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Shih-Chi Chao Gwo-Jang Wu Shu-Fu Huang Niann-Tzyy Dai Hsu-Kai Huang Mei-Fang Chou Yi-Ting Tsai Shiao-Pieng Lee Shih-Hurng Loh 《World journal of stem cells》2018,10(12):196-211
AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital(IRB No. B-106-09). Changes in the pH_i were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K~+/nigericin method. NH_4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power(β) was calculated from the ΔpH_i induced by perfusing different concentrations of(NH_4)_2SO_4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pH_i regulators and pluripotency markers.RESULTS In this study, our results indicated that(1) the steadystate pH_i value was found to be 7.5 ± 0.01(n = 20) and 7.68 ± 0.01(n =20) in HEPES and 5% CO_2/HCO_3~- buffered systems, respectively, which were much greater than that in normal adult cells(7.2);(2) in a CO_2/HCO_3~--buffered system, the values of total intracellular buffering power(β) can be described by the following equation: β_(tot) = 107.79(pH_i)~2-1522.2(pH_i) + 5396.9(correlation coefficient R~2 = 0.85), in the estimated pH_i range of 7.1- 8.0;(3) the Na~+/H~+ exchanger(NHE) and the Na~+/HCO_3~- cotransporter(NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively;(4) V-ATPase and some other unknown Na~+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1;(5) the Cl~-/OH~- exchanger(CHE) and the Cl~- /HCO_3 anion exchanger(AE) were found to be responsible for the weakening of intracellular proton loading;(6) besides the CHE and the AE, a Cl~--independent acid loading mechanism was functionally identified; and(7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pH i value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSION For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency. 相似文献
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Wang YC Nakagawa M Garitaonandia I Slavin I Altun G Lacharite RM Nazor KL Tran HT Lynch CL Leonardo TR Liu Y Peterson SE Laurent LC Yamanaka S Loring JF 《Cell research》2011,21(11):1551-1563
Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations. 相似文献
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Huan-Ting Lin Makoto Otsu Hiromitsu Nakauchi 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2013,368(1609)
In recent times, the epigenetic study of pluripotency based on cellular reprogramming techniques led to the creation of induced pluripotent stem cells. It has come to represent the forefront of a new wave of alternative therapeutic approaches in the field of stem cell therapy. Progress in drug development has saved countless lives, but there are numerous intractable diseases where curative treatment cannot be achieved through pharmacological intervention alone. Consequently, there has been an unfortunate rise in incidences of organ failures, degenerative disorders and cancers, hence novel therapeutic interventions are required. Stem cells have unique self-renewal and multilineage differentiation capabilities that could be harnessed for therapeutic purposes. Although a number of mature differentiated cells have been characterized in vitro, few have been demonstrated to function in a physiologically relevant context. Despite fervent levels of enthusiasm in the field, the reality is that other than the employment of haematopoietic stem cells, many other therapies have yet to be thoroughly proven for their therapeutic benefit and safety in application. This review shall focus on a discussion regarding the current status of stem cell therapy, the issues surrounding it and its future prospects with a general background on the regulatory networks underlying pluripotency. 相似文献
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Pluripotent stem cells are able to self-renew indefinitely and differentiate into all types of cells in the body. They can thus be an inexhaustible source for future cell transplantation therapy to treat degenerative diseases which currently have no cure. However, non-autologous cells will cause immune rejection. Induced pluripotent stem cell (iPSC) technology can convert somatic cells to the pluripotent state, and therefore offers a solution to this problem. Since the first generation of iPSCs, there has been an explosion of relevant research, from which we have learned much about the genetic networks and epigenetic landscape of pluripotency, as well as how to manipulate genes, epigenetics, and microRNAs to obtain iPSCs. In this review, we focus on the mechanism of cellular reprogramming and current methods to induce pluripotency. We also highlight new problems emerging from iPSCs. Better understanding of the fundamental mechanisms underlying pluripotenty and refining the methodology of iPSC generation will have a significant impact on future development of regenerative medicine. 相似文献
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S. L. Edwards J. C. Weakley A. W. Diamanduros J. B. Claiborne 《Journal of fish biology》2010,76(2):415-426
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Since mouse embryonic stem (ES) cells was first derived in 1981, the ability of this unprecedented cell type to self‐renew and differentiate without limit has revolutionized the discovery tools that are used to study gene functions and development. Furthermore, they have inspired others to hunt for similar cells from other species. The derivation of human ES cells in 1998 has accelerated these discoveries and has also widely provoked public interest, due to both the scientific significance of these cells for human tissue regeneration and the ethical disputes over the use of donated early human embryos. However, this is no longer a barrier, with the recent discovery of methods that can convert differentiated somatic cells into ES‐like cells or induced pluripotent stem (iPS) cells, by using defined reprogramming factors. This review attempts to summarize the progresses in the derivation of ES cells (as well as other embryo‐derived pluripotent cells) and iPS cells from various species. We will focus on the molecular and biological features of the cells, as well as the different determinants identified thus far to sustain their pluripotency. J. Cell. Biochem. 109: 16–25, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
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特定因子诱导多能干细胞 总被引：1，自引：0，他引：1
胚胎干细胞由于具有发育上的全能性,被认为是用于移植治疗的最佳来源。然而,由于人的胚胎干细胞直接运用引发免疫排斥以及触及伦理矛盾,人们一直在研发多能干细胞。2006年,多能干细胞的研究有了重大进展。首先,Yamanaka实验室构建用逆转录载体将候选因子导入成纤维细胞,而后检测多能性标志基因的表达。结果发现,四种因子Oct3/4、Sox2、c-Myc以及Klf4的组合产生了表达多能性标志基因才有的抗药性的克隆,意味着细胞获得了多能性。用这种方法筛选的细胞无论在形态和增殖分化能力方面均类似于干细胞,而且表达干细胞标志基因以及在体内外能向三个胚层的细胞类型分化,这种细胞被命名为诱导性多能干细胞(iPS细胞)。进一步,用更严格的筛选基因nanog得到的iPS能够嵌合到生殖系中。而后,运用改进的方法从人的成体成纤维细胞也可以得到iPS细胞。然而,这种方法得到的嵌合体小鼠存在肿瘤形成现象,可能是由于c-Myc逆转录病毒整合到了基因组。通过替代的方法,去掉c-Myc的iPS也能够获得。为了进一步降低肿瘤形成的几率,近来发展了一种不依赖于病毒的方法,用质粒载体作为介质。iPS进一步的研究热点在于安全性以及从更严格的医学角度提高诱导iPS的效率,其分子机理和相关的技术问题也有待解决和克服。 相似文献
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Eun Jin Son In Seok Moon Sung Huhn Kim Su Jin Kim Jae Young Choi 《Journal of cellular biochemistry》2009,107(5):965-972
Adequate regulation of endolymphatic pH is essential for maintaining inner ear function. The Na+–H+ exchanger (NHE) is a major determinant of intracellular pH (pHi), and facilitates Na+ and fluid absorption in various epithelia. We determined the functional and molecular expression of NHEs in cultured human endolymphatic sac (ES) epithelial cells and examined the effect of IFN‐γ on NHE function. Serial cultures of human ES epithelial cells were generated from tissue samples. The molecular expression of NHE1, ‐2, and ‐3 isoforms was determined by real‐time RT‐PCR. The functional activity of NHE isoforms was measured microfluorometrically using a pH‐sensitive fluorescent dye, 2′,7′‐bis(carbonylethyl)‐5(6)‐carboxyfluorescein (BCECF), and a NHE‐inhibitor, 3‐methylsulfonyl‐4‐piperidinobenzoyl guanidine methanesulfonate (HOE694). NHE1, ‐2, and ‐3 mRNAs were expressed in human ES epithelial cells. Functional activity of NHE1 and ‐2 was confirmed in the luminal membrane of ES epithelial cells by sequentially suppressing Na+‐dependent pHi recovery from intracellular acidification using different concentrations of HOE694. Treatment with IFN‐γ (50 nM for 24 h) suppressed mRNA expression of NHE1 and ‐2. IFN‐γ also suppressed functional activity of both NHE1 and ‐2 in the luminal membrane of ES epithelial cells. This study shows that NHEs are expressed in cultured human ES epithelial cells and that treatment with IFN‐γ suppresses the expression and functional activity of NHE1 and ‐2. J. Cell. Biochem. 107: 965–972, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
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哺乳动物的早期胚胎发育和干细胞多能性由转录因子构成的基因网络所调控。2003年,在胚胎干细胞中发现的重要转录因子NANOG位于基因网络调控中心,对胚胎第二次命运决定和基态多能性的建立至关重要。该文将在NANOG生物学特征的基础上,重点讨论其在早期胚胎发育、胚胎干细胞与诱导性多能干细胞中的功能。 相似文献
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Song Park Yonghun Sung Jain Jeong Minjee Choi Jinhee Lee Wookbong Kwon Soyoung Jang Si Jun Park Jae Young Kim Sung Hyun Kim Duhak Yoon Zae Young Ryoo Myoung Ok Kim 《Cell biochemistry and function》2017,35(7):392-400
Induced pluripotent stem (iPS) cells are important for clinical application and stem cell research. Although human melanoma‐associated antigen A2 (hMAGEA2) expression is known to affect differentiation in embryonic stem cells, its specific role in iPS cells remains unclear. To evaluate the function of hMAGEA2 and its characteristics in iPS cells, we produced hMAGEA2‐overexpressing iPS cells from hMAGEA2‐overexpressing transgenic mice. Although the iPS cells with overexpressed hMAGEA2 did not differ in morphology, their pluripotency, and self‐renewal related genes (Nanog, Oct3/4, Sox2, and Stat3), expression level was significantly upregulated. Moreover, hMAGEA2 contributed to the promotion of cell cycle progression, thereby accelerating cell proliferation. Through embryoid body formation in vitro and teratoma formation in vivo, we demonstrated that hMAGEA2 critically decreases the differentiation ability of iPS cells. These data indicate that hMAGEA2 intensifies the self‐renewal, pluripotency, and degree of proliferation of iPS cells, while significantly repressing their differentiation efficiency. Therefore, our findings prove that hMAGEA2 plays key roles in iPS cells. 相似文献
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Wobus AM 《BioEssays : news and reviews in molecular, cellular and developmental biology》2010,32(11):993-1002
Pluripotent stem cells have gained special attraction because of their almost unlimited proliferation and differentiation capacity in vitro. These properties substantiate the potential of pluripotent stem cells in basic research and regenerative medicine. Here three types of in vitro cultured pluripotent stem cells (embryonic carcinoma, embryonic stem and induced pluripotent stem cells) are compared in their historical context with respect to their different origin and properties. It became evident that tumourigenicity is an inherent property of pluripotent cells based on p53 down-regulation, expression of tumour-related genes and high telomerase activity that allow unlimited proliferation. In addition, culture-adapted genetic and epigenetic changes may induce tumourigenicity of pluripotent cells. The use of stem cells in regenerative medicine, however, requires non-malignant cell types and strategies that circumvent stages of malignancy.Reprogramming strategies of adult somatic cells that avoid the tumourigenic state of pluripotency may offer alternatives for future biomedical application. 相似文献
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Jianying Guo Dacheng Ma Rujin Huang Jia Ming Min Ye Kehkooi Kee Zhen Xie Jie Na 《蛋白质与细胞》2017,8(5):379-393
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Leqian Yu Yulei Wei Hai-Xi Sun Ahmed K. Mahdi Carlos A. Pinzon Arteaga Masahiro Sakurai Daniel A. Schmitz Canbin Zheng Emily D. Ballard Jie Li Noriko Tanaka Aoi Kohara Daiji Okamura Adrian A. Mutto Ying Gu Pablo J. Ross Jun Wu 《Cell Stem Cell》2021,28(3):550-567.e12
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Seung Bum Lee Sung-Hoon Han Min-Jung Kim Sehwan Shim Hye-Yun Shin Sun-Joo Lee 《Cell cycle (Georgetown, Tex.)》2017,16(21):2119-2127
Ionizing radiation causes not only targeted effects in cells that have been directly irradiated but also non-targeted effects in several cell generations after initial exposure. Recent studies suggest that radiation can enrich for a population of stem cells, derived from differentiated cells, through cellular reprogramming. Here, we elucidate the effect of irradiation on reprogramming, subjected to two different responses, using an induced pluripotent stem cell (iPSC) model. iPSCs were generated from non-irradiated cells, directly-irradiated cells, or cells subsequently generated after initial radiation exposure. We found that direct irradiation negatively affected iPSC induction in a dose-dependent manner. However, in the post-irradiated group, after five subsequent generations, cells became increasingly sensitive to the induction of reprogramming compared to that in non-irradiated cells as observed by an increased number of Tra1-81-stained colonies as well as enhanced alkaline phosphatase and Oct4 promoter activity. Comparative analysis, based on reducing the number of defined factors utilized for reprogramming, also revealed enhanced efficiency of iPSC generation in post-irradiated cells. Furthermore, the phenotypic acquisition of characteristics of pluripotent stem cells was observed in all resulting iPSC lines, as shown by morphology, the expression of pluripotent markers, DNA methylation patterns of pluripotency genes, a normal diploid karyotype, and teratoma formation. Overall, these results suggested that reprogramming capability might be differentially modulated by altered radiation-induced responses. Our findings provide that susceptibility to reprogramming in somatic cells might be improved by the delayed effects of non-targeted response, and contribute to a better understanding of the biological effects of radiation exposure. 相似文献
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《Cell Stem Cell》2022,29(5):744-759.e6
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Julie Cao Elizabeth S. Ng Don McNaughton Edouard G. Stanley Andrew G. Elefanty Mark J. Tobin Philip Heraud 《Journal of biophotonics》2014,7(10):767-781
Fourier transform infrared (FTIR) microspectroscopy was employed to elucidate the macromolecular phenotype of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and their differentiated progeny. Undifferentiated hESCs and hiPSC lines were found to be not clearly distinguishable from each other. However, although both hESC and hiPSC variants appeared to undergo similar changes during differentiation in terms of cell surface antigens, the derived cell types from all cell lines could be discriminated using FTIR spectroscopy. We foresee a possible future role for FTIR microspectroscopy as a powerful and objective investigative and quality control tool in regenerative medicine. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim) 相似文献