Histone Variant macroH2A1.1 Enhances Nonhomologous End Joining-dependent DNA Double-strand-break Repair and Reprogramming Efficiency of Human iPSCs.

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.

The Manufacture of Xeno- and Feeder-Free Clinical-Grade Human Embryonic Stem Cell Lines: First Step for Cell Therapy.

Human embryonic stem cells (hESCs) are increasingly used in clinical trials as they can change the outcome of treatment for many human diseases. They are used as a starting material for further differentiation into specific cell types and to achieve the desirable result of the cell therapy; thus, the quality of hESCs has to be taken into account. Therefore, current good manufacturing practice (cGMP) has to be implemented in the transport of embryos, derivation of inner cell mass to xeno-free, feeder-free and defined hESC culture, and cell freezing. The in-depth characterization of hESC lines focused on safety, pluripotency, differentiation potential and genetic background has to complement this process. In this paper, we show the derivation of three clinical-grade hESC lines, MUCG01, MUCG02, and MUCG03, following these criteria. We developed and validated the system for the manufacture of xeno-free and feeder-free clinical-grade hESC lines that present high-quality starting material suitable for cell therapy according to cGMP.

Clinical-Grade Human Pluripotent Stem Cells for Cell Therapy: Characterization Strategy.

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.

Integrative CUT&Tag-RNA-Seq analysis of histone variant macroH2A1-dependent orchestration of human induced pluripotent stem cell reprogramming.

Aim: Human induced pluripotent stem cells (iPSCs) are inefficiently derived from somatic cells by overexpression of defined transcription factors. Overexpression of H2A histone variant macroH2A1.1, but not macroH2A1.2, leads to increased iPSC reprogramming by unclear mechanisms. Materials & methods: Cleavage under targets and tagmentation (CUT&Tag) allows robust epigenomic profiling of a low cell number. We performed an integrative CUT&Tag-RNA-Seq analysis of macroH2A1-dependent orchestration of iPSCs reprogramming using human endothelial cells. Results: We demonstrate wider genome occupancy, predicted transcription factors binding, and gene expression regulated by macroH2A1.1 during reprogramming, compared to macroH2A1.2. MacroH2A1.1, previously associated with neurodegenerative pathologies, specifically activated ectoderm/neural processes. Conclusion: CUT&Tag and RNA-Seq data integration is a powerful tool to investigate the epigenetic mechanisms occurring during cell reprogramming.

Redox and Epigenetics in Human Pluripotent Stem Cells Differentiation.

Significance: Since their discovery, induced pluripotent stem cells (iPSCs) had generated considerable interest in the scientific community for their great potential in regenerative medicine, disease modeling, and cell-based therapeutic approach, due to their unique characteristics of self-renewal and pluripotency. Recent Advances: Technological advances in iPSC genome-wide epigenetic profiling led to the elucidation of the epigenetic control of cellular identity during nuclear reprogramming. Moreover, iPSC physiology and metabolism are tightly regulated by oxidation-reduction events that mainly occur during the respiratory chain. In theory, iPSC-derived differentiated cells would be ideal for stem cell transplantation as autologous cells from donors, as the risks of rejection are minimal. Critical Issues: However, iPSCs experience high oxidative stress that, in turn, confers a high risk of increased genomic instability, which is most often linked to DNA repair deficiencies. Genomic instability has to be assessed before iPSCs can be used in therapeutic designs. Future Directions: This review will particularly focus on the links between redox balance and epigenetic modifications-in particular based on the histone variant macroH2A1-that determine DNA damage response in iPSCs and derived differentiated cells, and that might be exploited to decrease the teratogenic potential on iPSC transplantation. Antioxid. Redox Signal. 34, 335-349.

Endothelial Progenitor Cells Produced From Human Pluripotent Stem Cells by a Synergistic Combination of Cytokines, Small Compounds, and Serum-Free Medium.

Human pluripotent stem cells (hPSCs) are a promising source of autologous endothelial progenitor cells (EPCs) that can be used for the treatment of vascular diseases. However, this kind of treatment requires a large amount of EPCs. Therefore, a highly efficient, robust, and easily reproducible differentiation protocol is necessary. We present a novel serum-free differentiation protocol that exploits the synergy of multiple powerful differentiation effectors. Our protocol follows the proper physiological pathway by differentiating EPCs from hPSCs in three phases that mimic in vivo embryonic vascular development. Specifically, hPSCs are differentiated into (i) primitive streak, which is subsequently turned into (ii) mesoderm, which finally differentiates into (iii) EPCs. This differentiation process yields up to 15 differentiated cells per seeded hPSC in 5 days. Endothelial progenitor cells constitute up to 97% of these derived cells. The experiments were performed on the human embryonic stem cell line H9 and six human induced pluripotent stem cell lines generated in our laboratory. Therefore, robustness was verified using many hPSC lines. Two previously established protocols were also adapted and compared to our synergistic three-phase protocol. Increased efficiency and decreased variability were observed for our differentiation protocol in comparison to the other tested protocols. Furthermore, EPCs derived from hPSCs by our protocol expressed the high-proliferative-potential EPC marker CD157 on their surface in addition to the standard EPC surface markers CD31, CD144, CD34, KDR, and CXCR4. Our protocol enables efficient fully defined production of autologous endothelial progenitors for research and clinical applications.

Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells.

New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.

DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs). METHODS: We measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. RESULTS: We demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages. CONCLUSIONS: Our observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.