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1.
hASCs [human ASCs (adipose derived stromal cells)] proliferate more rapidly in the presence of basic FGF-2 (fibroblast growth factor-2) and Dex (dexamethasone). We have examined the effects of expanding hASCs in media containing these two factors on their chondrogenic differentiation potential. Results show that the addition of FGF-2 and Dex to the expansion medium does not remarkably alter the chondrogenic potential of the cells induced by BMP-6 (bone morphogenetic protein-6), based on chondrogenic gene expression, sGAG (sulfated glycosaminoglycan) accumulation and immunohistochemical observation. This is in direct contrast to previously reported promotion of the osteogenic and adipogenic potential of hASCs by these two factors. Therefore, an expansion medium containing FGF-2, with or without Dex, is appropriate for the fast expansion of hASCs without compromising chondrogenic potential.  相似文献   

2.
We have previously demonstrated that adipose-derived stromal cells (ASCs) as well as bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. Both types are considered to include mesenchymal stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have also previously reported the plasticity of BSCs and ASCs. In this study, we focused on adipogenic differentiation in vitro by ASCs harvested from GFP transgenic mice. Moreover, preadipocytes and mature adipocytes were harvested at the same time, and the cells were cultured to compare them with ASCs. Inguinal fat pads from GFP transgenic mice were used for the isolation of ASCs, preadipocytes, and mature adipocytes. After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks. Adipogenic differentiation of ASCs was assessed by Oil Red O staining and the expression of the adipocyte specific peroxisome proliferative activated receptor gamma2 (PPAR-gamma2) gene. These ASCs stained positively, and expression of PPAR-gamma2 was detected. Moreover, we also tried to characterize the influence of sex differences on the adipogenic differentiation of ASCs harvested from both male and female mice. This was assessed by the expression levels of the PPAR-gamma2 gene using real-time PCR. The results showed that the expression levels of ASCs harvested from female mice were a maximum of 2.89 times greater than those harvested from male mice. This suggests that the adipogenic differentiation of ASCs is closely related to sex differences.  相似文献   

3.
In the current study, we investigated the effects of genistein on adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures and its potential signaling pathway. The terminal adipogenic differentiation was assessed by western-blotting analysis of adipogenic-specific proteins such as PPARgamma, C/EBPalpha, and aP2 and the formation of adipocytes. Treatment of mouse BMSC cultures with adipogenic cocktail resulted in sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated protein kinase (MAPK) family, at the early phase of adipogenesis (from days 3 to 9). Inhibition of ERK1/2 activation by PD98059, a specific MEK inhibitor, reversed the induced adipogenic differentiation. Genistein dose-dependently decreased the phosphorylation of ERK1/2 in mouse BMSC cultures. Genistein incubation for the entire culture period, as well as that applied during the early phase of the culture period, significantly inhibited the adipogenic differentiation of mouse BMSC cultures. While genistein was incubated at the late stage (after day 9), no inhibitory effect on adipogenic differentiation was observed. BMSC cultures treated with genistein in the presence of fibroblast growth factor-2 (FGF-2), an activator of the ERK1/2 signaling pathway, expressed normal levels of ERK1/2 activity, and, in so doing, are capable of undergoing adipogenesis. Our results suggest that activation of the ERK1/2 signaling pathway during the early phase of adipogenesis (from days 3 to 9) is essential to adipogenic differentiation of BMSC cultures, and that genistein inhibits the adipogenic differentiation through a potential downregulation of ERK1/2 activity at this early phase of adipogenesis.  相似文献   

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Adipose-derived stem cells (ASCs) have been successfully applied in treating bone defects both in animals and humans and promoted osteogenesis in vivo significantly. However, the mechanism of in vivo osteogenesis of ASCs was still little known, we hypothesized that this was mediated in part by interaction between implanted ASCs and local vein endothelial cells. In this study, human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVEC) were isolated and characterized. Cells were then either cultured alone or cocultured. Alkaline phosphatase (ALP) staining, quantitative measurement of ALP activity and Alizarin staining of hASCs cultured alone, HUVEC cultured alone and cells cocultured demonstrated that osteogenic differentiation of cocultured cells increased obviously. Osteocalcin (OC) expression of hASCs cocultured with HUVEC showed an obvious raise than hASCs cultured alone. HUVEC cultured alone showed BMP-2 secretion and increased with culturing time. Real-time PCR of the cocultured cells showed four osteogenic differentiation related genes raised with culturing time, while two adipogenic differentiation related genes showed a slightly decrease with culturing time. Results of our study with different culture models showed that in vitro osteogenesis of hASCs was enhanced by coculture with HUVEC which secreted BMP-2. This study not only provided us with an in vitro model of studying interaction between cells, but also helped us to understand the in vivo therapeutic mechanisms of ASCs.  相似文献   

7.
Chemical control of protein secretion using a small molecule approach provides a powerful tool to optimize tissue engineering strategies by regulating the spatial and temporal dimensions that are exposed to a specific protein. We placed fibroblast growth factor 2 (FGF-2) under conditional control of a small molecule and demonstrated greater than 50-fold regulation of FGF-2 release as well as tunability, reversibility, and functionality in vitro. We then applied conditional control of FGF-2 secretion to a cell-based, skeletal tissue engineering construct consisting of adipose stem cells (ASCs) on a biomimetic scaffold to promote bone formation in a murine critical-sized calvarial defect model. ASCs are an easily harvested and abundant source of postnatal multipotent cells and have previously been demonstrated to regenerate bone in critical-sized defects. These results suggest that chemically controlled FGF-2 secretion can significantly increase bone formation by ASCs in vivo. This study represents a novel approach toward refining protein delivery for tissue engineering applications.  相似文献   

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Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferator-activated receptor (PPAR-γ2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.  相似文献   

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Mesenchymal stem cells (MSC) that can differentiate to various connective tissue cells may be useful for autologous cell transplantation to defects of bone, cartilage, and tendon, if MSC can be expanded in vitro. However, a short life span of MSC and a reduction in their differentiation potential in culture have limited their clinical application. The purpose of this study is to identify a growth factor(s) involved in self-renewal of MSC and the maintenance of their multilineage differentiation potential. Fibroblast growth factor-2 (FGF-2) markedly increased the growth rate and the life span of rabbit, canine, and human bone marrow MSC in monolayer cultures. This effect of FGF-2 was more prominent in low-density cultures than in high-density cultures. In addition, all MSC expanded in vitro with FGF-2, but not without FGF-2, differentiated to chondrocytes in pellet cultures. The FGF+ MSC also retained the osteogenic and adipogenic potential throughout many mitotic divisions. These findings suggest that FGFs play a crucial role in self-renewal of MSC.  相似文献   

12.
Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.  相似文献   

13.
Cell number is an important determinant of adipose tissue mass, and the coordinated proliferation and differentiation of preadipocytes into mature lipid-laden adipocytes underpins the increased adipose tissue mass associated with obesity. Despite this, the molecular cues governing such adipose tissue expansion are poorly understood. We previously reported that fibroblast growth factor-1 (FGF-1) promotes both proliferation and differentiation of human preadipocytes and that the major adipogenic effect of FGF-1 occurs during proliferation, priming the cells for adipose conversion. In the current study, we examined whether this effect was linked to the mitogenic action of FGF-1 by investigating the mitogenic and adipogenic potential of other growth factors, platelet-derived growth factor (PDGF; AA and BB) and vascular endothelial growth factor. Although PDGF-AA and PDGF-BB showed comparable mitogenic potential to FGF-1, only FGF-1 treatment resulted in priming and subsequent differentiation. Pharmacological inhibition of FGF receptor (FGFR) tyrosine kinase activity, using the FGFR-specific inhibitors PD-173074 and SU-5402, revealed an obligate requirement for FGFR activity in these processes. A combination of biochemical and genetic approaches revealed an important role for FGFR1. Knock down of FGFR1 expression by small-interfering RNA reduced FGF-1-stimulated signaling events, proliferation, and priming. Together these data highlight the unique nature of the role of FGF-1 during the earliest stages of adipogenesis and establish a role for FGFR1 in human adipogenesis, identifying FGFR1 as a potential therapeutic target to reduce obesity.  相似文献   

14.
Yang X  Cai X  Wang J  Tang H  Yuan Q  Gong P  Lin Y 《Cell proliferation》2012,45(2):158-166
A reciprocal relationships between osteogenesis and adipogenesis has been observed in vitro and in vivo, and mechanical stretch has been believed to be a regulating factor of osteo-adipogenic axis differentiation of mesenchymal stem cells. In this study, rat adipose stem cells (ASCs) were isolated and cultured in adipogenic or normal medium. Their exposure to cyclic mechanical stretch (2000 με, 1 Hz) in the presence of adipogenic medium decreased mRNA and protein level of PPAR-γ, and increased Runx2 mRNA and protein levels as well as Pref-1 mRNA level, compared to static samples. ASCs cultured in normal medium without adipogenic induction did not show any significant change in mRNA expression of PPAR-γ, Runx2, nor Pref-1 irrespective of mechanical loading. Stretching induced phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) during the induction period. It was concluded that mechanical stretch inhibited adipogenesis and stimulated osteogenesis of these ASCs in the presence of adipogenic medium and that ERK1/2 activation may be involved in the mechanical stress-induced trans-differentiation.  相似文献   

15.

Background  

Potential therapeutic use of mesenchymal stem cells (MSCs) is likely to require large-scale in vitro expansion of the cells before transplantation. MSCs from adipose tissue can be cultured extensively until senescence. However, little is known on the differentiation potential of adipose stem cells (ASCs) upon extended culture and on associated epigenetic alterations. We examined the adipogenic differentiation potential of clones of human ASCs in early passage culture and upon senescence, and determined whether senescence was associated with changes in adipogenic promoter DNA methylation.  相似文献   

16.
Multiple sclerosis (MS), characterized by chronic inflammation, demyelination, and axonal damage, is a complicated neurological disease of the human central nervous system. Recent interest in adipose stromal/stem cell (ASCs) for the treatment of CNS diseases has promoted further investigation in order to identify the most suitable ASCs. To investigate whether MS affects the biologic properties of ASCs and whether autologous ASCs from MS-affected sources could serve as an effective source for stem cell therapy, cells were isolated from subcutaneous inguinal fat pads of mice with established experimental autoimmune encephalomyelitis (EAE), a murine model of MS. ASCs from EAE mice and their syngeneic wild-type mice were cultured, expanded, and characterized for their cell morphology, surface antigen expression, osteogenic and adipogenic differentiation, colony forming units, and inflammatory cytokine and chemokine levels in vitro. Furthermore, the therapeutic efficacy of the cells was assessed in vivo by transplantation into EAE mice. The results indicated that the ASCs from EAE mice displayed a normal phenotype, typical MSC surface antigen expression, and in vitro osteogenic and adipogenic differentiation capacity, while their osteogenic differentiation capacity was reduced in comparison with their unafflicted control mice. The ASCs from EAE mice also demonstrated increased expression of pro-inflammatory cytokines and chemokines, specifically an elevation in the expression of monocyte chemoattractant protein-1 and keratin chemoattractant. In vivo, infusion of wild type ASCs significantly ameliorate the disease course, autoimmune mediated demyelination and cell infiltration through the regulation of the inflammatory responses, however, mice treated with autologous ASCs showed no therapeutic improvement on the disease progression.  相似文献   

17.
In musculoskeletal tissues like bone, chemotherapy can impair progenitor cell differentiation and proliferation, resulting in decreased bone growth and mineralization throughout a patient?s lifetime. In the current study, we investigated the effects of chemotherapeutics on adipose-derived stem cell (ASC) function to determine whether this cell source could be a candidate for repairing, or even preventing, chemotherapy-induced tissue damage. Dose-dependent proliferation rates of ASCs and normal human fibroblasts (NHFs) were quantified after treatment with cytarabine (CY), etoposide (ETO), methotrexate (MTX), and vincristine (VIN) using a fluorescence-based assay. The influence of MTX on the multipotency of ASCs and freshly isolated stromal vascular fraction (SVF) cells was also evaluated using lineage-specific stains and spectrophotometry. ASC and NHF proliferation were equally inhibited by exposure to CY and ETO; however, when treated with MTX and VIN, ASCs exhibited greater resistance. This was especially apparent for MTX-treated samples, with ASC proliferation showing no inhibition for clinically relevant MTX doses ranging from 0.1 to 50 μM. Additional experiments revealed that the differentiation potential of ASCs was not affected by MTX treatment and that upregulation of dihydrofolate reductase possibly contributed to this response. Moreover, SVF cells, which include ASCs, exhibited similar resistance to MTX impairment, with respect to cellular proliferation, clonogenicity, and differentiation capability. Therefore, we have shown that the regenerative properties of ASCs resist the cytotoxicity of MTX, identifying these cells as a potential key for repairing musculoskeletal damage in patients undergoing chemotherapy.  相似文献   

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Adipose tissue is composed of lipid‐filled mature adipocytes and a heterogeneous stromal vascular fraction (SVF) population of cells. Similarly, the bone marrow (BM) is composed of multiple cell types including adipocytes, hematopoietic, osteoprogenitor, and stromal cells necessary to support hematopoiesis. Both adipose and BM contain a population of mesenchymal stromal/stem cells with the potential to differentiate into multiple lineages, including adipogenic, chondrogenic, and osteogenic cells, depending on the culture conditions. In this study we have shown that human adipose‐derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) populations display a common expression profile for many surface antigens, including CD29, CD49c, CD147, CD166, and HLA‐abc. Nevertheless, significant differences were noted in the expression of CD34 and its related protein, PODXL, CD36, CD 49f, CD106, and CD146. Furthermore, ASCs displayed more pronounced adipogenic differentiation capability relative to BMSC based on Oil Red staining (7‐fold vs. 2.85‐fold induction). In contrast, no difference between the stem cell types was detected for osteogenic differentiation based on Alizarin Red staining. Analysis by RT‐PCR demonstrated that both the ASC and BMSC differentiated adipocytes and osteoblast displayed a significant upregulation of lineage‐specific mRNAs relative to the undifferentiated cell populations; no significant differences in fold mRNA induction was noted between ASCs and BMSCs. In conclusion, these results demonstrate human ASCs and BMSCs display distinct immunophenotypes based on surface positivity and expression intensity as well as differences in adipogenic differentiation. The findings support the use of both human ASCs and BMSCs for clinical regenerative medicine. J. Cell. Physiol. 226: 843–851, 2011. © 2010 Wiley‐Liss, Inc.  相似文献   

19.
FGF-2对人骨髓间充质干细胞增殖和向成骨细胞分化的影响   总被引:4,自引:0,他引:4  
探讨体外培养条件下,成纤维细胞生长因子-2(FGF-2)和地塞米松(Dex)对第7代人骨髓间充质干细胞(MSCs)增殖和向成骨细胞分化的作用以及两者联合使用的效应。MSCs经含FGF-2或/和Dex的培养液作用后,于不同时间采用MTT法测定细胞增殖情况;对硝基苯磷酸(pNPP)法测定碱性磷酸酶(ALP)活性;ELISA法测定骨钙蛋白(OC)含量;茜素红S染色法对沉积的钙盐进行染色。发现:(1)FGF-2组细胞的生长速度为对照组的1.31倍,Dex/FGF-2组细胞的生长速度为FGF-2组的1.12倍。(2)Dex组的ALP活性、OC含量和细胞外基质钙盐沉积分别为对照组的17.0倍、2.12倍和10.56倍,并能形成成熟的羟基磷灰石(HA)结晶和骨结节;FGF-2组的ALP活性比对照组降低了76.7%,虽然OC含量、钙盐沉积增加,但不能形成成熟的HA结晶和骨结节;FGF-2对Dex诱导的ALP活性增加和HA结晶形成有拮抗作用。由此证明:(1)FGF-2可促进MSCs的增殖,Dex对MSCs的增殖无明显作用;Dex能增强FGF-2对MSCs的促增殖效应。(2)Dex可使MSCs分化为成熟的成骨细胞,是一个有效的成骨细胞分化诱导剂;FGF-2可使MSCs分化为未成熟的成骨细胞;FGF-2拮抗Dex诱导MSCs分化为成熟的成骨细胞。  相似文献   

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