Rational optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells with ultra-high efficiency and fast kinetics

Chen, Jiekai; Liu, Jing; Chen, You; Yang, Jiaqi; Chen, Jing; He, Li; Zhao, Xiangjie; Mo, Kunlun; Sui, Hong; Guo, Lin; Chu, Shilong; Wang, Deping; Ding, Ke; Pei, Duanqing

doi:10.1038/cr.2011.51

Cited by 85 publications

(96 citation statements)

References 49 publications

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“…Equally, naive stem cells from nonrodent models could revolutionize mammalian transgenics in a similar fashion to classically grown ESCs and the knockout mouse, allowing complex genetic modification techniques to be applied to a wide range of mammalian systems, including those more effectively modeling human physiology. Both vitamins A and C are known to improve the efficiency at which iPSCs can be generated from differentiated cells [37,60,61], and when cosupplemented, display additive and enhanced synergistic effects upon demethylation and reprogramming, respectively [37]. As such, it is likely that pluripotent cell media formulations developed in the future for regenerative medicine and mammalian transgenics will include optimized amounts of both vitamins.…”

Section: Vitamins a And C In Regenerative Medicine And Mammalian Transgenmentioning

confidence: 99%

Modulating Epigenetic Memory through Vitamins and TET: Implications for Regenerative Medicine and Cancer Treatment

Hore

2017

Epigenomics

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Vitamins A and C represent unrelated sets of small molecules that are essential to the human diet and have recently been shown to intensify erasure of epigenetic memory in naive embryonic stem cells. These effects are driven by complementary enhancement of the ten-eleven translocation (TET) demethylases -vitamin A stimulates TET expression, whereas vitamin C potentiates TET catalytic activity. Vitamin A and C cosupplementation synergistically enhances reprogramming of differentiated cells to the naive state, but overuse may exaggerate instability of imprinted genes. As such, optimizing their use in culture media will be important for regenerative medicine and mammalian transgenics. In addition, mechanistic perception of how these vitamins interact with the epigenome may be relevant for understanding cancer and improving patient treatment. Figure 1A), and both are associated with a long recorded history. Nightblindness, an early symptom of vitamin A deficiency, and its cure through liver consumption, was known to Egyptians in the prehistoric period [1]. Equally, the debilitating effects of scurvy (vitamin C deficiency) among sailors, and its treatment with oranges, was registered as far back as 1498 [2]. Vitamins A and C also have a lengthy and rich history in scientific literature; in 1753 James Lind described the first controlled clinical trial where various antiscorbutic treatments were tested on British seamen and in 1929, Frederick Gowland Hopkins was co-awarded the Nobel Prize in Medicine for the 'discovery of vitamins' following milk supplementation experiments in vitamin A deficient mice [3]. Eventual isolation of the respective compounds underlying both vitamins led to Nobel Prizes in Chemistry and Medicine in 1937 [4]. Since this time, a considerable amount of scientific progress (and at times conjecture) has been associated with vitamins A and C, and their respective biological effects touch disciplines as divergent as development and dermatology.One of the most recently discovered fields that vitamins A and C impact upon is epigenetics [5][6][7]. New research has shown that both vitamins drive active removal of DNA methylation in embryonic stem cells (ESCs) by enhancing the same family of ten-eleven translocation (TET) enzymes, and in doing so, help erase DNA methylation and the epigenetic memory encoded by it to improve the reprogramming of differentiated cells to an embryonic-like state. Here I review the effects of vitamins A and C on DNA methylation and epigenetic memory in stem cells, and outline their significance for the fields For reprint orders, please contact: reprints@futuremedicine.com

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Section: Vitamins a And C In Regenerative Medicine And Mammalian Transgenmentioning

confidence: 99%

Modulating Epigenetic Memory through Vitamins and TET: Implications for Regenerative Medicine and Cancer Treatment

Hore

2017

Epigenomics

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“…Furthermore, addition of small molecule inhibitors of both Gsk-3 and mitogen-activated protein kinase (ERK) (termed 2i cocktail) to culture media is sufficient to maintain pluripotency of mouse ESCs (16). Finally, the addition of Gsk-3 inhibitors have enhanced the efficiency of induced pluripotent stem cell (iPSC) derivation (17,18) and facilitated the derivation of ESCs from different strains of mice (19,20) and from different species (21,22). The mechanism by which Gsk-3 inhibition or loss-of-function promotes pluripotency is not completely resolved (20,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).…”

mentioning

confidence: 99%

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

Faulds

Egelston

Sedivy

et al. 2018

Journal of Biological Chemistry

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Glycogen synthase kinase-3 (Gsk-3) activity regulates multiple signal transduction pathways, and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of Gsk-3α and Gsk-3β or inhibition of Gsk-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for Gsk-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (m 6 A). Here, we describe an intersection between these components -the regulation of m 6 A by Gsk-3. We find that protein levels for the RNA demethylase, FTO (fat mass and obesity-associated protein), are elevated in Gsk-3α;Gsk-3β-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by Gsk-3, and mass spectrometry identified the sites on FTO that are phosphorylated in a Gsk-3-dependent fashion. Gsk-3 phosphorylation of FTO leads to polyubiquitination, but in Gsk-3 knockout ESCs, that process is impaired, resulting in elevated levels of FTO protein. As a consequence of altered FTO protein levels, mRNAs in Gsk-3 knockout ESCs have 50% less m 6 A than wild-type ESCs, and m 6 A-seq analysis reveals the specific mRNAs that have reduced m 6 A modifications. Taken together, we provide the first evidence for how m 6 A demethylation is regulated in mammalian cells, and sheds light onto a possible novel mechanism by which Gsk-3 activity regulates stem cell pluripotency.Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways (1). Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3β, and in general, Gsk-3 is a negative regulator of cellular signaling (2). Rare among kinases, Gsk-3 is active at a basal state, while pathway activation from upstream signaling cascades results in the inhibition of Gsk-3 activity (2). Gsk-3α and Gsk-3β together regulate signal transduction pathways such as Wnt, protein kinase A (PKA), Hedgehog, transforming growth factor-β (TGF-β), nuclear factor of activated T-cells (NF-AT) and phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling in a variety of biological settings (3)(4)(5).Gsk-3 activity can be inhibited through the use of small molecule inhibitors, such as SB- (6)(7)(8), and the clinically relevant mood-stabilizer lithium (9,10); however, a drawback to the use of small molecules to study Gsk-3 function is the potential for off-target effects (11). Cells in which Gsk-3α and Gsk-3β have been genetically deleted allows for a more confident assessment of Gsk-3-specific functions. Therefore, we utilize mouse embryonic stem cells (ESCs) deficient in both Gsk-3α and Gsk-3β (Gsk-3α -/-; Gsk-3β -/-), i.e., Gsk-3 double knockout (DKO), to assess Gsk-3-specific functions (12,13). One prominent phenotype of Gsk-3 DKO ESCs is their persistent pluripotency, assessed by their inability to differentiate into all three germs layers in a teratoma assay, as well as by analysis of global gene expression ...

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“…In addition to these markers, we also analyzed their impact on cell proliferation and determined that Fgf2 and, to a lesser degree, TTNPB affect cell proliferation as expected (supplemental Fig. S2C) (30).…”

Section: Chemically Induced Endoderm Progenitor Cells (Ciepcs) From Mmentioning

confidence: 99%

“…The starting rationale is to first inhibit TGF␤ signaling with RepSox (R) and then activate epithelial characteristics with BMP4 (B) based on our studies (28,29). In addition to RB, we also included Fgf2, CHIR, and Y27632 used previously in our reprogramming medium (30). Furthermore, through compound screening, we also determined that Forskolin (F) and TTNPB (T), two ingredients important for ciPSC generation (21), are very helpful in promoting the MET process.…”

Section: Chemical Induction Of Epithelial-like Cells (Elcs) From Mousmentioning

confidence: 99%

Chemical reprogramming of mouse embryonic and adult fibroblast into endoderm lineage

Cao

Chen

et al. 2017

Journal of Biological Chemistry

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We report here an approach to redirecting somatic cell fate under chemically defined conditions without transcription factors. We start by converting mouse embryonic fibroblasts to epithelial-like cells with chemicals and growth factors. Subsequent cell fate mapping reveals a robust induction of SOX17 in the resulting epithelial-like cells that can be further reprogrammed to endodermal progenitor cells. Interestingly, these cells can self-renew in vitro and further differentiate into albumin-producing hepatocytes that can rescue mice from acute liver injury. Our results demonstrate a rational approach to convert mouse embryonic fibroblasts to hepatocytes and suggest that this mechanism-driven approach may be generalized for other cells.

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Rational optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells with ultra-high efficiency and fast kinetics

Cited by 85 publications

References 49 publications

Modulating Epigenetic Memory through Vitamins and TET: Implications for Regenerative Medicine and Cancer Treatment

Modulating Epigenetic Memory through Vitamins and TET: Implications for Regenerative Medicine and Cancer Treatment

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

Chemical reprogramming of mouse embryonic and adult fibroblast into endoderm lineage

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