技术资料

We studied the immunoregulatory features of murine mesenchymal stem cells (MSCs) in vitro and in vivo. MSCs inhibited T-cell receptor (TCR)-dependent and -independent proliferation but did not induce apoptosis on T cells. Such inhibition was paired with a decreased interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and was partially reversed by interleukin-2 (IL-2). Thus,we used MSCs to treat myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice. We injected intravenously 1 x 10(6) MSCs before disease onset (preventive protocol) and at different time points after disease occurrence (therapeutic protocol). MSC administration before disease onset strikingly ameliorated EAE. The therapeutic scheme was effective when MSCs were administered at disease onset and at the peak of disease but not after disease stabilization. Central nervous system (CNS) pathology showed decreased inflammatory infiltrates and demyelination in mice that received transplants of MSCs. T-cell response to MOG and mitogens from MSC-treated mice was inhibited and restored by IL-2 administration. Upon MSC transfection with the enhanced green fluorescent protein (eGFP),eGFP(+) cells were detected in the lymphoid organs of treated mice. These data suggest that the immunoregulatory properties of MSCs effectively interfere with the autoimmune attack in the course of EAE inducing an in vivo state of T-cell unresponsiveness occurring within secondary lymphoid organs. View Publication

Although prolonged transgene expression in progenitor cells might be desirable for modified cell therapy,the viral promoter-based expression vector tends to promote transgene expression only for a limited period. Here,we examined the ability of cellular promoters from elongation factor-1alpha (EF-1alpha) and ubiquitin C to drive gene expression in hematopoietic TF-1 and mesenchymal progenitor cells. We compared the expression levels and duration of a model gene,interleukin-2,generated by the cellular promoters to those by the cytomegalovirus (CMV) promoter. The EF-1alpha and ubiquitin C promoters drove prolonged gene expression in hematopoietic TF-1 and mesenchymal progenitor cells,whereas the CMV promoter did not. At day 7 after transfection in TF-1 cells,the mRNA expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 118- and 56-fold higher,respectively,than those driven by the CMV promoter. Similarly,in mesenchymal progenitor cells,the expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 98- and 20-fold higher,respectively,than that driven by the CMV promoter-encoding plasmid. Moreover,the ubiquitin C promoter directed higher levels of green fluorescence protein expression in mesenchymal progenitor cells than did the CMV promoter. These results indicate that the use of cellular promoters such as those for EF-1alpha and ubiquitin C might direct prolonged gene expression in hematopoietic and mesenchymal progenitor cells. View Publication

BACKGROUND: Although current evidence suggests that only a minuscule number of osteoblast-lineage cells are present in peripheral blood,we hypothesized that such cells circulate but that their concentration has been vastly underestimated owing to the use of assays that required adherence to plastic. We further reasoned that the concentration of these cells is elevated during times of increased bone formation,such as during pubertal growth. METHODS: We used flow cytometry with antibodies to bone-specific proteins to identify circulating osteoblast-lineage cells in 11 adolescent males and 11 adult males (mean [+/-SD] age,14.5+/-0.7 vs. 37.7+/-7.6 years). Gene expression and in vitro and in vivo bone-forming assays were used to establish the osteoblastic lineage of sorted cells. RESULTS: Cells positive for osteocalcin and cells positive for bone-specific alkaline phosphatase were detected in the peripheral blood of adult subjects (1 to 2 percent of mononuclear cells). There were more than five times as many cells positive for osteocalcin in the circulation of adolescent boys (whose markers of bone formation were clearly increased as a result of pubertal growth) as compared with adult subjects (Ptextless0.001). The percentage of cells positive for osteocalcin correlated with markers of bone formation. Sorted osteocalcin-positive cells expressed osteoblastic genes,formed mineralized nodules in vitro,and formed bone in an in vivo transplantation assay. Increased values were also found in three adults with recent fractures. CONCLUSIONS: Osteoblast-lineage cells circulate in physiologically significant numbers,correlate with markers of bone formation,and are markedly higher during pubertal growth; therefore,they may represent a previously unrecognized circulatory component to the process of bone formation. View Publication

Bone marrow-derived mesenchymal stem cells (MSC) have been defined as primitive,undifferentiated cells,capable of self-renewal and with the ability to give rise to different cell lineages,including adipocytes,osteocytes,fibroblasts,chondrocytes,and myoblasts. MSC are key components of the hematopoietic microenvironment. Several studies,including some from our own group,suggest that important quantitative and functional alterations are present in the stroma of patients with myelodysplasia (MDS). However,in most of such studies the stroma has been analyzed as a complex network of different cell types and molecules,thus it has been difficult to identify and characterize the cell(s) type(s) that is (are) altered in MDS. In the present study,we have focused on the biological characterization of MSC from MDS. As a first approach,we have quantified their numbers in bone marrow,and have worked on their phenotypic (morphology and immunophenotype) and cytogenetic properties. MSC were obtained by a negative selection procedure and cultured in a MSC liquid culture medium. In terms of morphology,as well as the expression of certain cell markers,no differences were observed between MSC from MDS patients and those derived from normal marrow. In both cases,MSC expressed CD29,CD90,CD105 and Prolyl-4-hydroxylase; in contrast,they did not express CD14,CD34,CD68,or alkaline phosphatase. Interestingly,in five out of nine MDS patients,MSC developed in culture showed cytogenetic abnormalities,usually involving the loss of chromosomal material. All those five cases also showed cytogenetic abnormalities in their hematopoietic cells. Interestingly,in some cases there was a complete lack of overlap between the karyotypes of hematopoietic cells and MSC. To the best of our knowledge,the present study is the first in which a pure population of MSC from MDS patients is analyzed in terms of their whole karyotype and demonstrates that in a significant proportion of patients,MSC are cytogenetically abnormal. Although the reason of this is still unclear,such alterations may have an impact on the physiology of these cells. Further studies are needed to assess the functional integrity of MDS-derived MSC. View Publication

Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plastic-adherent mesenchymal stem cell able to generate tissues other than bone,increasing evidence suggests the existence of a more primitive cell that can differentiate to both hematopoietic and mesenchymal cells. We show here that the side population" (SP) of marrow stem cells View Publication

Mesenchymal stem cells (MSCs) have the capability for renewal and differentiation into various lineages of mesenchymal tissues. These features of MSCs attract a lot of attention from investigators in the context of cell-based therapies of several human diseases. Despite the fact that bone marrow represents the main available source of MSCs,the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferative/differentiation capacity with age. Thus,the search for possible alternative MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells and does not contain mesenchymal progenitors. However,MSCs circulate in the blood of preterm fetuses and may be successfully isolated and expanded. Where these cells home at the end of gestation is not clear. In this investigation,we have made an attempt to isolate MSCs from the subendothelial layer of umbilical cord vein using two standard methodological approaches: the routine isolation of human umbilical vein endothelial cell protocol and culture of isolated cells under conditions appropriate for bone-marrow-derived MSCs. Our results suggest that cord vasculature contains a high number of MSC-like elements forming colonies of fibroblastoid cells that may be successfully expanded in culture. These MSC-like cells contain no endothelium- or leukocyte-specific antigens but express alpha-smooth muscle actin and several mesenchymal cell markers. Therefore,umbilical cord/placenta stroma could be regarded as an alternative source of MSCs for experimental and clinical needs. View Publication

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid,beta-glycerophosphate,and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after,but not before,14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair. View Publication