scholarly journals Removing Reprogramming Roadblocks: Mbd3 Depletion Allows Deterministic iPSC Generation

2013 ◽  
Vol 13 (4) ◽  
pp. 379-381 ◽  
Author(s):  
Justin Brumbaugh ◽  
Konrad Hochedlinger
Keyword(s):  
Ipsc Generation

scholarly journals High Throughput Mechanobiological Screens Enable Mechanical Priming of Pluripotency in Mouse Fibroblasts

2018 ◽  
Author(s):  
Jason Lee ◽  
Miguel Ochoa ◽  
Pablo Maceda ◽  
Eun Yoon ◽  
Lara Samarneh ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Tumor Formation ◽  
Mechanical Forces ◽  
Mouse Fibroblasts ◽  
Cell Culture Systems ◽  
Ipsc Generation ◽  
Induced Pluripotent

Transgenic methods for direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) are effective in cell culture systems but ultimately limit the utility of iPSCs due to concerns of mutagenesis and tumor formation. Recent studies have suggested that some transgenes can be eliminated by using small molecules as an alternative to transgenic methods of iPSC generation. We developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. Using this system, we screened for optimized conditions to stimulate the activation of Oct-4 and other transcription factors to prime the development of pluripotency in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the priming of pluripotency of mouse fibroblasts in combination with mechanical loading. Taken together, our findings demonstrate the ability of mechanical forces to induce reprograming factors and support that biophysical conditioning can act cooperatively with small molecules to priming the induction pluripotency in somatic cells.

An Insight into Reprogramming Barriers to iPSC Generation

2019 ◽  
Vol 16 (1) ◽  
pp. 56-81 ◽  
Author(s):  
Krishna Kumar Haridhasapavalan ◽  
Khyati Raina ◽  
Chandrima Dey ◽  
Poulomi Adhikari ◽  
Rajkumar P. Thummer
Keyword(s):  
Ipsc Generation ◽  
Insight Into

Cell type of origin influences iPSC generation and differentiation to cells of the hematoendothelial lineage

2016 ◽  
Vol 365 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Jitrada Phetfong ◽  
Aungkura Supokawej ◽  
Methichit Wattanapanitch ◽  
Pakpoom Kheolamai ◽  
Yaowalak U-pratya ◽  
...  
Keyword(s):  
Cell Type ◽  
Ipsc Generation

scholarly journals BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

2016 ◽  
Vol 113 (46) ◽  
pp. 13057-13062 ◽  
Author(s):  
Yohei Hayashi ◽  
Edward C. Hsiao ◽  
Salma Sami ◽  
Mariselle Lancero ◽  
Christopher R. Schlieve ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Fibrodysplasia Ossificans Progressiva ◽  
Expression Change ◽  
Major Barrier ◽  
Smad Signaling ◽  
Signaling Axis ◽  
Ipsc Generation ◽  
Id Genes ◽  
P16 Ink4a ◽  
Induced Pluripotent

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes.

scholarly journals MYCL promotes iPSC-like colony formation via MYC Box 0 and 2 domains

2021 ◽  
Author(s):  
Chiaki Akifuji ◽  
Mio Iwasaki ◽  
Yuka Kawahara ◽  
Chiho Sakurai ◽  
Yusheng Cheng ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Proteome Analysis ◽  
Cytoskeletal Proteins ◽  
The Body ◽  
Germline Transmission ◽  
Phenotypic Difference ◽  
Reprogramming Efficiency ◽  
Ipsc Generation ◽  
Low Efficiency ◽  
Induced Pluripotent

Abstract Induced pluripotent stem cells (iPSCs) have the potential to differentiate into any cell in the body and thus have attractive regenerative medicine potential. Current iPSC generation protocols, however, have low efficiency and show variable quality among clones. This variability influences the efficiency and reproducibility of iPSC differentiation. Our previous study reported that MYC proteins (c-MYC and MYCL) are important for the reprogramming efficiency and germline transmission of iPSCs, but that MYCL can generate iPSC colonies more efficiently than c-MYC. However, why c-MYC and MYCL cause different reprogramming efficiencies is unknown. In this study, we found that MYC Box 0 (MB0) and MB2, two functional domains conserved in the MYC protein family, contribute to the phenotypic difference and promote iPSC generation in MYCL-induced reprogramming. Proteome analysis suggested that in MYCL-induced reprogramming, cell adhesion-related cytoskeletal proteins are regulated by the MB0 domain, while RNA processes are regulated by the MB2 domain. These findings provide a molecular explanation for why MYCL has higher reprogramming efficiency than c-MYC.

scholarly journals Dot1L methyltransferase activity is a barrier to acquisition of pluripotency but not transdifferentiation

2019 ◽  
Author(s):  
Coral K. Wille ◽  
Rupa Sridharan
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Cell Types ◽  
Transcriptional Elongation ◽  
Mrna Levels ◽  
Mesenchymal To Epithelial Transition ◽  
Ipsc Generation ◽  
Induced Pluripotent

ABSTRACTThe ability of pluripotent stem cells to be poised to differentiate into any somatic cell type is partly derived from a unique chromatin structure that is depleted for transcriptional elongation associated epigenetic modifications, primarily H3K79 methylation. Inhibiting the H3K79 methyltransferase, Dot1L, increases the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs) most potently at the mid-point of the process. Surprisingly, despite the enrichment of H3K79me2 on thousands of actively transcribed genes, Dot1L inhibition (Dot1Li) results in few changes in steady state mRNA levels during reprogramming. Dot1Li spuriously upregulates genes not involved in pluripotency and does not shutdown the somatic program. Depletion of the few genes that are downregulated, such as Nfix, enhances reprogramming efficiency in cooperation with Dot1Li. Contrary to the prevalent view, Dot1Li promotes iPSC generation beyond early phases of reprogramming such as the mesenchymal to epithelial transition and from already epithelial cell types including keratinocytes. Significantly, Dot1L inhibition does not enhance lineage conversion to neurons or muscle cells. Taken together, our results indicate that H3K79me is not a universal barrier of cell fate transitions but specifically protects somatic cells from reverting to the pluripotent state.

scholarly journals Xist Intron 1 Repression by TALE Transcriptional Factor Improves Somatic Cell Reprogamming in Mice

2018 ◽  
Author(s):  
Jindun Zhang ◽  
Xuefei Gao ◽  
Jian Yang ◽  
Xiaoying Fan ◽  
Wei Wang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Nuclear Transfer ◽  
Genomic Region ◽  
Embryonic Fibroblasts ◽  
Preimplantation Embryo Development ◽  
Intron 1 ◽  
Ipsc Generation ◽  
Critical Requirement ◽  
Induced Pluripotent

SummaryXist is the master regulator of X chromosome inactivation (XCI). In order to further understand the Xist locus in reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and in somatic cell nuclear transfer (SCNT), we tested transcription-factor-like effectors (TALE)-based designer transcriptional factors (dTFs), which were specific to numerous regions at the Xist locus. We report that the selected dTF repressor 6 (R6) binding the intron 1 of Xist, which did not affect Xist expression in mouse embryonic fibroblasts (MEFs), substantially improved the iPSC generation and the SCNT preimplantation embryo development. Conversely, the dTF activator targeting the same genomic region of R6 decreased iPSC formation, and blocked SCNT-embryo development. These results thus uncover the critical requirement for the Xist locus in epigenetic resetting, which is not directly related to Xist transcription. This may provide a unique route to improving the reprogramming.

scholarly journals Dot1L interaction partner AF10 safeguards cell identity during the acquisition of pluripotency

2020 ◽  
Author(s):  
Coral K. Wille ◽  
Edwin N. Neumann ◽  
Aniruddha J. Deshpande ◽  
Rupa Sridharan
Keyword(s):  
Protein Complexes ◽  
Expression Patterns ◽  
Interaction Domain ◽  
Cell Identity ◽  
Interacting Protein ◽  
Ipsc Generation ◽  
Transcriptional Changes ◽  
Differential Gene ◽  
Induced Pluripotent ◽  
H3 Acetylation

ABSTRACTHistone-modifying enzymes function as part of protein complexes that alter the accessibility of chromatin to elicit differential gene expression patterns. Dot1L, the sole histone H3 lysine (K) 79 (H3K79) methyltransferase, is associated with proteins that recognize specific histone modifications and target Dot1L to particular chromatin contexts. Here we find that depletion of the Dot1L interacting protein AF10, which recognizes unmodified H3K27, mimics Dot1L catalytic inhibition to increase the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs). AF10 deletion results in almost no steady state transcriptional changes yet is responsible for half of the Dot1L iPSC reprogramming phenotype. In contrast, reduced levels of Dot1L interactors AF9 and ENL that recognize H3 acetylation decrease iPSC generation. Despite the opposite effects in reprogramming of Dot1L interacting proteins with differing histone reader specificity, we find that the AF10-histone interaction domain is dispensable for increased iPSC generation. Instead, the AF10-Dot1L interaction domain that potentiates H3K79 di- and tri-methylation is a barrier to pluripotency acquisition. Taken together we reveal a key role for higher order gene body histone methylation in safeguarding cell identity.

scholarly journals AKT signaling is associated with epigenetic reprogramming via the upregulation of TET and its cofactor, alpha-ketoglutarate during iPSC generation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yoichi Sekita ◽  
Yuki Sugiura ◽  
Akari Matsumoto ◽  
Yuki Kawasaki ◽  
Kazuya Akasaka ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Primordial Germ Cells ◽  
Akt Signaling ◽  
Dna Demethylation ◽  
Cellular Reprogramming ◽  
Epigenetic Reprogramming ◽  
Cell Reprogramming ◽  
Somatic Cell Reprogramming ◽  
A Genome ◽  
Ipsc Generation

Abstract Background Phosphoinositide-3 kinase (PI3K)/AKT signaling participates in cellular proliferation, survival and tumorigenesis. The activation of AKT signaling promotes the cellular reprogramming including generation of induced pluripotent stem cells (iPSCs) and dedifferentiation of primordial germ cells (PGCs). Previous studies suggested that AKT promotes reprogramming by activating proliferation and glycolysis. Here we report a line of evidence that supports the notion that AKT signaling is involved in TET-mediated DNA demethylation during iPSC induction. Methods AKT signaling was activated in mouse embryonic fibroblasts (MEFs) that were transduced with OCT4, SOX2 and KLF4. Multiomics analyses were conducted in this system to examine the effects of AKT activation on cells undergoing reprogramming. Results We revealed that cells undergoing reprogramming with artificially activated AKT exhibit enhanced anabolic glucose metabolism and accordingly increased level of cytosolic α-ketoglutarate (αKG), which is an essential cofactor for the enzymatic activity of the 5-methylcytosine (5mC) dioxygenase TET. Additionally, the level of TET is upregulated. Consistent with the upregulation of αKG production and TET, we observed a genome-wide increase in 5-hydroxymethylcytosine (5hmC), which is an intermediate in DNA demethylation. Moreover, the DNA methylation level of ES-cell super-enhancers of pluripotency-related genes is significantly decreased, leading to the upregulation of associated genes. Finally, the transduction of TET and the administration of cell-permeable αKG to somatic cells synergistically enhance cell reprogramming by Yamanaka factors. Conclusion These results suggest the possibility that the activation of AKT during somatic cell reprogramming promotes epigenetic reprogramming through the hyperactivation of TET at the transcriptional and catalytic levels.

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