Case Report: Equine allogeneic umbilical cord blood mesenchymal stromal cells (CB-MSC) as adjunctive therapy in a foal with septic arthritis and osteomyelitis

SummaryBackgroundTendinopathies in racehorses are challenging to treat, and restoring normal tendon composition remains elusive despite extensive research. Equine multipotent mesenchymal stromal cells (MSC) have shown promise for tendon repair, and various sources of MSC have been described.ObjectivesThe objectives of the study were (1) to report the clinical efficacy of superficial digital tendon flexor tendinopathy treated with repeated injections of pooled frozen allogeneic umbilical cord blood mesenchymal stromal cells (CB‐MSC); (2) to confirm that the use of allogeneic CB‐MSC for treatment of superficial digital flexor tendon (SDFT) injuries through repeated injections does not induce adverse reactions.Study designProspective clinical study.MethodsFive Thoroughbreds, in speed training and racing, sustaining an SDFT lesion within 6 weeks prior to the treatment were included. Pooled frozen allogeneic umbilical CB‐MSC were delivered twice intralesionally 4 weeks apart. CD4+ and CD8+ lymphocyte populations were analysed by flow cytometry. Ultrasound and lameness rechecks were performed every 4 weeks for a year.ResultsAll three flat‐racing Thoroughbreds receiving CB‐MSC who adhered to the prescribed rehabilitation programme raced multiple times and remained reinjury‐free during the 12‐month follow‐up period. No serious adverse reactions were noted to the treatment. Four horses treated with CB‐MSC displayed an expansion of the needle tract at the injection site.Main limitationsThe study's main limitations include a small number of horses.ConclusionsThe novel repeated intralesional treatment using thawed pooled allogeneic equine CB‐MSC resulted in positive clinical outcomes, with all horses returning to racing without reinjury when following the rehabilitation programme.

Large Scale Manufacturing of GMP Grade Cord Blood Derived Megakaryocytes for Chemotherapy Induced Thrombocytopenia

Cord Blood Mesenchymal Stem Cells for Acute Renal Failure Repair.

Quantification and Immunophenotypic Characterization of Bone Marrow and Umbilical Cord Blood Mesenchymal Stem Cells by Multicolor Flow Cytometry

Allogeneic Mesenchymal Stem Cells for Treatment of Experimental Autoimmune Encephalomyelitis

Objective To study the modification of bone marrow and human umbilical cord blood mesenchymal stem cells with hBMP-2 gene and compare the modification results. Methods Bone marrow and human umbilical cord blood mesenchymal stem cells were separated and cultured by density gradient centrifugation and adherent culture. The recombinant hBMP-2 plasmid was transfected into bone marrow and human umbilical cord blood mesenchymal stem cells by high-performance non-liposome reagent X-treme GENE. The fluorescence intensity was measured by inverted fluorescence microscopy and the transfection efficiency was calculated. The hBMP was detected by qPCR. The expression of-2 and chondronectin in two kinds of cells was detected by immunohistochemistry. Results Bone marrow and human umbilical cord blood mesenchymal stem cells were successfully isolated and cultured. The two cells had different growth characteristics. The recombinant hBMP-2 plasmid mediated by high-performance non-liposome reagent was successfully transfected into bone marrow and human umbilical cord blood mesenchymal stem cells. The efficiency of transfection of bone marrow mesenchymal stem cells (18.44±5.94)% was lower than that of human umbilical cord blood mesenchymal stem cells (27.74±7.59)%, and the difference was statistically significant (t=3.027, P<0.05). The expression of hBMP-2, cartilage connectin and collagen type II were detected in both transfected cells. Conclusion hBMP-2 gene can effectively modify bone marrow and human umbilical cord blood mesenchymal stem cells and promote chondrocyte differentiation. Key words: BMP2 protein, human; Mesenchymal stem cells; Liposomes

Equine mesenchymal stromal cells (MSC) are commonly transported, chilled or frozen, to veterinary clinics. These MSC must remain viable and minimally affected by culture, transport, or injection processes. The safety of two carrier solutions developed for optimal viability and excipient use were evaluated in ponies, with and without allogeneic cord blood-derived (CB) MSC. We hypothesized that neither the carrier solutions nor CB-MSC would elicit measurable changes in clinical, hematological, or biochemical parameters. In nine ponies (study 1), a bolus of HypoThermosol® FRS (HTS-FRS), CryoStor® CS10 (CS10), or saline was injected IV (n = 3/treatment). Study 2, following a 1-week washout period, 5 × 107 pooled allogeneic CB-MSCs were administered IV in HTS-FRS following 24 h simulated chilled transport. Study 3, following another 1-week washout period 5 × 107 pooled allogeneic CB-MSCs were administered IV in CS10 immediately after thawing. Nine ponies received CB-MSCs in study 2 and 3, and three ponies received the cell carrier media without cells. CB-MSCs were pooled in equal numbers from five unrelated donors. In all studies, ponies were monitored with physical examination, and blood collection for 7 days following injection. CD4 and CD8 lymphocyte populations were also evaluated in each blood sample. In all three studies, physical exam, complete blood cell count, serum biochemistry, and coagulation panel did not deviate from established normal ranges. Proportions of CD4+ and CD8+ lymphocytes increased at 168 h postinjection in CB-MSC treatment groups regardless of the carrier solution. Decreases in CD4+/CD8+ double positive populations were observed at 24 and 72 h in CB-MSC-treated animals. There was no difference in viability between CB-MSCs suspended in HTS-FRS and CS10. HTS-FRS and CS10 used for low volume excipient injection of MSC suspensions were not associated with short-term adverse reactions. HTS-FRS and CS10 both adequately maintain CB-MSC viability following hypothermic or frozen simulated transport, respectively. CB-MSCs do not elicit clinical abnormalities, but allogeneic stimulation of CD4+ and CD8+ lymphocyte populations may occur. Future studies should include in vitro or in vivo evaluation of cell-mediated or adaptive immunity to autologous, identical allogeneic, or MSC originating from additional unrelated individuals in order to better characterize this response.

Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16(ink4a) protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-gamma secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E(2)-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications.

Objective To investigate the role of human umbilical cord blood(UCB)-derived mesenchymal stem cells(MSCs) in improving the neurological function of acute ischemic stroke model of rats and mechanisms. Methods The middle cerebral artery occulusion(MCAO) models in 82 rats were randomly divided into UCB- MSCs group(n= 41)and control group(n= 41).Human UCB- MSCs were isolated and cultured, and identified by nestin staining. The UCB- MSCs were transplanted into the ventricles of rats 48 h after modeling and marked.At 24th h, 7th day and 14 day after transplantation, the neurological functions were assessed by using improved neurological function in rats(m NSS).The proliferation of endogenous neural stem cells and expression of vascular endothelial growth factor(VEGF) were detected by using immunofluorescence method. Results UCB-MSCs were successfully separated in 42 parts.At 7th day and 14th day after transplantation, the function scores of rats in UCB- MSCs group were 6.76±0.42 and 4.27±0.49, which were statistically significantly lower than in the control group(P< 0.05).At 14th day after transplantation, the number of 5- bromo 2'- deoxyuridine(Brd U)/doublecortin(DCX)and Brd U/glial fibrillary acidic protein(GFAP) double labeled positive cells was 74.31±3.04 and 65.38±2.43 respectively, which were significantly greater than in the control group(P< 0.05).The expression of neurotrophic factor VEGF in UCB- MSCs group was 109.53±5.91, significantly higher than in the control group(P< 0.05). Conclusion UCB-MSCs transplantation could effectively promote neurological function recovery of acute ischemic stroke model possibly by promoting the proliferation of the endogenous neural stem cells and neurotrophic factor VEGF release. Key words: Human umbilical cord blood-derived mesenchymal stem cells; Ischemic stroke; Neurological function

Mitigating effects of hUCB-MSCs on the hematopoietic syndrome resulting from total body irradiation

Allogenic Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Are More Effective Than Bone Marrow Aspiration Concentrate for Cartilage Regeneration After High Tibial Osteotomy in Medial Unicompartmental Osteoarthritis of Knee

Colorectal cancer is the most common tumor of the gastrointestinal system. The conventional treatment options for colorectal cancer are troublesome for both patients and clinicians. Recently, mesenchymal stem cells (MSCs) have been the novel focus for cell therapy due to their migration to tumor sites. In this study, the apoptotic effect of MSCs on colorectal cancer cell lines has been aimed. HCT-116 and HT-29 were selected as the colorectal cancer cell lines. Human umbilical cord blood and Wharton's jelly were used as mesenchymal stem cell sources. To discriminate against the apoptotic effect of MSC on cancer, we also used peripheral blood mononuclear cells (PBMC) as a healthy control group. Cord blood-MSC and PBMC were obtained by ficoll-paque density gradient, and Wharton's jelly-MSC by explant method. Transwell co-culture systems were used as cancer cells or PBMC/MSCs at ratios of 1/5 and 1/10, with incubation times of 24h and 72h. The Annexin V/PI-FITC-based apoptosis assay was performed by flow cytometry. Caspase-3 and HTRA2/Omi proteins were measured by ELISA. For both ratios in both cancer cells, it was found that the apoptotic effect of Wharton's jelly-MSC was significantly higher in 72-h incubations (p < 0.006), whereas the effect of cord blood mesenchymal stem cell in 24-h incubations were higher (p < 0.007). In this study, we showed that human cord blood and tissue-derived MSCs treatment led to colorectal cancers to apoptosis. We anticipate that further in vivo studies may shed light on the apoptotic effect of MSC.

Potential Role of Immunosuppression in Therapeutic Effects Conferred by Cord Blood- Derived Mesenchymal Stem Cells.

Can the Therapeutic Advantages of Allogenic Umbilical Cord Blood???Derived Stem Cells and Autologous Bone Marrow???Derived Mesenchymal Stem Cells Be Combined and Synergized?

Objective To gain mesenchymal stem cells from humanumbilical cord blood and transfect the recombinant plasmid pIRES2-EGFP-hBMP-2 into human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) with liposome and to induce hUCB-MSCs osteoblastization. Methods Separated cells from human umbilical cord blood with density gradient centrifugation, and hUCB-MSCs were gained with adherent method, using flow cytometry to identify the surface markers of hUCB-MSCs. Transfected recombinate plasmid pIRES2-EGFP-hBMP-2 into the third generation hUCB-MSCs with X-treme GENE HP DNA Transfection reagent, then detected the intensity of EGFP. Collected the medium at different time after transfection and the hBMP-2 content in the medium was measured by ELISA. Use immunofluorescent to locate the existence of hBMP-2 within the cells and RT-PCR techniques to measure the transcription of the hBMP-2 gene. Two weeks after transfection, immunohistochemistry were used to detect the type Ⅱ collagen for discovering the possible changes of hUCB-MSCs. Results HUCB-MSCs could be isolated from blood in umbilical cord by density gradient centrifugation and the different ability of adherence. Flow cytometry showed that the hUCB-MSCs positively expressed CD90, CD105 and CD146, and did not express CD34, CD45 and Anti-HLA-DR. The recombinant plasmid pIRES2-EGFP-hBMP-2 was successfully fused into umbilical cord blood mesenchymal stem cells, and the fusion rate was (27.7±7.6)%. The ELISA test compared the expression of hBMP-2 between the experimental group and the control group, P<0.01 showed a statistically significant difference. RT-PCR results showed that the hBMP-2 gene was stably transcribed and the immunofluorescently labeled hBMP-2 protein showed red fluorescence. Immunohistochemical staining of type Ⅱ collagen showed that some cells were browned. RT-PCR results showed that the expression of CRLT1 in umbilical cord blood mesenchymal stem cells transfected with BMP-2 was higher than that in untransfected umbilical cord blood mesenchymal stem cells and human synovial fibrosis group (P<0.05). Conclusions Recombinat plasmid pIRES2-EGFP-BMP-2 coated with X-treme CENE can be successfully transfected into hUCB-MSCs. Both the marker gene and objectivegene can be transcribed and expressed in hUCB-MSCs, and cells can synthesis hBMP-2, which stimulates the hUCB-MSCs'differentiation towards to chondrocytes. Key words: Recombinant human bone morphogenetic protein-2; Mesenchymal stem cells; Gene editing; Liposomes