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1.
Klotz B Mentrup B Regensburger M Zeck S Schneidereit J Schupp N Linden C Merz C Ebert R Jakob F 《PloS one》2012,7(1):e29959
1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in fibroblast growth factor-23 (Fgf23) and klotho deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, β-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence- and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to β-galactosidase staining and P16 expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging. 相似文献
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Peters R Wolf MJ van den Broek M Nuvolone M Dannenmann S Stieger B Rapold R Konrad D Rubin A Bertino JR Aguzzi A Heikenwalder M Knuth AK 《PloS one》2010,5(12):e15689
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3.
Multipotent mesenchymal stromal (stem) cells (MMSCs) play an important role in the structural and functional balance in tissues and reparative remodeling. The wide range of soluble mediators synthesized by cMMSCs includes molecules with immunomodulatory, antiapoptotic, proangiogenic, supportive chemoattractive, and other effects. Microenvironmental factors, such as cell-to-cell contacts, the connective tissue matrix, and partial oxygen pressure, can significantly affect the profile of paracrine mediators synthesized by MMSCs and, respectively, change their biological activity. This review analyzes the recent data on the paracrine activity of MMSCs and its modulation in hypoxia, which is specific for their physiological tissue niches. 相似文献
4.
Putative stem cells have been isolated from various tissue fluids such as synovial fluid, amniotic fluid, menstrual blood, etc. Recently the presence of nestin positive putative mammary stem cells has been reported in human breast milk. However, it is not clear whether they demonstrate multipotent nature. Since human breast milk is a non-invasive source of mammary stem cells, we were interested in examining the nature of these stem cells. In this pursuit, we could succeed in isolating and expanding a mesenchymal stem cell-like population from human breast milk. These cultured cells were examined by immunofluorescent labeling and found positive for mesenchymal stem cell surface markers CD44, CD29, SCA-1 and negative for CD33, CD34, CD45, CD73 confirming their identity as mesenchymal stem cells. Cytoskeletal protein marker analysis revealed that these cells expressed mesenchymal stem cells markers, namely, nestin, vimentin, smooth muscle actin and also manifests presence of E-Cadherin, an epithelial to mesenchymal transition marker in their early passages. Further we tested the multipotent differentiation potential of these cells and found that they can differentiate into adipogenic, chondrogenic and oesteogenic lineage under the influence of specific differentiation cocktails. This means that these mesenchymal stem cells isolated from human breast milk could potentially be “reprogrammed” to form many types of human tissues. The presence of multipotent stem cells in human milk suggests that breast milk could be an alternative source of stem cells for autologous stem cell therapy although the significance of these cells needs to be determined. 相似文献
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Pei-Min Chen Men-Luh Yen Ko-Jiunn Liu Huey-Kang Sytwu B-Linju Yen 《Journal of biomedical science》2011,18(1):49
In recent years, a large number of studies have contributed to our understanding of the immunomodulatory mechanisms used by
multipotent mesenchymal stem cells (MSCs). Initially isolated from the bone marrow (BM), MSCs have been found in many tissues
but the strong immunomodulatory properties are best studied in BM MSCs. The immunomodulatory effects of BM MSCs are wide,
extending to T lymphocytes and dendritic cells, and are therapeutically useful for treatment of immune-related diseases including
graft-versus-host disease as well as possibly autoimmune diseases. However, BM MSCs are very rare cells and require an invasive
procedure for procurement. Recently, MSCs have also been found in fetal-stage embryo-proper and extra-embryonic tissues, and
these human fetal MSCs (F-MSCs) have a higher proliferative profile, and are capable of multilineage differentiation as well
as exert strong immunomodulatory effects. As such, these F-MSCs can be viewed as alternative sources of MSCs. We review here
the current understanding of the mechanisms behind the immunomodulatory properties of BM MSCs and F-MSCs. An increase in our
understanding of MSC suppressor mechanisms will offer insights for prevalent clinical use of these versatile adult stem cells
in the near future. 相似文献
6.
Proof of the efficacy of cell therapy by numerous studies and clinical trials inevitably has raised the question of improving the regulatory framework that governs its use. Particular attention should be paid to the genetic safety of cell preparations. The immune, genetic, and pharmacological modification and expansion of cells in vitro can lead to an undesired effect, which not only has reduced the healing, recovery, and regulatory potential of cell therapy, but also increased the risk of accumulating genetically aberrant cells and the oncogenic transformation of cell preparations. The article has presented the estimation of the parameters of the genetic stability of cultured multipotent mesenchymal stromal cells (MSCs) derived from bone marrow and adipose tissue. The study was conducted using classic methods of genotoxicology, i.e., the individual cells gel-electrophoresis (comet assay) and the micronucleus test. We described a basic level of DNA damage and the frequency of micronucleus, identified genetically instable cultures, and conducted the comparison of genetic variability of MSCs isolated from different tissues. 相似文献
7.
Hypoxia (5% O2) and the basic fibroblast growth factor (bFGF) were shown to reduce the doubling time of bone marrow mesenchymal stem cells (MSCs) in vitro, indicating an increase in cell proliferation. In addition, abcg2 expression at both the mRNA and protein levels increased in MSCs that were exposed to hypoxia and bFGF. The two factors acting together potentiated the effects of hypoxia in MSCs. Blocking the functional activity of ABCG2 led to a higher production of reactive oxygen species (ROS) in MSCs. Hypoxia and bFGF were tested for effects on the MSC protein profile. These findings, combined with the published data, implicate ABCG2 expression and function in maintaining the viability and proliferative activity of MSCs that are cultured in hypoxia, with ABCG2 playing a protective role. 相似文献
8.
Jarnail Singh Anita Mann D. Kumar J. S. Duhan P. S. Yadav 《In vitro cellular & developmental biology. Animal》2013,49(6):408-416
Recent findings have demonstrated umbilical cord, previously considered as a biomedical waste, as a source of stem cells with promising therapeutic applications in human as well as livestock species. The present study was carried out to isolate the umbilical cord matrix cells and culture for a prolonged period, cryopreserve these cells and test their post-thaw viability, characterize these cells for expression of stem cell markers and differentiation potential in vitro. The intact umbilical cord was taken out of the amniotic sac of a fetus and then incised longitudinally to remove umbilical vessels. Wharton’s jelly containing tissue was diced into small pieces and placed in tiny drops of re-calcified buffalo plasma for establishing their primary culture. Confluent primary culture was trypsinized and passaged with a split ratio of 1:2 for multiplication of cells. Cryopreservation of cells was performed at three different passages in cryopreservation medium containing 15%, 20% and 25% fetal bovine serum (FBS). A significant increase in post-thaw viability was observed in cells cryopreserved in freezing medium with higher concentration of FBS. After re-culturing, frozen-thawed cells started adhering, and spike formation occurred within 4–6 h with similar morphology to their parent representative cultures. The normal karyotype and positive expression of alkaline phosphatase and pluripotency genes OCT4, NANOG and SOX2 were observed at different passages of culture. When induced, these cells differentiated into adipogenic and osteogenic cells as confirmed by oil red O and alizarin red stains, respectively. This study indicates that buffalo umbilical cord matrix cells have stemness properties with mesenchymal lineage restricted differentiation and limited proliferation potential in vitro. 相似文献
9.
目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。 相似文献
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Xinghui Song Yanwei Li Xiao Chen Guoli Yin Qiong Huang Yingying Chen Guowei Xu Linlin Wang 《Genetics and molecular biology》2014,37(1):127-134
In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering. 相似文献
13.
Juan Han Hironori Okada Hideki Takai Youhei Nakayama Takahide Maeda Yorimasa Ogata 《Journal of cellular biochemistry》2009,107(6):1198-1204
In this work, we examined the culture condition of alveolar bone marrow multipotent mesenchymal stromal cells (ABMMSCs), aiming to apply regenerative therapy to older periodontitis patients. To better understand the character of cultured cells from alveolar bone marrow, the expression profiles of well‐known genes and their responses to the induction of osteogenic, chondrogenic, or adipogenic differentiation were examined. Using αMEM‐based culture, ABMMSCs could be obtained from older individuals than in previous reports. Interestingly, ABMMSCs expressing Klf4 were able to differentiate into osteoblasts. The prediction of differentiation potential by Klf4 could be a useful guide for further improvement of the culture conditions required to culture ABMMSCs derived from older individuals. J. Cell. Biochem. 107: 1198–1204, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
14.
Yusuke Aida Hidemi Kurihara Koichi Kato 《In vitro cellular & developmental biology. Animal》2018,54(6):468-476
Cementum is a calcified, avascular connective tissue that laminates the root of a tooth and plays a pivotal role in the development, homeostasis, and regeneration of a periodontal tissue. As a potential treatment for periodontal tissue defects in the patient with chronic periodontitis, much attention has been paid to tissue engineering combined with mesenchymal stem cells for regenerating periodontal tissues including cementum. However, limited information is available for the molecular factors that have impacts on the differentiation of mesenchymal stem cells into cementoblasts. Here, we focus on the effect of Wnt3a as a potential inducer and tested the effect of this protein in vitro using human bone marrow-derived mesenchymal stem cells. It was found that, when cells were cultured in an osteogenic medium containing Wnt3a, cementoblast-specific genes, such as cementum protein 1 and cementum attachment protein, as well as bone-related genes were significantly upregulated. These results suggest that Wnt3a promotes differentiation of the cells into cementoblast-like cells. Further experiments were carried out using inhibitors to gain deeper insights into molecular mechanisms underlying the observed differentiation. As a result, we conclude that Wnt3a-triggered differentiation into cementoblast-like cells is the consequence of the activation of the canonical Wnt signaling pathway with possible involvement of the non-canonical pathway. 相似文献
15.
In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life. 相似文献
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Kajiume T Yuge L Kawahara Y Yoshimoto R Sasaki A Ide T Asashima M Kataoka K Kobayashi M 《FEBS letters》2007,581(24):4645-4650
In this study we examined the effect of the specific gravity of culture medium on the frequency of hematopoietic stem cell (HSC) maintenance. We used a newly developed high-specific-gravity media. Bone marrow cells were isolated and cultured, and HSC activity was evaluated. The number of hematopoietic progenitor/stem cells was markedly higher in the medium with high specific gravity. In high-specific-gravity media, cells did not precipitate, maintenance of HSCs was increased, and there was a concomitant accumulation of beta-catenin. This novel technique for maintaining HSC populations provides an important new tool for studies in regenerative medicine. 相似文献
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Eimear Kennedy Ciaran J. Mooney Roya Hakimjavadi Emma Fitzpatrick Shaunta Guha Laura E. Collins Christine E. Loscher David Morrow Eileen M. Redmond Paul A. Cahill 《Cell and tissue research》2014,358(1):203-216
Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α–actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10+, Sox17+) and a glia marker (S100β+). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-β1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro. 相似文献