技术资料

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species,the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest,followed by an immunodepletion step using microbeads coated with CD11b,CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion,a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1),CD90,CD105 and CD73 cell surface markers,but negative for the hematopoietic surface markers CD14,CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic,osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time. View Publication

Autologous bone grafting represents the gold standard modality to treat atrophic non-unions by virtue of its osteoinductive and osteoconductive properties. The common harvest site is the iliac crest,but there are major concerns due to limited volume and considerable donor site morbidity. Alternative autologous bone graft can be harvested from the femoral bone cavity using a newly developed 'Reamer Irrigator Aspirator' (RIA). Osseous aspirated particles can be recovered with a filter and used as auto-graft. The purpose of this study was to compare the concentration and differentiation potential of mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) harvested with the RIA technique or from the iliac crest,respectively. RIA aspirate was collected from 26 patients undergoing intramedullary nailing of femur fractures. Iliac crest aspirate was collected from 38 patients undergoing bone graft transplantation. Concentration of MSC and EPC were assessed by means of the MSC colony assay,EPC culture assay and flowcytometry (CD34,CD133,VEGF-R2),respectively. Osteogenic differentiation of MSC's was measured by von Kossa staining. Patients in both groups did not significantly differ regarding their age,gender or pre-existing health conditions. In comparison to aspirates obtained from iliac crest the RIA aspirates from the femur contained a significantly higher percentage of CD34+ progenitor cells,a significantly higher concentration of MSC and a significantly higher concentration of early EPC. The percentage of late EPC did not differ between both sites. Moreover,the capability of MSC for calcium deposition was significantly enhanced in MSC obtained with RIA. Our results show that RIA aspirate is a rich source for different types of autologous progenitor cells,which can be used to accelerate healing of bone and other musculoskeletal tissues. View Publication

BACKGROUND: Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor,thereby causing relapse and patient death. Ewing's sarcoma,the second most common form of bone tumor in adolescents and young adults,follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved,even for patients with metastatic disease,but relapse remains frequent and is usually fatal. METHODOLOGY/PRINCIPAL FINDINGS: We have isolated a subpopulation of Ewing's sarcoma cells,from both human cell lines and human xenografts grown in immune deficient mice,which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity,sphere-formation,and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro,but this can be overcome by the ATP binding cassette transport protein inhibitor,verapamil. Importantly,these cells are not resistant to YK-4-279,a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy. View Publication

Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can differentiate into cell types of mesenchymal origin. Because of their immune properties and differentiation,potential MSCs are discussed for the use in tissue regeneration and tolerance induction in transplant medicine. This cell type can easily be obtained from the umbilical cord tissue (UCMSC) without medical intervention. Standard culture conditions include fetal bovine serum (FBS) which may not be approved for clinical settings. Here,we analyzed the phenotypic and functional properties of UCMSC under xeno-free (XF,containing GMP-certified human serum) and serum-free (SF) culture conditions in comparison with standard UCMSC cultures. Phenotypically,UCMSC showed no differences in the expression of mesenchymal markers or differentiation capacity. Functionally,XF and SF-cultured UCMSC have comparable adipogenic,osteogenic,and endothelial differentiation potential. Interestingly,the UCMSC-mediated suppression of T cell proliferation in an allogeneic mixed lymphocyte reaction (MLR) is more effective in XF and SF media than in standard FBS-containing cultures. Regarding the mechanism of action of MLR suppression,transwell experiments revealed that in neither UCMSC culture a direct cell-cell contact is necessary for inhibiting T cell proliferation,and that the major effector molecule is prostaglandin E₂ (PGE₂). Taken together,GMP-compliant growth media qualify for long-term cultures of UCMSC which is important for a future clinical study design in regenerative and transplant medicine. View Publication

Mesenchymal stromal cells (MSCs) may be derived from a variety of tissues,with human umbilical cord (UC) providing an abundant and noninvasive source. Human UC-MSCs share similar in vitro immunosuppressive properties as MSCs obtained from bone marrow and cord blood. However,the mechanisms and cellular interactions used by MSCs to control immune responses remain to be fully elucidated. In this paper,we report that suppression of mitogen-induced T cell proliferation by human UC-,bone marrow-,and cord blood-MSCs required monocytes. Removal of monocytes but not B cells from human adult PBMCs (PBMNCs) reduced the immunosuppressive effects of MSCs on T cell proliferation. There was rapid modulation of a number of cell surface molecules on monocytes when PBMCs or alloantigen-activated PBMNCs were cultured with UC-MSCs. Indomethacin treatment significantly inhibited the ability of UC-MSCs to suppress T cell proliferation,indicating an important role for PGE(2). Monocytes purified from UC-MSC coculture had significantly reduced accessory cell and allostimulatory function when tested in subsequent T cell proliferation assays,an effect mediated in part by UC-MSC PGE(2) production and enhanced by PBMNC alloactivation. Therefore,we identify monocytes as an essential intermediary through which UC-MSCs mediate their suppressive effects on T cell proliferation. View Publication

CONTEXT: The osteoanabolic properties of PTH may be due to increases in the number and maturity of circulating osteogenic cells. Hypoparathyroidism is a useful clinical model because this hypothesis can be tested by administering PTH. OBJECTIVE: The objective of the study was to characterize circulating osteogenic cells in hypoparathyroid subjects during 12 months of PTH (1-84) administration. DESIGN: Osteogenic cells were characterized using flow cytometry and antibodies against osteocalcin,an osteoblast-specific protein product,and stem cell markers CD34 and CD146. Changes in bone formation from biochemical markers and quadruple-labeled transiliac crest bone biopsies (0 and 3 month time points) were correlated with measurements of circulating osteogenic cells. SETTING: The study was conducted at a clinical research center. PATIENTS: Nineteen control and 19 hypoparathyroid patients were included in the study. INTERVENTION: Intervention included the administration of PTH (1-84). RESULTS: Osteocalcin-positive cells were lower in hypoparathyroid subjects than controls (0.7 ± 0.1 vs. 2.0 ± 0.1%; P textless 0.0001),with greater coexpression of the early cell markers CD34 and CD146 among the osteocalcin-positive cells in the hypoparathyroid subjects (11.0 ± 1.0 vs. 5.6 ± 0.7%; P textless 0.001). With PTH (1-84) administration,the number of osteogenic cells increased 3-fold (P textless 0.0001),whereas the coexpression of the early cell markers CD34 and CD146 decreased. Increases in osteogenic cells correlated with circulating and histomorphometric indices of osteoblast function: N-terminal propeptide of type I procollagen (R(2) = 0.4,P ≤ 0.001),bone-specific alkaline phosphatase (R(2) = 0.3,P textless 0.001),osteocalcin (R(2) = 0.4,P textless 0.001),mineralized perimeter (R(2) = 0.5,P textless 0.001),mineral apposition rate (R(2) = 0.4,P = 0.003),and bone formation rate (R(2) = 0.5,P textless 0.001). CONCLUSIONS: It is likely that PTH stimulates bone formation by stimulating osteoblast development and maturation. Correlations between circulating osteogenic cells and histomorphometric indices of bone formation establish that osteoblast activity is being identified by this methodology. View Publication

Mesenchymal stromal cells (MSCs) have important immunosuppressive properties,but the mechanisms and soluble factors involved in these effects remain unclear. We have studied prostaglandin-E2 (PGE2) as a possible candidate implied in adipose tissue-derived MSCs (Ad-MSCs) immunosuppressive properties over dendritic cells and T lymphocytes,compared to bone marrow derived MSCs (BM-MSCs). We found that both MSCs inhibited the maturation of myeloid-DCs and plasmocytoid-DCs. High levels of PGE2 were detected in DCs/MSCs co-cultures. Its blockade with indomethacin (IDM) allowed plasmocytoid-DCs but not myeloid-DCs maturation. Additionally,high levels of PGE2 were found in co-cultures in which Ad-MSCs or BM-MSCs inhibited activated T cells proliferation and pro-inflammatory cytokines production. PGE2 blockade by IDM preserved T lymphocytes proliferation but did not restore the pro-inflammatory cytokines secretion. However,an increased expression of transcription factors and cytokines genes involved in the Th1/Th2 differentiation pathway was detected in the T cells co-cultured with Ad-MSCs,but not with BM-MSCs. In conclusion,we propose that PGE2 is a soluble factor mediating most of the immunosuppressive effects of Ad-MSCs and BM-MSCs over p-DCs maturation and activated T lymphocytes proliferation and cytokine secretion. View Publication

This study aimed to determine the mechanisms responsible for long-term tissue damage following radiation injury. We irradiated p21-knockout (p21(-/-)) and wild-type (WT) mice and determined the long-term deleterious effects of this intervention on mesenchyme-derived tissues. In addition,we explored the mechanisms of radiation-induced mesenchymal stem cell (MSC) dysfunction in isolated bone marrow-derived cells. p21 expression was chronically elevated textgreater200-fold in irradiated tissues. Loss of p21 function resulted in a textgreater4-fold increase in the number of skin MSCs remaining after radiation. p21(-/-) mice had significantly less radiation damage,including 6-fold less scarring,40% increased growth potential,and 4-fold more hypertrophic chondrocytes in the epiphyseal plate (Ptextless0.01). Irradiated p21(-/-) MSCs had 4-fold increased potential for bone or fat differentiation,4-fold greater proliferation rate,and nearly 7-fold lower senescence as compared to WT MSCs (Ptextless0.01). Ectopic expression of p21 in knockout cells decreased proliferation and differentiation potential and recapitulated the WT phenotype. Loss of p21 function markedly decreases the deleterious effects of radiation injury in mesenchyme-derived tissues and preserves tissue-derived MSCs. In addition,p21 is a critical regulator of MSC proliferation,differentiation,and senescence both at baseline and in response to radiation. View Publication

AIMS: Endothelial progenitor cells (EPC) have been shown to repair pulmonary endothelium,although they can also migrate into the arterial intima and differentiate into smooth muscle-like (mesenchymal) cells contributing to intimal hyperplasia. The molecular mechanisms by which this process proceeds have not been fully elucidated. Here,we study whether genes involved in the endothelial-to-mesenchymal transition (EnMT) may contribute to the mesenchymal phenotype acquisition of EPC and we evaluate whether transforming growth factor β1 (TGFβ1) is involved in this process. METHODS AND RESULTS: Our results show that co-culture of EPC with smooth muscle cells (SMC) increases the expression of the mesenchymal cell markers α-smooth muscle actin,sm22-α,and myocardin,and decreases the expression of the endothelial cell marker CD31. In the same conditions,we also observed a concomitant increase in the gene expression of the EnMT-related transcription factors: slug,snail,zeb1,and endothelin-1. This indicates that mesenchymal phenotype acquisition occurred through an EnMT-like process. Inhibition of TGFβ receptor I (TGFβRI) downregulated snail gene expression,blocked the EnMT,and facilitated the differentiation of EPC to the endothelial cell lineage. Furthermore,TGFβRI inhibition decreased migration of EPC stimulated by SMC without affecting their functionality and adhesion capacity. CONCLUSION: These results indicate that EPC may differentiate into SMC-like cells through an EnMT-like process and that TGFβI plays an important role in the fate of EPC. View Publication

RATIONALE: Fibrocytes are progenitor cells characterized by the simultaneous expression of mesenchymal,monocyte,and hematopoietic stem cell markers. We previously documented their presence in lungs of patients with idiopathic pulmonary fibrosis. However,the mechanisms involved in their migration,subsequent homing,and local role remain unclear. Matrix metalloproteinases (MMPs) facilitate cell migration and have been implicated in the pathogenesis of pulmonary fibrosis. OBJECTIVES: To evaluate the expression and role of matrix metalloproteinases in human fibrocytes. METHODS: Fibrocytes were purified from CD14(+) monocytes and cultured for 8 days; purity of fibrocyte cultures was 95% or greater as determined by flow cytometry. Conditioned media and total RNA were collected and the expression of MMP-1,MMP-2,MMP-7,MMP-8,and MMP-9 was evaluated by real-time polymerase chain reaction. Protein synthesis was examined using a Multiplex assay,Western blot,fluorescent immunocytochemistry,and confocal microscopy. MMP-2 and MMP-9 enzymatic activities were evaluated by gelatin zymography. Migration was assessed using collagen I-coated Boyden chambers. Stromal cell-derived factor-1α and platelet-derived growth factor-B were used as chemoattractant with or without a specific MMP-8 inhibitor. MEASUREMENTS AND MAIN RESULTS: Fibrocytes showed gene and protein expression of MMP-2,MMP-9,MMP-8,and MMP-7. MMP-2 and MMP-9 enzymatic activities were also demonstrated by gelatin zymography. Likewise,we found colocalization of MMP-8 and MMP-7 with type I collagen in fibrocytes. Fibrocyte migration toward platelet-derived growth factor-B or Stromal cell-derived factor-1α in collagen I-coated Boyden chambers was significantly reduced by a specific MMP-8 inhibitor. CONCLUSIONS: Our findings reveal that fibrocytes express a variety of MMPs and that MMP-8 actively participates in the process of fibrocyte migration. View Publication

The endosteal niche is critical for the maintenance of hematopoietic stem cells (HSCs). However,it consists of a heterogeneous population in terms of differentiation stage and function. In this study,we characterized endosteal cell populations and examined their ability to maintain HSCs. Bone marrow endosteal cells were subdivided into immature mesenchymal cell-enriched ALCAM(-)Sca-1(+) cells,osteoblast-enriched ALCAM(+)Sca-1(-),and ALCAM(-)Sca-1(-) cells. We found that all 3 fractions maintained long-term reconstitution (LTR) activity of HSCs in an in vitro culture. In particular,ALCAM(+)Sca-1(-) cells significantly enhanced the LTR activity of HSCs by the up-regulation of homing- and cell adhesion-related genes in HSCs. Microarray analysis showed that ALCAM(-)Sca-1(+) fraction highly expressed cytokine-related genes,whereas the ALCAM(+)Sca-1(-) fraction expressed multiple cell adhesion molecules,such as cadherins,at a greater level than the other fractions,indicating that the interaction between HSCs and osteoblasts via cell adhesion molecules enhanced the LTR activity of HSCs. Furthermore,we found an osteoblastic marker(low/-) subpopulation in ALCAM(+)Sca-1(-) fraction that expressed cytokines,such as Angpt1 and Thpo,and stem cell marker genes. Altogether,these data suggest that multiple subsets of osteoblasts and mesenchymal progenitor cells constitute the endosteal niche and regulate HSCs in adult bone marrow. View Publication

BACKGROUND: Specific populations of highly tumorigenic cells are thought to exist in many human tumors,including pancreatic adenocarcinoma. However,the clinical significance of these tumor-initiating (ie,cancer stem) cells remains unclear. Aldehyde dehydrogenase (ALDH) activity can identify tumor-initiating cells and normal stem cells from several human tissues. We examined the prognostic significance and functional features of ALDH expression in pancreatic adenocarcinoma. METHODS: ALDH expression was analyzed by immunohistochemistry in 269 primary surgical specimens of pancreatic adenocarcinoma and examined for association with clinical outcomes and in paired primary tumors and metastatic lesions from eight pancreatic cancer patients who had participated in a rapid autopsy program. The clonogenic growth potential of ALDH-positive pancreatic adenocarcinoma cells was assessed in vitro by a colony formation assay and by tumor growth in immunodeficient mice (10-14 mice per group). Mesenchymal features of ALDH-positive pancreatic tumor cells were examined by using quantitative reverse transcription-polymerase chain reaction and an in vitro cell invasion assay. Gene expression levels and the invasive potential of ADLH-positive pancreatic cancer cells relative to the bulk cell population were examined by reverse transcription-polymerase chain reaction and an in vitro invasion assays,respectively. All statistical tests were two-sided. RESULTS: ALDH-positive tumor cells were detected in 90 of the 269 primary surgical specimens,and their presence was associated with worse survival (median survival for patients with ALDH-positive vs ALDH-negative tumors: 14 vs 18 months,hazard ratio of death = 1.28,95% confidence interval = 1.02 to 1.68,P = .05). Six (75%) of the eight patients with matched primary and metastatic tumor samples had ALDH-negative primary tumors,and in four (67%) of these six patients,the matched metastatic lesions (located in liver and lung) contained ALDH-positive cells. ALDH-positive cells were approximately five- to 11-fold more clonogenic in vitro and in vivo compared with unsorted or ALHD-negative cells,expressed genes consistent with a mesenchymal state,and had in vitro migratory and invasive potentials that were threefold greater than those of unsorted cells. CONCLUSIONS: ALDH expression marks pancreatic cancer cells that have stem cell and mesenchymal features. The enhanced clonogenic growth and migratory properties of ALDH-positive pancreatic cancer cells suggest that they play a key role in the development of metastatic disease that negatively affects the overall survival of patients with pancreatic adenocarcinoma. View Publication