With the capacities of multiple differentiation, immunoregulatory activities and easy handling for isolation and culture expansion, human bone marrow mesenchymal stem cells (MSCs) have been utilized in clinical trials for the prevention and treatment of graft-versus-host disease in allogeneic bone marrow transplantation, repair of bone and cartilage defects and treatment of cardiac infarction and liver injury. However, increasingly experimental data indicate that a great deal of issues, such as intra-neutralization of calf serum proteins into cultured MSCs, survival of engrafted cells and subsequent cell differentiation tendency, should be in stringent consideration before clinical trials are designed. In this paper, these issues that should be raised and solved in clinical trials with MSCs were reviewed.
Bone Marrow Transplantation ; adverse effects ; Clinical Trials as Topic ; Graft vs Host Disease ; prevention & control ; Humans ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Research Design

Objective: To investigate the role of human umbilical vein endothelial cells (HUVEC) in supporting hematopoiesis and the expansion of hematopoietic stem/progenitor cells in vitro. Methods: According to the fact that HUVEC supernatant has colony stimulating activity shown by methylcellulose colony-forming assay and HUVEC can maintain the survival of mononuclear cells for at least four weeks in vitro, CD34+ cells from umbilical cord blood were seeded with (HUVEC group) or without (control group) HUVEC monolayer. Every week cells were collected and counted, the frequency of CFU-GM was measured by using methylcellulose colony-forming assay, and the percentage of CD34+ and CD41a+ cells was measured by flow cytometry. Results: In control group，all the CD34+ cells died in two weeks. However, in HUVEC group,most nucleated cells and CD34+ cells were expanded by 68.1±14.8 fold and 6.6±1.4 fold，respectively at the third week while CFU-GM expansion reached its peak (5.7±2.1 fold) at the week 2. Moreover, the percentage of CD41a+ cells was enhanced significantly, reaching a maximum (15.6%) at the week 3. Conclusions:HUVEC can support hematopoiesis in vitro and expand the hematopoietic progenitor cells and CD41a+ cells in direct contact coculture.

This study was purposed to investigate the angiogenesis-promoting activities of human mesenchymal stem cells (hMSCs) modified by hepatocyte growth factor (HGF) and the underlying mechanisms. The hMSCs were transfected by recombinant adenoviral vector carrying human HGF gene and seeded onto the chicken chorioallantoic membrane. Three days later, the number of blood vessels was counted and their angiogenic response was compared with those of hMSCs of same generation, recombinant basic fibroblast growth factor (bFGF) and alpha-MEM as control. The expression levels of bFGF, VEGF, angiopoietin-1 and angiopoietin-2 were evaluated by RT-PCR assay. The results showed that gene-modified hMSCs exhibited greatest activity to promote angiogenesis while the angiogenic response was nearly same between groups treated by hMSCs and bFGF, all of which were significantly higher than that observed in control (p < 0.01). RT-PCR analysis revealed that hMSCs constitutively expressed multiple angiogenesis-associated growth factors and their levels seemed up-regulated by HGF gene transfer. It is concluded that HGF gene-modified hMSCs show a potent angiogenesis-promoting function and may be useful in the treatment of ischemic disorders.
Animals ; Cells, Cultured ; Chick Embryo ; Chickens ; Hepatocyte Growth Factor ; genetics ; Humans ; Mesenchymal Stromal Cells ; Neovascularization, Physiologic ; genetics ; Transfection

Hepatocyte growth factor (HGF) is a pleiotropic cytokine, its roles in the physiology and pathology of immune system, have been investigated thoroughly, great deal of data have been documented on its immunoregulatory activity. In this review, according to advance of study on HGF in recent years, the role of HGF in the immune regulation, such as immunoregulatory effects of HGF on T lymphocytes, B lymphocytes and dendritic cell, modulation of HGF on specific humoral and cellular immune response, control of acute GVHD and acceleration of myeloid and immunologic reconstitution in allogenetic bone marrow transplantation models, promotion of tissue repair and regeneration, and alleviation of immune rejection in allogeneic organ transplantation including the heart, liver and kidney transplantation, prevention of grafts from injury as well as applicably useful of HGF in the therapy of autoimmune disorders were summarized.
Animals ; Graft Rejection ; immunology ; prevention & control ; Graft vs Host Disease ; immunology ; prevention & control ; Graft vs Leukemia Effect ; immunology ; Hepatocyte Growth Factor ; physiology ; Humans ; Immunity, Cellular ; immunology

The aim of this study was to investigate if transfusion of mesenchymal stem cells (MSC) could exhibit beneficial effects on rheumatoid arthritis. Human bone marrow MSC were intraperitoneally injected into Wistar rats with collagen-induced arthritis at a dose of 10(7) on the next day (preventive group) or 2 weeks (treatment group) after collagen II induction, once a week for 2 weeks (preventive group) or 4 weeks (treatment group). The control group was given normal saline (NS) at corresponding time. The symptom scorings were documented weekly from the second week of the induction. On week 6, the hind joints of the rats were pathologically examined and the activation status of splenocytes was analyzed by flow cytometry. The results showed that all the rats developed arthritis and subsequent joint abnormality. On the sixth week, symptom scores of the rats that received MSC preventive (9.5 ± 0.5) or therapeutic (9.4 ± 0.6) infusions had no significant difference between each other, but were significantly greater than those of the NS controls (7.6 ± 0.6, P < 0.05). Consistently, pathological examination on the involved knees showed that the synovitis and arthritis scorings of MSC treated rats were greatly elevated compared with NS controls. Furthermore, the ratios of CD86(+) cells in the spleens of MSC prevention, MSC treatment and NS control groups were (4.16 ± 1.48), (4.06 ± 1.97) and (4.15 ± 2.04) respectively, while those of CD11b/c(+)CD86(+) cells were (1.04 ± 0.68), (0.95 ± 0.56) and (0.98 ± 0.44), all of which were significantly higher than those of healthy controls [(0.97 ± 0.18) and (0.30 ± 0.17), P < 0.05 for both parameters]. It is concluded that MSC infusion has little beneficial effects on collagen-induced arthritis in rats, conversely, MSC therapy aggravated the damage of the involved joints, its underlying mechanisms need to be further investigated.
Animals ; Arthritis, Experimental ; pathology ; prevention & control ; therapy ; Bone Marrow Cells ; Cells, Cultured ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Wistar ; Transplantation, Heterologous ; Treatment Outcome

The biological properties of cultured mesenchymal stem cells (MSC) have been intensively investigated, while there is still a paucity of information about the definite in vivo sites that harbor these stem cells due to the lack of specific surface markers. Previous data have demonstrated that human and murine MSC can be isolated from the compact bones. To investigate if it is the case for other species, the femurs from Wistar rats, Beagles, C57 mice and New Zealand rabbits were collected, minced and digested with collagenase type I. The digested bone fragments were seeded into the medium for human bone marrow culture after removal of the suspended cells in the digestion. The results showed that the fibroblast-like cells were observed to migrate from the bone fragments after several days of culture, and they gradually formed an adherent confluent layer. The adherent cells could be passaged and expressed homogenously the mesenchymal cell marker vimentin. Differentiation assays showed that these cells had the capacity to differentiate into osteoblasts and adipocytes. In conclusion, the results here provide new information for the further investigations on the in vivo biological features of MSC in the context of the simplicity of the compact bone structure.
Animals ; Bone and Bones ; cytology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Dogs ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Rabbits ; Rats ; Rats, Wistar

Recent reports have clearly demonstrated that bone marrow cells can be differentiated into neurons, suggesting the existence of cells with the differentiation capacity in the bone marrow cell population. It is well known that hematopoietic stem cells as well as mesenchymal stem cells (MSCs) can be transplanted and therefore, alternative of them might contribute to the process. In the present study it was addressed whether marrow MSCs could be coaxed into neuron-specific antigen bearing cells and if so, whether the differentiated cells possess the cytochemical features seen in neurons. The report here showed that high concentration of 2-mercaptoethanol (2-ME) could induce some of the MSCs into neuron-like cells expressing neurofilament (NF) and neuron specific enolase (NSE). The neuron-like cells were alkaline phosphotase positive while the others MSCs were kept negative. Cells treated with 2-ME were positive for alpha-naphthylacetate esterase and glycogen and negative for acetylchonlinesterase, which were similar with the results seen in untreated cells. Furthermore, Nissel body was not observed in treated cells shown by toluidine blue staining. Therefore, it is likely that the cells described here seem not belong to the neuronal lineage. These findings, however, reveal that human MSCs could alter their committed fates under some circumstances.

Endothelial cells are the critical cell component of hematopoietic microenvironment. For the purpose of facilitating the study on modulating effect of endothelial cells in hematopoiesis, a human umbilical vein cell line, IEC, was established. Primary human umbilical vein endothelial cells were transfected with the plasmid pSV(3neo) carried SV40 large T antigen by lipofection. The IEC cells expressed factor VIII and the UEA I-binding ratio was about 97%. The chromosome variation was existed in the cell line, with the karyotype 45, XX, -18, 18q(+). The cell line retained the proliferative capacity at least 25 passages. IEC cells stimulated the growth of granulocyte-macrophage progenitor cells in coculture of cord blood CD34(+) cells with IEC cells. It is concluded that IEC cell line possessed the biological features of endothelial cell and supported hematopoiesis in vitro.

Mesenchymal stem cells (MSCs) are a kind of adult stem cells which have the capability to differentiate into multiple cell types as well as self-renew continuously. Recent studies demonstrate that MSCs are low immunogenic and able to exert immunomodulatory function by various approaches, such as suppression of the lymphocyte proliferation, reduction of the dentritic cell generation, maturation and function, down-regulation of the CTL formation and enhancement of regulatory T-cell proportion. In vivo experiments show that MSC infusion can prolong the survival time of allo-skin graft in baboon and ameliorate experimental autoimmune encephalomyelitis in mice. Successful reports have been documented about clinical application of MSC in the management of graft-versus-host disease. In this review, the immunological characteristics and the immunomodulation functions in vitro and in vivo of MSC were summarized.
Animals ; Graft vs Host Disease ; prevention & control ; Humans ; Immunomodulation ; physiology ; Mesenchymal Stromal Cells ; immunology ; physiology ; T-Lymphocytes, Regulatory ; immunology

Our previous work has shown that mesenchymal stem cells (MSC) have little therapeutic effect on rat arthritis induced by collagen. This study was aimed to further investigate whether the MSC lysates exhibit beneficial effects on rheumatoid arthritis. Aliquots of cell lysates from 1×10(7) human bone marrow MSC were intraperitoneally injected into collagen-induced arthritis (CIA) Wistar rats weekly for 4 consecutive weeks. Methotrexate at a dose of 1 mg/kg or normal saline was served as positive and negative controls respectively. On week 4 the symptom scores were recorded and the hind joints of the rats were pathologically examined and X-ray examination was performed. The results showed that on week 4, the symptom scores of the rats that received MSC lysates (6.87 ± 0.83) and MTX (6.44 ± 1.13) were significantly lower than that of control rats (7.33 ± 0.77, P < 0.01). Meanwhile, pathological examination on the involved ankle showed that the synovitis and arthritis scores of MSC lysates and control groups were 2.28 ± 0.48 and 2.28 ± 0.55 respectively, significantly higher than that of MTX treatment rats (0.71 ± 0.48, P < 0.05). However, X-ray examination on the ankle joints showed that the injury score of control rats was 4 ± 0.57, greatly higher than those from MSC lysates (2.71 ± 0.75) and MTX treatment groups (2.57 ± 0.78, P < 0.05 for both groups). It is concluded that MSC lysate infusion has beneficial effects on CIA rat, but the effectiveness seems inferior to MTX.
Animals ; Arthritis, Experimental ; chemically induced ; therapy ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Collagen ; Humans ; Male ; Mesenchymal Stromal Cells ; cytology ; Methotrexate ; pharmacology ; Rats ; Rats, Wistar