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Detecting and Modulating ER Stress to Improve Generation of Induced Pluripotent Stem Cells

Part of the Methods in Molecular Biology book series (MIMB,volume 2454)

Abstract

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) has proven to be a powerful system creating new opportunities to interrogate molecular mechanisms controlling cell fate determination. Under standard conditions, the generation of iPSCs upon overexpression of OCT4, SOX2, KLF4, and c-MYC (OSKM) is generally slow and inefficient due to the presence of barriers that confer resistance to cell fate changes. Hyperactivated endoplasmic reticulum (ER) stress has emerged as a major reprogramming barrier that impedes the initial mesenchymal-to-epithelial transition (MET) step to form iPSCs from mesenchymal somatic cells. Here, we describe several systems to detect ER stress in the context of OSKM reprogramming and chemical interventions to modulate this process for improving iPSC formation.

Key words

  • Induced pluripotent stem cell
  • Cell fate change
  • Pluripotency
  • Endoplasmic reticulum stress
  • Unfolded protein response
  • Mesenchymal-to-epithelial transition

Alejandro Fuentes-Iglesias and Cristina Ameneiro should be considered as co-first authors.

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References

  1. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676. https://doi.org/10.1016/j.cell.2006.07.024

    CAS  CrossRef  PubMed  Google Scholar 

  2. Apostolou E, Stadtfeld M (2018) Cellular trajectories and molecular mechanisms of iPSC reprogramming. Curr Opin Genet Dev 52:77–85. https://doi.org/10.1016/j.gde.2018.06.002

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  3. Haridhasapavalan KK, Raina K, Dey C, Adhikari P, Thummer RP (2020) An insight into reprogramming barriers to iPSC generation. Stem Cell Rev Rep 16(1):56–81. https://doi.org/10.1007/s12015-019-09931-1

    CrossRef  PubMed  Google Scholar 

  4. Nishimura K, Fukuda A, Hisatake K (2019) Mechanisms of the metabolic shift during somatic cell reprogramming. Int J Mol Sci 20(9):2254. https://doi.org/10.3390/ijms20092254

    CAS  CrossRef  PubMed Central  Google Scholar 

  5. Plath K, Lowry WE (2011) Progress in understanding reprogramming to the induced pluripotent state. Nat Rev Genet 12(4):253–265. https://doi.org/10.1038/nrg2955

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Schiebinger G, Shu J, Tabaka M, Cleary B, Subramanian V, Solomon A, Gould J, Liu S, Lin S, Berube P, Lee L, Chen J, Brumbaugh J, Rigollet P, Hochedlinger K, Jaenisch R, Regev A, Lander ES (2019) Optimal-transport analysis of single-cell gene expression identifies developmental trajectories in reprogramming. Cell 176(4):928–943.e922. https://doi.org/10.1016/j.cell.2019.01.006

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Li R, Liang J, Ni S, Zhou T, Qing X, Li H, He W, Chen J, Li F, Zhuang Q, Qin B, Xu J, Li W, Yang J, Gan Y, Qin D, Feng S, Song H, Yang D, Zhang B, Zeng L, Lai L, Esteban MA, Pei D (2010) A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7(1):51–63. https://doi.org/10.1016/j.stem.2010.04.014

    CAS  CrossRef  PubMed  Google Scholar 

  8. Samavarchi-Tehrani P, Golipour A, David L, Sung HK, Beyer TA, Datti A, Woltjen K, Nagy A, Wrana JL (2010) Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7(1):64–77. https://doi.org/10.1016/j.stem.2010.04.015

    CAS  CrossRef  PubMed  Google Scholar 

  9. Hansson J, Rafiee MR, Reiland S, Polo JM, Gehring J, Okawa S, Huber W, Hochedlinger K, Krijgsveld J (2012) Highly coordinated proteome dynamics during reprogramming of somatic cells to pluripotency. Cell Rep 2(6):1579–1592. https://doi.org/10.1016/j.celrep.2012.10.014

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  10. Guallar D, Fuentes-Iglesias A, Souto Y, Ameneiro C, Freire-Agulleiro O, Pardavila JA, Escudero A, Garcia-Outeiral V, Moreira T, Saenz C, Xiong H, Liu D, Xiao S, Hou Y, Wu K, Torrecilla D, Hartner JC, Blanco MG, Lee LJ, Lopez M, Walkley CR, Wang J, Fidalgo M (2020) ADAR1-dependent RNA editing promotes MET and iPSC reprogramming by alleviating ER stress. Cell Stem Cell 27(2):300–314.e311. https://doi.org/10.1016/j.stem.2020.04.016

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  11. Hetz C, Zhang K, Kaufman RJ (2020) Mechanisms, regulation and functions of the unfolded protein response. Nat Rev Mol Cell Biol 21(8):421–438. https://doi.org/10.1038/s41580-020-0250-z

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  12. Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334(6059):1081–1086

    CAS  CrossRef  Google Scholar 

  13. Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13(2):89–102. https://doi.org/10.1038/nrm3270

    CAS  CrossRef  PubMed  Google Scholar 

  14. Simic MS, Moehle EA, Schinzel RT, Lorbeer FK, Halloran JJ, Heydari K, Sanchez M, Jullié D, Hockemeyer D, Dillin A (2019) Transient activation of the UPRER is an essential step in the acquisition of pluripotency during reprogramming. Sci Adv 5(4):eaaw0025. https://doi.org/10.1126/sciadv.aaw0025

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  15. Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7(12):1063–1066. https://doi.org/10.1038/sj.gt.3301206

    CAS  CrossRef  PubMed  Google Scholar 

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Acknowledgments

This research was funded by grants from the Spanish Agencia Estatal de Investigación, co-funded by the FEDER Program of the EU (BFU2016-80899-P and PID2019-105739GB-I00 to M.F. and RTI2018-096708-J-I00 to D.G.) (AEI/FEDER, UE); the Xunta de Galicia-Consellería de Cultura, Educación e Ordenación Universitaria (ED431F 2016/016 to M.F.) and the Fundación Ramón Areces (2016-PO025 to M.F.). M.F. and D.G. are recipients of a Ramón y Cajal (RYC-2014-16779) from the MINECO of Spain and Marie Skłodowska-Curie (MSCA-IF-EF-RI-895984) from the European Commission awards, respectively. A.F.-I. (MINECO, BES-2017-082007) and C.A. (Xunta de Galicia, ED481A-2020/046) are recipients of fellowships.

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Correspondence to Miguel Fidalgo .

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Fuentes-Iglesias, A., Ameneiro, C., Guallar, D., Fidalgo, M. (2021). Detecting and Modulating ER Stress to Improve Generation of Induced Pluripotent Stem Cells. In: Nagy, A., Turksen, K. (eds) Induced Pluripotent Stem (iPS) Cells. Methods in Molecular Biology, vol 2454. Humana, New York, NY. https://doi.org/10.1007/7651_2021_354

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  • DOI: https://doi.org/10.1007/7651_2021_354

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2118-9

  • Online ISBN: 978-1-0716-2119-6

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