Chromatin architecture reorganization during somatic cell reprogramming

Ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3) oxidize 5-methylcytosine (5mC) and generate 5-hydroxymethylcytosine (5hmC) marks on the genome. Each TET protein also interacts with specific binding partners and partly plays their role independent of catalytic activity. Although the basic role of TET enzymes is well established now, the molecular mechanism and specific contribution of their catalytic and noncatalytic domains remain elusive. Here, by combining in silico and biochemical screening strategy, we have identified a small molecule compound, C35, as a first-in-class TET inhibitor that specifically blocks their catalytic activities. Using this inhibitor, we explored the enzymatic function of TET proteins during somatic cell reprogramming. Interestingly, we found that C35-mediated TET inactivation increased the efficiency of somatic cell programming without affecting TET complexes. Using high-throughput mRNA sequencing, we found that by targeting 5hmC repressive marks in the promoter regions, C35-mediated TET inhibition activates the transcription of the BMP-SMAD-ID signaling pathway, which may be responsible for promoting somatic cell reprogramming. These results suggest that C35 is an important tool for inducing somatic cell reprogramming, as well as for dissecting the other biological functions of TET enzymatic activities without affecting their other nonenzymatic roles.

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Nonintegrating Human Somatic Cell Reprogramming Methods

Engineering and Application of Pluripotent Stem Cells - Advances in Biochemical Engineering/Biotechnology ◽

10.1007/10_2017_29 ◽

2017 ◽

pp. 1-21

Author(s):

Thorsten M. Schlaeger

Keyword(s):

Somatic Cell ◽

Cell Reprogramming ◽

Human Somatic Cell ◽

Somatic Cell Reprogramming

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Optimized Approaches for the Induction of Putative Canine Induced Pluripotent Stem Cells from Old Fibroblasts Using Synthetic RNAs

Animals ◽

10.3390/ani10101848 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1848

Author(s):

Mirae Kim ◽

Seon-Ung Hwang ◽

Junchul David Yoon ◽

Yeon Woo Jeong ◽

Eunhye Kim ◽

...

Keyword(s):

Stem Cells ◽

Somatic Cell ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Kinase Inhibitors ◽

Rna Virus ◽

Cell Reprogramming ◽

Somatic Cell Reprogramming ◽

Rna Transfection ◽

Induced Pluripotent

Canine induced pluripotent stem cells (ciPSCs) can provide great potential for regenerative veterinary medicine. Several reports have described the generation of canine somatic cell-derived iPSCs; however, none have described the canine somatic cell reprogramming using a non-integrating and self-replicating RNA transfection method. The purpose of this study was to investigate the optimal strategy using this approach and characterize the transition stage of ciPSCs. In this study, fibroblasts obtained from a 13-year-old dog were reprogrammed using a non-integrating Venezuelan equine encephalitis (VEE) RNA virus replicon, which has four reprogramming factors (collectively referred to as T7-VEE-OKS-iG and comprised of hOct4, hKlf4, hSox2, and hGlis1) and co-transfected with the T7-VEE-OKS-iG RNA and B18R mRNA for 4 h. One day after the final transfection, the cells were selected with puromycin (0.5 µg/mL) until day 10. After about 25 days, putative ciPSC colonies were identified showing TRA-1-60 expression and alkaline phosphatase activity. To determine the optimal culture conditions, the basic fibroblast growth factor in the culture medium was replaced with a modified medium supplemented with murine leukemia inhibitory factor (mLIF) and two kinase inhibitors (2i), PD0325901(MEK1/2 inhibitor) and CHIR99021 (GSK3β inhibitor). The derived colonies showed resemblance to naïve iPSCs in their morphology (dome-shaped) and are dependent on mLIF and 2i condition to maintain an undifferentiated phenotype. The expression of endogenous pluripotency markers such as Oct4, Nanog, and Rex1 transcripts were confirmed, suggesting that induced ciPSCs were in the late intermediate stage of reprogramming. In conclusion, the non-integrating and self-replicating VEE RNA replicon system can potentially make a great contribution to the generation of clinically applicable ciPSCs, and the findings of this study suggest a new method to utilize the VEE RNA approach for canine somatic cell reprogramming.

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