Functionalization of Ceramic Scaffolds with Exosomes from Bone Marrow Mesenchymal Stromal Cells for Bone Tissue Engineering.

The functionalization of bone substitutes with exosomes appears to be a promising technique to enhance bone tissue formation. This study investigates the potential of exosomes derived from bone marrow mesenchymal stromal cells (BMSCs) to improve bone healing and bone augmentation when incorporated into wide open-porous 3D-printed ceramic Gyroid scaffolds. We demonstrated the multipotent characteristics of BMSCs and characterized the extracted exosomes using nanoparticle tracking analysis and proteomic profiling. Through cell culture experimentation, we demonstrated that BMSC-derived exosomes possess the ability to attract cells and significantly facilitate their differentiation into the osteogenic lineage. Furthermore, we observed that scaffold architecture influences exosome release kinetics, with Gyroid scaffolds exhibiting slower release rates compared to Lattice scaffolds. Nevertheless, in vivo implantation did not show increased bone ingrowth in scaffolds loaded with exosomes, suggesting that the scaffold microarchitecture and material were already optimized for osteoconduction and bone augmentation. These findings highlight the lack of understanding about the optimal delivery of exosomes for osteoconduction and bone augmentation by advanced ceramic scaffolds.

Over expression of ubiquitin-conjugating enzyme E2O in bone marrow mesenchymal stromal cells partially attenuates acute myeloid leukaemia progression.

Bone marrow mesenchymal stromal cells (BM-MSCs) are implicated in the pathogenesis of acute myeloid leukaemia (AML). However, due to the high heterogeneity of AML the mechanism underlying the cross-talk between MSCs and leukaemia cells is not well understood. We found that mixed-lineage leukaemia-AF9 (MLL-AF9)-induced AML mice-derived MSCs had higher proliferative viability compared to wild-type mice-derived MSCs with ubiquitin-conjugating enzyme E2O (Ube2o) down-regulation. After overexpression of UBE2O in AML-derived MSCs, the growth capacity of MSCs was reduced with nuclear factor kappa B subunit 1 (NF-κB) pathway deactivation. In vitro co-culture assay revealed that UBE2O-overexpression MSCs suppressed the proliferation and promoted apoptosis of AML cells by direct contact. In vivo results revealed that the leukaemia burden was reduced and the overall survival of AML mice was prolonged, with decreased dissemination of leukaemia cells in BM, spleen, liver and peripheral blood. Additionally, subcutaneous tumorigenesis revealed that tumour growth was also suppressed in the UBE2O-overexpression MSCs group. In conclusion, UBE2O was expressed at a low level in MLL-AF9-induced AML mice-derived MSCs. Overexpression of UBE2O in MSCs suppressed their proliferation through NF-κB pathway deactivation, which resulted in AML suppression. Our study provides a theoretical basis for a BM microenvironment-based therapeutic strategy to control disease progression.

Role of mesenchymal stromal cells derivatives in diabetic foot ulcers: a controlled randomized phase 1/2 clinical trial.

BACKGROUND: Diabetes-related foot complications have been identified as the most common isolated cause of morbidity among patients with diabetes and the leading cause of amputation. Therefore, new strategies to stimulate skin regeneration may provide a novel therapeutic approach to reduce non-healing ulcer disease. Recently, we demonstrated in proof-of-concept in humans that administration of allogeneic bone marrow mesenchymal stromal cellss derivatives (allo-hBM-MSCDs) is effective in a similar way to the use of allogeneic bone marrow mesenchymal stromal cellss (allo-hBM-MSCs) in grade 2 diabetic foot ulcers (DFUs). AIM: To assess the safety and efficacy profile of the allo-hBM-MSCDs relative to the conventional approach (PolyMen® dressing) in 1/2 clinical trial phases in patients with grade 1 and 2 DFUs. METHODS: In the present study, we used 2 doses of allo-hBM-MSCDs (1 mL) or 1 dose of allo-hBM-MSCs (1 × 106 cells) intradermally injected around wounds and assessed their safety and effectiveness, relative to the conventional approach (PolyMem dressing). Allo-hBM-MSCDs and allo-hBM-MSCs were produced in a certified Good Manufacturing Practice-type Laboratory. Patients with grade 1 and 2 DFUs were randomized to receive allo-hBM-MSCDs (n=12), allo-hBM-MSCs (n=6) or conventional treatment (PolyMem dressing) (n=10). The wound-healing process was macroscopically evaluated until the complete closure of the ulcers. RESULTS: No adverse events were reported. Patients with grade 1 and 2 DFUs treated with either allo-hBM-MSCDs or allo-hBM-MSCs, achieved greater percentages of wound closure, enhanced skin regeneration in shorter times and a greater ulcer-free survival relative to the patients who received conventional treatment. Finally, through proteomic analysis, we elucidated the proteins and growth factors that are secreted by allo-hBM-MSCs and relevant to the wound-healing process. In addition, by combining proteomics with Gene Ontology analysis, we comprehensively classified secreted proteins on both biological process and molecular function. CONCLUSIONS: In this phase 1/2 trial, our cumulative results suggest that 2 doses of allo-hBM-MSCDs combined with a wound dressing are a safe and effective treatment for grade 1 and 2 DFUs.

Extracellular vesicles from bone marrow mesenchymal stromal cells of severe aplastic anemia patients attenuate hematopoietic functions of CD34+ hematopoietic stem and progenitor cells.

Mesenchymal stromal cells (MSC) regulate hematopoiesis in the bone marrow (BM) niche and extracellular vesicles (EVs) released by BM-MSC are important mediators of the cross-talk between BM-MSC and hematopoietic stem and progenitor cells (HSPC). We have previously demonstrated that BM-MSC of severe aplastic anemia (SAA) patients have an altered expression of hematopoiesis regulatory molecules. In the present study, we observed that CD34+ HSPC when cocultured with BM-MSC EVs from aplastic anemia patients exhibited a significant reduction in colony-forming units (p = .001), cell proliferation (p = .002), and increased apoptosis (p > .001) when compared to coculture with BM-MSC EVs from controls. Collectively, our results highlight that EVs derived from the BM-MSC of SAA patients impair the hematopoiesis supporting function of HSPC.

Libanoridin Isolated from Corydalis heterocarpa Inhibits Adipogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells.

Bone marrow adiposity is a complication in osteoporotic patients. It is a result of the imbalance between adipogenic and osteogenic differentiation of bone marrow cells. Phytochemicals can alleviate osteoporotic complications by hindering bone loss and decreasing bone marrow adiposity. Corydalis heterocarpa is a biennial halophyte with reported bioactivities, and it is a source of different coumarin derivatives. Libanoridin is a coumarin isolated from C. heterocarpa, and the effect of libanoridin on adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) was evaluated in the present study. Cells were induced to undergo adipogenesis, and their intracellular lipid accumulation and expression of adipogenic markers were observed under libanoridin treatment. Results showed that 10 µM libanoridin-treated adipocytes accumulated 44.94% less lipid compared to untreated adipocytes. In addition, mRNA levels of PPARγ, C/EBPα, and SREBP1c were dose-dependently suppressed with libanoridin treatment, whereas only protein levels of PPARγ were decreased in the presence of libanoridin. Fluorescence staining of adipocytes also revealed that cells treated with 10 µM libanoridin expressed less PPARγ compared to untreated adipocytes. Protein levels of perilipin and leptin, markers of mature adipocytes, were also suppressed in adipocytes treated with 10 µM libanoridin. Analysis of MAPK phosphorylation levels showed that treatment with libanoridin inhibited the activation of p38 and JNK MAPKs observed by decreased levels of phosphorylated p38 and JNK protein. It was suggested that libanoridin inhibited adipogenic differentiation of hBM-MSCs via suppressing MAPK-mediated PPARγ signaling. Future studies revealing the anti-adipogenic effects of libanoridin in vivo and elucidating its action mechanism will pave the way for libanoridin to be utilized as a nutraceutical with anti-osteoporotic properties.

Acoustophoresis Enables the Label-Free Separation of Functionally Different Subsets of Cultured Bone Marrow Stromal Cells.

Culture-expanded mesenchymal stromal cells (MSCs) are promising candidates for clinical cell-based therapies. MSC products are heterogeneous and we therefore investigated whether acoustophoresis, an ultrasound-based separation technology, could be used for the label-free enrichment of functionally different MSC populations. Acoustophoresis uses an ultrasonic standing wave field in a microchannel that differentially affects the movement of cells depending on their acoustophysical properties, such as size, density, and compressibility. Human bone marrow (BM) MSCs were generated by standard adherent culture in xeno-free medium and separated by microchip acoustophoresis. MSCs with up to 20% higher proliferation and 1.7-fold increased clonogenic potential were enriched in the side outlet of the chip compared to the input sample. These cells were significantly smaller (average diameter 14.5 ± 0.4 µm) compared to the center outlet fraction (average diameter 17.1 ± 0.6 µm) and expressed higher levels of genes related to proliferation and stem cell properties (i.e., Ki-67 [1.9-fold], Nanog1 [6.65-fold], Oct4 [2.9-fold], and CXCL12 [1.8-fold], n = 3) in the side outlet compared to input. Fractions of MSCs in G0 /G1 cell cycle phase were significantly enriched in the side fraction and an up to 2.8-fold increase of cells in S/G2 /M phases were observed in center fractions compared to side fractions and 1.3-fold increased compared to the input sample. Acoustophoresis did not compromise MSC phenotype, proliferation, clonogenic capacity, and viability (generally 87-98%), nor did it affect differentiation or immunomodulatory capacities. These results demonstrate that label-free acoustic separation can enrich functionally different MSC subsets which can potentially be employed to produce better-defined stromal cell products from cultured MSCs. Hence, acoustophoresis is a potentially promising separation technology to provide improved cell products for research and possible future clinical use. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Fetal bovine serum-derived exosomes regulate the adipogenic differentiation of human bone marrow mesenchymal stromal cells in a cross-species manner.

Fetal bovine serum (FBS) contains a large number of exosomes which may disturb the analysis of exosomes derived from cultured cells. We investigated the effect of FBS-derived exosomes (FBS-Exos) on the adipogenic differentiation of human bone marrow mesenchymal stromal cells (hBM-MSCs) and the underlying molecular mechanism. The uptake of FBS-Exos by hBM-MSCs could be detected by the laser confocal microscopy, and the treatment of exosomes resulted in the decreased lipid droplet formation and reduced expression of genes associated with adipogenic differentiation of hBM-MSCs. miR-1246 was identified as an abundant microRNA in FBS-Exos by public sequencing data identification and RT-qPCR validation. Moreover, miR-1246 overexpression in hBM-MSCs led to decreased adipogenic differentiation level, while miR-1246 knockdown in FBS-Exos attenuated the inhibitory effect on the adipogenic differentiation. Our results indicate that FBS-Exos inhibit the adipogenic differentiation of hBM-MSCs in a cross-species manner and miR-1246 transferred by FBS-Exos partly contributes to this effect.

Phase II multicenter randomized controlled clinical trial on the efficacy of intra-articular injection of autologous bone marrow mesenchymal stem cells with platelet rich plasma for the treatment of knee osteoarthritis.

BACKGROUND: Mesenchymal stromal cells are a safe and promising option to treat knee osteoarthritis as previously demonstrated in different clinical trials. However, their efficacy, optimal dose and addition of adjuvants must be determined. Here, we evaluated the clinical effects of a dose of 100 × 106 bone marrow mesenchymal stromal cells (BM-MSCs) in combination with Platelet Rich Plasma (PRGF®) as adjuvant in a randomized clinical trial. METHODS: A phase II, multicenter, randomized clinical trial with active control was conducted. Sixty patients diagnosed with knee OA were randomly assigned to 3 weekly doses of PRGF® or intraarticular administration of 100 × 106 cultured autologous BM-MSCs plus PRGF®. Patients were followed up for 12 months, and pain and function were assessed using VAS and WOMAC and by measuring the knee range of motion range. X-ray and magnetic resonance imaging analyses were performed to analyze joint damage. RESULTS: No adverse effects were reported after BM-MSC administration or during follow-up. According to VAS, the mean value (SD) for PRGF® and BM-MSC with PRGF® went from 5 (1.8) to 4.5 (2.2) (p = 0.389) and from 5.3 (1.9) to 3.5 (2.5) (p = 0.01), respectively at 12 months. In WOMAC, the mean (SD) baseline and 12-month overall WOMAC scores in patients treated with PRGF® was 31.9 (16.2) and 22.3 (15.8) respectively (p = 0.002) while that for patients treated with BM-MSC plus PRGF® was 33.4 (18.7) and 23.0 (16.6) (p = 0.053). Although statistical significances between groups have been not detected, only patients being treated with BM-MSC plus PRGF® could be considered as a OA treatment responders following OARSI criteria. X-ray and MRI (WORMS protocol) revealed no changes in knee joint space width or joint damage. CONCLUSIONS: Treatment with BM-MSC associated with PRGF® was shown to be a viable therapeutic option for osteoarthritis of the knee, with clinical improvement at the end of follow-up. Further phase III clinical trials would be necessary to confirm the efficacy. Trial registration Clinical Trials.gov identifier NCT02365142. Nº EudraCT: 2011-006036-23.

Intrinsic Angiogenic Potential and Migration Capacity of Human Mesenchymal Stromal Cells Derived from Menstrual Blood and Bone Marrow.

Several therapies are being developed to increase blood circulation in ischemic tissues. Despite bone marrow-derived mesenchymal stromal cells (bmMSC) are still the most studied, an interesting and less invasive MSC source is the menstrual blood, which has shown great angiogenic capabilities. Therefore, the aim of this study was to evaluate the angiogenic properties of menstrual blood-derived mesenchymal stromal cells (mbMSC) in vitro and in vivo and compared to bmMSC. MSC's intrinsic angiogenic capacity was assessed by sprouting and migration assays. mbMSC presented higher invasion and longer sprouts in 3D culture. Additionally, both MSC-spheroids showed cells expressing CD31. mbMSC and bmMSC were able to migrate after scratch wound in vitro, nonetheless, only mbMSC demonstrated ability to engraft in the chick embryo, migrating to perivascular, perineural, and chondrogenic regions. In order to study the paracrine effects, mbMSC and bmMSC conditioned mediums were capable of stimulating HUVEC's tube-like formation and migration. Both cells expressed VEGF-A and FGF2. Meanwhile, PDGF-B was expressed exclusively in mbMSC. Our results indicated that mbMSC and bmMSC presented a promising angiogenic potential. However, mbMSC seems to have additional advantages since it can be obtained by non-invasive procedure and expresses PDGF-B, an important molecule for vascular formation and remodeling.

Donor-derived marrow mesenchymal stromal cell co-transplantation following a haploidentical hematopoietic stem cell transplantation trail to treat severe aplastic anemia in children.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is associated with an increased risk of graft failure and severe graft-versus-host disease (GVHD). Recent studies have shown that mesenchymal stromal cells (MSCs) display potent immunosuppressive effects and can support normal hematopoiesis. In a multi-center trial, we co-transplanted culture-expanded donor-derived bone marrow MSCs (BM-MSCs) into 35 children with severe aplastic anemia (SAA) undergoing haplo-HSCT. All 35 patients (100%) achieved hematopoietic reconstitution and showed sustained full donor chimerism. The median time for myeloid engraftment was 14 days (range 10-22 days), while that for platelet engraftment was 18 days (range 9-36 days). The incidence of grade II-IV acute GVHD and chronic GVHD was 25.71 and 22.86%, respectively. The overall survival rate was 85.71% with a median of 22 months (range 3.5-37 months). The combined transplantation of haploidentical HSCs and BM-MSCs into children with SAA without an HLA-identical sibling donor is relatively safe and may represent an effective new therapy to improve survival rates and reduce the risk of graft failure.

Study of the Osteoindictive Properties of Protein-Modified Polylactide Scaffolds.

Bone marrow mesenchymal stromal cells are multipotent and can differentiate into cells of various tissues, which determines their high importance for clinical application. We performed an in vitro study of the osteogenic potential of mesenchymal stromal cells cultured on intact polylactide scaffolds or scaffolds modified with collagen I or fibrin. Scanning electron microscopy showed that the cells formed osteogenic nodules or osteogenic nodules on both intact and fibrin-modified polylactide scaffolds. Spectrophotometric detection of alkaline phosphatase activity on days 7 and 11 showed that mesenchymal stromal cell grown on intact polylactide scaffolds and on scaffolds modified with collagen type I or fibrin more intensively synthesized alkaline phosphatase than in the control (culture plastic). This dependence increases in the presence of osteogenic differentiation factors in the medium. After long-term culturing (4 weeks), the presence of calcium deposits detected by alizarin red staining confirmed the osteoinductive properties of intact and protein-modified polylactide scaffolds. These findings suggest that polylactide scaffolds and collagen I increase the osteogenic differentiation potential of mesenchymal stromal cells.

Microtubule defects in mesenchymal stromal cells distinguish patients with Progressive Supranuclear Palsy.

Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease whose etiopathogenesis remains elusive. The intraneuronal accumulation of hyperphosphorylated Tau, a pivotal protein in regulating microtubules (MT), leads to include PSP into tauopathies. Pathological hallmarks are well known in neural cells but no word yet if PSP-linked dysfunctions occur also in other cell types. We focused on bone marrow mesenchymal stromal cells (MSCs) that have recently gained attention for therapeutic interventions due to their anti-inflammatory, antiapoptotic and trophic properties. Here, we aimed to investigate MSCs biology and to disclose if any disease-linked defect occurs in this non-neuronal compartment. First, we found that cells obtained from patients showed altered morphology and growth. Next, Western blotting analysis unravelled the imbalance in α-tubulin post-translational modifications and in MT stability. Interestingly, MT mass is significantly decreased in patient cells at baseline and differently changes overtime compared to controls, suggesting their inability to efficiently remodel MT cytoskeleton during ageing in culture. Thus, our results provide the first evidence that defects in MT regulation and stability occur and are detectable in a non-neuronal compartment in patients with PSP. We suggest that MSCs could be a novel model system for unravelling cellular processes implicated in this neurodegenerative disorder.

Bone marrow-derived stem/stromal cells (BMSC) 3D microtissues cultured in BMP-2 supplemented osteogenic induction medium are prone to adipogenesis.

Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.

Improvement of the folliculogenesis by transplantation of bone marrow mesenchymal stromal cells in mice with induced polycystic ovary syndrome.

BACKGROUND: Many studies have reported that inflammation and oxidative stress are involved in the pathogenesis of polycystic ovary syndrome (PCOS). Bone marrow mesenchymal stromal cells (BM-MSCs) have anti-oxidant and anti-inflammation properties. In this study, we investigate the beneficial effect of stem cell therapy on folliculogenesis in mice with induced PCOS METHODS: Mouse model of PCOS was performed through daily injection of testosterone enanthate (1 mg/100 g/body weight subcutaneous (s.c).) for a period of 5 weeks. Naval Medical Research Institute (NMRI) mice (21 days old) were divided into three groups: control, PCOS and PCOS + BM-MSCs. BM-MSCs were labeled with Hoechst 33342 (0.5 µg/mL) and then injected into the mice (106/animal, via the tail vein) at 1 and 14 days after PCOS confirmation. Mice were humanely killed at 2 weeks after last transplantation. Ovarian stereological studies were done. Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), testosterone, interleukin (IL)-6 and tumor necrosis factor (TNF)-α serum levels were measured. The levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) in serum were analyzed. Apoptotic index for ovarian follicles was assessed using Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). CD31 expression in ovarian vessels was assessed with the immunohistochemistry. RESULTS: There was a significant increase in the total volume of ovary, cortex, number of antral follicles, volume of oocyte and zona pellucida thickness, and there was a significant decrease in the primary and preantral follicles number in the PCOS + BM-MSCs group compared with the PCOS group. There was a significant increase in the serum level of FSH and TAC and a significant decrease in the serum level of testosterone, LH, MDA and percentage of TUNEL-positive apoptotic cells in the PCOS + BM-MSCs group in comparison with the PCOS group. DISCUSSION: BM-MSC transplantation improves folliculogenesis in mice with induced PCOS. BM-MSC therapy can be an operative treatment for PCOS via anti-inflammatory, anti-oxidant and anti-apoptotic properties.

Human mesenchymal stromal cell therapy for damaged cochlea repair in nod-scid mice deafened with kanamycin.

BACKGROUND: Kanamycin, mainly used in the treatment of drug-resistant-tuberculosis, is known to cause irreversible hearing loss. Using the xeno-transplant model, we compared both in vitro and in vivo characteristics of human mesenchymal stromal cells (MSCs) derived from adult tissues, bone marrow (BM-MSCs) and adipose tissue (ADSCs). These tissues were selected for their availability, in vitro multipotency and regenerative potential in vivo in kanamycin-deafened nod-scid mice. METHODS: MSCs were isolated from informed donors and expanded ex vivo. We evaluated their proliferation capacity in vitro using the hexosaminidase assay, the phenotypic profile using flow-cytometry of a panel of surface antigens, the osteogenic potential using alkaline phosphatase activity and the adipogenic potential using oil-red-O staining. MSCs were intravenously injected in deafened mice and cochleae, liver, spleen and kidney were sampled 7 and 30 days after transplantation. The dissected organs were analyzed using lectin histochemistry, immunohistochemistry, polymerase chain reaction (PCR) and dual color fluorescence in situ hybridization (DC-FISH). RESULTS: MSCs showed similar in vitro characteristics, but ADSCs appeared to be more efficient after prolonged expansion. Both cell types engrafted in the cochlea of damaged mice, inducing regeneration of the damaged sensory structures. Several hybrid cells were detected in engrafted tissues. DISCUSSION: BM-MSCs and ADSCs showed in vitro characteristics suitable for tissue regeneration and fused with resident cells in engrafted tissues. The data suggest that paracrine effect is the prevalent mechanism inducing tissue recovery. Overall, BM-MSCs and ADSCs appear to be valuable tools in regenerative medicine for hearing loss recovery.

Effect of Concentration of Collagen Gel on Functional Activity of Bone Marrow Mesenchymal Stromal Cells.

Collagen I gels with protein concentrations of 1, 2, and 3.5 mg/ml were prepared and embedded in a porous polylactide scaffold to reduce their contraction. Concentration of the gel did not affect its degradation. Collagen gels promoted the formation of cell networks. The cells in the collagen gel with a concentration of 1 mg/ml embedded in polylactide scaffold had elongated spindle-like shape, in contrast to flattened cells in collagen gel of the same concentration not embedded in the scaffold. Stabilization of the collagen gel in the polylactide scaffold promoted active synthesis of laminin and fibronectin by cells as soon as on day 5 of culturing in comparison with that in free collagen substrate.

Intra-articular injection of two different doses of autologous bone marrow mesenchymal stem cells versus hyaluronic acid in the treatment of knee osteoarthritis: multicenter randomized controlled clinical trial (phase I/II).

BACKGROUND: Mesenchymal stromal cells are a promising option to treat knee osteoarthritis. Their safety and usefulness must be confirmed and the optimal dose established. We tested increasing doses of bone marrow mesenchymal stromal cells (BM-MSCs) in combination with hyaluronic acid in a randomized clinical trial. MATERIALS: A phase I/II multicenter randomized clinical trial with active control was conducted. Thirty patients diagnosed with knee OA were randomly assigned to intraarticularly administered hyaluronic acid alone (control), or together with 10 × 10(6) or 100 × 10(6) cultured autologous BM-MSCs, and followed up for 12 months. Pain and function were assessed using VAS and WOMAC and by measuring the knee motion range. X-ray and magnetic resonance imaging analyses were performed to analyze joint damage. RESULTS: No adverse effects were reported after BM-MSC administration or during follow-up. BM-MSC-administered patients improved according to VAS during all follow-up evaluations and median value (IQR) for control, low-dose and high-dose groups change from 5 (3, 7), 7 (5, 8) and 6 (4, 8) to 4 (3, 5), 2 (1, 3) and 2 (0,4) respectively at 12 months (low-dose vs control group p = 0.005 and high-dose vs control group p < 0.009). BM-MSC-administered patients were also superior according to WOMAC, although improvement in control and low-dose patients could not be significantly sustained beyond 6 months. On the other hand, the BM-MSC high-dose group exhibited an improvement of 16.5 (12, 19) points at 12 months (p < 0.01). Consistent with WOMAC and VAS values, motion ranges remained unaltered in the control group but improved at 12 months with BM-MSCs. X-ray revealed a reduction of the knee joint space width in the control group that was not seen in BM-MSCs high-dose group. MRI (WORMS protocol) showed that joint damage decreased only in the BM-MSC high-dose group, albeit slightly. CONCLUSIONS: The single intraarticular injection of in vitro expanded autologous BM-MSCs together with HA is a safe and feasible procedure that results in a clinical and functional improvement of knee OA, especially when 100 × 10(6) cells are administered. These results pave the way for a future phase III clinical trial. CLINICAL TRIALS: gov identifier NCT02123368. Nº EudraCT: 2009-017624-72.

Functional Restoration of Amyotrophic Lateral Sclerosis Patient-Derived Mesenchymal Stromal Cells Through Inhibition of DNA Methyltransferase.

Alteration of DNA methylation is highly associated with aging and neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). Remedying these aberrant methylation patterns may serve to improve these diseases. Previously, we reported that human bone marrow mesenchymal stromal cells isolated from ALS patients (ALS-MSCs) have functionally decreased stem cell potency, and excessively express DNA methyltransferases (DNMTs). In this study, we examined the correlation between excessive DNMT expression and functional decline in ALS-MSCs. The DNMT inhibitor RG108 was used for this. RG108-treated ALS-MSCs exhibit increased expression of the anti-senescence genes TERT, VEGF, and ANG, and decreased expression of the senescence-related genes ATM and p21. The activity of SA-ß-galactosidase and the expression of senescence proteins p53 and p16 were reduced in RG108-treated ALS-MSCs. The abilities of cell migration and protection against oxidative damage were improved in the treated ALS-MSCs. In neuronal differentiation experiments, the treated MSCs more effectively differentiated into neuron-like cells. These results suggest that ALS-MSC function can be restored by inhibiting excessively expressed DNMTs, an approach that may ultimately provide better efficacy in stem cell therapy.

Harvest tissue source does not alter the protective power of stromal cell therapy after intestinal ischemia and reperfusion injury.

BACKGROUND: Transplantation of mesenchymal stromal cells (MSCs) may be a novel treatment for intestinal ischemia. The optimal stromal cell source that could yield maximal protection after injury, however, has not been identified. We hypothesized that (1) MSCs would increase survival and mesenteric perfusion, preserve intestinal histologic architecture, and limit inflammation after intestinal ischemia and reperfusion (I/R) injury, and (2) MSCs harvested from different sources of tissue would have equivalent protective properties to the intestine after I/R inury. METHODS: Adult male mice were anesthetized, and a midline laparotomy was performed. The intestines were eviscerated, the small bowel mesenteric root was identified, and baseline intestinal perfusion was determined using laser Doppler imaging. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 min with a noncrushing clamp. After ischemia, the clamp was removed and the intestines were allowed to recover. Before abdominal closure, 2 × 10(6) human umbilical cord-derived MSCs, bone marrow-derived MSCs, or keratinocytes in 250 µL of phosphate-buffered saline vehicle were injected into the peritoneum. Animals were allowed to recover for 12 or 24 h (perfusion, histology, and inflammatory studies) or 7 d (survival studies). Survival data was analyzed using the log-rank test. Perfusion was expressed as a percentage of the baseline, and 12- and 24-h data was analyzed using one-way analysis of variance and the Student t-test. Nonparametric data was compared using the Mann-Whitney U-test. A P value of <0.05 was considered statistically significant. RESULTS: All MSCs increased 7-d survival after I/R injury and were superior to vehicle and keratinocytes (P < 0.05). All MSCs increased mesenteric perfusion more than vehicle at 12 and 24 h after injury (P < 0.05). All MSCs provided superior perfusion compared with keratinocytes at 24 h after injury (P < 0.05). Administration of each MSC line improved intestinal histology after I/R injury (P < 0.05). Multiple proinflammatory chemokines were downregulated after the application of MSCs, suggesting a decreased inflammatory response after MSC therapy. CONCLUSIONS: Transplantation of MSCs after intestinal I/R injury, irrespective of a tissue source, significantly increases survival and mesenteric perfusion and at the same time limits intestinal damage and inflammation. Further studies are needed to identify the mechanism that these cells use to promote improved outcomes after injury.

Autologous transplantation of mesenchymal stromal cells tends to prevent progress of interstitial fibrosis in a rhesus Macaca mulatta monkey model of chronic kidney disease.

BACKGROUND AIMS: Chronic kidney disease (CKD) attributed to cisplatin is well documented. Mesenchymal stromal cells (MSCs) are proven to be renotropic. Although they have been shown to improve function in CKD and reduce fibrosis in different experimental rodent models, their efficiency in primates is unknown. The present study aimed to evaluate the prevention of CKD and reduction of fibrosis in monkeys treated with MSCs after cisplatin nephrotoxicity. METHODS: We induced CKD in adult rhesus Macaca mulatta monkeys by means of intravenous administration of cisplatin. Autologous MSCs were transplanted by means of intrarenal arterial injections to assess the adverse effects of cisplatin in two CKD models: preventative and stable. Preventative CKD monkeys (n = 3) underwent cell transplantation 4 days after the cisplatin injection. The stable CKD monkeys (n = 2) underwent cell transplantation 6 months after the cisplatin injection. Non-treated (n = 4) and normal saline-injected animals (n = 3) comprised the control and vehicle groups, respectively. We followed the animals for survival rate, serum biochemistry, urine analysis and histopathological indices. RESULTS: In the preventive CKD model, MSC transplantation tended to improve some renal functions but significantly reduced the histopathologic score compared with the vehicle and control groups. In the stable CKD model, MSCs did not ameliorate renal function or pathological score. CONCLUSIONS: These results suggest that MSCs tend to delay progression of CKD and fibrosis but do not reduce established interstitial fibrosis in this unique primate model of cisplatin-induced nephrotoxicity.