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1.
Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non-adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as "dedifferentiation." To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post-mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up-regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications.  相似文献   

2.
The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57–4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90‐negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90‐positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100‐positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell‐based cartilage repair treatments. J. Cell. Physiol. 222: 411–420, 2010. © 2009 Wiley‐Liss, Inc.  相似文献   

3.
Tissue engineering efforts for the fabrication of cartilage substitutes head toward applicability in osteoarthritis (OA). Progenitor cells can be harvested from the osteoarthritic joint itself, resembling multipotent mesenchymal stromal cells (MSC). Our objective was to analyze MSC characteristics of those cells in respect to the OA‐related damage of their harvest site. OA cartilage was obtained from six patients during alloarthroplastic knee surgery, sample grading was done according to Outerbridge's classification. Upon enzymatic dissociation, primary chondrocytes were expanded in two‐dimensional monolayer culture. At distinct cell passages, the process of dedifferentiation was phenotypically monitored; cell surface expression of classical MSC markers was analyzed by flow cytometry. Cells were subjected to chondrogenesis and osteogenesis after their fourth passage. At third passage, 95% of cells became positive for cluster of differentiation (CD)105 and further subclassification revealed that the majority of them were positive for both CD73 and CD90. CD105+CD73+CD90+ phenotype meets thus the minimal surface antigen criteria for MSC definition. More than one‐third of dedifferentiated chondrocytes displayed a coexpression of CD9+CD166+CD90+ and to a lesser extent CD105+CD73+CD44+, irrespective of the stage of the original cartilage degradation. Finally, we could successfully demonstrate a redifferentiation of these progenitors into sulfated glycosaminoglycan producing cells. The basic level of alkaline phosphatase activity could not be enhanced upon osteogenic differentiation. In conclusion, chondrogenic progenitors derived from OA cartilages with low or high Outerbridge's grade can be seen as a potential cellular source for cartilage replacement. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013  相似文献   

4.
OBJECTIVE: To test the hypothesis that dedifferentiated adult human cartilage chondrocytes (HAC) are a true multipotent primitive population. METHODS: Studies to characterize dedifferentiated HAC included cell cycle and quiescence analysis, cell fusion, flow-FISH telomere length assays, and ABC transporter analysis. Dedifferentiated HAC were characterized by flow cytometry, in parallel with bone marrow mesenchymal stem cells (MSC) and processed lipoaspirate (PLA) cells. The in vitro differentiation potential of dedifferentiated HAC was studied by cell culture under several inducing conditions, in multiclonal and clonal cell populations. RESULTS: Long-term HAC cultures were chromosomically stable and maintained cell cycle dynamics while showing telomere shortening. The phenotype of dedifferentiated HAC was quite similar to that of human bone marrow MSC. In addition, this population expressed human embryonic stem cell markers. Multiclonal populations of dedifferentiated HAC differentiated to chondrogenic, osteogenic, adipogenic, myogenic, and neurogenic lineages. Following VEGF induction, dedifferentiated HAC expressed characteristics of endothelial cells, including AcLDL uptake. A total of 53 clonal populations of dedifferentiated HAC were efficiently expanded; 17 were able to differentiate to chondrogenic, osteogenic, and adipogenic lineages. No correlation was observed between telomere length or quiescent population and differentiation potential in the clones assayed. CONCLUSION: Dedifferentiated HAC should be considered a human multipotent primitive population.  相似文献   

5.
The aim of this study was to assess the effect of extracellular matrix (ECM) deposited by synovium-derived stem cells (SDSCs) on articular chondrocyte expansion and maintenance of differentiation status and redifferentiation capacity. Passage 0 (P0) pig articular chondrocytes were expanded for six passages on plastic flasks (Plastic), SDSC-derived ECM (ECM), or substrate switching from either Plastic to ECM (PtoE) or ECM to Plastic (EtoP). Cell morphology, gene expression profiles, and immunophenotypes at each passage were used to characterize differentiation status of expanded cells. Chondrocytes at P0, P2, and P6 were assessed for redifferentiation capacity in a pellet culture system treated with either TGF-β1- or serum-containing medium for 14 days, using histology, immunohistochemistry, biochemistry, Western blot, and real-time PCR. We found that ECM not only greatly enhanced chondrocyte expansion but also delayed dedifferentiation of expanded chondrocytes. Intriguingly, compared to a dramatic decrease in CD90+/CD105+ cells and CD90+ cells, CD105+ cells dramatically increased when chondrocytes were plated on Plastic; on the contrary, ECM expansion dramatically increased CD90+ cells and delayed the decrease of CD90+/CD105+ cells. Interestingly, expanded chondrocytes on ECM also acquired a strong redifferentiation capacity, particularly in the pellets treated with TGF-β1. In conclusion, the ratio of CD90 to CD105 may serve as a marker indicative of proliferation and redifferentiation capacity of dedifferentiated chondrocytes. ECM deposited by SDSCs provides a tissue-specific three-dimensional microenvironment for ex vivo expansion of articular chondrocytes while retaining redifferentiation capacity, suggesting that ECM may provide a novel approach for autologous chondrocyte-based cartilage repair.  相似文献   

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Mesenchymal stem cells (MSC) are able to transdifferentiate into cells with different functional phenotypes and considered as a promising resource for regenerative therapy. MSC derived from different tissues vary in their differentiation potential and in some cases express tissue specific markers indicating a kinship between mesenchymal and parenchymal phenotypes in the same tissue. It is possible that homorganic MSC can be more effectively induced to tissue specific differentiation and preferable for cell therapy of this organ as compared with bone marrow derived cells being commonly used for this purpose. Using bladder tissue explants, we prepared primary MSC cultures from the fetal (MSC-BF) and adult syngenic BALB/c mice and characterized their abilities during long-term passaging. In contrast to the cells from adult mice, the MSC-BF cells have the ability for a sustained growth in vitro, clonogenicity and differentiation into adipose and bone cells. Similar to the bone marrow MSC, MSC-BF express the mesenchymal markers CD29, CD44, CD49f, CD90, CD105 but not the leukocyte common antigen CD45. In normal conditions, MSC-BF produce such urothelial markers as CK14 and FOXA1 although their expression level is by far lower than in the bladder tissue. The hypomethylating agent, 5-azacytidine, induces in MSC-BF the expression of the urothelial differentiation activator PPARγ and the functional urothelium markers UP1a, UP1b, UP3a, UP3b. The data obtained suggest that MSC-BF can be epigenetically reprogrammed into urothelium by the 5-azacytidine treatment, and this may offer the novel strategy for cell therapy of bladder diseases.  相似文献   

8.
Markers of stemness in equine mesenchymal stem cells: a plea for uniformity   总被引:1,自引:0,他引:1  
Mesenchymal stromal cells (MSC) are a very promising subpopulation of adult stem cells for cell-based regenerative therapies in veterinary medicine. Despite major progress in the knowledge on adult stem cells during recent years, a proper identification of MSC remains a challenge. In human medicine, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) recently proposed three criteria to define MSC. Firstly, cells must be plastic-adherent when maintained under standard culture conditions. Secondly, MSC must express CD73, CD90 and CD105, and lack expression of CD34, CD45, CD14 or CD11b, CD79α or CD19 and MHC class II antigens. Thirdly, MSC must be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro. Successful isolation and differentiation of equine MSC from different sources such as bone marrow, fat tissue, umbilical cord blood, Wharton's Jelly or peripheral blood has been widely reported. However, their unequivocal immunophenotyping is hampered by the lack of a single specific marker and the limited availability of monoclonal anti-horse antibodies, which are two major factors complicating successful research on equine MSC. Detection of gene expression on mRNA level is hereby a valuable alternative, although the need still exists to test several antibody clones in search for cross-reactivity. To date, commercial antibodies recognizing equine epitopes are only available for CD13, CD44 and MHC-II. Moreover, as the expression of certain adult stem cell markers may differ between species, it is mandatory to define a set of CD markers which can be uniformly applied for the identification of equine MSC.  相似文献   

9.
Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.  相似文献   

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脐血CD-34单个核细胞来源间充质干细胞研究   总被引:2,自引:0,他引:2  
目的 :探讨分离培养脐血CD-3 4 细胞来源间充质干细胞 (MSC)及研究其生物学特征。方法 :取足月妊娠健康产妇胎儿脐血 ,分离其中单个核细胞 (MNC) ,去除CD 3 4 细胞 ,体外用低糖型DMEM培养基培养。观察细胞形态、测定生长曲线、利用流式细胞仪对培养细胞进行表型测定、细胞周期分析、体外诱导分化实验以及检测造血因子的表达情况。结果 :脐血CD-3 4 细胞中可培养出间充质干细胞 ,可诱导向成骨和脂肪细胞分化并表达IL 6、SCF和SDF 1等造血生长因子。结论 :从足月妊娠健康产妇脐血CD-3 4 细胞可分离培养出间充质干细胞 ,具有与其它来源MSC类似的表型及分化潜能 ,在体外传代可保持其低分化状态并表达造血因子 ,可作为组织工程的种子细胞和具有促进造血作用  相似文献   

14.
Background aimsMesenchymal stromal cells (MSC) exhibit non-specific hematopoietic cell and/or stromal cell markers (e.g. CD73, CD105 and CD166) that have been used to identify MSC by flow cytometry. Because a neural glial antigen, NG2 (a progenitor cell marker in the central nervous system), is expressed by several tissue cells originating in the mesenchyme but not hematopoietic cells, it might be useful for isolating and identifying MSC. We investigated NG2 expression on culture-expanded MSC by flow cytometry.MethodsHuman bone marrow (BM) samples taken from 12 donors were cultured for MSC to be used in up to nine serial passages. Using flow cytometry, the neural glial antigen NG2 and commonly used MSC markers CD73, CD105 and CD166, were analyzed on the surface of culture-expanded MSC. The multipotential differentiation of the MSC was examined by adipogenic and osteogenic induction.ResultsThe percentage of cells positive for NG2 was similar to the percentages of cells positive for CD73, CD105 and CD166 in all passages of BM samples. The mean fluorescent intensities of NG2 did not change with culture passage. The MSC was successfully differentiated into adipogenic and osteogenic lines. The cells showed no karyotypic abnormalities.ConclusionsNG2 seems to be a promising marker for investigating the biology of MSC.  相似文献   

15.

Background

Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC.

Methods and Findings

UCB-derived mononuclear cells (MNC) or selected CD34+ cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34+ selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin+, CD133+ and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages.Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies.

Conclusions

This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice.  相似文献   

16.
This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC’s were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.  相似文献   

17.
Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.  相似文献   

18.
Mesenchymal stem cells (MSCs) can not only support the expansion of hematopoietic stem cells in vitro, but also alleviate complications and accelerate recovery of hematopoiesis during hematopoietic stem cell transplantation. However, it proved challenging to culture MSCs from umbilical cord blood (UCB) with a success rate of 20–30%. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In this work, fractional factorial design was applied to study the effect of cell inoculated density, combination and dose of cytokines, and presence of serum and stromal cells. The cultured UCB‐MSC‐like cells were characterized by flow cytometry and their multilineage differentiation potentials were tested. The optimal protocol was identified achieving above 90% successful outcome: 2 × 106 cells/mL mononuclear cells inoculated in Iscove's modified Dulbecco's medium supplied with 10% FBS, 15 ng/mL IL‐3, and 5 ng/mL Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Moreover, the UCB‐MSC‐like cells expressed MSC surface markers of CD13, CD29, CD105, CD166, and CD44 positively, and CD34, CD45, and human leukocyte antigens‐DR (HLA‐DR) negatively. Meanwhile, these cells could differentiate into osteoblasts, chondrocytes, and adipocytes similarly to MSCs derived from bone marrow. In conclusion, we have developed an efficient protocol for the primary culture of UCB‐MSCs by adding suitable cytokines into the culture system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009  相似文献   

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The differentiated phenotype of chondrocyte is rapidly lost during in vitro culture by a process designated "dedifferentiation." In this study, we investigate the roles of protein kinase C (PKC) and extracellular signal-regulated protein kinase (ERK) in the maintenance of the differentiated chondrocyte phenotype. Chondrocytes isolated from rabbit articular cartilage underwent dedifferentiation upon serial monolayer culture with cessation of type II collagen expression and proteoglycan synthesis, which was reversed by culturing dedifferentiated cells in alginate gel. The expression pattern of PKC alpha was essentially the same as that of type II collagen during de- and redifferentiation, in that expression was decreased during dedifferentiation and increased during redifferentiation. In contrast to PKC alpha, ERK activity increased 15-fold during dedifferentiation. This enhanced activity was terminated during redifferentiation. Down-regulation of PKC alpha in passage 0 chondrocytes resulted in dedifferentiation. However, overexpression of PKC alpha did not affect type II collagen levels, suggesting that PKC alpha expression is not sufficient to maintain the differentiated phenotype. However, inhibition of ERK by PD98059 enhanced type II collagen expression and proteoglycan synthesis in passage 0 cells, retarded dedifferentiation during monolayer cultures, and reversed dedifferentiation caused by down-regulation of PKC. Unlike PKC-dependent ERK regulation of chondrogenesis, PKC and ERK independently modulated chondrocyte dedifferentiation, as confirmed by observations that PKC down-regulation and ERK inhibition did not alter ERK phosphorylation and PKC expression, respectively. In addition, expression of N-cadherin, alpha-catenin, and beta-catenin, which are oppositely regulated to type II collagen during phenotype alterations, were modulated by PKC and ERK during chondrogenesis but not dedifferentiation, supporting distinct mechanisms for the regulation of chondrocyte differentiation and maintenance of differentiated phenotype by these two protein kinases.  相似文献   

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