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Retinoic acid (RA), a metabolite of retinol, is one of the most biologically active forms of retinoid and plays vital roles in embryonic development and in the regulation of cell proliferation and differentiation. Knowing that liposomes simulate cell membranes and that hydrogel is an ideal delivery vehicle for topical medicine, liposome-hydrogel is a novel preparation that has synergistic advantages over each component separately. Our objective was to investigate the characteristics of RA liposome-hydrogel. For quality control of the RA-loaded liposomes, we measured their morphology, particle size, Zeta-potential, and entrapment efficiency. Then we determined the viscosity of RA liposome-hydrogel. Next, the diffusion through mouse skin was explored, followed by investigation of the mRNA expression levels of Ker18, REX1, and α-FP using Q-PCR. The results showed that RA liposome-hydrogel penetrates the mouse skin effectively. The permeation rates were: Qn (%) of RA liposome-hydrogel < Qn(%) of RA-loaded liposome < Qn (%) of RA. The mRNA expression levels were dose-dependent and the effective dose decreased between vehicles due to their different release rates. F9 mouse teratocarcinoma stem cells were an ideal model to explore the mechanism of RA liposome-hydrogel in stem cell differentiation.
O ácido retinóico (RA) é um metabolito de retinol. Ele também é uma das formas mais biologicamente ativas de retinóide. Desempenha papel vital no desenvolvimento embrionário e na regulação da proliferação e diferenciação celular. Sabendo-se que lipossomas simulam a membrana das células e que hidrogel é um sistema ideal para o medicamento tópico, o lipossoma-hidrogel é uma nova preparação, que apresenta vantagens sinérgicas em relação a cada um dos componentes separados. Nosso objetivo foi investigar as características de RA lipossoma-hidrogel. A fim de controlar a qualidade do lipossoma carregado com RA, medimos morfologia, tamanho das partículas, potencial zeta e eficiência de retenção. Em seguida, determinou-se a viscosidade de RA lipossoma-hidrogel. Em seguida, avaliou-se a sua difusão através da pele de camundongos, seguida da investigação dos níveis da expressão de mRNA de Ker18, REX e de α-FP, utilizando-se Q-PCR. Os resultados mostraram que RA lipossoma-hidrogel pode penetrar na pele do camundongo de forma eficaz. As taxas de permeação foram: Qn (%) de RA lipossoma-hidrogel<Qn(%) de lipossoma RA- carregado <Qn (%) de RA. Os níveis de expressão de mRNA foram dependentes de dose e a dose efetiva diminuiu entre os veículos devido às diferentes taxas de liberação, As células estaminais de teratocarcinoma F9 de camundongo mostraram-se como modelo ideal para explorar o mecanismo de diferenciaçãode células tronco pelo RA lipossoma-hidrogel.
Sujet(s)
Trétinoïne/analyse , Tératocarcinome , 33783/classification , Liposomes/classification , DiffusionRÉSUMÉ
BACKGROUND:As mesenchymal stem cells are commonly used as seed cells in studies of regenerative medicine and tissue engineering, the regulatory mechanism of their biological characteristics is a current research focus. OBJECTIVE:To summarize the regulations of Wnt signaling pathway on proliferation, senescence and differentiation of mesenchymal stem cells. METHODS:PubMed database and CNKI database were retrieved by computer using the key words of“mesenchymal stem cells, Wnt signaling pathway, proliferation, senescence, differentiation”in Chinese and English, respectively, between 2002 and 2014. Final y, 44 articles were included in result analysis. RESULTS AND CONCLUSION:Wnt signaling pathway is widely involved in the regulations of the biological characteristics of mesenchymal stem cells. Canonical Wnt signaling pathway reveals a bi-directional regulation effect on cellproliferation and osteogenic differentiation, and enhances senescence and neural differentiation, but inhibits adipogenic differentiation;non-canonical Wnt signaling pathway enhances senescence and osteogenic differentiation, and inhibits proliferation and adipogenic differentiation of mesenchymal stem cells, but it takes no part in neural differentiation of mesenchymal stem cells. So the regulations of Wnt signaling pathway on the biological characteristics of mesenchymal stem cells can be used as the new therapeutic targets of bone tissue engineering, nerve injury repair, and so on.
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BACKGROUND:Synovial mesenchymal stem cells have the ability of multilineage differentiation in vitro, which are expected to be seed cells for the treatment of cartilage defects in cartilage tissue engineering. Appropriate growth factors are critical for the chondrocyte differentiation of synovial mesenchymal stem cells. OBJECTIVE:To study the role of secreted factors by chondrocytes to induce chondrogenesis of synovial mesenchymal stem cells. METHODS:The synovial mesenchymal stem cells and chondrocytes were harvested from rat knee joints and cultured through the digestion method. The supernatant was col ected from chondrocytes, and centrifuged, filtered and cryopreserved. The third passage synovial mesenchymal stem cells centrifuged as pel ets were cultured in the chondrocyte supernatant for 21 days. And the cells morphology was examined and the type II col agen and aggrecan were detected through immunohistochemistry and RT-PCR. RESULTS AND CONCLUSION:The synovial mesenchymal stem cellpel ets cultured in the chondrocyte supernatant became cartilage-like tissue after 21 days. The type II col agen was detected positively in the matrix of synovial mesenchymal stem cellpel et immunohistochemical y. RT-PCR examination showed that the type II col agen and aggrecan expressed in the synovial mesenchymal stem cellpel et cultured in the chondrocyte supernatant. It suggested that synovial mesenchymal stem cellcould be induced to differentiate into chondrocytes depending on soluble factors secreted by chondrocytes.
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BACKGROUND:Traditional Chinese medicine shares wide application in clinical treatment because of its high security. At present, a lot of studies have been reported, in which, traditional Chinese medicine or monomers are used for in vitro differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells. OBJECTIVE:To summarize and analyze the research progress of Chinese medicine to induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells and to looking prospect for its application. METHODS:CNKI database was searched for the articles about Chinese medicine-induced differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, published from January 2001 to January 2013. The key words were“Chinese medicine, bone marrow mesenchymal stem cells, cardiomyocyte”in Chinese and“bone marrow mesenchymal stem cells, myocardial cells”in English. Obsolete or repetitive articles were excluded. Final y, 36 articles were included in result analysis. RESULTS AND CONCLUSION:Different Chinese medicine formulations were used as inducers to induce differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, including Panax notoginseng saponins, salvianolic acid B, Jiawei Danshen Yin, icari n, astragaloside. Then, the induced cells were examined by immunohistochemistry and reverse transcription-PCR. The results showed that the induced cells were positive for Desmin, cardiac troponin I and major histocompatibility complex. Positive cells were fusiform and fibroblast-like morphology, which indicates a role in promoting proliferation and differentiation. These findings show that Chinese medicine-induced differentiation of bone marrow mesenchymal stem cells into cardiomyocytes has become a worldwide research hotspot, providing a theoretical basis for clinical treatment of ischemic heart diseases.
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BACKGROUND:Bone marrow mesenchymal stem cells have potential to self-renewal and multi-lineage differentiation. But after a long period of culture in vitro, the proliferation and differentiation capacities of bone marrow mesenchymal stem cells gradual y loss, the mechanism underlying which is not clear now. OBJECTIVE:To observe the expression of autophagy-related gene Beclin-1 in differentiation from human bone marrow mesenchymal stem cells into neuron-like cells in vitro. METHODS:The changes of morphological characteristics of neuron-like cells differentiated from human bone marrow mesenchymal stem cells induced by epidermal growth factor were observed. The expression of neuron-specific enolase and glial fibril ary acidic protein in treated and untreated human bone marrow mesenchymal stem cells were detected using immunocytochemistry. The Beclin-1 protein expressions were detected by western blot before and after induction. RESULTS AND CONCLUSION:After being induced, human bone marrow mesenchymal stem cells presented classical neuron-like morphology;the expressions of neuron-specific enolase and glial fibril ary acidic protein were 78.7%and 8.1%, respectively. The expression of Beclin-1 protein was changed correspondingly during the induction, which increased after 30 minutes of induction and decreased gradual y after 1 hour of induction. Human bone marrow mesenchymal stem cells could be induced into neuron-like cells in vitro by epidermal growth factor. Autophagy-related gene was highly expressed in the induction of early differentiation and the expression gradual y reduced until it remained at a low level during the differentiation.
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OBJECTIVE:To investigate the paracrine effects of bone marrow mesenchymal stem cells on osteoblast biological function. METHODS:Bone marrow mesenchymal stem cells were isolated using standard Ficol-Paquedensity gradient centrifugation. Mesenchymal stem cellconditioned medium was prepared to cultivate osteoblasts, MG63. Proliferation of MG63 cells was analyzed by cellcounting kit-8. Migration of MG63 cells was analyzed by cellscratch method. Alkaline phosphatase activity of MG63 cells was analyzed by microplate test kit. Real-time PCR was performed to evaluate osteoblast differentiation markers, alkaline phosphatase, col agen type I and osteocalcin. Alizarin red staining was performed to evaluate osteoblast mineralization. RESULTS AND CONCLUSION:The cells were strongly positive for CD44, CD73 and CD90, but negative for CD34. MG63 cells cultured in the conditioned medium showed better proliferation and migration than those cultured in the Dulbecco’s modified Eagle’s medium. The activity and mRNA expression of alkaline phosphatase were much higher after induction of 4, 7 days (P<0.01). There was no significant difference in expression of col agen type I and osteocalcin after induction of 4 days, but they were significantly higher than those in the control group after induction of 7 days (P<0.05). Alizarin red staining showed that the number of calcium nodules was increased and the mineral apposition was enhanced after induction of 21 days with the conditioned medium. These findings suggest that the paracrine substance of bone marrow mesenchymal stem cells can significantly promote osteoblast proliferation, migration, differentiation and mineralization.
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BACKGROUND:Transforming growth factor-βhas been shown to exert an obvious induction effect on the differentiation of bone marrow mesenchymal stem cells into chondrocytes. Cyclical tensile strain simulates mechanical environment of chondrocytes in the body, and plays an important regulatory role in cellproliferation and differentiation. OBJECTIVE:To discuss the synergy of transforming growth factor-βand cyclical tensile strain in inducing the differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells. METHODS:A total of 10 2-month-old New Zealand rabbits were selected. Bone needle was used to penetrate the medul ary cavity of bone. 3.0-4.0 mL of bone marrow was extracted for isolation and culture of bone marrow mesenchymal stem cells. Passage 3 cells were randomly assigned to four groups:blank, transforming growth factor-β, cyclical tensile strain and cyclical tensile strain+transforming growth factor-βgroups. After 1, 3 and 6 days, cells were obtained. General morphology was observed using safranin O staining. Glycosaminoglycan levels were detected by alcian blue staining. Matrix metal oproteinase-13 and tissue inhibitor of metal oproteinase-1 levels in supernatant were measured using ELISA. Type II col agen, matrix metal oproteinase-13 and tissue inhibitor of metal oproteinase-1 mRNA relative expression was detected using RT-PCR. RESULTS AND CONCLUSION:Safranin O staining showed fusiform or irregular triangular cells. cellnumber and matrix secretion increased in each experimental group than in blank group. Glycosaminoglycan levels in the supernatant were greater in the transforming growth factor-βand cyclical tensile strain+transforming growth factor-βgroups than in the blank group (P<0.05). Type II col agen mRNA relative expression was higher in the cyclical tensile strain+transforming growth factor-βgroup than in the blank group (P<0.05). Results indicated that transforming growth factor-βand cyclical tensile strain could induce the differentiation of bone marrow mesenchymal stem cells into chondrocytes, showing an apparent cooperative action.
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BACKGROUND:Recent studies have found that stem cellpluripotency and differentiation is regulated by many long non-coding RNAs (LncRNAs). The expression and effect of LncRNA AK089560 during differentiation of stem cells is unclear. OBJECTIVE:To investigate the expression of LncRNA AK089560 in mesenchymal stem cells C3H10T1/2 undergoing osteogenic and adipogenic differentiation. METHODS:Osteogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by recombinant human bone morphogenetic protein-2 and evaluated using alkaline phosphatase staining. The adipogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by three factors (dexamethasone, indomethacin and insulin) and evaluated by oil red O staining. The dynamical expression of LncRNA AK089560 was detected by qRT-PCR assay. The AK089560 secondary structure was predicted using RNAfold software. The relationship between AK089560 and neighboring protein-coding genes was analyzed using UCSC genome browser and visualized by fancyGENE online software. RESULTS AND CONCLUSION:Over 70%of C3H10T1/2 cells were positive for alkaline phosphatase after osteogenic induction and more than 80%of the cells positive for oil red O staining after adipogenic induction. qRT-PCR results showed that the expression of LncRNA AK089560 at days 2, 4, 6 of both osteogenic and adipogenic differentiation was significantly decreased compared with the control group (P<0.05). Bioinformatics analysis showed that there was a stem-loop structure for AK089560 and sense overlap relationship between AK089560 and protein-encoding gene Sema3a. These findings indicate that LncRNA AK089560 expression is reduced during osteogenic differentiation and adipogenic differentiation, showing that AK089560 may be involved in regulating the multi-directional differentiation of stem cells.
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BACKGROUND:The discovery and concept of pulp tissue-derived stem cells is beneficial to the understanding of tooth development and regeneration and repair mechanisms from the cellular level. OBJECTIVE:To understand the induced differentiation capacity and induced conditions in vitro of human dental pulp stem cells into neuron-like cells. METHODS:Pulp tissue was separated from human healthy third molars. Single cellsuspensions were prepared and seeded into 6-wel plates containing alpha-modified minimum essential medium supplemented with 15%fetal bovine serum. Subconfluent cultures (first passage) of colony forming cells were induced with butylhydroxy anisole, forskolin,β-mercaptoethanol, basic fibroblast growth factor. RESULTS AND CONCLUSION:Immunofluorescence and reverse transcription-PCR assay showed that human dental pulp stem cells positively expressed stro-1, Col-I, dentin sialoprotein after 2 weeks of induction. Nestin and neuron-specific enolase were strongly expressed, but the gingival fibroblasts were negatively expressed. It indicates that adult stem cells in human dental pulp have a high neuron-like celldifferentiation potential under a certain inductive condition.
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BACKGROUND:Human amniotic membrane-derived mesenchymal stem cells (AMSCs) are considered to be one kind of adult stem cells that can be easily obtained in large quantities without using an invasive method. Because of their low immunogenicity, anti-inflammatory properties, multipotency of differentiation and without ethical issue, human amniotic membrane-derived mesenchymal stem cells have been proposed as a good candidate to be used in celltherapy and regenerative medicine. However, the biological properties and the differentiation capacity of human amniotic membrane-derived mesenchymal stem cells are stil poorly characterized. OBJECTIVE:To establish a practical method for isolation and purification of human amniotic membrane-derived mesenchymal stem cells, and to study the biological characteristics and dopaminergic neural-like celldifferentiation potential of the human amniotic membrane-derived mesenchymal stem cells. METHODS:Human amniotic membrane-derived mesenchymal stem cells were disassociated and isolated from the amniotic membrane by trypsin and col agenase based enzymic digestion, and purified by percol mediated density gradient centrifugation. Expressions of surface antigens and transcription factors of the human amniotic membrane-derived mesenchymal stem cells were determined by flow cytometry and western blot assays. Based on the osteogenic and adipogenic induction, the multipotent differentiation capability of human amniotic membrane-derived mesenchymal stem cells was determined. Induction of neural celldifferentiation of human amniotic membrane-derived mesenchymal stem cells was conducted in Neurabasal conditioning medium with ATRA supplement. Neural cellassociated bio-markers were determined by immunofluoresence staining and confocal microscope. RESULTS AND CONCLUSION:In this study, we performed a practical method to isolate and purify human amniotic membrane-derived mesenchymal stem cells and amniotic epithelial cells simultaneously, with high cells yield. We demonstrated a group of constitutive expressions of neural antigens and embryonic associated transcription factor proteins (OCT-4, SOX-2 and KLF4) in fresh isolated human amniotic membrane-derived mesenchymal stem cells as wel as in human amniotic membrane-derived mesenchymal stem cells after in vitro passage, which suggested that the human amniotic membrane-derived mesenchymal stem cells not only possessed intrinsic tendency to neural celldifferentiation, but also maintained their stem cellcharacteristics after in vitro passage. We stimulated the human amniotic membrane-derived mesenchymal stem cells in the neurobasal-A and B27 based conditioning medium to induce neural celldifferentiation. The induced human amniotic membrane-derived mesenchymal stem cells displayed an up-regulation of expression in panel of neural and dopaminergic associate molecules (β-tubulin III, neuron-specific nuclear protein, tyrosine hydroxylase, glial fibril ary acidic protein, myelin basic protein and nestin) by flow cytometry and immunofluorescence staining, which demonstrated the multipotent differentiation capability and dopaminergic neuron-like differentiation potential of the human amniotic membrane-derived mesenchymal stem cells.
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BACKGROUND:Studies have demonstrated that bone marrow mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells under certain condition, which provides a new idea for the treatment of end-sate liver diseases, such as acute hepatic failure. OBJECTIVE:To review the discovery, isolation and culture, induction and differentiation, and application prospects of bone marrow mesenchymal stem cells. METHODS:A computer-based online research of CNKI and PubMed databases was performed to col ect articles about bone marrow mesenchymal stem cells and their differentiation into hepatocyte-like cells published between 1999 and 2014. The key words were“hepatocyte-like cells, bone marrow mesenchymal stem cells, celldifferentiation”in Chinese and English. Final y, 52 articles were included in result analysis. RESULTS AND CONCLUSION:Currently, the most important issue in liver tissue engineering is to find a kind of seed cells with stable traits and liver-specific functions. Mature hepatocytes are difficult to be harvested, and immune rejection and difficulty in in vitro culture also severely limit the development of liver transplantation. Bone marrow mesenchymal stem cells characterized as multiple differentiation, rapid self-renewal, easy to be cultured and proliferated have been considered as the most promising source of cells. Increasing studies have shown that bone marrow mesenchymal stem cells can differentiate into liver cells in vitro and in vivo, and differentiated cells have the synthesis and secretion functions same as hepatocytes. But how to proliferate such a large number of cells, to maintain the good differentiation potential of cells, to optimize the culture conditions in vitro as wel as mechanisms of induction and differentiation mechanisms and clinical security need further studies.
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BACKGROUND:The biological function of human periodontal ligament stem cells is a hot area of research in the treatment of periodontal disease. Human periodontal ligament cells are one of the end cells derived from human periodontal ligament stem cells;meanwhile, it can also provide supports to the development of human periodontal ligament stem cells. However, few studies are reported about the difference of biological characteristics between human periodontal ligament stem cells and human periodontal ligament cells. OBJECTIVE:To compare the differences of biological characteristics between human periodontal ligament stem cells and human periodontal ligament cells. METHODS:The human periodontal ligament stem cells and human periodontal ligament cells were isolated and purified using tissue explant method and cellclone method, respectively, and then were observed under light microscope to compare the differences of morphology. cellproliferation curves of human periodontal ligament stem cells and human periodontal ligament cells were drawn respectively with cellcounting kit 8 assay. Flow cytometry analysis was used to detect their cellcircles and their surface markers expressions. The alkaline phosphatase gene, proliferating cellnuclear antigen gene and Scleraxis gene of human periodontal ligament stem cells and human periodontal ligament cells were detected by Real-time PCR assay.RESULTS AND CONCLUSION:The human periodontal ligament stem cells and human periodontal ligament cells showed a notable difference in morphology under the light microscope observation. During the first 5 days, the cellproliferation curve of human periodontal ligament stem cells was lower than that of human periodontal ligament cells, but 5 days later, the curve of human periodontal ligament stem cells was significantly higher than that of human periodontal ligament cells. The cellcircles of human periodontal ligament stem cells and human periodontal ligament cells were 41.1%and 23.9%, respectively. The surface markers of human periodontal ligament stem cells and human periodontal ligament cells were similar, but their expression rates had significant difference. The expressions of alkaline phosphatase gene, proliferating cellnuclear antigen gene and Scleraxis gene of human periodontal ligament stem cells were significantly higher than those of human periodontal ligament cells. The above results suggest that human periodontal ligament stem cells have much stronger potential ability than human periodontal ligament cells in osteogensis and cellproliferation.
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BACKGROUND:Bio-Gide col agen membranes show a good biocompatibility with stem cells. But the research on the osteogenetic differentiation of bone marrow mesenchymal stem cells cultured on the Bio-Gide col agen membranes is rarely reported. OBJECTIVE:To observe the effect of Bio-Gide col agen membranes on the proliferation and the osteogenetic differentiation of bone marrow mesenchymal stem cells. METHODS:Bone marrow mesenchymal stem cells from rabbits were isolated and cultured by using the whole bone marrow adherence method in vitro. Passage 3 bone marrow mesenchymal stem cells were selected and seeded on the Bio-Gide col agen membrane pretreated petri dish (experimental group) and simple petri dish (control group). The proliferation of bone marrow mesenchymal stem cells was detected by cellCounting Kit-8 at 1, 4, 7, 14 days. The supernatant of the cells cultured in osteogenic differentiation medium were col ected to detect the activity of alkaline phosphatase at 1, 4, 7, 14 days. RESULTS AND CONCLUSION:The number of bone marrow mesenchymal stem cells in the two groups was increased with the increasing time, and the control group had more cells than the experimental group at 7 days (P<0.05). There was no significant difference between the two groups at other time points. The alkaline phosphatase activity was increased with the increasing culture time, and the experimental group had a higher activity than the control group at 14 days (P<0.05). There was no significant difference between the two groups at other time points. Experimental findings indicate that Bio-Gide col agen membranes can promote the proliferation and the osteogenetic differentiation of bone marrow mesenchymal stem cells.
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BACKGROUND:Urothelial cells are important seeding cells for urinary tissue engineering, but they are difficult to proliferate in vitro. Several studies have shown that bone marrow mesenchymal stem cells can differentiate into urothelial cells, but how these cells functions in vivo in epithelium generation after implantation, and the application of these cells in tissue engineering, are rarely studied. OBJECTIVE:To explore the isolation and proliferation of rabbit bone marrow mesenchymal stem cells that are induced into urothelial cells in combination with rabbit bladder acellular matrix to construct tissue-engineered grafts, and to assess the effect of the induced cells as seeding cells. METHODS:Twelve 8-week-old male New Zealand white rabbits were chosen to obtain bone marrow samples through tibia puncture, and to isolate bone marrow mesenchymal stem cells by density gradient centrifugation. Then the fourth or fifth generation of bone marrow mesenchymal stem cells were cultured in conditioned medium for 2 weeks, and then identified by PCR and immunofluorescence. After that, the induced cells were seeded on rabbit bladder acellular matrix to construct tissue-engineered grafts for bladder repairing. Another 12 rabbits served as control group, and urothelial cells combined with bladder acellular matrix was used for bladder repairing. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells were successful y cultured and proliferated in vitro. After induction, PCR detection suggested that stem cellmarker (CD44) expression decreased, and epithelial cellmarker (UP1a) expression increased in the induced cells. Immunofluorescence staining demonstrated that the induced cells rather than bone marrow mesenchymal stem cells were positive for specific urothelial marker, UP1a. A stable continuous epithelial layer was observed on tissue-engineered grafts constructed by induced cells after 2 weeks, similar to the grafts built by urothelial cells. Induced bone marrow mesenchymal stem cells can differentiate into urothelial cells that can be used as seeding cells for urinary tissue engineering, which may be another choice out of urothelial cells.
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BACKGROUND:A variety of embryonic stem cells induction and differentiation systems have been established so far, while the research that promotes embryonic stem cells to differentiate into hematopoietic stem cells is stil at an initial stage, and the induction efficiency needs to be improved. OBJECTIVE:To active the Wnt/β-catenin signal pathway in mouse embryonic stem cells with exogenous win3a as an inducer, and then to observe whether the activation of this pathway wil promote the directional differentiation of embryonic stem cells into hematopoietic progenitor cells. METHODS:The ES-E14TG2a mouse stem cells were cultured with the exogenous wnt3a (100 μg/L) for 21 days, the content ofβ-catenin was tested by cellimmunofluorescence and western blot, and expression of Wnt downstream target gene was detected by quantitative reverse transcription PCR to determine the activation of Wnt/β-catenin signal pathway. Single-layer adherent culture method was used to induce the directional differentiate of above-mentioned cells into hematopoietic stem cells, and detection of hematopoietic development associated surface marker CD34+/Sca-1+was achieved by flow cytometry;meanwhile, the expression of hematopoietic associated gene was measured by quantitative reverse transcription PCR. RESULTS AND CONCLUSION:We found thatβ-catenin accumulated in ES-E14TG2a mouse stem cells after cultured with wnt3a (100 μg/L) for 21 days;the expressions of Wnt downstream target genes such as Pitx2, Frizzled, Sox17 and Oct4 showed the different degrees in increase, meaning the activation of Wnt/β-catenin signal pathway. Furthermore, during the time that we used single-layer adherent culture method to induce hematopoietic stem cells, the CD34+/Sca-1+cells accounted for 20.2%of total cells at day 14, and control cells only accounted for 11.9%. Again, expression quantity of hematopoietic associated gene BMP4, FLK2 and CD34 increased while Smad5 was suppressed significantly. Our data suggest that sustaining action by wnt3a wil active Wnt/β-catenin signal pathway, and also promote the directional differentiation of ES-E14TG2a mouse stem cells into hematopoietic progenitor cells.
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BACKGROUND:Adult stem cells are capable of proliferation and differentiation, which are an important part of tissue engineering. Although several studies have demonstrated that human adipose-derived stem cells obtained from elective liposuction procedures represent the characters of mesenchymal stem cells and can be induced to differentiate into myocardial cells, less is known about the characters of extrapericardial adipose-derived stem cells and their differentiation into myocardial cells. OBJECTIVE:To explore the culture of extrapericardial adipose-derived stem cells in vitro and their ability to differentiate into myocardial cells. METHODS:The extrapericardial adipose tissues were obtained and then isolated, cultured and passaged in vitro. The expressions of cellsurface specific antigens (CD44 and CD90) were detected by immunofluorescence assay at different time periods, and the fluorescence intensity was compared. After induced and cultured with myocardium induction medium for an optimal period,α-actin and cardiac troponin T expressions were detected by immunocytochemical detection and the phenotypic identifications were performed. RESULTS AND CONCLUSION:During the in vitro culture, extrapericardial adipose-derived stem cells adhered to culture flask wal and proliferated strongly. The cells appeared to be shuttle-shaped and exhibit a vortex alignment. They were positive for CD44 and CD90, and the fluorescence intensity reached the peak at passages 3 and 4, but negative for CD34 and CD54. After induced and cultured with myocardium induction medium, the differentiated cells were muscle-like cells, the expression ofα-actin and cardiac troponin T were positive, and the number of cells reached the peak at passages 3 and 4. These findings suggested that the extrapericardial adipose-derived stem cells from human represent the morphological and cellular immunological characters of mesenchymal stem cells, and they can be induced to differentiate into myocardial cells. In addition, there is the best induction period.
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BACKGROUND:Uric acid as an endogenous antioxidant has garnered increasing attentions because of its anti-oxidation, anti-DNA damage and neuroprotective effects. OBJECTIVE:To observe the effect of uric acid at different concentrations on the neural differentiation of bone marrow mesenchymal stem cells. METHODS:Bone marrow mesenchymal stem cells were isolated, purified and cultured in vitro. The morphology change was observed. The third passages of bone marrow mesenchymal stem cells were induced to differentiate to neuron-like cells by induced liquid containing four different concentrations of uric acid (0 mmol/L as control group, 0.2 mmol/L, 0.4 mmol/L, 0.8 mmol/L) for 24 hours. Then, after second intervention for 1 hour, cells were detected by Nissl staining and specific markers were detected by immunohistochemistry method. RESULTS AND CONCLUSION:After induction, the cellbody shrank, forming processes and connections. Nissl body was found in the cytoplasm. The positive rates of neuron-specific enolase were significantly higher in uric acid groups of different concentrations compared to the control group (P<0.05);moreover, the positive rates of neuron-specific enolase were increased as the increase in concentrations of uric acid (P<0.05). The positive rates of Nestin were decreased in uric acid groups of different concentrations compared to the control group (P<0.05). After 4 hours of induction, cells fel off significantly. In a certain period of time, uric acid can promote differentiation of bone marrow mesenchymal stem cells into neuron-like cells in a certain concentration-dependent manner in vitro.
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BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) can be induced to differentiate into neuron-like cells directional y. Accordingly, BMSCs can be used as seed cells theoretical y in constructing tissue-engineered peripheral nerves. OBJECTIVE:Using combination of two cytokines to induce BMSCs differentiating into neuron-like cells directional y, and further to discusse its application in peripheral nerve injury. METHODS:BMSCs were isolated and purificated from the bone marrow of Wistar rats by using the differential adherence method. Basic fibroblast growth factor and epidermal growth factor were used to induce the BMSCs differentiating into neuron-like cells. The morphological change was observed and the neuronal specific markers were detected by immunohistochemistry technique. The morphological and immunohistological changes were also studied after the induce agent were removed. RESULTS AND CONCLUSION:With presence of morphological and immunohistochemical features of nerve cells induced by neurotrophic factors, BMSCs exhibited two or more processes that were interconnected as a meshwork;cellnucleus and nucleus could be observed with strong light refraction of cytoplasm. After immunohistochemical staining, neuroln specific enolase, neurofilament protein and synaptophysin protein positive cells were detected. A great amount of cells reversed to their original fibroblast-like morphology, and the expression of the three above-mentioned proteins decreased as the induce agent withdrawn. Our study showed that BMSCs can be induced to differentiate into neuron-like cells, but the transdifferentiation is a short-time reversible phenomenon.
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BACKGROUND:There have been a large number of reports on establishing induced pluripotent stem celllines, but studies concerning large-scale in vitro induced differentiation of induced pluripotent stem cels into hematopoietic progenitor cels stil have a lack of in-depth discussion. OBJECTIVE:To develop methods to induce differentiation of induced pluripotent stem cels into hematopoietic progenitor cels in vitro. METHODS: Using the method of infection with lentivirus particles containing four transcriptionfactor genes, which are Oct4, Sox2, Nanog and Lin28, human skin fibroblasts are transduced into induced pluripotent stem cels. In the induced differentiation system, Y-27632, a kind of tyrosine kinase inhibitor-ROCK (p160-Rho-associated coiled-coil kinase), was added, which obviously suppressed apoptosis of cels. Based on conditioned medium with OP9 cels, a differentiation system of inducing induced pluripotent stem cels differentiating into hematopoietic progenitor cels was established. RESULTS AND CONCLUSION:(1) Apoptosis of induced pluripotent stem cels at the first three passages was very obvious, and the cels were difficult in a large-scale expansion. After Y-27632 was added, the apoptosis of embryonic stem cels was obviously inhibited. (2) During embryoid body differentiation, induced pluripotent stem cels cultured in OP9 conditional growth medium differentiated into hematopoietic progenitor celsin vitro that were positive for CD34.
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BACKGROUND:Our previous studies have demonstrated that melatonin inhibits the adipogenic differentiation of bone marrow mesenchymal stem cels. However, the mechanism underlying the effect of melatonin on adipogenesis is stil unknown. OBJECTIVE:To determine whether melatonin can inhibit the adipogenesis of bone marrow mesenchymal stem cels through retinoid-related orphan receptor α. METHODS:Bone marrow mesenchymal stem cels were isolated and purified by density gradient centrifugation combined with cellattachment culture. The phenotype was investigated by flow cytometry. Then, cels were induced for adipogenic differentiation with melatonin, CGP52608 and normal adipose tissue, respectively. The levels of retinoid-related orphan receptor α mRNA and protein were investigated by real-time RT-PCR and western blot assay, respectively. Further, the effect of retinoid-related orphan receptor α on the dipogenic differentiation of bone marrow mesenchymal stem cels was investigated by CGP52608. RESULTS AND CONCLUSION:The primary isolated bone marrow mesenchymal stem cels were spindle-shaped fibroblast-like cels. These cels did not express hematopoietic stem cels markers: CD34 and CD45; and highly expressed MSC markers: CD29, CD44, and CD10. The result of RT-PCR demonstrated that melatonin nuclear receptor, retinoid-related orphan receptor α, was highly expressed in bone marrow mesenchymal stem cels and the expression of retinoid-related orphan receptor α was further enhanced by melatonin in a dose-dependent manner, which was confirmed at protein level by western blot assay. During adiogenesis, the expression of retinoid-related orphan receptor αmRNA was up-regulated in the early stage, but maintained at a low level in the mild-later stage. While the retinoid-related orphan receptor α was activated by agonist CGP52608, the adipogenic differentiation of bone marrow mesenchymal stem cels was inhibited, which was similar to the inhibitory effect of melatonin. Therefore, melatonin inhibited the adipogenic differentiation of bone marrow mesenchymal stem cels through retinoid-related orphan receptor α, suggesting that melatonin plays an important role in the differentiation of adipocytes.