The Experts below are selected from a list of 5981706 Experts worldwide ranked by ideXlab platform
Lizhen Jiang - One of the best experts on this subject based on the ideXlab platform.
-
human amniotic epithelial cell feeder layers maintain ips cell pluripotency by inhibiting endogenous dna methyltransferase 1
Experimental and Therapeutic Medicine, 2013Co-Authors: Qing Chen, Lizhen Jiang, Yongyi Huang, Qin HuangAbstract:Maintaining induced pluripotent stem (iPS) cells in an undifferentiated, self-renewing state during long-term cultivation is, at present, a major challenge. We previously showed that human amniotic epithelial cells (HuAECs) were able to provide a good source of feeder cells for mouse and human embryonic or spermatogonial stem cells; however, the epigenetic mechanisms have not been elucidated. In the present study, mouse embryonic fibroblasts (MEFs) and HuAECs were compared as feeder layers for the long-term culture of human iPS cells. The HuAEC feeders allowed human iPS cells to maintain a high level of alkaline phosphatase (AP) activity and to express key stem cell markers during long-term subculture whereas the MEF feeders did not,. Moreover, the HuAEC feeders significantly affected the cell cycle regulation of the iPS cells, maintaining them in the resting stage and the early stage of DNA synthesis (G0/G1 stage). Furthermore, the CpG islands of the Nanog and Oct4 promoters were hypomethylated, while the Nanog- and Oct4-specific loci exhibited higher levels of histone H3 acetylation and lower levels of H3K27 trimethylation in iPS cells cultured on HuAECs compared with those cultured on MEFs. The DNA methyltransferase 1 (DNMT1) expression in iPS cells cultured on HuAECs was shown to be lower than in those cultured on MEFs. In addition, DNMT1-silenced human iPS cells were able to maintain pluripotency over long-term culture on MEFs. In combination, these results suggest that endogenous DNMT1 expression in human iPS cells may be regulated by HuAEC feeder cells and that Nanog and Oct4 are crucial components required for the maintenance of iPS cells in an undifferentiated, proliferative state, capable of self-renewal.
-
low microrna 199a expression in human amniotic epithelial cell feeder layers maintains human induced pluripotent stem cell pluripotency via increased leukemia inhibitory factor expression
Acta Biochimica et Biophysica Sinica, 2012Co-Authors: Qing Chen, Qin Huang, Yongyi Huang, Lizhen JiangAbstract:Human-induced pluripotent stem (iPS) cells share the same key properties as embryonic stem cells, and may be generated from patient- or disease-specific sources, which makes them attractive for personalized medicine, drug screens, or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state is a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, as they express endogenous leukemia inhibitory factor (LIF) at high levels. Here, we examined the effect of exogenous microRNA-199a regulation on endogenous LIF expression in HuAECs, and in turn on human iPS cell pluripotency. We found that HuAECs feeder cells transfected with microRNA-199a mutant expressed LIF at high levels, allowing iPS to maintain a high level of alkaline phosphatase activity in long-term culture and form teratomas in severe combined immunodeficient mice. The expression of stem cell markers was increased in iPS cultured on HuAECs feeder cells transfected with the microRNA-199a mutant, compared with iPS cultured on HuAECs transfected with microRNA-199a or mouse embryo fibroblasts. Taken together, these results suggested that LIF expression might be regulated by microRNA-199a, and LIF was a crucial component in feeder cells, and also was required for maintenance of human iPS cells in an undifferentiated, proliferative state capable of self-renewal.
-
human amniotic epithelial cell feeder layers maintain human ips cell pluripotency via inhibited endogenous microrna 145 and increased sox2 expression
Experimental Cell Research, 2012Co-Authors: Weiwei Cheng, Qin Huang, Yongyi Huang, Lizhen JiangAbstract:Abstract Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal.
-
high efficiency of reprogramming cd34 cells derived from human amniotic fluid into induced pluripotent stem cells with oct4
Stem Cells and Development, 2012Co-Authors: Te Liu, Gang Zou, Yongtao Gao, Xinrong Zhao, Hui Wang, Qin Huang, Lizhen Jiang, Lihe Guo, Weiwei ChengAbstract:Although many techniques can be used to generate multitype-induced pluripotent stem (iPS) cells from multitype seed cells, improving the efficiency and shortening the period of cell reprogramming remain troublesome issues. In this study, to generate iPS cells, CD34⁺ cells, isolated from human amniotic fluid cells (HuAFCs) by flow cytometry, were infected with retroviruses carrying only one reprogramming factor (Oct4) and cultured on human amniotic epithelial cell (HuAEC) feeder layers. Approximately 4 to 5 days after viral infection, some embryonic stem cell (ESC)-like colonies appeared among the feeder cells. These colonies were positive for alkaline phosphatase and expressed high levels of ESC pluripotent markers (Nanog, Sox2, Oct4, CD133, and Rex1). Moreover, these iPS cells exhibited high levels of telomerase activity and had normal karyotypes. Additionally, these cells could differentiate into cell types from all 3 germ layers in vivo and in teratomas. In summary, we report a novel way of iPS generation that uses CD34⁺ HuAFCs as seed cells. Using this method, we can generate human iPS cells with greater efficiency and safety (the oncogenic factors, c-Myc and Klf4, were not used), and using the minimum number of reprogramming factors (only one factor, Oct4). Besides, HuAECs were used as feeder layers to culture human iPS cells, which could not only avoid contamination with heterogeneous proteins, but also maintain iPS cells in a self-renewing and undifferentiated state for a long time.
Karl Tryggvason - One of the best experts on this subject based on the ideXlab platform.
-
long term self renewal of human pluripotent stem cells on human recombinant laminin 511
Nature Biotechnology, 2010Co-Authors: Sergey Rodin, Anna Domogatskaya, Susanne Strom, Emil M Hansson, Kenneth R Chien, Jose Inzunza, Outi Hovatta, Karl TryggvasonAbstract:We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on alpha6beta1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes.
-
long term self renewal of human pluripotent stem cells on human recombinant laminin 511
Nature Biotechnology, 2010Co-Authors: Sergey Rodin, Anna Domogatskaya, Susanne Strom, Emil M Hansson, Kenneth R Chien, Jose Inzunza, Outi Hovatta, Karl TryggvasonAbstract:We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on α6β1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes.
James A Thomson - One of the best experts on this subject based on the ideXlab platform.
-
Nanog and transcriptional networks in embryonic stem cell pluripotency
Cell Research, 2007Co-Authors: Guangjin Pan, James A ThomsonAbstract:Several extrinsic signals such as LIF, BMP and Wnt can support the self-renewal and pluripotency of embryonic stem (ES) cells through regulating the “pluripotent genes.” A unique homeobox transcription factor, Nanog, is one of the key downstream effectors of these signals. Elevated level of Nanog can maintain the mouse ES cell self-renewal independent of LIF and enable human ES cell growth without feeder cells. In addition to the external signal pathways, intrinsic transcription factors such as FoxD3, P53 and Oct4 are also involved in regulating the expression of Nanog. Functionally, Nanog works together with other key pluripotent factors such as Oct4 and Sox2 to control a set of target genes that have important functions in ES cell pluripotency. These key factors form a regulatory network to support or limit each other's expression level, which maintains the properties of ES cells.
-
basic fgf and suppression of bmp signaling sustain undifferentiated proliferation of human es cells
Nature Methods, 2005Co-Authors: Ruthann M Peck, James A Thomson, Xuezhu Feng, Tenneille LudwigAbstract:Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.
Weiwei Cheng - One of the best experts on this subject based on the ideXlab platform.
-
human amniotic epithelial cell feeder layers maintain human ips cell pluripotency via inhibited endogenous microrna 145 and increased sox2 expression
Experimental Cell Research, 2012Co-Authors: Weiwei Cheng, Qin Huang, Yongyi Huang, Lizhen JiangAbstract:Abstract Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal.
-
high efficiency of reprogramming cd34 cells derived from human amniotic fluid into induced pluripotent stem cells with oct4
Stem Cells and Development, 2012Co-Authors: Te Liu, Gang Zou, Yongtao Gao, Xinrong Zhao, Hui Wang, Qin Huang, Lizhen Jiang, Lihe Guo, Weiwei ChengAbstract:Although many techniques can be used to generate multitype-induced pluripotent stem (iPS) cells from multitype seed cells, improving the efficiency and shortening the period of cell reprogramming remain troublesome issues. In this study, to generate iPS cells, CD34⁺ cells, isolated from human amniotic fluid cells (HuAFCs) by flow cytometry, were infected with retroviruses carrying only one reprogramming factor (Oct4) and cultured on human amniotic epithelial cell (HuAEC) feeder layers. Approximately 4 to 5 days after viral infection, some embryonic stem cell (ESC)-like colonies appeared among the feeder cells. These colonies were positive for alkaline phosphatase and expressed high levels of ESC pluripotent markers (Nanog, Sox2, Oct4, CD133, and Rex1). Moreover, these iPS cells exhibited high levels of telomerase activity and had normal karyotypes. Additionally, these cells could differentiate into cell types from all 3 germ layers in vivo and in teratomas. In summary, we report a novel way of iPS generation that uses CD34⁺ HuAFCs as seed cells. Using this method, we can generate human iPS cells with greater efficiency and safety (the oncogenic factors, c-Myc and Klf4, were not used), and using the minimum number of reprogramming factors (only one factor, Oct4). Besides, HuAECs were used as feeder layers to culture human iPS cells, which could not only avoid contamination with heterogeneous proteins, but also maintain iPS cells in a self-renewing and undifferentiated state for a long time.
Qin Huang - One of the best experts on this subject based on the ideXlab platform.
-
human amniotic epithelial cell feeder layers maintain ips cell pluripotency by inhibiting endogenous dna methyltransferase 1
Experimental and Therapeutic Medicine, 2013Co-Authors: Qing Chen, Lizhen Jiang, Yongyi Huang, Qin HuangAbstract:Maintaining induced pluripotent stem (iPS) cells in an undifferentiated, self-renewing state during long-term cultivation is, at present, a major challenge. We previously showed that human amniotic epithelial cells (HuAECs) were able to provide a good source of feeder cells for mouse and human embryonic or spermatogonial stem cells; however, the epigenetic mechanisms have not been elucidated. In the present study, mouse embryonic fibroblasts (MEFs) and HuAECs were compared as feeder layers for the long-term culture of human iPS cells. The HuAEC feeders allowed human iPS cells to maintain a high level of alkaline phosphatase (AP) activity and to express key stem cell markers during long-term subculture whereas the MEF feeders did not,. Moreover, the HuAEC feeders significantly affected the cell cycle regulation of the iPS cells, maintaining them in the resting stage and the early stage of DNA synthesis (G0/G1 stage). Furthermore, the CpG islands of the Nanog and Oct4 promoters were hypomethylated, while the Nanog- and Oct4-specific loci exhibited higher levels of histone H3 acetylation and lower levels of H3K27 trimethylation in iPS cells cultured on HuAECs compared with those cultured on MEFs. The DNA methyltransferase 1 (DNMT1) expression in iPS cells cultured on HuAECs was shown to be lower than in those cultured on MEFs. In addition, DNMT1-silenced human iPS cells were able to maintain pluripotency over long-term culture on MEFs. In combination, these results suggest that endogenous DNMT1 expression in human iPS cells may be regulated by HuAEC feeder cells and that Nanog and Oct4 are crucial components required for the maintenance of iPS cells in an undifferentiated, proliferative state, capable of self-renewal.
-
low microrna 199a expression in human amniotic epithelial cell feeder layers maintains human induced pluripotent stem cell pluripotency via increased leukemia inhibitory factor expression
Acta Biochimica et Biophysica Sinica, 2012Co-Authors: Qing Chen, Qin Huang, Yongyi Huang, Lizhen JiangAbstract:Human-induced pluripotent stem (iPS) cells share the same key properties as embryonic stem cells, and may be generated from patient- or disease-specific sources, which makes them attractive for personalized medicine, drug screens, or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state is a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, as they express endogenous leukemia inhibitory factor (LIF) at high levels. Here, we examined the effect of exogenous microRNA-199a regulation on endogenous LIF expression in HuAECs, and in turn on human iPS cell pluripotency. We found that HuAECs feeder cells transfected with microRNA-199a mutant expressed LIF at high levels, allowing iPS to maintain a high level of alkaline phosphatase activity in long-term culture and form teratomas in severe combined immunodeficient mice. The expression of stem cell markers was increased in iPS cultured on HuAECs feeder cells transfected with the microRNA-199a mutant, compared with iPS cultured on HuAECs transfected with microRNA-199a or mouse embryo fibroblasts. Taken together, these results suggested that LIF expression might be regulated by microRNA-199a, and LIF was a crucial component in feeder cells, and also was required for maintenance of human iPS cells in an undifferentiated, proliferative state capable of self-renewal.
-
human amniotic epithelial cell feeder layers maintain human ips cell pluripotency via inhibited endogenous microrna 145 and increased sox2 expression
Experimental Cell Research, 2012Co-Authors: Weiwei Cheng, Qin Huang, Yongyi Huang, Lizhen JiangAbstract:Abstract Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal.
-
high efficiency of reprogramming cd34 cells derived from human amniotic fluid into induced pluripotent stem cells with oct4
Stem Cells and Development, 2012Co-Authors: Te Liu, Gang Zou, Yongtao Gao, Xinrong Zhao, Hui Wang, Qin Huang, Lizhen Jiang, Lihe Guo, Weiwei ChengAbstract:Although many techniques can be used to generate multitype-induced pluripotent stem (iPS) cells from multitype seed cells, improving the efficiency and shortening the period of cell reprogramming remain troublesome issues. In this study, to generate iPS cells, CD34⁺ cells, isolated from human amniotic fluid cells (HuAFCs) by flow cytometry, were infected with retroviruses carrying only one reprogramming factor (Oct4) and cultured on human amniotic epithelial cell (HuAEC) feeder layers. Approximately 4 to 5 days after viral infection, some embryonic stem cell (ESC)-like colonies appeared among the feeder cells. These colonies were positive for alkaline phosphatase and expressed high levels of ESC pluripotent markers (Nanog, Sox2, Oct4, CD133, and Rex1). Moreover, these iPS cells exhibited high levels of telomerase activity and had normal karyotypes. Additionally, these cells could differentiate into cell types from all 3 germ layers in vivo and in teratomas. In summary, we report a novel way of iPS generation that uses CD34⁺ HuAFCs as seed cells. Using this method, we can generate human iPS cells with greater efficiency and safety (the oncogenic factors, c-Myc and Klf4, were not used), and using the minimum number of reprogramming factors (only one factor, Oct4). Besides, HuAECs were used as feeder layers to culture human iPS cells, which could not only avoid contamination with heterogeneous proteins, but also maintain iPS cells in a self-renewing and undifferentiated state for a long time.