Marrow-derived Mesenchymal Stem Cells Group Research Articles

Effects of Bone Marrow and Adipose-Derived Mesenchymal Stem Cells on microRNA Expressions in Acute Alkaline Corneal Burn.

Purpose: The aim of this study was to investigate the microRNA (miRNA) expressions of the corneal tissue after an alkaline burn and to compare the efficiency of adipose- and bone marrow-derived mesenchymal stem cells (MSCs) on expressions. Methods: Thirty-two rats were divided into 4 groups. No intervention was made in the control group. A chemical burn was created by applying 4 μL NaOH soaked in 6 mm filter paper to the right eye of each animal in the other groups. Whereas only subconjunctival 0.1 mL phosphate-buffered saline (PBS) was injected to in the group 1, 2 × 106 adipose- or bone marrow-derived MSC in 0.1 mL PBS was injected subconjunctivally to the animals in the remaining groups (groups 2 and 3, respectively). Tissue samples were collected for miRNA analysis on the third day after the burn. Results: When group 1 was compared with the control group, the expression of 3 of 93 miRNAs increased significantly, whereas the expression of 50 miRNAs decreased significantly. Significant changes in miRNA expressions were observed when group 1 was compared with groups 2 and 3. Although a significant change was observed in the expression of 6 miRNAs in the adipose-derived MSC group, it was found that the expression of 65 miRNAs significantly changed in the bone marrow-derived MSC group. Conclusion: This study shows that there are significant changes in some miRNA expressions after corneal alkaline burn and these changes can be reversed with the subconjunctival injection of MSCs.

The efficacy of interfollicular epidermal stem cells versus bone marrow-derived mesenchymal stem cells in cutaneous wound healing in diabetic rats

Objective: Many sources of stem cells, such as bone marrow stem cells, embriyonic stem cells, adipose-derived stem cells, epidermal stem cells were used extensively for diabetic wound healing. This study investigated whether interfollicular epidermal stem cell versus bone marrow-derived mesenchymal stem cell effectiveness in the enhancement of diabetic wound healing. Methods: The streptozotocin-induced Spraque-Dowley rats with 5 mm punch biopsy were used. Rats were divided into three groups: group I diabetic controls receiving no stem cells; group II, rats receiving bone marrow-derived mesenchymal stem cells; group III, rats receiving interfollicular epidermal stem cells. Wound healing was assessed clinically that regarding healing time and wound size. Results: Clinical results showed that wound size was significantly reduced in mesenchymal and interfollicular epidermal stem cell-treated groups as compared with controls. Complete wound-healing times were 19.4±2.85 days in bone marrow-derived mesenchymal stem cells group versus 20.3±3.45 days in interfollicular epidermal stem cells group and 24.7±4.17 in the control group. In the measurement of the wound area, there were no significant differences between the bone marrow-derived mesenchymal stem cells group (P=0.115) and interfollicular epidermal stem cells group (P=0.085). Conclusions: Interfollicular epidermal stem cells were found as effective as bone marrow-derived mesenchymal cells in the treatment of the diabetic wound.

Bone marrow-derived mesenchymal stem cells attenuate tubulointerstitial injury through multiple mechanisms in UUO model.

Current evidence supports the use of bone marrow-derived mesenchymal stem cells (MSCs) for a diverse range of clinical applications, and many studies have shown that MSCs have renal-protective effects, but the mechanism is not well understood. Therefore, in this study, we aim to further identify whether MSCs can attenuate renal fibrosis by decreasing tubulointerstitial injury in a unilateral ureteral obstruction (UUO) model. In this study, we cultured MSCs and then transplanted them into a UUO model through the tail vein. Histology, cell proliferation, peritubular capillary (PTC) loss and myofibroblast markers were examined on days 3, 7 and 14 after surgery. We demonstrated that renal interstitial fibrosis in the MSC group was significantly attenuated compared with the UUO and DMEM groups. Moreover, MSC treatment inhibited the loss of PTCs and increased parenchymal cell proliferation. In addition, UUO-induced activation and proliferation of myofibroblasts were suppressed by MSC infusion. Furthermore, MSCs attenuated tubulointerstitial infiltration of macrophages in UUO mice. Tubulointerstitial damage plays a very important role in the progression of chronic kidney disease (CKD). PTC loss, macrophage recruitment, and myofibroblast activation are directly correlated with the development of renal tubulointerstitial fibrosis. Our results suggest that MSC infusion in the UUO model is a promising therapeutic strategy for promoting kidney repair.

Platelet rich concentrate enhances mesenchymal stem cells capacity to repair focal cartilage injury in rabbits

BackgroundIt has been previously suggested that the use of regenerative promoters, which include bone marrow-derived mesenchymal stem cells (MSCs) or natural growth factors supplement such as platelet-rich concentrate (PRC) could promote cartilage regeneration. However, the notion that the concurrent use of both promoters may provide a synergistic effect that improves the repair outcome of focal cartilage injury has not been previously demonstrated. This study was thus conducted to determine whether the concomitant use of PRC could further enhance the reparative potential of MSCs encapsulated in alginate transplanted into focal cartilage injury in rabbits. MethodsArtifically created full thickness cartilage defects were made on the weight-bearing region of medial femoral condyles in bilateral knees of New Zealand White rabbits (N = 30). After one month, the right knee was treated with either i) PRC (n = 10), ii) MSCs (n = 10), or, iii) a combination of PRC and MSCs (PRC + MSC) (n = 10), all encapsulated in alginate. The left knee remained untreated (control). Rabbits were sacrificed at 3 and 6 months after treatment. Cartilage tissue regeneration was accessed using ICRS morphologic scoring, histologic grading by O'Driscoll scoring, immunohistochemical staining and quantitative analysis of glycosaminoglycans (GAG) per total protein content. ResultsAt 3 months, transplantation using PRC alone was equally effective as MSCs in inducing the repair of cartilage defects. However, PRC + MSC resulted in significantly higher ICRS and O’Driscoll scores (p < 0.05) as compared to other groups. The regenerated tissues from the PRC + MSC group also had stronger staining for Safranin-O and collagen type II. By 6 months, in addition to superior ICRS and O’Driscoll scores as well as stronger staining, glycosaminoglycan per total protein content was also significantly higher (p < 0.05) in the PRC + MSC group (3.4 ± 0.3 μg/mg) as compared to the MSC (2.6 ± 0.2 μg/mg) or PRC (2.1 ± 0.2 μg/mg) groups. ConclusionPRC enhances the reparative effects of MSC in treating focal articular cartilage injuries.

Mesenchymal stem cell therapy of hepatocellular carcinoma in rats: Detection of cell homing and tumor mass by magnetic resonance imaging using iron oxide nanoparticles.

Bone marrow-derived mesenchymal stem cells (MSCs) are reported to improve hepatic fibrosis, and may impact the signaling mechanisms leading to the induction of hepatocellular carcinoma (HCC) in animal models of liver cirrhosis. The aim of this study was to clarify and explain the therapeutic role played by MSCs in hepatic cirrhosis and HCC by tracking them using nanoparticles. Liver cirrhosis and HCC were established in rats with the use of carbon tetrachloride and diethylnitrosamine injection. Magnetic resonance imaging (MRI) was used to track nanoparticlelabeled MSCs in the intact animal following injection and to monitor the changes in the hepatic parenchyma. Labeling of MSCs with iron oxide nanoparticles did not adversely affect their viability and proliferation. MRI indicated a significant reduction in tumor mass in the labeled MSCs group compared to the control group. Histopathologic examination of the liver, following MSCs treatment, showed an apparently normal looking liver with no evidence of neoplastic cellular changes. The biochemical results support these findings. This work documents that MSCs could be labeled with nanoparticles and traced in normal and cirrhotic liver and in liver with HCC in animals using MRI. MRI monitors the homing and localization of MSCs in the liver. MSCs infusion in animal models of cirrhosis and carcinoma may prove to be useful in limiting the cirrhotic process. Also, it may have a possible therapeutic potential on the carcinogenic process.

Bone marrow-derived mesenchymal stem cells (MSCs) to enhance nerve regeneration and functional recovery in vascular composite allotransplantation (VCA)

Abstract Introduction Optimizing nerve regeneration and mitigating denervation atrophy of target muscles are the keys to achieving successful VCA such as limb and face transplants. Methods Lewis rat was anesthetized, sciatic or individual sciatic nerve branches (tibial, peroneal and sural) of the right hind-limb were transected and surgically repaired. MSC (5 million; passage ≤6) or vehicle were administered locally and intravenously. Results Rat MSCs expanded ex vivo were CD29+, CD90+, CD34−, CD31−, CD45low, MHC Class I+, Class II−, and were pluripotent. Total sensory function recovery at 2 weeks post sciatic or individual nerve repair (SNR or INR) with or without MSC was ~ 1.2 on a scale of Grade 0–3 (0=No function; 3=Normal); by 12 weeks it was 2.6–2.8 (n≥6/group). Peroneal sensory function recovery was as early as 1 week but not tibial or sural. MSC treatment accelerated sensory function onset. At 8 weeks post-INR, the sciatic nerve function index (SFI), a measure of motor function (0=Normal; −100=Nonfunctional) was −34 and −77 in MSC and vehicle groups, respectively (n≥9); with SNR it was −72 and −92 in MSC and vehicle groups, respectively. Long-term motor function (24 weeks) was apparent in MSC treated INR (SFI −63) but not in SNR (SFI −100) model. Gastrocnemius muscle atrophy was lower (P ≤0.05) in INR compared to SNR model. Significant reduction in axonal area (P ≤0.002), g ratio/axonal myelination (P ≤0.05), reduced nerve fiber density, and increased degenerating axons in the distal segment of the transected/repaired tibial nerve was observed compared to the contralateral naive tibial nerve. Histology and molecular studies are ongoing. Conclusion MSC therapy appears to promote peripheral nerve regeneration and functional recovery.

Abstract 11150: Hyperbaric Oxygenation Enhances Transplanted Cell Graft Survival and Functional Recovery in Critical Limb Ischemia

Introduction: One of the major limitations in the application of stem-cell therapy to repair ischemic tissue is the low survival of transplanted cells, possibly due to poor oxygenation. Also the effect of hyperbaric oxygen therapy (HBOT) on bone marrow-derived mesenchymal stem cells (MSCs) is poorly understood. Hypothesis: We hypothesized that HBOT could be used as an adjuvant treatment to augment stem-cell therapy. Therefore, this study aimed to evaluate the effect of HBOT on the engraftment of bone marrow-derived MSCs transplanted in critical limb ischemia (CLI). Methods: Sixty-nine consecutive CLI patients refractory to peripheral revascularization therapy were enrolled. MSC transplantation was performed in all patients. HBOT (given 100% oxygen under 2.8 atmosphere absolute, for 60 min) was administered to 36 patients with and 33 patients without, by 2 weeks after those therapies. These patients were followed up by an outpatient clinic after limb survival. The primary endpoint was tissue oxygen tension of blood flow recovery, and secondary endpoint was adverse event rate (death and major limb amputation), analyzed by intention-to-treat. Results: Mean follow up was 11.4 ± 0.6 years. Limb survival rate was 84.1% and survival was 76.8%. The effect of HBOT was independent of background conditions, such as diabetes and hemodialysis (NS, respectively, Breslow-Day analysis). After 4 weeks, recovery of skin oxygen tension was significantly increased in MSC+HBO group compared to that in MSC group (22.7±16.3 to 34.5±21.4 vs 24.4±15.4 to 28.3±19.6, p = 0.045, odds ratio; 0.194). Surprisingly, in diabetic patients, MSC+HBOT group had significantly lower adverse event rate compared to MSC group (2 vs. 10 cases, p = 0.043, log-rank test, figure). Conclusions: HBOT appears to be a potential and clinically viable non-invasive adjuvant treatment for CLI stem-cell therapy.

The Effect of Purified Human Bone Marrow–Derived Mesenchymal Stem Cells on Rotator Cuff Tendon Healing in an Athymic Rat

To evaluate the ability of purified human bone marrow-derived mesenchymal stem cells (MSCs) to augment healing of an acute small- to medium-sized rotator cuff repair in a small-animal model, evaluating the structure and composition of the healing tendon-bone interface with histologic and biomechanical analyses. Fifty-two athymic rats underwent unilateral detachment and transosseous repair of the supraspinatus tendon augmented with either fibrin glue (control group) or fibrin glue with 106 human MSCs (experimental group) applied at the repair site. Flow cytometry verified the stem cell phenotype of the cells as CD73+, CD90+, CD105+, CD14-, CD34-, and CD45-. Rats were killed at 2 and 4weeks, with 10 from each group used for biomechanical testing and 3 for histologic analysis. Safranin O staining identified increased fibrocartilage formation at the repair site at 2weeks in the human MSC group (18.6% ± 2.9% vs 9.1% ± 1.6%, P= .026). Picrosirius staining identified decreased energy (36.88 ± 4.99J vs 54.97 ± 8.33J, P= .04) and increased coherence in the human MSC group (26.96% ± 15.32% vs 14.53% ± 4.10%, P= .05), indicating improved collagen orientation. Biomechanical testing showed a significant increase in failure load (11.5 ± 2.4N vs 8.5 ± 2.4N, P= .002) and stiffness (7.1 ± 1.2N/mm vs 5.7 ± 2.1N/mm, P < .001) in the experimental group compared with the control group at 2weeks. These effects dissipated by 4weeks, with no significant differences in fibrocartilage formation (35% ± 5.0% vs 26.6% ± 0.6%, P= .172) or biomechanical load to failure (24.6 ± 7.1N vs 21.5 ± 4.1N, P=.361) or stiffness (13.5 ± 3.1N/mm vs 16.1 ± 5.6N/mm, P= .384). All failures occurred at the bone-tendon interface. Rotator cuff repair augmentation with purified human MSCs improved early histologic appearance and biomechanical strength of the repair at 2weeks, although the effects dissipated by 4weeks with no significant differences between groups. Human MSCs may improve early rotator cuff healing during the first 2weeks after repair.

Proteome Analysis the Differences of the Mesenchymal Stem Cells and Extracorporeal Shock Wave Therapy Enhancing Diabetic Wound Healing

PURPOSE: Non-healing foot ulcers in patients with diabetes are the leading cause of complications. Our studies have demonstrated topical application of bone marrow-derived mesenchymal stem cells (MSCs) and extracorporeal shock wave therapy (ESWT) both could enhance the diabetic wound healing in a rodent STZ-induced diabetes model.1,2 However, the details mechanisms how MSCs and ESWT increased the wound healing is still unclear. Therefore, this study investigated the topical wounding margin tissue between the MSC group and ESWT group and diabetic controls by using 2D proteome study. MATERIALS AND METHODS: A dorsal skin defect (area, 6×5cm) in a streptozotocin-induced diabetes Wistar rats as a rodent model was used. Group I consisted of diabetic control receiving no MSC; group II, rats received 2 sessions of MSC treatment on day 7 and 10 post-wounding. Group III, rats received 2 sessions of ESWT treatment on day 3 and 7 post-wounding. Each group were assessed at least three wounding-edge skin tissue samples. The proteomic study of skin tissue after different treatment was analyzed. The spots of interest were subjected to in-gel trypsin digestion and MALDI-TOF mass spectrometry in order to elucidate the peptide mass fingerprints. RESULTS: Representative two-dimensional gel electrophoretograms of skin tissue protein in diabetic wound control, post-MSC and post-ESWT treatments. The proteomic study of wounding–edge skin tissue in MSC group had significantly higher abundance of the alpha-2-HS-glycoprotein and vitamin D-binding protein as compared to that in controls. MSC group had significantly lower levels of the Serine protease inhibitor A3N, Serpin B5, tropomyosinα-1 chain, Haptoglobin, as compared to the diabetic controls. In ESWT group had significantly higher abundance of the hemopexin, alpha-2-HS-glycoprotein and vitamin D-binding protein as compared to that in controls. ESWT group had significantly lower levels of the Serine protease inhibitor A3N, Leukocyte elastase inhibitor A, Haptoglobin, Structural maintenance of chromosomes protein 3 and Catechol O-methyltransferase as compared to the diabetic controls without ESWT.Figure 1: Mass spectrometric characteristics of the positive identified proteins in the peri-wounding skin tissue at day 3 post-mesenchymal stem cells treatment.Figure 2: Mass spectrometric characteristics of the positive identified proteins in the peri-wounding skin tissue post- extracorporeal shock wave therapy.CONCLUSION: This proteomic study demonstrated there were some protein indeed up-regulation (alpha-2-HS-glycoprotein and vitamin D-binding protein) or down-regulation (Serine protease inhibitor A3N, Haptoglobin) between MSCs treatment group and ESWT group. Further studies are needed to elucidate whether modulate these proteins could increase diabetic wound healing.

Mesenchymal Stem Cells Overexpressing Angiotensin-Converting Enzyme 2 Rescue Lipopolysaccharide-Induced Lung Injury

Bone marrow-derived mesenchymal stem cells (MSCs), which have beneficial effects in acute lung injury (ALI), can serve as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2), a counterregulatory enzyme of ACE that degrades angiotensin (Ang) II into Ang 1-7, has a protective role against ALI. Because ACE2 expression is severely reduced in the injured lung, a therapy targeted to improve ACE2 expression in lung might attenuate ALI. We hypothesized that MSCs overexpressing ACE2 would have further benefits in lipopolysaccharide (LPS)-induced ALI mice, when compared with MSCs alone. MSCs were transduced with ACE2 gene (MSC-ACE2) by a lentiviral vector and then infused into wild-type (WT) and ACE2 knockout (ACE2(-/y)) mice following an LPS-induced intratracheal lung injury. The results demonstrated that the lung injury of ALI mice was alleviated at 24 and 72 h after MSC-ACE2 transplantation. MSC-ACE2 improved the lung histopathology and had additional anti-inflammatory effects when compared with MSCs alone in both WT and ACE2(-/y) ALI mice. MSC-ACE2 administration also reduced pulmonary vascular permeability, improved endothelial barrier integrity, and normalized lung eNOS expression relative to the MSC group. The beneficial effects of MSC-ACE2 could be attributed to its recruitment into the injured lung and enhanced local expression of ACE2 protein without changing the serum ACE2 levels after MSC-ACE2 transplantation. The biological activity of the increased ACE2 protein decreased the Ang II amount and increased the Ang 1-7 level in the lung when compared with the ALI and MSC-only groups, thereby inhibiting the detrimental effects of accumulating Ang II. Therefore, compared to MSCs alone, the administration of MSCs overexpressing ACE2 resulted in a further improvement in the inflammatory response and pulmonary endothelial function of LPS-induced ALI mice. These additional benefits could be due to the degradation of Ang II that accompanies the targeted overexpression of ACE2 in the lung.

Delayed post-treatment with bone marrow-derived mesenchymal stem cells is neurorestorative of striatal medium-spiny projection neurons and improves motor function after neonatal rat hypoxia-ischemia.

Perinatal hypoxia-ischemia is a major cause of striatal injury and may lead to cerebral palsy. This study investigated whether delayed administration of bone marrow-derived mesenchymal stem cells (MSCs), at one week after neonatal rat hypoxia-ischemia, was neurorestorative of striatal medium-spiny projection neurons and improved motor function. The effect of a subcutaneous injection of a high-dose, or a low-dose, of MSCs was investigated in stereological studies. Postnatal day (PN) 7 pups were subjected to hypoxia-ischemia. At PN14, pups received treatment with either MSCs or diluent. A subset of high-dose pups, and their diluent control pups, were also injected intraperitoneally with bromodeoxyuridine (BrdU), every 24h, on PN15, PN16 and PN17. This permitted tracking of the migration and survival of neuroblasts originating from the subventricular zone into the adjacent injured striatum. Pups were euthanized on PN21 and the absolute number of striatal medium-spiny projection neurons was measured after immunostaining for DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32), double immunostaining for BrdU and DARPP-32, and after cresyl violet staining alone. The absolute number of striatal immunostained calretinin interneurons was also measured. There was a statistically significant increase in the absolute number of DARPP-32-positive, BrdU/DARPP-32-positive, and cresyl violet-stained striatal medium-spiny projection neurons, and fewer striatal calretinin interneurons, in the high-dose mesenchymal stem cell (MSC) group compared to their diluent counterparts. A high-dose of MSCs restored the absolute number of these neurons to normal uninjured levels, when compared with previous stereological data on the absolute number of cresyl violet-stained striatal medium-spiny projection neurons in the normal uninjured brain. For the low-dose experiment, in which cresyl violet-stained striatal medium-spiny neurons alone were measured, there was a lower statistically significant increase in their absolute number in the MSC group compared to their diluent controls. Investigation of behavior in another cohort of animals showed that delayed administration of a high-dose of bone marrow-derived MSCs, at one week after neonatal rat hypoxia-ischemia, improved motor function on the cylinder test. Thus, delayed therapy with a high- or low-dose of adult MSCs, at one week after injury, is effective in restoring the loss of striatal medium-spiny projection neurons after neonatal rat hypoxia-ischemia and a high-dose of MSCs improved motor function.

Investigation of the immune response to autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses

Mesenchymal stem cells have demonstrated immunomodulatory capabilities as well as modest efficacy in animal models of joint injury, warranting further study as a potential treatment of joint disease. The goal of the study was to investigate the blood and synovial immune and histologic response to intra-articular injection of autologous, allogeneic, and xenogeneic bone marrow-derived mesenchymal stem cells (MSC) in horses. The study group consisted of 6 five-year-old Thoroughbred mares that had been injected previously with 15 million, genetically modified autologous, allogeneic, or xenogeneic MSC into the fetlock joints. One group of autologous cells was genetically modified to permit MSC biolocalization in the synovium. To assess response to the injection, synovial biopsies were obtained via arthroscopy 60 days after MSC injection for gross, histologic and molecular analyses. Peripheral blood mononuclear cells were isolated from each horse 120 days after MSC injection and co-cultured with a monolayer of each MSC group to permit quantification of activated CD4+ lymphocytes and cytokine release (ELISA) upon re-exposure to MSC.Arthroscopic examination revealed normal synovium with no grossly detrimental effect to the synovium or cartilage. Intra-articular MSC produced a persistent mononuclear infiltrate for at least 60 days, mostly perivascular, identified as CD3+ lymphocytes. An immune response (significant increase in CD4+ lymphocytes) was detected upon re-exposure to xenogeneic but not to allogeneic or autologous MSC. An inflammatory cytokine release from peripheral blood mononuclear cell/MSC co-cultures was present in all MSC groups but was significantly greater in the xenogeneic group. In conclusion, intra-articular injection of MSC, regardless of cell origin, incited a persistent mononuclear synovitis demonstrating a sustained biologic influence of these cells. Allogeneic cells did not elicit a detectable immune response upon re-exposure using our methods.

Abstract 231: Bone Marrow-Derived Mesenchymal Stem Cells from Amputated Limbs of Patients with Critical Limb Ischemia Provide Stromal Support for Endothelial Cells

Critical limb ischemia (CLI) often results in amputation, but autologous cellular therapy using bone marrow-derived mesenchymal stem cells (MSCs) may be able to prevent this outcome. However, the quality of MSCs in the CLI population is not clear. Amputated CLI limbs contain reservoirs of MSCs that can be used to characterize their stromal capacity. We hypothesize that MSC stromal capacity is relatively preserved in ischemic limbs compared to ones from healthy donors. Methods Healthy donor MSCs (hdMSCs, n=2) and ischemic MSCs (iMSCs, n=3) from amputated limbs of non-diabetic CLI patients were cultured. Human microdermal ECs were commercially obtained. Co-culture pellets (1:1 ratio) of hdMSC:EC, iMSC:EC, and EC alone were formed and cultured in 3-D fibrin hydrogels in EC growth media. Images of the pellets and invasion areas were captured up to 5 days. The invasion area was quantified as the difference between the total and pellet area normalized to initial pellet area. Experiments were performed in quadruplicates and co-culture experiments were duplicated. Results iMSC:EC co-culture provided similar stromal support as the healthy donor cohort. Both MSC groups were significantly greater than the ECs alone following day 2 (see figure, *P&lt;0.0005, ANOVA). Both MSC groups also exhibited significant increase in invasion area with time (P&lt;0.01) while the ECs did not (P&gt;0.5). Conclusion This is the first study to demonstrate that iMSCs provide similar stromal support to ECs as healthy hdMSCs, thus iMSCs may be of sufficient quality for use in autologous therapy. We are currently quantifying secretome expressions of the iMSCs to provide insights into mechanisms of the stromal support.

Neural transdifferentiation of human bone marrow mesenchymal stem cells on hydrophobic polymer-modified surface and therapeutic effects in an animal model of ischemic stroke

Human bone marrow-derived mesenchymal stem cells (MSCs) have multi-lineage differentiation potential and can become cells of mesodermal and neural lineages. These stem cells thus hold considerable clinical promise for the treatment of neurodegenerative diseases. For successful regeneration of damaged neural tissues, directed differentiation of neural or neuronal precursor cells from MSCs and integration of transplanted cells are pivotal factors. We induced MSCs into neurogenesis using a modified protocol. The therapeutic potency of the resulting neural progenitor cells in a rat model of ischemic stroke was analyzed. Using a highly hydrophobic diphenylamino-s-triazine-bridged p-phenylene (DTOPV)-coated surface and adopting a procedure for propagation of neural stem cells, we efficiently converted MSCs into neurosphere-like cellular aggregates (NS-MSCs). The spherical cells were subsequently induced to differentiate into neural cells expressing neuroectodermal markers. To determine whether these cells had neuronal fates and induced neuro-protective effects in vivo, NS-MSCs were intra-cerebrally administered to rats 48h after permanent middle cerebral artery occlusion (pMCAo). The results showed a remarkable attenuation of ischemic damage with significant functional recovery, although the cells were not fully incorporated into the damaged tissues on post-operative day 26. Improvement in the NS-MSC-transplanted rats was faster than in the MSC group and suppression of inflammation was likely the key factor. Thus, our culture system using the hydrophobic surface of a biocompatible DTOPV coating efficiently supported neural cell differentiation from MSCs. Neural-primed MSCs exhibited stronger therapeutic effects than MSCs in rat brains with pMCAo.

Differentiation of GFP-Bcl-2-engineered mesenchymal stem cells towards a nucleus pulposus-like phenotype under hypoxia in vitro

Differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into a nucleus pulposus-like phenotype under hypoxia has been proposed as a potential therapeutic approach for intervertebral disc degeneration. However, limited cell viability under hypoxic conditions has restricted MSC differentiation capacity and thus restricted its clinical application. In this study, we genetically modified MSCs with an anti-apoptotic GFP-Bcl-2 gene and evaluated cell survival and functional improvement under hypoxia in vitro. Rat bone marrow MSCs were transfected by lentiviral vectors with the GFP-Bcl-2 gene (GFP-Bcl-2-MSCs). Cell proliferation and apoptosis were assessed, and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate phenotypic and biosynthetic activities. In addition, Alcian blue staining was used to detect the formation of sulfated glycosaminoglycans (GAGs) in the differentiated cells. We found that the Bcl-2 gene protected MSCs against apoptosis. We also observed that Bcl-2 over-expression reduced apoptosis by 40.61% in non-transfected MSCs and 38.43% in vector-MSCs to 18.33% in Bcl-2-MSCs. At 3days, the number of viable Bcl-2-MSCs was approximately two times higher than the number of MSCs or vector-MSCs under hypoxic conditions. RT-PCR showed higher expression of chondrocyte-related genes (Sox-9, aggrecan and type II collagen) in GFP-Bcl-2-MSCs cultured under hypoxia. The accumulation of proteoglycans in the pellet was 86% higher in GFP-Bcl-2-MSCs than in the control groups. Furthermore, the ratio of proteoglycans/collagen II in GFP-Bcl-2-MSCs was 6.2-fold higher compared to the MSC and vector-MSC groups, which denoted a nucleus pulposus-like differentiation phenotype. Our findings support the hypothesis that anti-apoptotic gene-modified MSCs can differentiate into cells with a nucleus pulposus-like phenotype in vitro, which may have value for the regeneration of intervertebral discs using cell transplantation therapy.

Acceleration of Wound Healing with Stem Cell–Derived Growth Factors

Recently, it has been revealed that bone marrow-derived mesenchymal stem cells (MSCs) accelerate the healing of skin wounds. Although the proliferative capacity of MSCs decreases with age, MSCs secrete many growth factors. The present study examined the effect of mesenchymal stem cell-conditioned medium (MSC-CM) on wound healing. The wound-healing process was observed macroscopically and histologically using an excisional wound-splinting mouse model, and the expression level of hyaluronic acid related to the wound healing process was observed to evaluate the wound-healing effects of MSC, MSC-CM, and control (phosphate-buffered saline). The MSC and MSC-CM treatments accelerated wound healing versus the control group. At 7 days after administration, epithelialization was accelerated, thick connective tissue had formed in the skin defect area, and the wound area was reduced in the MSC and MSC-CM groups versus the control group. At 14 days, infiltration of inflammatory cells was decreased versus 7 days, and the wounds were closed in the MSC and MSC-CM groups, while a portion of epithelium was observed in the control group. At 7 and 14 days, the MSC and MSC-CM groups expressed significantly higher levels of hyaluronic acid versus the control group (P < .05). The expression level of hyaluronic acid was lower at 14 days than at 7 days in all three groups. Both the MSC and MSC-CM groups accelerated wound healing versus the control group to a similar degree. Accordingly, it is suggested that the MSC-CM contains growth factor derived from stem cells, is able to accelerate wound healing as well as stem cell transplantation, and may become a new therapeutic method for wound healing in the future.

Inflammatory effects of autologous, genetically modified autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses

To compare the clinical and inflammatory joint responses to intra-articular injection of bone marrow-derived mesenchymal stem cells (MSC) including autologous, genetically modified autologous, allogeneic, or xenogeneic cells in horses. Six five-year-old Thoroughbred mares had one fetlock joint injected with Gey's balanced salt solution as the vehicle control. Each fetlock joint of each horse was subsequently injected with 15 million MSC from the described MSC groups, and were assessed for 28 days for clinical and inflammatory parameters representing synovitis, joint swelling, and pain. There were not any significant differences between autologous and genetically modified autologous MSC for synovial fluid total nucleated cell count, total protein, interleukin (IL)-6, IL-10, fetlock circumference, oedema score, pain-free range-of-motion, and soluble gene products that were detected for at least two days. Allogeneic and xenogeneic MSC produced a greater increase in peak of inflammation at 24 hours than either autologous MSC group. Genetically engineered MSC can act as vehicles to deliver gene products to the joint; further investigation into the therapeutic potential of this cell therapy is warranted. Intra-articular MSC injection resulted in a moderate acute inflammatory joint response that was greater for allogeneic and xenogeneic MSC than autologous MSC. Clinical management of this response may minimize this effect.

Protective effect of transplanted bone marrow-derived mesenchymal stem cells on pancreatitis-associated lung injury in rats

Severe acute pancreatitis (SAP) is initiated by the premature activation of digestive enzymes within the pancreatic acinar cells, leading to self-digestion and inflammatory responses in pancreatic ductal cells, thus giving rise to systemic inflammatory response syndrome (SIRS). The most common and serious SIRS is pancreatitis-associated lung injury, and inflammatory mediators play an important role in its pathogenesis. Bone marrow-derived mesenchymal stem cells (MSCs) are differentiated into alveolar endothelial cells to replace the damaged alveolar endothelial cells and inhibit inflammatory response in the injured lung tissues. In this study, we aimed to investigate the therapeutic effect of bone marrow-derived MSCs in rats with pancreatitis-associated lung injury. Experimental SAP was induced by a retrograde injection of 5% sodium taurocholate into the biliopancreatic duct of 75 male Sprague-Dawley rats, which were divided into the SAP group (n=25), the MSC group (n=25) and the sham-operated group (n=25) to explore the pathology and function of lung tissues and the regulation of inflammatory mediators. Pulmonary edema was estimated by measuring water content in the lung tissues. Pulmonary myeloperoxidase (MPO) activity was detected using spectrophotometry. Serum amylase was detected using the Automatic Biochemistry Analyzer. Tumor necrosis factor-α (TNF-α) and substance P (SP) mRNA levels were determined by quantitative reverse transcriptase-polymerase chain reaction. Our results showed that serum amylase activity was significantly decreased in the MSC group compared to the SAP group. Pulmonary edema was significantly diminished (p<0.05) in the MSC group compared to the SAP group. Typical acute lung injury was observed in the SAP group, and the pathological changes were mild in the MSC group. The expression of TNF-α and SP mRNA in lung tissue was diminished in the MSC group compared to the SAP group. In conclusion, MSC transplantation attenuates pulmonary edema and inflammation, and reduces the mRNA expression of TNF-α and SP in pancreatitis-associated lung injury.

Immunomodulatory Effects of Bone Marrow-Derived Mesenchymal Stem Cells in a Swine Hemi-Facial Allotransplantation Model

BackgroundIn this study, we investigated whether the infusion of bone marrow-derived mesenchymal stem cells (MSCs), combined with transient immunosuppressant treatment, could suppress allograft rejection and modulate T-cell regulation in a swine orthotopic hemi-facial composite tissue allotransplantation (CTA) model.Methodology/Principal FindingsOutbred miniature swine underwent hemi-facial allotransplantation (day 0). Group-I (n = 5) consisted of untreated control animals. Group-II (n = 3) animals received MSCs alone (given on days −1, +1, +3, +7, +14, and +21). Group-III (n = 3) animals received CsA (days 0 to +28). Group-IV (n = 5) animals received CsA (days 0 to +28) and MSCs (days −1, +1, +3, +7, +14, and +21). The transplanted face tissue was observed daily for signs of rejection. Biopsies of donor tissues and recipient blood sample were obtained at specified predetermined times (per 2 weeks post-transplant) or at the time of clinically evident rejection. Our results indicated that the MSC-CsA group had significantly prolonged allograft survival compared to the other groups (P<0.001). Histological examination of the MSC-CsA group displayed the lowest degree of rejection in alloskin and lymphoid gland tissues. TNF-α expression in circulating blood revealed significant suppression in the MSC and MSC-CsA treatment groups, as compared to that in controls. IHC staining showed CD45 and IL-6 expression were significantly decreased in MSC-CsA treatment groups compared to controls. The number of CD4+/CD25+ regulatory T-cells and IL-10 expressions in the circulating blood significantly increased in the MSC-CsA group compared to the other groups. IHC staining of alloskin tissue biopsies revealed a significant increase in the numbers of foxp3+T-cells and TGF-β1 positive cells in the MSC-CsA group compared to the other groups.ConclusionsThese results demonstrate that MSCs significantly prolong hemifacial CTA survival. Our data indicate the MSCs did not only suppress inflammation and acute rejection of CTA, but also modulate T-cell regulation and related cytokines expression.

Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model.Bone marrow-derived mesenchymal stem cells (Lin−CD105+) were harvested from GFP+-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP+-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay.We were able to visualize perivascular homing of MSCs in vivo. After 4days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls.In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.