Generation and characterization of human emryonic stem cells with increased expression of HIF-2a



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Abstract

The HIF-2 a subunit is involved in regulation of transcription factors, controlling the self-renewal of human pluripotent stem cells, embryonic development of the cardiovascular system and the regulation of angiogenesis by transcriptional activation of angiogenic cascades in physiological and pathological processes. Currently, modulation of HIF-2a expression is considered as a promising strategy for the treatment of ischemic and cancer diseases. However, the problem of choosing the optimal methods of effective regulation of HIF-2a remains. The aim of this study is to obtain human embryonic stem cells with increased expression of HIF-2a at normal oxygen concentration due to silencing of INT6, the regulator of HIF-2a. In this study, we obtained genetically modified human embryonic stem cells with increased expression of HIF-2a under atmospheric oxygen conditions. The approach used is based on a CRISPR/Cas9-mediated deletion of a part of the INT6 gene, an HIF-2a inhibitor. A study of the resulting genetically modified human embryonic stem cells will contribute to an understanding of the connection between hypoxia and pluripotency. Obtaining endothelial derivatives of pluripotent stem cells with increased expression of HIF-2a and enhanced regenerative potential may become the basis for the development of promising strategies for treatment of ischemic diseases.

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About the authors

M. K Zhiven

Institute of Cytology and Genetics, the Siberian Branch of the RAS; E.N. Meshalkin National Medical Research Center

Email: zhiven92@mail.ru

I. S Zakharova

Institute of Cytology and Genetics, the Siberian Branch of the RAS; E.N. Meshalkin National Medical Research Center

A. I Shevchenko

Institute of Cytology and Genetics, the Siberian Branch of the RAS; E.N. Meshalkin National Medical Research Center

E. A Elisaphenko

Institute of Cytology and Genetics, the Siberian Branch of the RAS; E.N. Meshalkin National Medical Research Center

K. E Orishchenko

Institute of Cytology and Genetics, the Siberian Branch of the RAS

S. M Zakian

Institute of Cytology and Genetics, the Siberian Branch of the RAS; E.N. Meshalkin National Medical Research Center

References

  1. Cassavaugh J., Lounsbury K.M. Hypoxia-mediated biological control. J. Cell. Biochem. 2011; 112(3): 735-44.
  2. Semenza G.L. Hypoxia-inducible factors in physiology and medicine. Cell 2012; 148(3): 399-408.
  3. Semenza G.L. Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 2003; 3(10): 721-32.
  4. Semenza G.L. Oxygen Sensing, Hypoxia-Inducible Factors, and Disease Pathophysiology. Annu. Rev. Pathol. Mech. Dis. 2014; 9(1): 47-71.
  5. Manalo D.J., Rowan A., Lavoie T. et al. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood 2005; 105(2): 659-69.
  6. Hashimoto T., Shibasaki F. Hypoxia-inducible factor as an angiogenic master switch. Front. Pediatr. 2015; 3: 33.
  7. Slemc L., Kunej T. Transcription factor HIF1A: downstream targets, associated pathways, polymorphic hypoxia response element (HRE) sites, and initiative for standardization of reporting in scientific literature. Tumour Biol. 2016; 37(11): 51-61.
  8. Dong X., Sun B., Zhao X. et al. Expression of relative-protein of hypoxia-inducible factor-1 a in vasculogenesis of mouse embryo. J. Biol. Res. 2014; 21(1): 4.
  9. Greijer A.E., van der Groep P., Kemming D. et al. Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1). J. Pathol. 2005; 206(3): 291-304.
  10. Zimna A., Kurpisz M. Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological Angiogenesis: Applications and Therapies. Biomed. Res. Int. 2015; 2015: 549412.
  11. Schellinge I.N., Cordasic N., Panesar J. et al. Hypoxia inducible factor stabilization improves defective ischemia-induced angiogenesis in a rodent model of chronic kidney disease. Kidney Int. 2017; 91(3): 616-27.
  12. Tang N., Wang L., Esko J. et al. Loss of HIF-1alpha in endothelial cells disrupts a hypoxia-driven VEGF autocrine loop necessary for tumorigenesis. Cancer Cell 2004; 6(5): 485-95.
  13. Kaelin Jr.W.G., Ratcliffe Sir P.J., Semenza G.L. Press release: The Nobel Prize in Physiology or Medicine 2019, https://www.nobelprize.org/ prizes/medicine/2019/press-release/.
  14. Vink A., Schoneveld A.H., Lamers D. et al. HIF-1 alpha expression is associated with an atheromatous inflammatory plaque phenotype and upregulated in activated macrophages. Atherosclerosis 2007; 195(2): 69-75.
  15. Talks K.L., Turley H., Gatter K.C. et al. The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am. J. Pathol. 2000; 157(2): 411-21.
  16. Duscher D., Januszyk M., Maan Z.N. et al. Comparison of the Hydroxylase Inhibitor Dimethyloxalylglycine and the Iron Chelator Deferoxamine in Diabetic and Aged Wound Healing. Plast. Reconstr. Surg. 2017; 139(3): 695-706.
  17. Chen L., Uchida K., Endler A. Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. J. Biol. Chem. 2007; 282(17): 12707-16.
  18. Chen L., Uchida K., Endler A. et al. Mammalian tumor suppressor INT6 specifically targets hypoxia inducible factor 2 alpha for dеgradation by hypoxia-and pVHL-independent regulation. J. Biol. Chem. 2007; 282(17): 12707-16.
  19. Hashimoto T., Chen L., Kimura H. et al. Silencing of eIF3e promotes blood perfusion recovery after limb ischemia through stabilization of hypoxia-inducible factor 2a activity. J. Vasc. Surg. 2016; 64(1): 219-26.
  20. Cowan C.A., Klimanskaya I., McMahon J. et al. Derivation of Embryonic Stem-Cell Lines from Human Blastocysts. N. Engl. J. Med. 2004; 350(13): 1353-6.
  21. Lagarkova M.A., Volchkov P.Y., Lyakisheva A.V. Diverse epigenetic profile of novel human embryonic stem cell lines. Cell Cycle 2006; 5(4): 416-20.
  22. Prokhorovich M.A., Lagar’kova M.A., Shilov A.G. Cultures of hESM human embryonic stem cells: Chromosomal aberrations and karyotype stability. Bull. Exp. Biol. Med. 2007; 144(1): 126-9.
  23. Linden T., Katschinski D., Eckhardt M. The antimycotic ciclopirox olamine induces HIF-1 alpha stability, VEGF expression, and angiogenesis. FASEB J. 2003; 17(6): 761-3.
  24. Yang L., Shen L., Li G. Silencing of hypoxia inducible factor-1 a gene attenuated angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice. Atherosclerosis 2016; 252: 40-9.
  25. Chu C.Y., Jin Y.T., Zhang W. et al. CA IX is upregulated in CoCl2-induced hypoxia and associated with cell invasive potential and a poor prognosis of breast cancer. Int. J. Oncol. 2016; 48(1): 271-80.
  26. Wang G.L., Semenza G.L. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J. Biol. Chem. 1993; 268(29): 21513-8.
  27. Gunshin H., Mackenzie B., Berger U.V. et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997; 388(6641): 482-8.
  28. Taniguchi C.M., Miao Y.R., Diep A.N. et al. PHD inhibition mitigates and protects against radiation-induced gastrointestinal toxicity via HIF2. Sci. Transl. Med. 2014; 6(236): 236-64.
  29. Yuan Q., Bleiziffer O., Boos A.M. et al. PHDs inhibitor DMOG promotes the vascularization process in the AV loop by HIF-1a up-regulation and the preliminary discussion on its kinetics in rat. BMC Biotechnol. 2014; 14(1): 112.
  30. Cong L., Ran F.A., Cox D. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339(6121): 819-23.
  31. Медведев С.П., Шевченко А.И., Сухих Г.Т. и соавт. Протокол иммунофлуоресцентного окрашивания. В: Власов В.В., редактор. Индуцированные плюрипотентные стволовые клетки. 1-e издание. Новосибирск: Издательство СО РАН; 2011: 133-7.
  32. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-AACT method. Methods 2001; 25(4): 402-8.
  33. Живень М.К., Захарова И.С., Смирнова А.М. и соавт. CRISPR/ Cas9-опосредованное получение генетически модифицированной линии плюрипотентных стволовых клеток человека, экспрессирующих HIF - фактор, индуцируемый гипоксией. В: Деев Р.В., Павлов И.П., ред. Материалы Международного конгресса CRISPR-2018; 2018 сент. 10-14; Новосибирск; Гены и Клетки 2018, приложение 2. стр. 30.
  34. Singh M., Chaudhry P., Parent S. et al. Ubiquitin-Proteasomal Degradation of COX-2 in TGF-p Stimulated Human Endometrial Cells Is Mediated Through Endoplasmic Reticulum Mannosidase I. Endocrinology 2012; 153(1): 426-37.
  35. Dunwoodie S.L. The Role of Hypoxia in Development of the Mammalian Embryo. Developmental Cell 2009; 17(6): 755-73.
  36. Jauniaux E., Watson A., Ozturk O. In-vivo measurement of intrauterine gases and acid-base values early in human pregnancy. Hum. Reprod. 1999; 14(11): 2901-4.
  37. Rodesch F., Simon P., Donner C. Oxygen measurements in endometrial and trophoblastic tissues during early pregnancy. Obstet. Gynecol. 1992; 80(2): 283-5.
  38. Ezashi T., Das P., Roberts R.M. Low O2 tensions and the prevention of differentiation of hES cells. PNAS USA 2005; 102(13): 4783-8.
  39. Forsyth N.R., Musio A., Vezzoni P.W. Physiologic Oxygen Enhances Human Embryonic Stem Cell Clonal Recovery and Reduces Chromosomal Abnormalities. Cloning Stem Cells 2006; 8(1): 16-23.
  40. Forristal C.E., Wright K.L., Hanley N.A. Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions. Reproduction 2010; 139(1): 85-97.
  41. Lengner C.J., Gimelbrant A.A., Erwin J.A. et al. Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell 2010; 141(5): 872-83.
  42. Mutoh T., Sanosaka T., Ito K. Oxygen levels epigenetically regulate fate switching of neural precursor cells via hypoxia-inducible factor 1 a-Notch signal interaction in the developing brain. Stem Cells 2012; 30(3): 561-9.
  43. Studer L., Csete M., Lee S.H. et al. Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J. Neurosci. 2000; 20(19): 7377-83.
  44. Ross H.H., Sandhu M.S., Cheung T.F. et al. In vivo intermittent hypoxia elicits enhanced expansion and neuronal differentiation in cultured neural progenitors. Exp. Neurol. 2012; 235(1): 238-45.
  45. Morrison S.J., Csete M., Groves A.K. Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J. Neurosci. 2000; 20(19): 7370-6.
  46. Behbahan I.S., Duan Y., Lam A. et al. New Approaches in the Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells toward Hepatocytes. Stem Cell Reviews and Reports 2011; 7(3): 748-59.
  47. Si-Tayeb K., Noto F.K., Nagaoka M. et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatol-ogy 2010; 51(1): 297-305.
  48. Bae D., Mondragon-Teran P., Hernandez D. et al. Hypoxia enhances the generation of retinal progenitor cells from human induced pluripotent and embryonic stem cells. Stem Cells Dev. 2012; 21(8): 1344-55.
  49. Salvagiotto G., Burton S., Daigh C.A. A defined, feeder-free, serum-free system to generate In Vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs. PLoS One 2011; 6(3): e17829.
  50. Yoshida Y., Takahashi K., Okita K. Hypoxia Enhances the Generation of Induced Pluripotent Stem Cells. Cell Stem Cell 2009; 5(3): 237-41.
  51. Sesen J., Casaos J., Scotland S.J et al. The Bad, the Good and eIF3e/ INT6. Front. Biosci. 2017; 22: 1-20.
  52. Petruzzelli R., Christensen D.R., Parry K.L. HIF-2a Regulates NANOG Expression in Human Embryonic Stem Cells following Hypoxia and Reoxygenation through the Interaction with an Oct-Sox Cis Regulatory Element. PLoS One 2014; 9(10): e108309.
  53. Sugimoto K., Matsuura T., Nakazono A. Effects of hypoxia inducible factors on pluripotency in human iPS cells. Microsc. Res. Tech. 2018; 81(7): 749-54.
  54. Zhdanov A.V., Okkelman I.A., Collins W.J. A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression. Biochim. Biophys. Acta 2015; 1847(10): 1254-66.

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