Encapsulated mesenchymal stromal cells for in vivo transplantation

Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in vitro and in vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 h post-injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post-SCI indicated that as few as 5 × 10(4) encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.     

Co-transplantation of islets with mesenchymal stem cells in microcapsules demonstrates graft outcome can be improved in an isolated-graft model of islet transplantation in mice

Background aimsCo-transplantation of islets with mesenchymal stem cells (MSCs) has been shown to improve graft outcome in mice, which has been partially attributed to the effects of MSCs on revascularization and preservation of islet morphology. Microencapsulation of islets provides an isolated-graft model of islet transplantation that is non-vascularized and prevents islet aggregation to preserve islet morphology. The aim of this study was to investigate whether MSCs could improve graft outcome in a microencapsulated/isolated-graft model of islet transplantation.MethodsMouse islets and kidney MSCs were co-encapsulated in alginate, and their function was assessed in vitro. A minimal mass of 350 syngeneic islets encapsulated alone or co-encapsulated with MSCs (islet+MSC) were transplanted intraperitoneally into diabetic mice, and blood glucose concentrations were monitored. Capsules were recovered 6 weeks after transplantation, and islet function was assessed.ResultsIslets co-encapsulated with MSCs in vitro had increased glucose-stimulated insulin secretion and content. The average blood glucose concentration of transplanted mice was significantly lower by 3 weeks in the islet+MSC group. By week 6, 71% of the co-encapsulated group were cured compared with 16% of the islet-alone group. Capsules recovered at 6 weeks had greater glucose-stimulated insulin secretion and insulin content in the islet+MSC group.ConclusionsMSCs improved the efficacy of microencapsulated islet transplantation. Using an isolated-graft model, we were able to eliminate the impact of MSC-mediated enhancement of revascularization and preservation of islet morphology and demonstrate that the improvement in insulin secretion and content is sustained in vivo and can significantly improve graft outcome.     

Improved motor function in dko mice by intravenous transplantation of bone marrow-derived mesenchymal stromal cells

Background aimsWe explored the potential therapeutic value of transplanting bone marrow (BM)-derived mesenchymal stromal cells (MSC) into utrophin/dystrophin-deficient double knock-out (dko) mice, a murine model of Duchenne muscular dystrophyMethodsMSC from male rats were isolated and transplanted into female dko mice via the caudal vein. Behavior and locomotor function were later evaluated, along with the expression of dystrophin and utrophin in the sarcolemma of myofiber tissues. The presence of grafted cells was confirmed via polymerase chain reaction for the sex-determining region of the Y-chromosomeResultsLocomotor activity improved significantly (P < 0.05) from 5 to 15 weeks after cell transplantation, as measured by traction, rotating rod and running wheel tests. We also found that the expression of dystrophin and utrophin increased significantly (P < 0.05) and progressively in the sarcolemma from 5 to 15 weeks after transplantation. The median lifespan of mice in the normal group (74.1 weeks) was significantly (P < 0.001) higher than those in the control (22.0 weeks) and transplantation (35.0 weeks) groups, and the median lifespan of mice in the transplantation group was significantly (P < 0.001) higher than that in the control groupConclusionsResults of this study demonstrate that BM MSC have potential value in xenogeneic transplantation therapy for muscular dystrophy.     

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

Treatment of diabetic wounds with fetal murine mesenchymal stromal cells enhances wound closure

The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF ^E6-EGFP cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF ^E6-EGFP cells expressed tendon/ligament phenotype-related genes such as Scleraxis (Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF ^E6-EGFP cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF ^E6-EGFP cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF ^E6-EGFP cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies. This work was supported by a Grant-in Aid for the High-Tech Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the AGU High-Tech Research Center Project, the 2003-Multidisciplinary Research Project from MEXT, and grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

Senescence is heterogeneous in mesenchymal stromal cells: Kaleidoscopes for cellular aging

Comment on: Wagner W, et al. Aging 2010; 2:224-30.     

The antiproliferative effect of mesenchymal stem cells is a fundamental property shared by all stromal cells

Although it has been widely demonstrated that human mesenchymal stem cells exert potent immunosuppressive effects, there is little information as to whether more mature mesenchymal stromal cells (SC) share the same property. Accordingly, we set out to test the ability of SC from different human tissues to inhibit the proliferation of PBMC following polyclonal stimuli. Chondrocytes, as well as fibroblasts from synovial joints, lung, and skin, were used as a source of SC. Irrespective of their differentiation potential and/or content of progenitor cells, SC from all tissues exhibited antiproliferative functions. This was in marked contrast to parenchymal cells. Although SC did not interfere with early T lymphocyte activation, they arrested stimulated T cells in the G(0)/G(1) phase of the cell cycle and rescued them from apoptosis. In addition, IFN-gamma and TNF-alpha production were reduced. We observed that the inhibitory effect is ultimately mediated by soluble factors, the production of which requires SC to be licensed in an inflammatory environment by cell contact. We conclude that the immunosuppressive effect of mesenchymal cells is not confined to multipotent stem cells, but is a fundamental characteristic of all stroma. Our data suggest that SC, appropriately licensed, regulate T cell homeostasis.     

N-Cadherin is expressed on human hematopoietic progenitor cells and mediates interaction with human mesenchymal stromal cells

Specific cell–cell junctions between hematopoietic stem cells (HSC) and their niche have been shown to regulate stem cell function. N-cadherin was suggested to play a central role in this process, whereas other studies indicated that it did not play an essential role in the murine model. We have analyzed the role of N-cadherin for interaction between hematopoietic progenitor cells (HPC) and supportive mesenchymal stromal cells (MSC) in a human–human setting. Expression of N-cadherin and of cadherin-11 (osteoblast cadherin) was analyzed in HPC by quantitative RT-PCR, Western blot, and flow cytometry. N-cadherin and cadherin-11 were expressed in HPC at a moderate level, whereas they were not detectable in differentiated cells. Confocal laser scanning microscopy revealed that N-cadherin and β-catenin are colocalized at the junction of HPC and MSC. siRNA knockdown of N-cadherin or cadherin-11 as well as treatment with the blocking function antibody decreased adhesive interaction of HPC to MSC. Furthermore, knockdown of N-cadherin or blocking function antibody impaired maintenance of long-term culture-initiating cells (LTC-IC) on coculture of HPC and MSC. These results indicate that N-cadherin is involved in the bidirectional interaction of human HPC with their cellular determinants in the niche.     

Effect of hTIMP-1 overexpression in human umbilical cord mesenchymal stem cells on the repair of pancreatic islets in type-1 diabetic mice

Mesenchymal stem cells (MSCs) have been suggested for pancreatic islet repair in Type 1 diabetes mellitus (T1DM). This study aimed to investigate the effect of human umbilical cord MSCs (hUC-MSCs) transfected with tissue inhibitors of matrix metalloproteinase (TIMP)-1 on the regeneration of β-cell islets in vitro and in vivo. hUC-MSCs were isolated, cultured, and transfected with lentiviruses for the overexpression of hTIMP-1. An in vitro coculture system of hUC-MSCs and streptozotocin-induced islets was established to examine the morphology, apoptosis, and insulin secretion of the cocultured islets. Diabetic mouse models were injected with lenti-TIMP-1-enhanced green fluorescent protein (EGFP)-hUC-MSCs to test the effect of hTIMP-1 on insulin levels and glucose tolerance in vivo. The expression of insulin and glucagon was evaluated by immunofluorescence staining. The results showed that coculture with hUC-MSCs or Lenti-TIMP-1-EGFP-hUC-MSCs improved islet viability rates. Lenti-TIMP-1-EGFP-hUC-MSC coculture increased the insulin and C-peptide secretion function of the cultured islets and increased the secretion of tumor necrosis factor-β1, interleukin-6, IL-10, and hTIMP-1. hUC-MSCs, especially those transfected with Lenti-hTIMP-1-EGFP, showed a strong protective effect in diabetic mice by alleviating weight loss and improving glucose and insulin metabolism. In addition, transplantation rescued islet histology and function in vivo. The overexpression of TIMP-1 by hUC-MSCs seems to exert beneficial effects on pancreatic islet cells. In conclusion, this study may provide a new perspective on the development of hUC-MSC-based cell transplantation therapy for T1DM.     

Cell therapy with autologous mesenchymal stromal cells in post-traumatic syringomyelia

Background aims

Recently, clinical studies show that cell therapy with mesenchymal stromal cells (MSCs) improves the sequelae chronically established in paraplegic patients, being necessary to know which of them can obtain better benefit.

Biodegradable biliary stents coated with mesenchymal stromal cells in a porcine choledochojejunostomy model

Background aimsRoux en y anastomosis is a preferred method of biliary reconstruction in liver transplantation that involves living donors or pediatric patients. However, biliary stricture is a frequent and serious complication, accounting for up to 40% of biliary complications in these patients. Previously, we demonstrated that extraluminal delivery of adipose-derived (AD) mesenchymal stromal cells (MSCs) decreased peri-biliary fibrosis and increased neo-angiogenesis in a porcine model of duct-to-duct biliary anastomosis. In this study, we used a porcine model of Roux en y anastomosis to evaluate the beneficial impact of a novel intraluminal MSC delivery system.MethodsNine animals were divided into three groups: no stent (group 1), bare stent (group 2) and stent coated with AD-MSCs (group 3). All animals underwent cholecystectomy with roux en y choledochojejunostomy. Two animals per group were followed for 4 weeks and one animal per group was followed for 8 weeks. Cholangiograms and blood were sampled at baseline and the end of study. Biliary tissue was collected and examined by Masson trichrome staining and immunohistochemical staining for MSC markers (CD34 and CD44) and for neo-angiogenesis (CD31).ResultsTwo of three animals in group 1 developed an anastomotic site stricture. No strictures were observed in the animals of group 2 or group 3. CD34 and CD44 staining showed that AD-MSCs engrafted successfully at the anastomotic site by intraluminal delivery (group 3). Furthermore, biliary tissue from group 3 showed significantly less fibrosis and increased angiogenesis compared with the other groups.ConclusionsIntraluminal delivery of AD-MSCs resulted in successful biliary engraftment of AD-MSCs as well as reduced peri-biliary fibrosis and increased neo-angiogenesis.     

Isolation of self antigen-reactive cells from inflamed islets of nonobese diabetic mice using CD4high expression as a marker.

The low precursor frequency of Ag-reactive CD4+ T cells has been a barrier to the study of CD4+ T cell responses to conventional Ags as well as CD4+ T cell responses to autoantigens recognized during the course of an autoimmune disease. We have recently reported that all "conventional Ag" reactive CD4+ T cells are contained within the subpopulation expressing high levels of the CD4 molecule, termed CD4high. We have identified a CD4high population in the islets of Langerhans of prediabetic nonobese diabetic (NOD) mice that is extremely potent in transferring disease. As few as 500 CD4high islet-infiltrating CD4+ T cells transferred insulin-dependent diabetes mellitus to CD8 reconstituted NOD-SCID mice within 30 days of transfer. In contrast, CD4high T cells isolated from either NOD spleen or salivary glands did not transfer insulin-dependent diabetes mellitus into similar CD8-reconstituted NOD-SCID recipients. These data indicate that the precursor frequency of NOD islet-reactive, pathogenic CD4+ T cells is much higher in the prediabetic NOD pancreas than in these other organs. The islet-infiltrating CD4high T cells displayed selected memory markers, by cell surface analysis, and displayed a Th 1 phenotype by RNase protection assay, but had a marked decrease in IL-4 mRNA determined by quantitative real time PCR when compared with the less pathogenic CD4normal islet-infiltrating T cells. Use of the CD4high marker to select Ag activated T cells represents a tool to isolate and study pathogenic CD4+ T cells from autoimmune lesions in which the Ag has not been previously defined.     

Anti-inflammatory roles of mesenchymal stromal cells during acute Streptococcus pneumoniae pulmonary infection in mice

Background

Pneumonia is the fourth leading cause of death worldwide, and Streptococcus pneumoniae is the most commonly associated pathogen. Increasing evidence suggests that mesenchymal stromal cells (MSCs) have anti-inflammatory roles during innate immune responses such as sepsis. However, little is known about the effect of MSCs on pneumococcal pneumonia.

Mesenchymal stromal cells enhance wound healing by ameliorating impaired metabolism in diabetic mice

Background aimsMesenchymal stromal cells (MSCs) have been documented to improve delayed wound healing in diabetes, but the underlying mechanism remains obscure. We aimed to investigate whether the therapeutic effects on wounds was associated with metabolic alterations by paracrine action of MSCs.MethodsMSCs from mice with high-fat diet/streptozotocin–induced diabetes or wild-type C57BL/6 mice were evaluated for their paracrine potential in vitro using enzyme-linked immunosorbent assay and immunohistochemical staining assay. MSCs were then evaluated for their therapeutic potential in vivo using an excisional cutaneous wound model in mice with diabetes. Metabolic alterations and glucose transporter four (GLUT4) as well as PI3K/Akt signaling pathway expression after wounding were also examined.ResultsMSCs from normal mice expressed even more insulin-like growth factor-1 (IGF-1) than mice with diabetes, suggesting putative paracrine action. Furthermore, compared with IGF-1 knockdown MSCs, normal MSCs markedly accelerated wound healing, as revealed by higher wound closure rate and better healing quality at 21 days post-wound. By contrast, MSCs administration increased the level of insulin as well as GLUT4 and PI3K/Akt signaling pathway expression but repressed the biochemical indexes of glucose and lipid, resulting in obvious metabolic improvement.ConclusionsThese findings suggest that IGF-1 is an important paracrine factor that mediates the therapeutic effects of MSCs on wound healing in diabetes, and the benefits of MSCs may be associated with metabolism improvements, which would provide a new target for treatment.     

Identification of alpha-enolase as a potential immunogenic molecule during allogeneic transplantation of human adipose-derived mesenchymal stromal cells

Background aimsGiven their low immunogenicity, immunoregulatory effects and multiple differentiation capacity, mesenchymal stromal cells (MSCs) have the potential to be used for “off-the-shelf” cell therapy to treat various diseases. However, the allorejection of MSCs indicates that they are not fully immune-privileged. In this study, the authors investigated the immunogenicity of human adipose-derived MSCs (Ad-MSCs) and identified potential immunogenic molecules.MethodsTo evaluate the immunogenicity of human Ad-MSCs in vivo, cells were transplanted into humanized mice (hu-mice), then T-cell infiltration and clearance of human Ad-MSCs were observed by immunofluorescence and bioluminescence imaging. One-way mixed lymphocyte reaction and flow cytometry were performed to evaluate the immunogenicity of human Ad-MSCs in vitro. High-throughput T-cell receptor (TCR) repertoire sequencing and mass spectrometry were applied to identified potential immunogenic molecules.ResultsThe authors observed that allogeneic Ad-MSCs recruited human T cells and caused faster clearance in hu-mice than non-humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The proliferation and activation of T cells were significantly enhanced during in vitro co-culture with human Ad-MSCs. In addition, the level of HLA-II expression on human Ad-MSCs was dramatically increased after co-culture with human peripheral blood mononuclear cells (PBMCs). High-throughput sequencing was applied to analyze the TCR repertoire of the Ad-MSC-recruited T cells to identify dominant TCR CDR3 sequences. Using synthesized TCR CDR3 peptides, the authors identified several potential immunogenic candidates, including alpha-enolase (ENO1). The ENO1 expression level of Ad-MSCs significantly increased after co-culture with PBMCs, whereas ENO1 inhibitor (ENOblock) treatment decreased the expression level of ENO1 and Ad-MSC-induced proliferation of T cells.ConclusionsThe authors’ findings improve the understanding of the immunogenicity of human Ad-MSCs and provide a theoretical basis for the safe clinical application of allogeneic MSC therapy.