Abstract
Advances in induced pluripotent stem cell (iPSC) technology provide a renewable source of cells for tissue regeneration and therefore hold great promise for cell replacement therapy. However, immune rejection of allograft due to human leukocyte antigen (HLA) mismatching remains a major challenge. Considerable efforts have been devoted to overcoming the immunogenicity of allograft transplantation. One of the approaches is an elimination of HLA molecules on the surface of allogeneic cells using genome editing technology to generate universal stem cells. Here, we present a simple and effective genome editing approach to knockout the β-2-immunoglobulin (B2M) gene, which encodes B2M protein that forms a heterodimer with HLA class I proteins, in induced pluripotent stem cells (iPSCs) leading to HLA class I (HLA-I) depletion. We also describe detailed procedures for validation of the B2M-knockout iPSCs using flow cytometry, and genotypic analysis for potential off-target regions. Our protocol is also applicable for knocking out other genes in iPSCs and other cell types.
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References
Mao AS, Mooney DJ (2015) Regenerative medicine: current therapies and future directions. Proc Natl Acad Sci U S A 112(47):14452–14459. https://doi.org/10.1073/pnas.1508520112
Mahdi BM (2013) A glow of HLA typing in organ transplantation. Clin Transl Med 2(1):6–6. https://doi.org/10.1186/2001-1326-2-6
Wang Y, Singh NK, Spear TT, Hellman LM, Piepenbrink KH, McMahan RH, Rosen HR, Vander Kooi CW, Nishimura MI, Baker BM (2017) How an alloreactive T-cell receptor achieves peptide and MHC specificity. Proc Natl Acad Sci 114(24):E4792. https://doi.org/10.1073/pnas.1700459114
Singh VK, Kalsan M, Kumar N, Saini A, Chandra R (2015) Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev Biol 3:2–2. https://doi.org/10.3389/fcell.2015.00002
Diecke S, Jung SM, Lee J, Ju JH (2014) Recent technological updates and clinical applications of induced pluripotent stem cells. Korean J Intern Med 29(5):547–557. https://doi.org/10.3904/kjim.2014.29.5.547
Ferreira LMR, Mostajo-Radji MA (2013) How induced pluripotent stem cells are redefining personalized medicine. Gene 520(1):1–6. https://doi.org/10.1016/j.gene.2013.02.037
Doss MX, Sachinidis A (2019) Current challenges of iPSC-based disease modeling and therapeutic implications. Cells 8(5):403. https://doi.org/10.3390/cells8050403
Taylor Craig J, Peacock S, Chaudhry Afzal N, Bradley JA, Bolton Eleanor M (2012) Generating an iPSC bank for HLA-matched tissue transplantation based on known donor and recipient HLA types. Cell Stem Cell 11(2):147–152. https://doi.org/10.1016/j.stem.2012.07.014
Gourraud PA, Gilson L, Girard M, Peschanski M (2012) The role of human leukocyte antigen matching in the development of multiethnic “haplobank” of induced pluripotent stem cell lines. Stem Cells (Dayton, Ohio) 30(2):180–186. https://doi.org/10.1002/stem.772
Adli M (2018) The CRISPR tool kit for genome editing and beyond. Nat Commun 9(1):1911. https://doi.org/10.1038/s41467-018-04252-2
Pickar-Oliver A, Gersbach CA (2019) The next generation of CRISPR–Cas technologies and applications. Nat Rev Mol Cell Biol 20(8):490–507. https://doi.org/10.1038/s41580-019-0131-5
Ceccaldi R, Rondinelli B, D’Andrea AD (2016) Repair pathway choices and consequences at the double-strand break. Trends Cell Biol 26(1):52–64. https://doi.org/10.1016/j.tcb.2015.07.009
Wang H, La Russa M, Qi LS (2016) CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem 85(1):227–264. https://doi.org/10.1146/annurev-biochem-060815-014607
Torikai H, Mi T, Gragert L, Maiers M, Najjar A, Ang S, Maiti S, Dai J, Switzer KC, Huls H, Dulay GP, Reik A, Rebar EJ, Holmes MC, Gregory PD, Champlin RE, Shpall EJ, Cooper LJN (2016) Genetic editing of HLA expression in hematopoietic stem cells to broaden their human application. Sci Rep 6(1):21757. https://doi.org/10.1038/srep21757
Han X, Wang M, Duan S, Franco PJ, Kenty JH, Hedrick P, Xia Y, Allen A, Ferreira LMR, Strominger JL, Melton DA, Meissner TB, Cowan CA (2019) Generation of hypoimmunogenic human pluripotent stem cells. Proc Natl Acad Sci U S A 116(21):10441–10446. https://doi.org/10.1073/pnas.1902566116
Xu H, Wang B, Ono M, Kagita A, Fujii K, Sasakawa N, Ueda T, Gee P, Nishikawa M, Nomura M, Kitaoka F, Takahashi T, Okita K, Yoshida Y, Kaneko S, Hotta A (2019) Targeted disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced immune compatibility. Cell Stem Cell 24(4):566–578.e567. https://doi.org/10.1016/j.stem.2019.02.005
Wang D, Quan Y, Yan Q, Morales JE, Wetsel RA (2015) Targeted disruption of the β2-microglobulin gene minimizes the immunogenicity of human embryonic stem cells. Stem Cells Transl Med 4(10):1234–1245. https://doi.org/10.5966/sctm.2015-0049
Gornalusse GG, Hirata RK, Funk SE, Riolobos L, Lopes VS, Manske G, Prunkard D, Colunga AG, Hanafi L-A, Clegg DO, Turtle C, Russell DW (2017) HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat Biotechnol 35(8):765–772. https://doi.org/10.1038/nbt.3860
Deuse T, Hu X, Gravina A, Wang D, Tediashvili G, De C, Thayer WO, Wahl A, Garcia JV, Reichenspurner H, Davis MM, Lanier LL, Schrepfer S (2019) Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol 37(3):252–258. https://doi.org/10.1038/s41587-019-0016-3
Wattanapanitch M, Ritthaphai A, Park C, Boonkaew B, Netsrithong R, Pattanapanyasat K, Limsiri P, Vatanashevanopakorn C (2018) Generation of a human induced pluripotent stem cell line (MUSIi001-A) from caesarean section scar fibroblasts using Sendai viral vectors. Stem Cell Res 27:105–108. https://doi.org/10.1016/j.scr.2018.01.013
Acknowledgments
This study was supported by a grant from Thailand Research Fund (grant no. RSA6280090), Siriraj Research Fund, Faculty of Medicine Siriraj Hospital, Mahidol University (grant number (IO) R016333015), the National Research Council of Thailand (NRCT): NRCT-RGJ63012-126 and the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (Grant No. B05F630080). NT is supported by Siriraj Graduate Scholarship, Faculty of Medicine Siriraj Hospital, Mahidol University. MW is supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University.
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Thongsin, N., Wattanapanitch, M. (2021). CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs). 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_352
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DOI: https://doi.org/10.1007/7651_2021_352
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