101 research outputs found
A Complex Role for FGF-2 in Self-Renewal, Survival, and Adhesion of Human Embryonic Stem Cells
The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor-2 (FGF-2), which activates FGF receptors (FGFRs) and stimulates the mitogen-activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin-like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF-2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF-2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation-related genes and downregulation of stem cell genes. Thus, exogenous FGF-2 reinforced the pluripotency maintenance program of intracrine FGF-2 signaling. Consistent with this hypothesis, expression of endogenous FGF-2 decreased during hESC differentiation and FGF-2 knockdown-induced hESC differentiation. In addition, FGF-2 signaling via FGFR2 activated MAPK kinase/extracellular signal-regulated kinase and AKT kinases, protected hESC from stress-induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self-renewal, cell survival, and adhesion by exogenous and endogenous FGF-2 may synergize to maintain the undifferentiated growth of hESCs. Stem Cells 2009;27:1847β185
Concise Review: The Evolution of human pluripotent stem cell culture: From feeder cells to synthetic coatings
Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells and embryonic stem cells, in an undifferentiated state typically depend on the support of feeder cells such as mouse embryonic fibroblasts (MEFs) or an extracellular matrix such as Matrigel. Culture conditions that depend on these undefined support systems limit our ability to interpret mechanistic studies aimed at resolving how hPSCs interact with their extracellular environment to remain in a unique undifferentiated state and to make fateβchanging lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel ultimately hinder our ability to use pluripotent stem cells to treat debilitating human diseases. Many of these obstacles have been overcome by the development of synthetic coatings and bioreactors that support hPSC expansion and selfβrenewal within defined culture conditions that are free from xenogeneic contamination. The establishment of defined culture conditions and synthetic matrices will facilitate studies to more precisely probe the molecular basis of pluripotent stem cell selfβrenewal and differentiation. When combined with threeβdimensional cultures in bioreactors, these systems will also enable largeβscale expansion for future clinical applications. S TEM C ells 2013;31:1β7Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94687/1/1260_ftp.pd
Zinc Maintains Embryonic Stem Cell Pluripotency and Multilineage Differentiation Potential via AKT Activation
[EN] Embryonic stem cells (ESCs) possess remarkable abilities, as they can differentiate into all cell types (pluripotency) and be self-renewing, giving rise to two identical cells. These characteristics make ESCs a powerful research tool in fundamental embryogenesis as well as candidates for use in regenerative medicine. Significant efforts have been devoted to developing protocols to control ESC fate, including soluble and complex cocktails of growth factors and small molecules seeking to activate/inhibit key signaling pathways for the maintenance of pluripotency states or activate differentiation. Here we describe a novel method for the effective maintenance of mouse ESCs, avoiding the supplementation of complex inhibitory cocktails or cytokines, e.g., LIF. We show that the addition of zinc to ESC cultures leads to a stable pluripotent state that shares biochemical, transcriptional and karyotypic features with the classical LIF treatment. We demonstrate for the first time that ESCs maintained in long-term cultures with added zinc, are capable of sustaining a stable ESCs pluripotent phenotype, as well as differentiating efficiently upon external stimulation. We show that zinc promotes long-term ESC self-renewal (>30 days) via activation of ZIP7 and AKT signaling pathways. Furthermore, the combination of zinc with LIF results in a synergistic effect that enhances LIF effects, increases AKT and STAT3 activity, promotes the expression of pluripotency regulators and avoids the expression of differentiation markers.PR acknowledges support from the Spanish Ministry of Science, Innovation and Universities (RTI2018-096794), and Fondo Europeo de Desarrollo Regional (FEDER). CIBER-BBN was an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. MS-S acknowledges support from the UK Engineering and Physical Sciences Research Council (EPSRC - EP/P001114/1).Mnatsakanyan, H.; Sabater I Serra, R.; SalmerΓ³n SΓ‘nchez, M.; Rico Tortosa, PM. (2019). Zinc Maintains Embryonic Stem Cell Pluripotency and Multilineage Differentiation Potential via AKT Activation. Frontiers in Cell and Developmental Biology. 7:1-17. https://doi.org/10.3389/fcell.2019.00180S1177Anzellotti, A. I., & Farrell, N. P. (2008). Zinc metalloproteins as medicinal targets. Chemical Society Reviews, 37(8), 1629. doi:10.1039/b617121bArmstrong, L., Hughes, O., Yung, S., Hyslop, L., Stewart, R., Wappler, I., β¦ Lako, M. (2006). The role of PI3K/AKT, MAPK/ERK and NFΞΊΞ² signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis. Human Molecular Genetics, 15(11), 1894-1913. doi:10.1093/hmg/ddl112Bechard, M., & Dalton, S. (2009). Subcellular Localization of Glycogen Synthase Kinase 3Ξ² Controls Embryonic Stem Cell Self-Renewal. Molecular and Cellular Biology, 29(8), 2092-2104. doi:10.1128/mcb.01405-08Ten Berge, D., Kurek, D., Blauwkamp, T., Koole, W., Maas, A., Eroglu, E., β¦ Nusse, R. (2011). Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells. Nature Cell Biology, 13(9), 1070-1075. doi:10.1038/ncb2314Cardone, M. H. (1998). Regulation of Cell Death Protease Caspase-9 by Phosphorylation. Science, 282(5392), 1318-1321. doi:10.1126/science.282.5392.1318Chen, S., Do, J. T., Zhang, Q., Yao, S., Yan, F., Peters, E. C., β¦ Ding, S. (2006). Self-renewal of embryonic stem cells by a small molecule. Proceedings of the National Academy of Sciences, 103(46), 17266-17271. doi:10.1073/pnas.0608156103Clevers, H. (2016). Modeling Development and Disease with Organoids. Cell, 165(7), 1586-1597. doi:10.1016/j.cell.2016.05.082Cohen, L., Sekler, I., & Hershfinkel, M. (2014). The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon. Cell Death & Disease, 5(6), e1307-e1307. doi:10.1038/cddis.2014.262Czechanski, A., Byers, C., Greenstein, I., Schrode, N., Donahue, L. R., Hadjantonakis, A.-K., & Reinholdt, L. G. (2014). Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains. Nature Protocols, 9(3), 559-574. doi:10.1038/nprot.2014.030Doble, B. W. (2003). GSK-3: tricks of the trade for a multi-tasking kinase. Journal of Cell Science, 116(7), 1175-1186. doi:10.1242/jcs.00384Eiselleova, L., Matulka, K., Kriz, V., Kunova, M., Schmidtova, Z., Neradil, J., β¦ Dvorak, P. (2009). A Complex Role for FGF-2 in Self-Renewal, Survival, and Adhesion of Human Embryonic Stem Cells. Stem Cells, 27(8), 1847-1857. doi:10.1002/stem.128Evans, M. (2011). Discovering pluripotency: 30 years of mouse embryonic stem cells. Nature Reviews Molecular Cell Biology, 12(10), 680-686. doi:10.1038/nrm3190Evseenko, D., Zhu, Y., Schenke-Layland, K., Kuo, J., Latour, B., Ge, S., β¦ Crooks, G. M. (2010). Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells. Proceedings of the National Academy of Sciences, 107(31), 13742-13747. doi:10.1073/pnas.1002077107Fang, X., Yu, S. X., Lu, Y., Bast, R. C., Woodgett, J. R., & Mills, G. B. (2000). Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A. Proceedings of the National Academy of Sciences, 97(22), 11960-11965. doi:10.1073/pnas.220413597Fischer, A. H., Jacobson, K. A., Rose, J., & Zeller, R. (2008). Hematoxylin and Eosin Staining of Tissue and Cell Sections. Cold Spring Harbor Protocols, 2008(6), pdb.prot4986-pdb.prot4986. doi:10.1101/pdb.prot4986Franke, T. F. (2008). PI3K/Akt: getting it right matters. Oncogene, 27(50), 6473-6488. doi:10.1038/onc.2008.313Hamatake, M., Iguchi, K., Hirano, K., & Ishida, R. (2000). Zinc Induces Mixed Types of Cell Death, Necrosis, and Apoptosis, in Molt-4 Cells. Journal of Biochemistry, 128(6), 933-939. doi:10.1093/oxfordjournals.jbchem.a022844Hamilton, W. B., & Brickman, J. M. (2014). Erk Signaling Suppresses Embryonic Stem Cell Self-Renewal to Specify Endoderm. Cell Reports, 9(6), 2056-2070. doi:10.1016/j.celrep.2014.11.032Heo, J., Lee, J.-S., Chu, I.-S., Takahama, Y., & Thorgeirsson, S. S. (2005). Spontaneous differentiation of mouse embryonic stem cells in vitro: Characterization by global gene expression profiles. Biochemical and Biophysical Research Communications, 332(4), 1061-1069. doi:10.1016/j.bbrc.2005.04.173Hogstrand, C., Kille, P., Nicholson, R. I., & Taylor, K. M. (2009). Zinc transporters and cancer: a potential role for ZIP7 as a hub for tyrosine kinase activation. Trends in Molecular Medicine, 15(3), 101-111. doi:10.1016/j.molmed.2009.01.004Hu, J., Yang, Z., Wang, J., Yu, J., Guo, J., Liu, S., β¦ Cheng, J. (2016). Zinc Chloride Transiently Maintains Mouse Embryonic Stem Cell Pluripotency by Activating Stat3 Signaling. PLOS ONE, 11(2), e0148994. doi:10.1371/journal.pone.0148994Huang, L., Kirschke, C. P., Zhang, Y., & Yu, Y. Y. (2005). The ZIP7 Gene (Slc39a7) Encodes a Zinc Transporter Involved in Zinc Homeostasis of the Golgi Apparatus. Journal of Biological Chemistry, 280(15), 15456-15463. doi:10.1074/jbc.m412188200Jaenisch, R., & Young, R. (2008). Stem Cells, the Molecular Circuitry of Pluripotency and Nuclear Reprogramming. Cell, 132(4), 567-582. doi:10.1016/j.cell.2008.01.015Jeong, C.-H., Cho, Y.-Y., Kim, M.-O., Kim, S.-H., Cho, E.-J., Lee, S.-Y., β¦ Dong, Z. (2010). Phosphorylation of Sox2 Cooperates in Reprogramming to Pluripotent Stem Cells. STEM CELLS, 28(12), 2141-2150. doi:10.1002/stem.540Jo, H., Mondal, S., Tan, D., Nagata, E., Takizawa, S., Sharma, A. K., β¦ Luo, H. R. (2012). Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death. Proceedings of the National Academy of Sciences, 109(26), 10581-10586. doi:10.1073/pnas.1202810109Johnson-Farley, N. N., Patel, K., Kim, D., & Cowen, D. S. (2007). Interaction of FGF-2 with IGF-1 and BDNF in stimulating Akt, ERK, and neuronal survival in hippocampal cultures. Brain Research, 1154, 40-49. doi:10.1016/j.brainres.2007.04.026Kattman, S. J., Witty, A. D., Gagliardi, M., Dubois, N. C., Niapour, M., Hotta, A., β¦ Keller, G. (2011). Stage-Specific Optimization of Activin/Nodal and BMP Signaling Promotes Cardiac Differentiation of Mouse and Human Pluripotent Stem Cell Lines. Cell Stem Cell, 8(2), 228-240. doi:10.1016/j.stem.2010.12.008Kennedy, S. G., Wagner, A. J., Conzen, S. D., Jordan, J., Bellacosa, A., Tsichlis, P. N., & Hay, N. (1997). The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes & Development, 11(6), 701-713. doi:10.1101/gad.11.6.701Kim, E., Kim, M., Woo, D.-H., Shin, Y., Shin, J., Chang, N., β¦ Lee, J. (2013). Phosphorylation of EZH2 Activates STAT3 Signaling via STAT3 Methylation and Promotes Tumorigenicity of Glioblastoma Stem-like Cells. Cancer Cell, 23(6), 839-852. doi:10.1016/j.ccr.2013.04.008Kunath, T., Saba-El-Leil, M. K., Almousailleakh, M., Wray, J., Meloche, S., & Smith, A. (2007). FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development, 134(16), 2895-2902. doi:10.1242/dev.02880Lanner, F., & Rossant, J. (2010). The role of FGF/Erk signaling in pluripotent cells. Development, 137(20), 3351-3360. doi:10.1242/dev.050146Li, W., & Ding, S. (2010). Small molecules that modulate embryonic stem cell fate and somatic cell reprogramming. Trends in Pharmacological Sciences, 31(1), 36-45. doi:10.1016/j.tips.2009.10.002Lin, Y., Yang, Y., Li, W., Chen, Q., Li, J., Pan, X., β¦ Wang, Y.-J. (2012). Reciprocal Regulation of Akt and Oct4 Promotes the Self-Renewal and Survival of Embryonal Carcinoma Cells. Molecular Cell, 48(4), 627-640. doi:10.1016/j.molcel.2012.08.030Llames, S., GarcΓa-PΓ©rez, E., Meana, Γ., Larcher, F., & del RΓo, M. (2015). Feeder Layer Cell Actions and Applications. Tissue Engineering Part B: Reviews, 21(4), 345-353. doi:10.1089/ten.teb.2014.0547Manning, B. D., & Cantley, L. C. (2007). AKT/PKB Signaling: Navigating Downstream. Cell, 129(7), 1261-1274. doi:10.1016/j.cell.2007.06.009Morgani, S. M., Canham, M. A., Nichols, J., Sharov, A. A., Migueles, R. P., Ko, M. S. H., & Brickman, J. M. (2013). Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions. Cell Reports, 3(6), 1945-1957. doi:10.1016/j.celrep.2013.04.034Murakami, M., & Hirano, T. (2008). Intracellular zinc homeostasis and zinc signaling. Cancer Science, 99(8), 1515-1522. doi:10.1111/j.1349-7006.2008.00854.xMurray, P., & Edgar, D. (2001). The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF). Differentiation, 68(4-5), 227-234. doi:10.1046/j.1432-0436.2001.680410.xNair, G., Abranches, E., Guedes, A. M. V., Henrique, D., & Raj, A. (2015). Heterogeneous lineage marker expression in naive embryonic stem cells is mostly due to spontaneous differentiation. Scientific Reports, 5(1). doi:10.1038/srep13339Niwa, H. (2007). How is pluripotency determined and maintained? Development, 134(4), 635-646. doi:10.1242/dev.02787Niwa, H., Burdon, T., Chambers, I., & Smith, A. (1998). Self-renewal of pluripotent embryonic stem cells is mediated via activation ofΒ STAT3. Genes & Development, 12(13), 2048-2060. doi:10.1101/gad.12.13.2048Ohtsuka, S., & Dalton, S. (2007). Molecular and biological properties of pluripotent embryonic stem cells. Gene Therapy, 15(2), 74-81. doi:10.1038/sj.gt.3303065Oshimori, N., & Fuchs, E. (2012). The Harmonies Played by TGF-Ξ² in Stem Cell Biology. Cell Stem Cell, 11(6), 751-764. doi:10.1016/j.stem.2012.11.001Pera, M. F., & Tam, P. P. L. (2010). Extrinsic regulation of pluripotent stem cells. Nature, 465(7299), 713-720. doi:10.1038/nature09228Rose-John, S. (2002). GP130 stimulation and the maintenance of stem cells. Trends in Biotechnology, 20(10), 417-419. doi:10.1016/s0167-7799(02)02056-5Saxe, J. P., Tomilin, A., SchΓΆler, H. R., Plath, K., & Huang, J. (2009). Post-Translational Regulation of Oct4 Transcriptional Activity. PLoS ONE, 4(2), e4467. doi:10.1371/journal.pone.0004467Schugar, R. C., Robbins, P. D., & Deasy, B. M. (2007). Small molecules in stem cell self-renewal and differentiation. Gene Therapy, 15(2), 126-135. doi:10.1038/sj.gt.3303062Shao, Y., Wolf, P. G., Guo, S., Guo, Y., Gaskins, H. R., & Zhang, B. (2017). Zinc enhances intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells. The Journal of Nutritional Biochemistry, 43, 18-26. doi:10.1016/j.jnutbio.2017.01.013Singhal, P. K., Sassi, S., Lan, L., Au, P., Halvorsen, S. C., Fukumura, D., β¦ Seed, B. (2015). Mouse embryonic fibroblasts exhibit extensive developmental and phenotypic diversity. Proceedings of the National Academy of Sciences, 113(1), 122-127. doi:10.1073/pnas.1522401112Stewart, M. H., Bendall, S. C., & Bhatia, M. (2008). Deconstructing human embryonic stem cell cultures: niche regulation of self-renewal and pluripotency. Journal of Molecular Medicine, 86(8), 875-886. doi:10.1007/s00109-008-0356-9Tamm, C., Pijuan GalitΓ³, S., & AnnerΓ©n, C. (2013). A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing. PLoS ONE, 8(12), e81156. doi:10.1371/journal.pone.0081156Taylor, K. M., Hiscox, S., Nicholson, R. I., Hogstrand, C., & Kille, P. (2012). Protein Kinase CK2 Triggers Cytosolic Zinc Signaling Pathways by Phosphorylation of Zinc Channel ZIP7. Science Signaling, 5(210), ra11-ra11. doi:10.1126/scisignal.2002585Taylor, K. M., Vichova, P., Jordan, N., Hiscox, S., Hendley, R., & Nicholson, R. I. (2008). ZIP7-Mediated Intracellular Zinc Transport Contributes to Aberrant Growth Factor Signaling in Antihormone-Resistant Breast Cancer Cells. Endocrinology, 149(10), 4912-4920. doi:10.1210/en.2008-0351Tremml, G., Singer, M., & Malavarca, R. (2008). Culture of Mouse Embryonic Stem Cells. Current Protocols in Stem Cell Biology, 5(1). doi:10.1002/9780470151808.sc01c04s5Wang, E. S., Reyes, N. A., Melton, C., Huskey, N. E., Momcilovic, O., Goga, A., β¦ Oakes, S. A. (2015). Fas-Activated Mitochondrial Apoptosis Culls Stalled Embryonic Stem Cells to Promote Differentiation. Current Biology, 25(23), 3110-3118. doi:10.1016/j.cub.2015.10.020Watanabe, S., Umehara, H., Murayama, K., Okabe, M., Kimura, T., & Nakano, T. (2006). Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells. Oncogene, 25(19), 2697-2707. doi:10.1038/sj.onc.1209307Weinberger, L., Ayyash, M., Novershtern, N., & Hanna, J. H. (2015). Dynamic stem cell states: naive to primed pluripotency in rodents and humans. doi:10.1101/030676West, J. A., Park, I.-H., Daley, G. Q., & Geijsen, N. (2006). In vitro generation of germ cells from murine embryonic stem cells. Nature Protocols, 1(4), 2026-2036. doi:10.1038/nprot.2006.303Wichterle, H., Lieberam, I., Porter, J. A., & Jessell, T. M. (2002). Directed Differentiation of Embryonic Stem Cells into Motor Neurons. Cell, 110(3), 385-397. doi:10.1016/s0092-8674(02)00835-8Xu, Z., Robitaille, A. M., Berndt, J. D., Davidson, K. C., Fischer, K. A., Mathieu, J., β¦ Moon, R. T. (2016). Wnt/Ξ²-catenin signaling promotes self-renewal and inhibits the primed state transition in naΓ―ve human embryonic stem cells. Proceedings of the National Academy of Sciences, 113(42), E6382-E6390. doi:10.1073/pnas.1613849113Yamaguchi, H., & Wang, H.-G. (2001). The protein kinase PKB/Akt regulates cell survival and apoptosis by inhibiting Bax conformational change. Oncogene, 20(53), 7779-7786. doi:10.1038/sj.onc.1204984Yamasaki, S., Sakata-Sogawa, K., Hasegawa, A., Suzuki, T., Kabu, K., Sato, E., β¦ Hirano, T. (2007). Zinc is a novel intracellular second messenger. Journal of Cell Biology, 177(4), 637-645. doi:10.1083/jcb.200702081Yiangou, L., Ross, A. D. B., Goh, K. J., & Vallier, L. (2018). Human Pluripotent Stem Cell-Derived Endoderm for Modeling Development and Clinical Applications. Cell Stem Cell, 22(4), 485-499. doi:10.1016/j.stem.2018.03.016Yu, J. S. L., & Cui, W. (2016). Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination. Development, 143(17), 3050-3060. doi:10.1242/dev.137075Zhou, J., Wulfkuhle, J., Zhang, H., Gu, P., Yang, Y., Deng, J., β¦ Zhang, Y. (2007). Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance. Proceedings of the National Academy of Sciences, 104(41), 16158-16163. doi:10.1073/pnas.0702596104Ziomek, C. A., Lepire, M. L., & Torres, I. (1990). A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity. Journal of Histochemistry & Cytochemistry, 38(3), 437-442. doi:10.1177/38.3.168934
Human Fetal Liver Stromal Cells That Overexpress bFGF Support Growth and Maintenance of Human Embryonic Stem Cells
In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells β bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2), and transforming growth factor Ξ² (TGF-Ξ²), thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically
Neural Stem Cells Achieve and Maintain Pluripotency without Feeder Cells
Background: Differentiated cells can be reprogrammed into pluripotency by transduction of four defined transcription factors. Induced pluripotent stem cells (iPS cells) are expected to be useful for regenerative medicine as well as basic research. Recently, the report showed that mouse embryonic fibroblasts (MEF) cells are not essential for reprogramming. However, in using fibroblasts as donor cells for reprogramming, individual fibroblasts that had failed to reprogram could function as feeder cells. Methodology/Principal Finding: Here, we show that adult mouse neural stem cells (NSCs), which are not functional feeder cells, can be reprogrammed into iPS cells using defined four factors (Oct4, Sox2, Klf4, and c-Myc) under feeder-free conditions. The iPS cells, generated from NSCs expressing the Oct4-GFP reporter gene, could proliferate for more than two months (passage 20). Generated and maintained without feeder cells, these iPS cells expressed pluripotency markers (Oct4 and Nanog), the promoter regions of Oct4 and Nanog were hypomethylated, could differentiated into to all three germ layers in vitro, and formed a germline chimera. These data indicate that NSCs can achieve and maintain pluripotency under feeder-free conditions. Conclusion/Significance: This study suggested that factors secreted by feeder cells are not essential in the initial/early stages of reprogramming and for pluripotency maintenance. This technology might be useful for a human system, as
FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self-Renewal
SummaryPrecise self-renewal of the germ cell lineage is fundamental to fertility and reproductive success. The early precursors for the germ lineage, primordial germ cells (PGCs), survive and proliferate in several embryonic locations during their migration to the embryonic gonad. By elucidating the active signaling pathways in migratory PGCs inΒ vivo, we were able to create culture conditions that recapitulate this embryonic germ cell environment. In defined medium conditions without feeder cells, the growth factors FGF2, insulin, and Activin A, signaling through their cognate-signaling pathways, were sufficient for self-renewal of germline-competent PGCs. Forced expression of constitutively active MEK1, AKT, and SMAD3 proteins could replace their respective upstream growth factors. Unexpectedly, we found that BMP4 could replace Activin A in non-clonal growth conditions. These defined medium conditions identify the key molecular pathways required for PGC self-renewal and will facilitate efforts in biobanking of chicken genetic resources and genome editing
The Role of RNA Polymerase II Contiguity and Long-Range Interactions in the Regulation of Gene Expression in Human Pluripotent Stem Cells
The eukaryotic nucleus is a highly complex structure that carries out multiple functions primarily needed for gene expression, and among them, transcription seems to be the most fundamental. Diverse approaches have demonstrated that transcription takes place at discrete sites known as transcription factories, wherein RNA polymerase II (RNAP II) is attached to the factory and immobilized while transcribing DNA. It has been proposed that transcription factories promote chromatin loop formation, creating long-range interactions in which relatively distant genes can be transcribed simultaneously. In this study, we examined long-range interactions between the POU5F1 gene and genes previously identified as being POU5F1 enhancer-interacting, namely, CDYL, TLE2, RARG, and MSX1 (all involved in transcriptional regulation), in human pluripotent stem cells (hPSCs) and their early differentiated counterparts. As a control gene, RUNX1 was used, which is expressed during hematopoietic differentiation and not associated with pluripotency. To reveal how these long-range interactions between POU5F1 and the selected genes change with the onset of differentiation and upon RNAP II inhibition, we performed three-dimensional fluorescence in situ hybridization (3D-FISH) followed by computational simulation analysis. Our analysis showed that the numbers of long-range interactions between specific genes decrease during differentiation, suggesting that the transcription of monitored genes is associated with pluripotency. In addition, we showed that upon inhibition of RNAP II, long-range associations do not disintegrate and remain constant. We also analyzed the distance distributions of these genes in the context of their positions in the nucleus and revealed that they tend to have similar patterns resembling normal distribution. Furthermore, we compared data created in vitro and in silico to assess the biological relevance of our results
Kinetic analysis of deviation from the undifferentiated state in colonies of human induced pluripotent stem cells on feeder layers
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