技术资料

CREB-binding protein (CREBBP) is important for the cell-autonomous regulation of hematopoiesis,including the stem cell compartment. In the present study,we show that CREBBP plays an equally pivotal role in microenvironment-mediated regulation of hematopoiesis. We found that the BM microenvironment of Crebbp(+/-) mice was unable to properly maintain the immature stem cell and progenitor cell pools. Instead,it stimulates myeloid differentiation,which progresses into a myeloproliferation phenotype. Alterations in the BM microenvironment resulting from haploinsufficiency of Crebbp included a marked decrease in trabecular bone that was predominantly caused by increased osteoclastogenesis. Although CFU-fibroblast (CFU-F) and total osteoblast numbers were decreased,the bone formation rate was similar to that found in wild-type mice. At the molecular level,we found that the known hematopoietic modulators matrix metallopeptidase-9 (MMP9) and kit ligand (KITL) were decreased with heterozygous levels of Crebbp. Lastly,potentially important regulatory proteins,endothelial cell adhesion molecule 1 (ESAM1) and cadherin 5 (CDH5),were increased on Crebbp(+/-) endothelial cells. Our findings reveal that a full dose of Crebbp is essential in the BM microenvironment to maintain proper hematopoiesis and to prevent excessive myeloproliferation. View Publication

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However,HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C,in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29,CD73,CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2),OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also,the cells showed high expression of MSC markers CD29,CD73,CD44 and CD105,but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion,HUCB is a good source of MSC using this new technique. 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

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication

It is well accepted that umbilical cord blood has been a source for hematopoietic stem cells. However,controversy exists as to whether cord blood can serve as a source of mesenchymal stem cells,which can differentiate into cells of different connective tissue lineages such as bone,cartilage,and fat,and little success has been reported in the literature about the isolation of such cells from cord blood. Here we report a novel method to obtain single cell-derived,clonally expanded mesenchymal stem cells that are of multilineage differentiation potential by negative immunoselection and limiting dilution. The immunophenotype of these clonally expanded cells is consistent with that reported for bone marrow mesenchymal stem cells. Under appropriate induction conditions,these cells can differentiate into bone,cartilage,and fat. Surprisingly,these cells were also able to differentiate into neuroglial- and hepatocyte-like cells under appropriate induction conditions and,thus,these cells may be more than mesenchymal stem cells as evidenced by their ability to differentiate into cell types of all 3 germ layers. In conclusion,umbilical cord blood does contain mesenchymal stem cells and should not be regarded as medical waste. It can serve as an alternative source of mesenchymal stem cells to bone marrow. View Publication

We reconstituted type I collagen nanofibers prepared by electrospin technology and examined the morphology,growth,adhesion,cell motility,and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) on three nano-sized diameters (50-200,200-500,and 500-1,000 nm). Results from scanning electron microscopy showed that cells on the nanofibers had a more polygonal and flattened cell morphology. MTS (3-[4,5-dimethythiazol-2-yl]-5-[3-carboxy-methoxyphenyl]-2-[4-sul-fophenyl]-2H-tetrazolium compound) assay demonstrated that the MSCs grown on 500-1,000-nm nanofibers had significantly higher cell viability than the tissue culture polystyrene control. A decreased amount of focal adhesion formation was apparent in which quantifiable staining area of the cytoplasmic protein vinculin for the 200-500-nm nanofibers was 39% less compared with control,whereas the area of quantifiable vinculin staining was 45% less for both the 200-500-nm and 500-1,000-nm nanofibers. The distances of cell migration were quantified on green fluorescent protein-nucleofected cells and was 56.7%,37.3%,and 46.3% for 50-200,200-500,and 500-1,000 nm,respectively,compared with those on the control. Alkaline phosphatase activity demonstrated no differences after 12 days of osteogenic differentiation,and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed comparable osteogenic gene expression of osteocalcin,osteonectin,and ostepontin between cells differentiated on polystyrene and nanofiber surfaces. Moreover,single-cell RT-PCR of type I collagen gene expression demonstrated higher expression on cells seeded on the nanofibers. Therefore,type I collagen nanofibers support the growth of MSCs without compromising their osteogenic differentiation capability and can be used as a scaffold for bone tissue engineering to facilitate intramembranous bone formation. Further efforts are necessary to enhance their biomimetic properties. View Publication

Implanted materials,such as medical devices,provoke the body to initiate an inflammatory reaction,known as the foreign body reaction (FBR),which causes several complications for example in hip prostheses,silicone implants,peritoneal dialysis catheters and left ventricular assist devices. FBR is initiated by macrophage adherence and results in granulation tissue formation. The early immunobiology and development of this tissue is not completely understood,but there are indications from related myofibroblast-forming diseases such as vascular repair and fibrosis that primitive stem cells also play a role in the formation of FBR-tissue. To investigate this,acellular photo-oxidized bovine pericardium patches were implanted intraperitoneally in rats and retrieved at time-points ranging from 6h to 7 days. A significant fraction of Sca-1(+) (6h-2 days),c-kit(+),CD34(+) and CD271(+) (2-3 days) stem/progenitor cells were detected. Colony-forming and differentiation capacity of the primitive stem cells into adipo-,osteo-,and myofibroblasts were shown. The presence of these primitive cells and their myofibroblastic differentiation potential were also confirmed at RNA level. The identification of specific primitive cells during FBR may have important implications for the inflammatory responses to inert materials and their use in tissue prostheses. View Publication

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft-versus-host disease,leading to a speedy recovery of hematopoiesis. To meet this clinical demand,a fast MSC expansion method is required. In the present study,we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors,including stem cell factor and interleukin-3 and -6. After 8 days of culture,total cell numbers,Stro-1(+)CD44(+)CD34(-) MSCs,and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-,29-,and 8-fold,respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin,whereas markers associated with lineage differentiation,including osteocalcin (osteogenesis),type II collagen (chondrogenesis),and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis),were not detected. Upon induction,the bioreactor-expanded MSCs were able to differentiate into osteoblasts,chondrocytes,and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs. View Publication