =0.45 versus baseline) in the late group. The treatment effect of BM-MNC as estimated by ANCOVA was 1.25 (95% confidence interval, −1.83 to 4.32; P =0.42) for the early therapy group and 0.55 (95% confidence interval, −2.61 to 3.71; P =0.73) for the late therapy group. Conclusions— Among patients with ST-segment elevation myocardial infarction and LV dysfunction after successful reperfusion, intracoronary infusion of BM-MNC at either 5 to 7 days or 3 to 4 weeks after acute myocardial infarction did not improve LV function at 4-month follow-up. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00355186.

527 The Effects of Bone Marrow Derived Mononuclear Cells on CTO Neovascularization and Morphology

Chronic total occlusions (CTO) are extremely common and cause significant morbidity and possibly mortality to patients with coronary artery disease. Composition of CTO evolves over time but CTO mainly contain cholesterol-rich lipids, collagen, proteoglycans and microvessels. We have previously reported a correlation between the extent of intraluminal microvessels and successful CTO guidewire crossing. Recently, we have found that VEGF injections into CTO increase intraluminal neovascularization. Bone marrow-derived mononuclear cells (BM-MNC) have been reported to increase neovascularization in ischemic hindlimb and heart. In this study we sought to study the effects of BM-MNC on CTO intraluminal neovascularization and morphology. BM-MNC were isolated from male white New Zealand rabbits and characterized by immunostaining, flowcytometry and functional studies. Twelve weeks femoral artery CTO were injected with autologous BM-MNC (n=19) or saline (n=13) using finecross catheters. After 3 weeks, rabbits were euthanized and microfil was injected into CTO through the abdominal aorta. CTO were dissected and fixed in formalin for Micro-CT scanning and histology. To confirm recruitment of BM-MNC into CTO, cells were labeled with Qtracker 655 before injection and animals were sacrificed at 4 hrs or 1 week. Light microscopy showed that cultured BM-MNC consisted of spindle-shaped and round cells that gave rise to capillary-like structures. At one week, the cells expressed CD34 (endothelial progenitor cell surface marker), CD31 (endothelial cell (EC) surface marker), eNOS and caveolin-1 (EC markers), CD14 (monocyte marker) and RAM-11 (macrophage marker). The cells took up acLDL and formed capillary-like structures on Matrigel, indicating functional characteristics of EC. These results confirm that the cultured BM-MNC populations contained a mixture of cells from hematopoietic, mesenchymal and endothelial origin. In vivo studies showed that Qtracker labeled cells were present in the proximal section of CTO at 4 hrs and in the more distal sections at 1 week. Micro-CT analysis showed significant increase in relative blood volume in the treated CTO compared to controls (7.46±1.12 % vs. 4.35±0.70 %, p<0.05). Histology results also showed (Fig 1) increased neovascularization but calcification was observed in 6 out of 19 treated CTO that was not evident in control CTO. Increased CTO neovascularization was achieved by BM-MNC treatment, although the presence of calcification within treated CTO may be a limitation of this therapeutic approach.

Analysis of molecular changes after autologous cell therapy in swine myocardial infarction tissue can reveal novel targets for future therapy

Although several studies have demonstrated a functional recovery of infarcted myocardial tissue after cell therapy, little is known about the molecular mechanisms behind it. The aim of this study was to characterize the effect of cell therapy at the molecular level to screen for novel target candidates for future therapy of infarcted myocardial tissue. We used a swine acute myocardial infarction model evoked by transient occlusion of the circumflex coronary artery. Autologous bone marrow-derived mononuclear cells (BMMCs) or saline were injected intramyocardially or into the circumflex coronary artery. Samples for protein and RNA analysis were collected from the infarction area and healthy myocardium after a 3 week recovery period and analysed by two-dimensional gel electrophoresis (2DE) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Proteomic screening detected 13 protein spots which were altered after infarction but had been restored by BMMC treatment. The identification of seven proteins by mass spectrometry revealed that five proteins with decreased expression after infarction corresponded to mitochondrial proteins involved in energy metabolism. Their restored levels after BMMC treatment indicate their involvement in the recovery of heart function. In contrast, the elevated levels of α-crystallin B chain and cathepsin D after infarction suggest an involvement in the pathological mechanisms causing a decreased heart function. This study reveals that cell therapy with BMMCs after myocardial infarction causes restoration of several altered protein levels after 3 weeks and identifies potential marker proteins involved in the pathology of infarction.

Endothelial Nitric Oxide Synthase Overexpression Restores the Efficiency of Bone Marrow Mononuclear Cell-Based Therapy

Bone marrow-derived mononuclear cells (BMMNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. However, cardiovascular risk factors, including diabetes mellitus and hypercholesterolemia, lead to the abrogation of BMMNCs proangiogenic potential. NO has been shown to be critical for the proangiogenic function of BMMNCs, and increased endothelial NO synthase (eNOS) activity promotes vessel growth in ischemic conditions. We therefore hypothesized that eNOS overexpression could restore both the impaired neovascularization response and decreased proangiogenic function of BMMNCs in clinically relevant models of diabetes and hypercholesterolemia. Transgenic eNOS overexpression in diabetic, atherosclerotic, and wild-type mice induced a 1.5- to 2.3-fold increase in postischemic neovascularization compared with control. eNOS overexpression in diabetic or atherosclerotic BMMNCs restored their reduced proangiogenic potential in ischemic hind limb. This effect was associated with an increase in BMMNC ability to differentiate into cells with endothelial phenotype in vitro and in vivo and an increase in BMMNCs paracrine function, including vascular endothelial growth factor A release and NO-dependent vasodilation. Moreover, although wild-type BMMNCs treatment resulted in significant progression of atherosclerotic plaque in ischemic mice, eNOS transgenic atherosclerotic BMMNCs treatment even had antiatherogenic effects. Cell-based eNOS gene therapy has both proangiogenic and antiatherogenic effects and should be further investigated for the development of efficient therapeutic neovascularization designed to treat ischemic cardiovascular disease.

Bone marrow mononuclear cells shift bioactive lipid pattern in injured kidney towards tissue repair in rats with unilateral ureteral obstruction

Bioactive lipids are important in tissue injury and regeneration. Ceramide (Cer) is known for its pro-apoptotic action and sphingosine-1-phosphate (S1P) for inducing proliferation and cell survival; diacylglycerol (DAG) and lysophosphatidic acid (LPA) are involved in various signalling pathways including modulation of ion transport. LPA signalling through its receptor LPA(1) is also related to the progression of fibrosis. This study investigated the modulation of lipid signalling pathways induced by administration of bone marrow-derived mononuclear cells (BMMC) in chronic kidney disease. Unilateral ureteral obstruction (UUO) was followed by intravenous injection of ∼2 × 10(7) BMMC. Controls were UUO group treated with buffered solution and sham-operated group. Animals were killed 14 days after surgery, and lipid phosphorylation assays and immunoblotting were performed on the kidney homogenates. More DAG was available in the UUO rats (2.4 ± 0.4 and 2.4 ± 0.3 vs 1.0 ± 0.2 pmol (32)PA mg(-)(1) min(-)(1), in UUO and UUO + BMMC vs SHAM). Sphingosine kinase was 150 ± 12% more active in UUO + BMMC than in UUO and SHAM. Cer levels were 76 ± 7% lower in the UUO + BMMC than UUO. LPA receptor type 1 (LPA(1)) expression was 169 ± 7% higher in the UUO group than in UUO + BMMC and SHAM. BMMC maintain control levels of Ca(2+)-ATPase expression altered by UUO by 40%. BMMC infusion modulated diverse lipid signalling pathways and protein expression, shifted sphingolipid metabolism toward a regenerative pattern and favourably reduced the levels of a receptor involved in the progression of tissue fibrosis. These results strengthen the benefits of BMMC treatment and give insight into its paracrine mechanisms of action.

Arteriogenesis: Noninvasive Quantification with Multi–Detector Row CT Angiography and Three-dimensional Volume Rendering in Rodents

To evaluate two-dimensional (2D) multi-detector row computed tomographic (CT) angiography and three-dimensional (3D) volume rendering for depiction of patterns of arterial growth and quantification of blood vessel density and volume. The institutional animal care and use committee approved this study. The right femoral artery and its branches were ligated and excised in 16 inbred Lewis rats; animals were randomly assigned to receive 70 microL Dulbecco's modified Eagle's medium (DMEM) or 1.5 x 10(7) bone marrow-derived mononuclear cells (BMC) from isogenic donor rats in 70 microL DMEM. At 2 weeks, CT angiography was performed with injection of 0.45 mL barium sulfate suspension at 0.7 mL/min, followed by silver staining. Number of blood vessels, area, mean area, volume, and blood vessel size distribution derived from digitally subtracted 2D CT angiographic sections were quantified; 3D images were reconstructed. Two-way analysis of variance and paired and unpaired Student t tests were performed. CT angiography showed two patterns of arterial growth: collateral arterial formation and branching arteriogenesis. Two-way analysis of variance indicated that differences within subjects (ischemic vs nonischemic legs) and between subjects (BMC vs DMEM treatment) were significant for total blood vessel area, total blood vessel volume, and mean of blood vessel area (P < .001). In the BMC group, there were significantly more arteries (mean, 241.6 +/- 77.0 [standard deviation] vs 196.4 +/- 75.2, P = .028), but mean cross-sectional area of these arteries was smaller in ischemic versus nonischemic legs (5.4 mm(2) +/- 1.2 vs 6.8 mm(2) +/- 1.3, P = .006). Total arterial area and volume did not differ significantly between ischemic and nonischemic legs. BMC injection had a substantial effect on arteriogenesis, with normalization of total arterial area and volume in the BMC group; this effect was successfully depicted.

Effect of Intracoronary Transplantation of Autologous Bone Marrow-Derived Mononuclear Cells on Outcomes of Patients With Refractory Chronic Heart Failure Secondary to Ischemic Cardiomyopathy

Recent studies have indicated that stem cell implantation increases cardiac function by repairing damaged myocardium. We investigated whether intracoronary transplantation of autologous bone marrow-derived mononuclear cells (BMMCs) confers beneficial effects in patients with refractory chronic heart failure. Twenty-eight patients received standard heart failure medication and BMMC transplantation (BMMC treatment) or standard medication only (controls). BMMCs were harvested from each patient. Clinical manifestations, biochemical assays, rhythm studies, echocardiograms, and positron emission tomograms were recorded. Fourteen patients with cell grafting had symptomatic relief of heart failure within 3 days. Left ventricular ejection fraction increased by 9.2% and 10.5% at 1 week and 3 months after the procedure, respectively, versus baseline (p < 0.01 for the 2 comparisons). Left ventricular end-systolic volume decreased by 30.7% after 3 months (p < 0.01). Brain natriuretic peptide levels at days 3 and 7 after cell infusion significantly decreased by 69.2% and 70.4%, respectively, whereas atrial natriuretic peptide levels increased by 30.1% at day 7. Positron emission tomographic analysis showed a significant increase in cell viability of 10.3% in the infarcted zone. No patient died in the BMMC-treated group at 6-month follow-up. In contrast, heart failure did not improve in any control patient. Left ventricular ejection fraction decreased by 7.2% after 3 months. Two control patients died from heart failure within 6 months. In conclusion, this is the first demonstration in humans that intracoronary BMMC transplantation is a feasible and safe therapeutic strategy to decrease symptoms, increase cardiac function, and possibly prolong life in patients with end-stage heart failure refractory to standard medical therapy.