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1.
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   

2.
Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days’ culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days’ culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-β signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.  相似文献   

3.
Synovium is considered a candidate source of cells for cartilage tissue engineering. Compared with mesenchymal stem cells (MSCs) from other sources, synovium-derived stem cells (SDSCs) have a higher capacity for chondrogenic differentiation. Our objective was to define cocktails of growth factors that support the growth and chondrogenic differentiation of SDSCs in chemically defined medium. We established a fast and highly selective technique of negative isolation of SDSC populations. The individual and combined effects of three growth factors-transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (FGF-2)-were evaluated in serum-free pellet cultures of SDSCs for the chondrogenesis of SDSCs using histology, biochemical analysis, and real-time RT-PCR. In vitro studies identified TGF-beta1 as the key factor for both the growth and chondrogenesis of SDSCs. The highest rates of SDSC growth were observed with the synergistic interaction of all three factors. With respect to chondrogenic differentiation of SDSCs, the interaction of TGF-beta1 and IGF-I applied simultaneously was superior to the sequential application of these two factors or any other combination of growth factors studied. Based on these findings, we propose a two-step protocol for the derivation of chondrogenic SDSCs: a cocktail of TGF-beta1, IGF-I, and FGF-2 is applied first to induce cell growth followed by a cocktail of TGF-beta1 and IGF-I applied to induce chondrogenesis.  相似文献   

4.
Human adipose derived stem cells (hASCs) can be easily isolated and their plasticity has been well characterized. Several TGF-β superfamily ligands can direct hASCs towards chondrocytes. However, these ligands are difficult to purify and expensive. We have developed a library of Activin/BMP2 chimeric ligands (AB2 ligands) by systematically mixing their sequence segments and have tested their chondrogenic potential in hASCs. Cells cultured in monolayer or in a pellet culture system were incubated with a chemically defined medium supplemented with the chimeric ligands for 4 or 6 weeks and showed higher expression levels of type II collagen, aggrecan, and Sox9 mRNAs when compared with control and non-treated cells. Moreover, toluidine blue, alcian blue, and Masson's trichrome staining was markedly increased in treated cells, both in cell pellet and monolayer assays. In addition, immunohistochemical staining for detection of type I collagen, type II collagen, and Sox 9 demonstrated the acquisition of a chondrogenic phenotype in both culture systems. We present here an inexpensive and robust protocol for differentiation of hASCs towards chondrocytes in a reproducible and highly efficient manner. The AB2 ligands employed are easily produced and have properties that may become useful in cell therapy.  相似文献   

5.
Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.  相似文献   

6.
The differentiated phenotype of chondrocytes from hyaline cartilage is gradually lost during expansion in monolayers. Chondrocytes can reexpress their differentiated phenotype by transfer into an environment that prevents cell flattening, but serially passaged cells never completely recover their chondrogenic potential. We report that chondrocytes expanded (up to 2000-fold) in the presence of fibroblast growth factor 2 (FGF-2) dedifferentiated, but fully maintained their potential for redifferentiation in response to environmental changes. After seeding onto three-dimensional polymer scaffolds, chondrocytes expanded in the presence of FGF-2 formed cartilaginous tissue that was histologically and biochemically comparable to that obtained using primary chondrocytes, in contrast to chondrocytes expanded to the same degree but in the absence of FGF-2. The presence of FGF-2 inhibited the formation of thick F-actin structures, which otherwise formed during monolayer expansion, were maintained during tissue cultivation, and were associated with reduced ability of chondrocytes to reexpress their differentiated phenotype. This study provides evidence that FGF-2 maintains the chondrogenic potential during chondrocyte expansion in monolayers, possibly due to changes in the architecture of F-actin elements and allows more efficient utilization of harvested tissue for cartilage tissue engineering.  相似文献   

7.
8.
Adipose-derived stem cells (ASCs) are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue. Isolated ASCs are typically expanded in monolayer on standard tissue culture plastic with a basal medium containing 10% fetal bovine serum. However, recent data suggest that altering the monolayer expansion conditions by using suspension culture plastic, adding growth factors to the medium, or adjusting the seeding density may affect the self-renewal rate, multipotency, and lineage-specific differentiation potential of the ASCs. We hypothesized that variation in any of these expansion conditions would influence the chondrogenic potential of ASCs. ASCs were isolated from human liposuction waste tissue and expanded through two passages with different tissue culture plastic, feed medium, and cell seeding densities. Once expanded, the cells were cast in an agarose gel and subjected to identical chondrogenic culture conditions for 7 days, at which point cell viability, radiolabel incorporation, and gene expression were measured. High rates of matrix synthesis upon chondrogenic induction were mostly associated with smaller cells, as indicated by cell width and area on tissue culture plastic, and it appears that expansion in a growth factor supplemented medium is important in maintaining this morphology. All end-point measures were highly dependent on the specific monolayer culture conditions. These results support the hypothesis that monolayer culture conditions may "prime" the cells or predispose them towards a specific phenotype and thus underscore the importance of early culture conditions in determining the growth and differentiation potential of ASCs.  相似文献   

9.
Handorf AM  Li WJ 《PloS one》2011,6(7):e22887
Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG.  相似文献   

10.
Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. Results. hSDFs, hASCs and WI38 exhibited a similar spindle‐like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell‐associated gene expressions by performing real‐time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5‐fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell‐derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. Conclusions. These findings suggest that (i) so‐called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony‐forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.  相似文献   

11.
12.
Osteogenesis and the production of composite osteochondral tissues were investigated using human adult adipose‐derived stem cells and polyglycolic acid (PGA) mesh scaffolds under dynamic culture conditions. For osteogenesis, cells were expanded with or without osteoinduction factors and cultured in control or osteogenic medium for 2 weeks. Osteogenic medium enhanced osteopontin and osteocalcin gene expression when applied after but not during cell expansion. Osteogenesis was induced and mineralized deposits were present in tissues produced using PGA culture in osteogenic medium. For development of osteochondral constructs, scaffolds seeded with stem cells were precultured in either chondrogenic or osteogenic medium, sutured together, and cultured in dual‐chamber stirred bioreactors containing chondrogenic and osteogenic media in separate compartments. After 2 weeks, total collagen synthesis was 2.1‐fold greater in the chondroinduced sections of the composite tissues compared with the osteoinduced sections; differentiation markers for cartilage and bone were produced in both sections of the constructs. The results from the dual‐chamber bioreactor highlight the challenges associated with achieving simultaneous chondrogenic and osteogenic differentiation in tissue engineering applications using a single stem‐cell source. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013  相似文献   

13.

Introduction  

Infrapatellar fat pad (IPFP) is a possible source of stem cells for the repair of articular cartilage defects. In this study, adherent proliferative cells were isolated from digests of IPFP tissue. The effects of the expansion of these cells in fibroblast growth factor-2 (FGF-2) were tested on their proliferation, characterisation, and chondrogenic potential.  相似文献   

14.
We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 G93A-dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.  相似文献   

15.
Heat Shock Proteins (HSP) are molecular chaperones activated upon cellular stress/stimuli. HSP gene expression is regulated by Heat Shock Factors (HSF). We have recently demonstrated a functional role for heat shock factor-2 (HSF-2) in fibroblast growth factor-2 (FGF-2)-induced RANK ligand (RANKL), a critical osteoclastogenic factor expression on stromal/preosteoblast cells. In the present study, we show that FGF-2 treatment did not induce RANKL expression in HSF-2-/-stromal/preosteoblast cells. Interestingly, HSF-2 deficiency resulted in rapid induction of alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in these cells. Furthermore, FGF-2 did not induce osteoclast formation in co-culture of normal mouse spleen cells and HSF-2-/-stromal/preosteoblast cells. Electron microscopy analysis demonstrated that osteoclasts from HSF-2-/-mice have poorly developed ruffled borders. These data further confirm that HSF-2 plays an important role in FGF-2-induced RANKL expression in stromal/preosteoblast cells. HSF-2 deficiency has pleotropic effects on gene expression during osteoblast differentiation and osteoclastogenesis in the bone microenvironment. Novel therapeutic agents that modulate HSF-2 activation may have therapeutic utility against increased levels of FGF-2 and bone destruction associated with pathologic conditions.  相似文献   

16.
Fibroblast growth factor-2 (FGF-2) stimulates periodontal regeneration by a broad spectrum of effects on periodontal ligament (PDL) cells, such as proliferation, migration, and production of extracellular matrix. A critical factor in the success of periodontal regeneration is the rapid resolution of inflammatory responses in the tissue. We explored an anti-inflammatory effect of FGF-2 during periodontal regeneration and healing. We found that FGF-2 on mouse periodontal ligament cells (MPDL22) markedly downregulated CD40 expression, a key player of inflammation. In addition, FGF-2 inhibited CD40 signaling by the non-canonical nuclear factor-kappa B2 (NFκB2) pathway, resulting in decreased production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which have the potential to recruit immune cells to inflamed sites. Furthermore, in vivo treatment of FGF-2 enhanced healing of skin wounds by counteracting the CD40-mediated inflammation. These results reveal that FGF-2 has an important function as a negative regulator of inflammation during periodontal regeneration and healing.  相似文献   

17.
Adult stem cells are becoming the best option for regenerative medicine because they have low tumourigenic potential and permit autologous transplantation, even without in vitro culture. Our objectives were to evaluate the effects of exogenous nucleosides on the proliferation of hASCs (human adipose‐derived stem cells), with or without co‐treatment with 5‐aza (5‐azacytidine), and to analyse the expression of lamin A/C during cardiomyocyte differentiation of these cells. We isolated hASCs from human lipoaspirates that were positive for mesenchymal stem cell markers. We found that 5‐aza induces a dose‐dependent inhibition of hASC proliferation [IC50 (inhibitory concentration 50): 5.37 μM], whereas exogenous nucleosides significantly promote the proliferation of hASCs and partially revert the antiproliferative effect of the drug. Multipotentiality of isolated hASCs was confirmed by adipogenic, osteogenic and cardiomyogenic induction. 5‐Aza‐induced cells expressed cardiac troponins I and T and myosin light chain 2, myocardial markers that were directly correlated with lamin A/C expression. Our results support the importance of the nucleoside supplementation of media to improve conditions for the expansion and maintenance of hASCs in culture. In addition, the quantification of lamin A/C expression appears to be a good marker for the characterization of cardiomyocyte differentiation of stem cells that has rarely been used.  相似文献   

18.
Adult human articular chondrocytes were expanded in a medium with 10% serum (CTR) or further supplemented with different mitogens (i.e., EGF, PDGFbb, FGF-2, TGF beta 1, or FGF-2/TGF beta 1). Cells were then induced to redifferentiate in 3D pellets using serum-supplemented medium (SSM), serum-free medium (SFM), or SFM supplemented with factors inducing differentiation of chondroprogenitor cells (i.e., TGF beta 1 and/or dexamethasone). All factors tested during expansion enhanced chondrocyte proliferation and dedifferentiation, as assessed by the mRNA ratios of collagen type II to type I (CII/CI) and aggrecan to versican (Agg/Ver), using real-time PCR. FGF-2/TGF beta 1-expanded chondrocytes displayed the lowest doubling times, CII/CI and Agg/Ver ratios, averaging, respectively, 50, 0.2 and 15% of CTR-expanded cells. Redifferentiation in pellets was more efficient in SFM than SSM only for EGF-, PDGFbb- or FGF-2-expanded chondrocytes. Upon supplementation of SFM with TGF beta and dexamethasone (SFM TD), CII/CI ratios decreased 4.4-fold for EGF- and PDGFbb-expanded chondrocytes, but increased 96-fold for FGF-2/TGF beta 1-expanded cells. Chondrocytes expanded with FGF-2/TGF beta 1 and redifferentiated in SFM TD expressed the largest mRNA amounts of CII and aggrecan and generated cartilaginous tissues with the highest accumulation of glycosaminoglycans and collagen type II. Our results provide evidence that growth factors during chondrocyte expansion not only influence cell proliferation and differentiation, but also the cell potential to redifferentiate and respond to regulatory molecules upon transfer into a 3D environment.  相似文献   

19.
20.
Recent advances in hematopoietic stem cells (HSCs) expansion by growth factors including angiopoietin-like proteins (Angptls) have opened up the possibility to use HSCs in regenerative medicine. However, the unavailability of true in vitro HSCs expansion by these growth factors has limited the understanding of the cellular and molecular mechanism of HSCs expansion. Here, we report the functional role of mouse Angptls 1, 2, 3, 4, 6 and 7 and growth factors SCF, TPO, IGF-2 and FGF-1 on purified mouse bone-marrow (BM) Lineage-Sca-1+(Lin-Sca-1+) HSCs. The recombinant retroviral transduced- CHO-S cells that secrete Angptls in serum-free medium were used alone or in combination with growth factors (SCF, TPO, IGF-2 and FGF-1). None of the Angptls stimulated HSC proliferation, enhanced or inhibited HSCs colony formation, but they did support the survival of HSCs. By contrast, any of the six Angptls together with saturating levels of growth factors dramatically stimulated a 3- to 4.5-fold net expansion of HSCs compared to stimulation with a combination of those growth factors alone. These findings lead to an understanding of the basic function of Angptls on signaling pathways for the survival as well as expansion of HSCs in the bone marrow niche.  相似文献   

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