DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.

• Keywords
• DNA damage, cell reprogramming, induced pluripotent stem cells, macroH2A1.1,
• MeSH
• DNA MeSH
• Endothelial Cells metabolism   MeSH
• Histones * metabolism   MeSH
• Induced Pluripotent Stem Cells * metabolism   MeSH
• Humans MeSH
• Mice MeSH
• DNA Repair MeSH
• X-ray Repair Cross Complementing Protein 1 genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• Histones * MeSH
• MACROH2A1 protein, human MeSH Browser
• X-ray Repair Cross Complementing Protein 1 MeSH
• XRCC1 protein, human MeSH Browser

Human pluripotent stem cells have the potential to change the way in which human diseases are cured. Clinical-grade human embryonic stem cells and human induced pluripotent stem cells have to be created according to current good manufacturing practices and regulations. Quality and safety must be of the highest importance when humans' lives are at stake. With the rising number of clinical trials, there is a need for a consensus on hPSCs characterization. Here, we summarize mandatory and 'for information only' characterization methods with release criteria for the establishment of clinical-grade hPSC lines.

• Keywords
• cGMP, cell therapy, characterization, clinical, hESC, hPSCs, hiPSC, human embryonic stem cells, human induced pluripotent stem cells, human pluripotent stem cells,
• MeSH
• Bacteria MeSH
• Cell- and Tissue-Based Therapy methods   MeSH
• Endotoxins MeSH
• Induced Pluripotent Stem Cells MeSH
• Humans MeSH
• Human Embryonic Stem Cells MeSH
• Mycoplasma MeSH
• Pluripotent Stem Cells * MeSH
• Viruses MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Endotoxins MeSH

Hypoxia is involved in the regulation of stem cell fate, and hypoxia-inducible factor 1 (HIF-1) is the master regulator of hypoxic response. Here, we focus on the effect of hypoxia on intracellular signaling pathways responsible for mouse embryonic stem (ES) cell maintenance. We employed wild-type and HIF-1α-deficient ES cells to investigate hypoxic response in the ERK, Akt, and STAT3 pathways. Cultivation in 1% O2 for 24 h resulted in the strong dephosphorylation of ERK and its upstream kinases and to a lesser extent of Akt in an HIF-1-independent manner, while STAT3 phosphorylation remained unaffected. Downregulation of ERK could not be mimicked either by pharmacologically induced hypoxia or by the overexpression. Dual-specificity phosphatases (DUSP) 1, 5, and 6 are hypoxia-sensitive MAPK-specific phosphatases involved in ERK downregulation, and protein phosphatase 2A (PP2A) regulates both ERK and Akt. However, combining multiple approaches, we revealed the limited significance of DUSPs and PP2A in the hypoxia-mediated attenuation of ERK signaling. Interestingly, we observed a decreased reactive oxygen species (ROS) level in hypoxia and a similar phosphorylation pattern for ERK when the cells were supplemented with glutathione. Therefore, we suggest a potential role for the ROS-dependent attenuation of ERK signaling in hypoxia, without the involvement of HIF-1.

• MeSH
• Down-Regulation MeSH
• Hypoxia-Inducible Factor 1, alpha Subunit metabolism   MeSH
• Mitogen-Activated Protein Kinase Kinases metabolism   MeSH
• Mouse Embryonic Stem Cells metabolism   MeSH
• Mice MeSH
• Reactive Oxygen Species metabolism   MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Hypoxia-Inducible Factor 1, alpha Subunit MeSH
• Mitogen-Activated Protein Kinase Kinases MeSH
• Reactive Oxygen Species MeSH