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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
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Yung‐Ching Chen King‐Chuen Wu Bu‐Miin Huang Edmund Cheung So Yang‐Kao Wang 《Journal of cellular and molecular medicine》2018,22(5):2896-2907
Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high‐density culture performed with TGF‐β‐driven chondrogenic induction medium. Treatment of the Midazolam dose‐dependently inhibited chondrogenesis, examined using Alcian blue‐stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor‐β‐induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam‐induced congenital malformations of the musculoskeletal system through PBR. 相似文献
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Guifang Gao Xiao‐Fei Zhang Karen Hubbell Xiaofeng Cui 《Biotechnology and bioengineering》2017,114(1):208-216
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Zhang X Hirai M Cantero S Ciubotariu R Dobrila L Hirsh A Igura K Satoh H Yokomi I Nishimura T Yamaguchi S Yoshimura K Rubinstein P Takahashi TA 《Journal of cellular biochemistry》2011,112(4):1206-1218
Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB‐MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper‐exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB‐MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB‐MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM‐MSC) and adipose tissue (AT‐MSC) as reference controls, we observed that CB‐MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 109 cells required for cell therapies. CB‐MSC showed karyotype stability after prolonged expansion. Functionally, CB‐MSC could be more readily induced to differentiate into chondrocytes than could BM‐MSC and AT‐MSC. CB‐MSC showed immunosuppressive activity equal to that of BM‐MSC and AT‐MSC. Collectively, our data indicate that viable CB‐MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system. J. Cell. Biochem. 112: 1206–1218, 2011. © 2011 Wiley‐Liss, Inc. 相似文献
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Chiu‐Mei Hsueh Hung‐Ming Lin Te‐Yu Tseng Yao‐De Huang Hsuan‐Shu Lee Chen‐Yuan Dong 《Journal of biophotonics》2019,12(2)
Second‐order susceptibility (SOS) microscopy is used to image and characterize chondrogenesis in cultured human mesenchymal stem cells. SOS analysis shows that the SOS tensor ratios can be used to characterize type I and II collagens in living tissues and that both collagen types are produced at the onset of chondrogenesis. Time‐lapse analysis shows a modulation of extracellular matrix results in a higher rate in increase of type II collagen, as compared to type I collagen. With time, type II collagen content stabilizes at the composition of 70% of total collagen content. SOS microscopy can be used to continuously and noninvasively monitor the production of collagens I and II. With additional development, this technique can be developed into an effective quality control tool for monitoring extracellular matrix production in engineered tissues. 相似文献
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Shirasawa S Sekiya I Sakaguchi Y Yagishita K Ichinose S Muneta T 《Journal of cellular biochemistry》2006,97(1):84-97
There are increasing reports that mesenchymal stem cells (MSCs) are present in various tissues other than bone marrow, including synovium. Here we investigated the optimal conditions for in vitro chondrogenesis of human synovium-derived MSCs and compared these cells with bone marrow-derived MSCs, especially in terms of their chondrogenesis potential. Synovium and bone marrow were harvested from six donors during knee operations for ligament injuries. Digested synovium cells or nucleated cells from bone marrow were expanded clonally. A pellet culture system was used for chondrogenesis, and the best combination of up to three cytokines of the seven assessed. Synovium-derived MSCs plated at a lower density expanded more rapidly. Contrary to previous reports, a combination of TGFbeta and dexamethasone was not sufficient to induce chondrogenesis. However, addition of BMP2 to TGFbeta and dexamethasone dramatically increased cartilage pellet size and the synthesis of cartilage matrix. The cartilage pellets were also analyzed by electron microscopy and immunohistology. DNA content per pellet decreased during chondrogenesis, indicating the pellet increased its size through the accumulation of newly synthesized extracellular matrix. Sequential chondrogenic gene expression was demonstrated by RT-PCR. Synovium-derived MSCs looked similar to the bone marrow-derived MSCs in their surface epitopes and proliferation potential; however, cartilage pellets from synovium were significantly larger than those from bone marrow in patient-matched comparisons. We demonstrated that the combination of TGFbeta, dexamethasone, and BMP2 was optimal for in vitro chondrogenesis of synovium-derived MSCs and that the synovium-derived MSCs have a greater chondrogenesis potential than bone marrow-derived MSCs. 相似文献
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This review describes the freeze-dried mesenchymal stem cells (MSCs) and their ability to restore damaged tissues and organs. An analysis of the literature shows that after the lyophilization MSCs retain >80% of paracrine factors and that the mechanism of their action on the restoration of damaged tissues and organs is similar to the mechanism of action of paracrine factors in fresh and cryopreserved mesenchymal stem cells. Based on the own materials, the use of paracrine factors of freeze-dried MSCs in vivo and in vitro for the treatment of various diseases of organs and tissues has shown to be effective. The study also discusses about the advantages and disadvantages of freeze-dried MSCs versus cryopreserved MSCs. However, for the effective use of freeze-dried MSCs in clinical practice, a more detailed study of the mechanism of interaction of paracrine factors of freeze-dried MSCs with target cells and tissues is required. It is also necessary to identify possible other specific paracrine factors of freeze-dried MSCs. In addition, develop new therapeutic strategies for the use of freeze-dried MSCs in regenerative medicine and tissue bioengineering. 相似文献
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目的:比较骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞3种间充质干细胞的成软骨分化潜能,为软骨组织工程中种子细胞的选择提供实验依据。方法:采用贴壁法分别分离提取兔骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞3种间充质干细胞,并进行传代培养,绘制3种间充质干细胞的生长曲线并比较其倍增时间。将3种间充质干细胞成软骨诱导14 d后,行甲苯胺蓝染色及II型胶原免疫组化染色以观测3种细胞成软骨分化能力。结果:脂肪间充质干细胞的倍增时间短于骨髓间充质干细胞,滑膜间充质干细胞的倍增时间最短;3种细胞成软骨诱导14 d后均产生糖胺聚糖和II型胶原,且组与组之间II型胶原表达水平的差异有统计学意义,骨髓间充质干细胞组高于脂肪间充质干细胞组(P0.01),滑膜间充质干细胞组高于骨髓间充质干细胞组(P0.01)。结论:在一定的培养条件下,3种间充质干细胞均有一定的成软骨细胞分化潜能,滑膜间充质干细胞最快的增殖速度及最强的成软骨分化潜能。 相似文献
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Schmitt B Ringe J Häupl T Notter M Manz R Burmester GR Sittinger M Kaps C 《Differentiation; research in biological diversity》2003,71(9-10):567-577
Human bone marrow-derived mesenchymal stem cells (MSCs) have been shown to differentiate into distinct mesenchymal tissues including bone and cartilage. The capacity of MSCs to replicate undifferentiated and to mature into cartilaginous tissues suggests these cells as an attractive cell source for cartilage tissue engineering. Here we show that the stimulation of human bone marrow-derived MSCs with recombinant bone morphogenetic protein-2 (BMP2) results in chondrogenic lineage development under serum-free conditions. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining of cartilage-specific type II collagen revealed the deposition of typical cartilage extracellular matrix components. Semi-quantitative real-time gene expression analysis of characteristic chondrocytic matrix genes, such as cartilage link protein, cartilage oligomeric matrix protein, aggrecan, and types I, II, and IX collagen, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with BMP2 and transforming growth factor-beta3 (TGFbeta3). Histologic staining of mineralized extracellular matrix with von Kossa, immunostaining of type X collagen (typical for hypertrophic chondrocytes), and gene expression analysis of osteocalcin and adipocyte-specific fatty acid binding protein (aP2) further documented that BMP2 induced chondrogenic lineage development and not osteogenesis and/or adipogenesis in human MSCs. These results suggest BMP2 as a promising candidate for tissue engineering approaches regenerating articular cartilage on the basis of mesenchymal progenitors from bone marrow. 相似文献
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《Cytotherapy》2014,16(6):776-788
Background aimsPrevious studies have demonstrated that porcine synovial membrane stem cells can adhere to a cartilage defect in vivo through the use of a tissue-engineered construct approach. To optimize this model, we wanted to compare effectiveness of tissue sources to determine whether porcine synovial fluid, synovial membrane, bone marrow and skin sources replicate our understanding of synovial fluid mesenchymal stromal cells or mesenchymal progenitor cells from humans both at the population level and the single-cell level. Synovial fluid clones were subsequently isolated and characterized to identify cells with a highly characterized optimal phenotype.MethodsThe chondrogenic, osteogenic and adipogenic potentials were assessed in vitro for skin, bone marrow, adipose, synovial fluid and synovial membrane–derived stem cells. Synovial fluid cells then underwent limiting dilution analysis to isolate single clonal populations. These clonal populations were assessed for proliferative and differentiation potential by use of standardized protocols.ResultsPorcine-derived cells demonstrated the same relationship between cell sources as that demonstrated previously for humans, suggesting that the pig may be an ideal preclinical animal model. Synovial fluid cells demonstrated the highest chondrogenic potential that was further characterized, demonstrating the existence of a unique clonal phenotype with enhanced chondrogenic potential.ConclusionsPorcine stem cells demonstrate characteristics similar to those in human-derived mesenchymal stromal cells from the same sources. Synovial fluid–derived stem cells contain an inherent phenotype that may be optimal for cartilage repair. This must be more fully investigated for future use in the in vivo tissue-engineered construct approach in this physiologically relevant preclinical porcine model. 相似文献
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白血病细胞中不同启动子驱动外源基因表达能力差异分析 总被引：2，自引：0，他引：2
为了分析不同启动子在白血病细胞中驱动外源基因表达能力的差异,文章选择了4种含不同启动子EF1α、PGK、Ubiquitin和CMV驱动的GFP报告基因的慢病毒载体,用以感染4种不同的白血病细胞株——NB4、HL60、Kasumi和THP1,利用荧光显微镜、流式细胞仪和荧光定量PCR的方法检测其GFP表达效率,发现EF1α启动子驱动GFP表达的能力最强,CMV最弱,PGK和Ubiquitin则介于两者之间。该结果提示在白血病细胞中研究基因功能时,应根据不同的研究需求选择最为合适的启动子。 相似文献
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Guillot PV De Bari C Dell'Accio F Kurata H Polak J Fisk NM 《Differentiation; research in biological diversity》2008,76(9):946-957
Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC. 相似文献
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