Mesenchymal stem cell (MSC) transplantation has been explored as a new clinical approach to repair injured tissue. A growing corpus of studies have highlighted two important aspects of MSC therapy: (1) MSCs can modulate T-cell mediated immunological responses, and (2) systemically administered MSCs home to sites of ischemia or injury. In this review, we describe the known mechanisms of immunomodulation and homing of MSCs. First, we examine the low immunogenicity of MSCs and their antigen presentation capabilities. Next, we discuss the paracrine interactions between MSCs and innate (dendritic cells (DC)) and adaptive immune cells (T lymphocytes) with a focus on prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO) and toll-like receptor (TLR) signaling pathways. We transition to outline the steps of activation, rolling/adhesion, and transmigration of MSCs into target tissues during inflammatory or ischemic conditions. These aspects of MSC grafts - immunomodulation and homing - are contextualized to understand a reported side-effect of MSC therapy, cancer development.
Immunosuppression; T-cell proliferation; Stem cell migration; IFN-γ; NFκB
Mesenchymal stem cells (MSCs) can suppress dendritic cells (DCs) maturation and function, mediated by soluble factors, such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), and nitric oxide (NO). Interleukin-10 (IL-10) is a common immunosuppressive cytokine, and the downstream signaling of the JAK-STAT pathway has been shown to be involved with DCs differentiation and maturation in the context of cancer. Whether IL-10 and/or the JAK-STAT pathway play a role in the inhibitory effect of MSCs on DCs maturation remains controversial. In our study, we cultured MSCs and DCs derived from rat bone marrow under different culturing conditions. Using Transwell plates, we detected by ELISA that the level of IL-10 significantly increased in the supernatants of MSC-DC co-cultures at 48 hours. The cell immunofluorescence assay suggested that the MSCs secreted more IL-10 than the DCs in the co-cultures. Adding exogenous IL-10 to the DCs monoculture or MSC-DC co-cultures stimulated IL-10 and led to a decrease in IL-12, and lower expression of the DCs surface markers CD80, CD86, OX62, MHC-II and CD11b/c. Supplementing the culture with an IL-10 neutralizing antibody (IL-10NA) showed precisely the opposite effect of adding IL-10. Moreover, we demonstrated that the JAK-STAT signaling pathway is involved in inhibiting DCs maturation. Both JAK1 and STAT3 expression and IL-10 secretion decreased markedly after adding a JAK inhibitor (AG490) to the co-culture plate. We propose that there is an IL-10 positive feedback loop, which may explain our observations of elevated IL-10 and enhanced JAK1 and STAT3 expression. Overall, we demonstrated that MSCs inhibit the maturation of DCs through the stimulation of IL-10 secretion, and by activating the JAK1 and STAT3 signaling pathway.
The ability of mesenchymal stromal cells (MSCs) to suppress immune responses combined with their potential to actively participate in tissue repair provides a strong rationale for the use of MSCs as a new treatment option in diseases characterized by inflammation and severe tissue damage, such as Crohn's disease (CD) and perianal fistulas. Multiple studies have shown that MSCs suppress a range of immune cells, such as dendritic cells (DC), naïve and effector T cells, and natural killer (NK) cells. Recently published papers attribute the immunosuppressive capacity of MSCs to soluble factors produced by MSCs, such as prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO). Promising results are obtained from phase I and II clinical trials with autologous and allogeneic MSCs as treatment for refractory CD and perianal fistulas; however the question remains: what are the molecular mechanisms underlying the immunomodulating properties of MSCs? This paper highlights the present knowledge on the immunosuppressive effects of MSCs and its complexity in relation to CD and perianal fistulas.
AIM: To investigate the effect of human umbilical cord stem cells, both mesenchymal and hematopoietic (CD34+), in the treatment of arthritis.
METHODS: Mesenchymal stem cells (MSCs) and hematopoietic (CD34+) stem cells (HSC) were isolated from human umbilical cord blood obtained from the umbilical cord of healthy pregnant donors undergoing full-term normal vaginal delivery. MSC, HSC, methotrexate (MTX) and sterile saline were injected intra-articularly into the rat hindpaw with complete freunds adjuvant (CFA) induced arthritis after the onset of disease (day 34), when arthritis had become well established (arthritis score ≥ 2). Arthritic indices were evaluated and the levels of interleukin (IL)-1, tumor necrosis factor (TNF)-α and interferon (IFN)-γ and anti-inflammatory cytokine IL-10 in serum were determined using enzyme-linked immunosorbent assay. Animals of all groups were sacrificed 34 d after beginning treatment, except positive control (PC) which was sacrificed at 10, 21 and 34 d for microscopic observation of disease progression. We used hematoxylin, eosin and Masson’s trichrome stains for histopathological examination of cartilage and synovium.
RESULTS: The mean arthritis scores were similar in all groups at 12 and 34 d post immunization, with no statistical significant difference. Upon the injection of stem cells (hematopoietic and mesenchymal), the overall arthritis signs were significantly improved around 21 d after receiving the injection and totally disappeared at day 34 post treatment in MSC group. Mean hindpaw diameter (mm) in the MSC rats was about half that of the PC and MTX groups (P = 0.007 and P = 0.021, respectively) and 0.6 mm less than the HSC group (P = 0.047), as indicated by paw swelling. Associated with these findings, serum levels of TNF-α, IFN-γ and IL-1 decreased significantly in HSC and MSC groups compared to PC and MTX groups (P < 0.05), while the expression of IL-10 was increased. Histopathological examination with H and E stain revealed that the MTX treated group showed significant reduction of leucocytic infiltrate and hypertrophy of the synovial tissue with moderate obliteration of the joint cavity. Stem cells treated groups (both hematopoietic CD34+ and mesenchymal), showed significant reduction in leucocytic infiltrate and hypertrophy of the synovial tissue with mild obliteration of the joint cavity. With Masson’s trichrome, stain sections from the PC group showed evidence of vascular edema of almost all vessels within the synovium in nearly all arthritic rats. Vacuoles were also visible in the outer vessel wall. The vessel became hemorrhagic and finally necrotic. In addition, there was extensive fibrosis completely obliterating the joint cavity. The mean color area percentage of collagen in this group was 0.324 ± 0.096, which was significantly increased when compared to the negative control group. The mean color area percentage of collagen in hematopoietic CD34+ and mesenchymal groups was 0.176 ± 0.0137 and 0.174 ± 0.0197 respectively, which showed a marked decrement compared to the PC group, denoting a mild increase in synovial tissue collagen fibers.
CONCLUSION: MSC enhance the efficacy of CFA-induced arthritis treatment, most likely through the modulation of the expression of cytokines and amelioration of pathological changes in joints.
Complete freunds adjuvant-induced arthritis; Human umbilical mesenchymal stem cell; Hematopoietic stem cell; CD34+
Previous in vivo studies have shown that mesenchymal stem cell (MSC) transplantation significantly improves the condition of a number of autoimmune diseases including autoimmune cerebrospinal meningitis, multiple sclerosis, glomerulonephritis and systemic lupus erythematosus.
To investigate the immunoregulatory effect of stem cell transplantation, human umbilical cord MSCs were co-cultured with peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA). Orphan nuclear receptor gamma (ROR-γ) mRNA and protein expression was detected with real-time PCR and Western blotting. Interleukin (IL)-17, IL-6 and tumor necrosis factor (TNF-α) in the cell culture supernatant were measured using a flow cytometric bead capture method.
After 72 hours of co-culture, the mRNA and protein expression levels of ROR-γ in co-cultured PBMCs were decreased compared with that in PBMC of RA patients cultured alone (p < 0.05). Moreover, the decrement was positively related to the disease activity of RA (p < 0.05). Decreased secretion of IL-17, TNF-α and IL-6 were also found in co-culture supernatants of PBMCs from patients with severe and moderate disease activity, but not in supernatant from PBMCs cultured alone. The decreased cytokine expression levels were positively correlated to the concentrations of MSCs. In contrast, PBMCs from healthy controls or patients with mild RA did not show significant differences in ROR-γ expression or cytokine secretion following co-culture with MSCs as compared with those cultured alone.
In vitro co-culture with MSCs down-regulated the inflammatory response of PBMCs from RA patients with severe disease activity, but had no significant effect on PBMCs from healthy controls or patients with mild disease activity, suggesting that the immunoregulatory role of MSCs may associate with the occurrence of inflammatory mediators.
Mesenchymal stem cell; Peripheral blood mononuclear cells; Rheumatoid arthritis; T helper 17 cells
In human skeletal muscle, myoblasts represent the main population of myogenic progenitors. We previously showed that, beside their myogenic differentiation capacities, myoblasts also differentiate towards osteogenic and chondrogenic lineages, some properties generally considered being hallmarks of mesenchymal stem cells (MSCs). MSCs are also characterized by their immunosuppressive potential, through cell-cell contacts and soluble factors, including prostaglandin E-2 (PGE-2), transforming growth factor-β1 (TGF-β1), interleukine-10, or indoleamine 2,3-dioxygenase. We and others also reported that Galectin-1 (Gal-1) and Semaphorin-3A (Sema-3A) were involved in MSCs-mediated immunosuppression. Here, we show that human myoblasts induce a significant and dose-dependant proliferation inhibition, independently of PGE-2 and TGF-β1. Our experiments revealed that myoblasts, in culture or in situ in human muscles, expressed and secreted Gal-1 and Sema-3A. Furthermore, myoblasts immunosuppressive functions were reverted by using blocking antibodies against Gal-1 or Sema-3A. Together, these results demonstrate an unsuspected immunosuppressive effect of myoblasts that may open new therapeutic perspectives.
Mesenchymal stem cells (MSCs) have acquired great interests for their potential use in the clinical therapy of many diseases because of their functions including multiple lineage differentiation, low immunogenicity and immunosuppression. Many studies suggest that MSCs are strongly immunosuppressive in vitro and in vivo. MSCs exert a profound inhibitory effect on the proliferation of T cells, B cells, dendritic cells and natural killer cells. In addition, several soluble factors have been reported to involved in the immunosuppressive effects by MSCs such as TGF-β, HGF, PGE2, IDO and iNOS. These results suggest that MSCs can be used in the therapy of immune disorder diseases, prevention of organ transplantation rejection and tissue injury. In recent study, we demonstrated that MSCs in tumor inflammatory microenvironment might be elicited of immunosuppressive function. Thus, the application of MSCs in cancer therapy might have negative effect by helping tumor cells escaping from the immune surveillance.
Mesenchymal stem cells; Immunosuppression; Tumor growth
The immunological and homing properties of mesenchymal stem cells (MSCs) provide a potentially attractive treatment for arthritis. The objective of this study was to determine effects of genetic disparity on the immunosuppressive potential of MSCs in vitro and in vivo within collagen induced arthritis (CIA).
The ability of DBA/1, FVB and BALB/c MSC preparations to impact the cytokine release profile of CD3/CD28 stimulated DBA/1 T cells was assessed in vitro. The effect of systemically delivered MSCs on the progression of CIA and cytokine production was assessed in vivo.
All MSC preparations suppressed the release of TNFα and augmented the secretion of IL-4 and IL-10 by stimulated DBA/1 T-cells. However, assessment of the ratio of IFNγ to IL-4 production indicated that the more genetically distant BALB/c MSCs had significantly less immunosuppressive capacity. Systemic delivery of BALB/c MSC resulted in an exacerbation of CIA disease score in vivo and a higher erosive disease burden. This was not seen after treatment with syngeneic or partially mismatched MSCs. An increase in serum levels of IL-1β was observed up to 20 days post treatment with allogeneic MSCs. An initial elevation of IL-17 in these treatment groups persisted in those treated with fully mismatched BALB/c MSCs. Over the course of the study, there was a significant suppression of serum IL-17 levels in groups treated with syngeneic MSCs.
These data demonstrate a significant difference in the immunosuppressive properties of syngeneic and allogeneic MSCs in vitro and in vivo, which needs to be appreciated when developing MSC based therapies for inflammatory arthritis.
Background: In diseased joints, the catabolic environment results in progressive joint damage. Mesenchymal stem cells (MSCs) can have immunomodulatory effects by secreting anti-inflammatory factors. To exert these effects, MSCs need to be triggered by pro-inflammatory cytokines. To explore the potential of MSCs as a treatment for diseased joints, we studied the effect of synovial fluid (SF) from donors with different joint diseases and donors without joint pathology on the immunomodulatory capacities of human MSCs in vitro. We hypothesized that SF of diseased joints influences the immunomodulatory effects of MSCs. Materials and Methods: MSCs were cultured in medium with SF of six osteoarthritis (OA) or six rheumatoid arthritis (RA) donors and three donors without joint pathology were used as control. Gene expressions of IL-6, HGF, TNFa, TGFb1, and indoleamine 2,3-dioxygenase (IDO) were analyzed. l-kynurenine concentration in conditioned medium (CM) by MSCs with SF was determined as a measure of IDO activity by MSCs. Furthermore, the effect of CM with SF on proliferation of activated lymphocytes was analyzed. Results: Addition of SF significantly up-regulated the mRNA expression of IL-6 and IDO in MSCs. SF(OA) induced significantly higher expression of IDO than SF(control), although no difference in IDO activity of the MSCs could be shown with a l-kynurenine assay. Medium conditioned by MSCs with SF(OA or RA) suppressed activated lymphocyte proliferation in vitro more than medium conditioned by MSCs without SF or with SF(control). Discussion: SF can influence the expression of genes involved in immunomodulation by MSCs and the effect on lymphocyte proliferation. We found indications for disease-specific differences between SFs but the variation between donors, even within one disease group was high. These data warrant further research to examine the potential application of MSC therapy in arthritic joints.
MSC; osteoarthritis; rheumatoid arthritis; synovial fluid; immunomodulation
Mesenchymal stem cells (MSCs) possess self-renewal and multipotential differentiation abilities, and they are thought to be one of the most reliable stem cell sources for a variety of cell therapies. Recently, cell therapy using MSCs has been studied as a novel therapeutic approach for cancers that show refractory progress and poor prognosis. MSCs from different tissues have different properties. However, the effect of different MSC properties on their application in anticancer therapies has not been thoroughly investigated. In this study, to characterize the anticancer therapeutic application of MSCs from different sources, we established two different kinds of human MSCs: umbilical cord blood-derived MSCs (UCB-MSCs) and adipose-tissue-derived MSCs (AT-MSCs). We used these MSCs in a coculture assay with primary glioblastoma multiforme (GBM) cells to analyze how MSCs from different sources can inhibit GBM growth. We found that UCB-MSCs inhibited GBM growth and caused apoptosis, but AT-MSCs promoted GBM growth. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling assay clearly demonstrated that UCB-MSCs promoted apoptosis of GBM via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL was expressed more highly by UCB-MSCs than by AT-MSCs. Higher mRNA expression levels of angiogenic factors (vascular endothelial growth factor, angiopoietin 1, platelet-derived growth factor, and insulin-like growth factor) and stromal-derived factor-1 (SDF-1/CXCL12) were observed in AT-MSCs, and highly vascularized tumors were developed when AT-MSCs and GBM were cotransplanted. Importantly, CXCL12 inhibited TRAIL activation of the apoptotic pathway in GBM, suggesting that AT-MSCs may support GBM development in vivo by at least two distinct mechanisms—promoting angiogenesis and inhibiting apoptosis. The opposite effects of AT-MSCs and UCB-MSCs on GBM clearly demonstrate that differences must be considered when choosing a stem cell source for safety in clinical application.
Mesenchymal stem cells (MSCs) are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP) and brain-derived neurotropfic factor (BDNF) plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs) with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells.
Using microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83%) and only minimal cell damage than when conventional liposome-based reagent (<20%) or established electroporation methods were used (30-40%). More importantly, microporation did not affect the immunophenotype of hUCB-MSCs, their proliferation activity, ability to differentiate into mesodermal and ectodermal lineages, or migration ability towards cancer cells. In addition, the BDNF gene could be successfully transfected into hUCB-MSCs, and BDNF expression remained fairly constant for the first 2 weeks in vitro and in vivo. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their in vitro differentiation into neural cells.
Taken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.
The therapeutic efficacy of human mesenchymal stem cells (hMSCs) for the treatment of hypoxic-ischemic diseases is closely related to level of hypoxia in the damaged tissues. To elucidate the potential therapeutic applications and limitations of hMSCs derived from human umbilical cords, the effects of hypoxia on the morphology and proliferation of hMSCs were analyzed.
After treatment with DFO and CoCl2, hMSCs were elongated, and adjacent cells were no longer in close contact. In addition, vacuole-like structures were observed within the cytoplasm; the rough endoplasmic reticulum expanded, and expanded ridges were observed in mitochondria. In addition, DFO and CoCl2 treatments for 48 h significantly inhibited hMSCs proliferation in a concentration-dependent manner (P < 0.05). This treatment also increased the number of cells in G0/G1 phase and decreased those in G2/S/M phase.
The hypoxia-mimetic agents, DFO and CoCl2, alter umbilical cord-derived hMSCs morphology and inhibit their proliferation through influencing the cell cycle.
Due to their immunosuppressive properties, human mesenchymal stem cells (hMSC) represent a promising tool for cell-based therapies of autoimmune diseases such as multiple sclerosis (MS). Mouse MSC (mMSC) have been used extensively to characterize and optimize route of administration, motility, cellular targets, and immunosuppressive mechanisms in mouse models of autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE). Tryptophan (trp) catabolism by indolamine-2,3-dioxygenase 1 (IDO1) is a chief endogenous metabolic pathway that tightly regulates unwanted immune responses through depletion of trp and generation of immunosuppressive kynurenines (kyn). IDO1 activity contributes to the immunosuppressive phenotype of hMSC. Here, we demonstrate that although IDO1 is inducible in bone marrow-derived mMSC by proinflammatory stimuli such as interferon-g (IFN-g) and ligands of toll-like receptors (TLR), it does not lead to catabolism of trp in vitro. This failure to catabolize trp is not due to defective TLR signaling as demonstrated by induction of interleukin 6 (IL-6) by TLR activation. While mMSC suppressed the activation of antigen-specific myelin oligodendrocyte glycoprotein (MOG)-reactive T-cell receptor (TCR) transgenic T-helper (TH) cells in co-culture, neither pharmacologic inhibition nor genetic ablation of IDO1 reversed this suppressive effect. Finally, systemic administration of both, IDO1-proficient and phenotypically identical IDO1-deficient mMSC, equally resulted in amelioration of EAE. mMSC, unlike hMSC, do not display IDO1-mediated suppression of antigen-specific T-cell responses.
Stem cell transplantation is a promising therapeutic strategy for the treatment of stroke. Mesenchymal stem cells (MSCs) are a potential cell source for clinical application because they can be easily obtained and cultivated with a high proliferative capacity. The safety and efficacy of cell therapy depends on the mode of cell administration. To determine the therapeutic potential of intrathecal administration of MSCs by lumbar puncture (LP), we administrated human umbilical cord blood-derived MSCs (hUCB-MSCs) intrathecally into the lumbar spinal cord or intravenously into the tail vein in a rat model of stroke, and then investigated whether hUCB-MSCs could enter the brain, survive, and improve post-stroke neurological functional recovery.
hUCB-MSCs (1.0 × 106) were administrated three days after stroke induced by occlusion of the middle cerebral artery. The presence of hUCB-MSCs and their survival and differentiation in the brain tissue of the rats was examined by immunohistochemistry. Recovery of coordination of movement after administration of hUCB-MSCs was examined using a Rotarod test and adhesive-removal test on the 7th, 14th, 21st, and 28th days after ischemia. The volume of ischemic lesions seven days after the experimental procedure was evaluated using 2-3-5-triphenyltetrazolium (TTC) staining.
Rats receiving hUCB-MSCs intrathecally by LP had a significantly higher number of migrated cells within the ischemic area when compared with animals receiving cells intravenously. In addition, many of the cells administered intrathecally survived and a subset of them expressed mature neural-lineage markers, including the mature neuron marker NeuN and glial fibrillary acidic protein, typical of astrocytes. Animals that received hUCB-MSCs had significantly improved motor function and reduced ischemic damage when compared with untreated control animals. Regardless of the administration route, the group treated with 1 × 106 hUCB-MSCs showed better neurological recovery, without significant differences between the two treatment groups. Importantly, intrathecal administration of 5 × 105 hUCB-MSCs significantly reduced ischemic damage, but not in the intravenously treated group. Furthermore, the cells administered intrathecally survived and migrated into the ischemic area more extensively, and differentiated significantly into neurons and astrocytes.
Together, these results indicate that intrathecal administration of MSCs by LP may be useful and feasible for MSCs treatment of brain injuries, such as stroke, or neurodegenerative disorders.
Mesenchymal stem cells (MSCs) are self-renewing, multipotent progenitor cells with multilineage potential to differentiate into cell types of mesodermal origin, such as adipocytes, osteocytes, and chondrocytes. In addition, MSCs can migrate to sites of inflammation and exert potent immunosuppressive and anti-inflammatory effects through interactions between lymphocytes associated with both the innate and adaptive immune system. Along with these unique therapeutic properties, their ease of accessibility and expansion suggest that use of MSCs may be a useful therapeutic approach for various disorders. In the clinical setting, MSCs are being explored in trials of various conditions, including orthopedic injuries, graft versus host disease following bone marrow transplantation, cardiovascular diseases, autoimmune diseases, and liver diseases. Furthermore, genetic modification of MSCs to overexpress antitumor genes has provided prospects for clinical use as anticancer therapy. Here, we highlight the currently reported uses of MSCs in clinical trials and discuss their efficacy as well as their limitations.
Clinical trial; Tissue therapy; Graft vs host disease; Mesenchymal stromal cells
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease primarily involving the synovium. Evidence in recent years has suggested that the bone marrow (BM) may be involved, and may even be the initiating site of the disease. Abnormalities in haemopoietic stem cells' (HSC) survival, proliferation and aging have been described in patients affected by RA and ascribed to abnormal support by the BM microenvironment. Mesenchymal stem cells (MSC) and their progeny constitute important components of the BM niche. In this study we test the hypothesis that the onset of inflammatory arthritis is associated with altered self-renewal and differentiation of bone marrow MSC, which alters the composition of the BM microenvironment.
We have used Balb/C Interleukin-1 receptor antagonist knock-out mice, which spontaneously develop RA-like disease in 100% of mice by 20 weeks of age to determine the number of mesenchymal progenitors and their differentiated progeny before, at the start and with progression of the disease.
We showed a decrease in the number of mesenchymal progenitors with adipogenic potential and decreased bone marrow adipogenesis before disease onset. This is associated with a decrease in osteoclastogenesis. Moreover, at the onset of disease a significant increase in all mesenchymal progenitors is observed together with a block in their differentiation to osteoblasts. This is associated with accelerated bone loss.
Significant changes occur in the BM niche with the establishment and progression of RA-like disease. Those changes may be responsible for aspects of the disease, including the advance of osteoporosis. An understanding of the molecular mechanisms leading to those changes may lead to new strategies for therapeutic intervention.
Various source-derived mesenchymal stem cells (MSCs) have been considered for cell therapeutics in incurable diseases. To characterize MSCs from different sources, we compared human bone marrow (BM), adipose tissue (AT), and umbilical cord blood-derived MSCs (UCB-MSCs) for surface antigen expression, differentiation ability, proliferation capacity, clonality, tolerance for aging, and paracrine activity. Although MSCs from different tissues have similar levels of surface antigen expression, immunosuppressive activity, and differentiation ability, UCB-MSCs had the highest rate of cell proliferation and clonality, and significantly lower expression of p53, p21, and p16, well known markers of senescence. Since paracrine action is the main action of MSCs, we examined the anti-inflammatory activity of each MSC under lipopolysaccharide (LPS)-induced inflammation. Co-culture of UCB-MSCs with LPS-treated rat alveolar macrophage, reduced expression of inflammatory cytokines including interleukin-1α (IL-1α), IL-6, and IL-8 via angiopoietin-1 (Ang-1). Using recombinant Ang-1 as potential soluble paracrine factor or its small interference RNA (siRNA), we found that Ang-1 secretion was responsible for this beneficial effect in part by preventing inflammation. Our results demonstrate that primitive UCB-MSCs have biological advantages in comparison to adult sources, making UCB-MSCs a useful model for clinical applications of cell therapy.
umbilical cord blood; bone marrow; adipo tissue; mesenchymal stem cell; expansion; senescence; anti-inflammation; angiopoietin-1; cell therapy
The application of mesenchymal stem cells (MSCs) in treating rheumatoid arthritis (RA) has been made possible by the immunosuppressive and differentiation abilities of these cells. A non-invasive means of assessing cell integration and bio-distribution is fundamental in evaluating the risks and success of this therapy, thereby enabling clinical translation. This paper defines the use of superparamagnetic iron oxide nanoparticles (SPIONs) in conjunction with magnetic resonance imaging (MRI) to image and track MSCs in vivo within a murine model of RA.
Murine MSCs (mMSCs) were isolated, expanded and labelled with SiMAG, a commercially available particle. In vitro MRI visibility thresholds were investigated by labelling mMSCs with SiMAG with concentrations ranging from 0 to 10 μg/ml and resuspending varying cell doses (103 to 5 × 105 cells) in 2 mg/ml collagen prior to MR-imaging. Similarly, in vivo detection thresholds were identified by implanting 3 × 105 mMSCs labelled with 0 to 10 μg/ml SiMAG within the synovial cavity of a mouse and MR-imaging. Upon RA induction, 300,000 mMSCs labelled with SiMAG (10 μg/ml) were implanted via intra-articular injection and joint swelling monitored as an indication of RA development over seven days. Furthermore, the effect of SiMAG on cell viability, proliferation and differentiation was investigated.
A minimum particle concentration of 1 μg/ml (300,000 cells) and cell dose of 100,000 cells (5 and 10 μg/ml) were identified as the in vitro MRI detection threshold. Cell viability, proliferation and differentiation capabilities were not affected, with labelled populations undergoing successful differentiation down osteogenic and adipogenic lineages. A significant decrease (P < 0.01) in joint swelling was measured in groups containing SiMAG-labelled and unlabelled mMSCs implying that the presence of SPIONs does not affect the immunomodulating properties of the cells. In vivo MRI scans demonstrated good contrast and the identification of SiMAG-labelled populations within the synovial joint up to 7 days post implantation. This was further confirmed using histological analysis.
We have been able to monitor and track the migration of stem cell populations within the rheumatic joint in a non-invasive manner. This manuscript goes further to highlight the key characteristics (biocompatible and the ability to create significant contrast at realistic doses within a clinical relevant system) demonstrated by SiMAG that should be incorporated into the design of a new clinically approved tracking agent.
5-Azacytidine (5-Aza) induces differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. However, the underlying mechanisms are not well understood. Our previous work showed that 5-Aza induces human bone marrow-derived MSCs to differentiate into cardiomyocytes. Here, we demonstrated that 5-Aza induced cardiac differentiation of human umbilical cord-derived MSCs (hucMSCs) and explored the potential signaling pathway. Our results showed that hucMSCs had cardiomyocyte phenotypes after 5-Aza treatment. In addition, myogenic cells differentiated from hucMSCs were positive for mRNA and protein of desmin, β-myosin heavy chain, cardiac troponin T, A-type natriuretic peptide, and Nkx2.5. Human diploid lung fibroblasts treated with 5-Aza expressed no cardiac-specific genes. 5-Aza did not induce hucMSCs to differentiate into osteoblasts. Further study revealed that 5-Aza treatment activated extracellular signal related kinases (ERK) in hucMSCs, but protein kinase C showed no response to 5-Aza administration. U0126, a specific inhibitor of ERK, could inhibit 5-Aza-induced expression of cardiac-specific genes and proteins in hucMSCs. Increased phosphorylation of signal transducers and activators of transcription 3, and up-regulation of myocyte enhancer-binding factor-2c and myogenic differentiation antigen in 5-Aza-treated hucMSCs were also suppressed by U0126. Taken together, these results suggested that sustained activation of ERK by 5-Aza contributed to the induction of the differentiation of hucMSCs into cardiomyocytes in vitro.
Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P<0.01). This was associated with significant enhancement of mitochondrial membrane potential (P<0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P< 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture.
cell viability; intercellular transfer; mesenchymal stromal cells; umbilical cord blood
Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the two widely studied and characterized adult stem cells. Thus far, MSCs were obtained from the bone marrow, which is a painful procedure. Therefore, MSCs from less common sources like cord blood, adipose tissue, tooth pulp, and so on, have been the subject of research. The purpose of this study is to explore the possibility of finding MSCs from a less controversial, easy, and abundant source, such as the umbilical cord, for potential regenerative medicine applications.
Study Design and Methods:
Five bone marrow samples (BM), seventy cord blood units (CB), and four umbilical cord matrix (CM) samples have been used for the study. Expanded MSCs were checked for biomarker expression by flow cytometry and were also checked for their differentiation to mesodermal and ectodermal lineages.
MSCs could be isolated from 100% BM and CM samples, as compared to only 6% of CB samples. The fold expansion of the mesenchymal stem cells observed in CB (CB-MSCs) was distinctly higher as compared to BM (BM-MSCs) and CM (CM-MSCs). MSCs isolated from all the three sources expressed a characteristic mesenchymal phenotype of CD45 − /vWF − /CD14 − /CD31 − /CD73 + /CD105 + /SSEA4 + /CD29 + /CD44 + /HLAABC +, whereas, the HLA DR was conspicuously absent in CM-MSCs and CB-MSCs. Although osteogenic, chondrogenic, and neural differentiation was observed in MSCs from all sources, adipogenic differentiation was observed only in BM-MSCs.
CM-MSCs are a dependable source of an unlimited number of MSCs for autologous and allogenic use in regenerative medicine.
Mesenchymal stem cells; bone marrow; umbilical cord blood; umbilical cord
Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow–derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton’s jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated.
STUDY DESIGN AND METHODS
Umbilical cord–derived MSCs (UC-MSCs) were cultured from the Wharton’s jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay.
Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays.
These data suggest the potential therapeutic application of Wharton’s jelly–derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation.
The regenerative abilities and the immunosuppressive properties of mesenchymal stromal cells (MSCs) make them potentially the ideal cellular product of choice for treatment of autoimmune and other immune mediated disorders. Although the usefulness of MSCs for therapeutic applications is in early phases, their potential clinical use remains of great interest. Current clinical evidence of use of MSCs from both autologous and allogeneic sources to treat autoimmune disorders confers conflicting clinical benefit outcomes. These varied results may possibly be due to MSC use across wide range of autoimmune disorders with clinical heterogeneity or due to variability of the cellular product. In the light of recent genome wide association studies (GWAS), linking predisposition of autoimmune diseases to single nucleotide polymorphisms (SNPs) in the susceptible genetic loci, the clinical relevance of MSCs possessing SNPs in the critical effector molecules of immunosuppression is largely undiscussed. It is of further interest in the allogeneic setting, where SNPs in the target pathway of MSC's intervention may also modulate clinical outcome. In the present review, we have discussed the known critical SNPs predisposing to disease susceptibility in various autoimmune diseases and their significance in the immunomodulatory properties of MSCs.
Numerous studies have shown the benefits of mesenchymal stem cells (MSCs) on the repair of spinal cord injury (SCI) model and on behavioral improvement, but the underlying mechanisms remain unclear. In this study, to investigate possible mechanisms by which MSCs contribute to the alleviation of neurologic deficits, we examined the potential effect of human umbilical cord blood-derived MSCs (hUCB-MSCs) on the endogenous cell proliferation and oligogenesis after SCI. SCI was injured by contusion using a weight-drop impactor and hUCB-MSCs were transplanted into the boundary zone of the injured site. Animals received a daily injection of bromodeoxyuridine (BrdU) for 7 days after treatment to identity newly synthesized cells of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells was evident. Behavior analysis revealed that locomotor functions of hUCB-MSCs group were restored significantly and the cavity volume was smaller in the MSCs-transplanted rats compared to the control group. In MSCs-transplanted group, TUNEL-positive cells were decreased and BrdU-positive cells were significantly increased rats compared with control group. In addition, more of BrdU-positive cells expressed neural stem/progenitor cell nestin and oligo-lineage cell such as NG2, CNPase, MBP and glial fibrillary acidic protein typical of astrocytes in the MSC-transplanted rats. Thus, endogenous cell proliferation and oligogenesis contribute to MSC-promoted functional recovery following SCI.
Mesenchymal stem cells (MSCs) hold great therapeutic potential. However, MSCs undergo replication senescence during the in vitro expansion process. Wharton's jelly from the human umbilical cord harbors a large number of MSCs. In this study, we hypothesized that Wharton's jelly would be beneficial for in vitro expansion of MSCs. Wharton's jelly extract (WJEs), which is mainly composed of extracellular matrix and cytokines, was prepared as coating substrate. Human MSCs were isolated and cultured on WJE-coated plates. Although the proliferation capacity of cells was not augmented by WJE in early phase culture, adynamic growth in late-phase culture was clearly reduced, suggesting that the replicative senescence of MSCs was efficiently slowed by WJE. This was confirmed by β-galactosidase staining and telomere length measurements of MSCs in late-phase culture. In addition, the decreased differentiation ability of MSCs after long-term culture was largely ameliorated by WJE. Reactive oxygen species (ROS), p53, and p16INK4a/pRb expression increased with passaging. Analysis at the molecular level revealed that WJE-based culture efficiently suppressed the enhancement of intracellular ROS, p53, and p16INK4a/pRb in MSCs. These data demonstrated that WJE provided an ideal microenvironment for MSCs culture expansion in vitro preserved MSC properties by delaying MSCs senescence, and allowed large numbers of MSCs to be obtained for basic research and clinical therapies.