PMID- 26285657 OWN - NLM STAT- MEDLINE DCOM- 20151216 LR - 20220321 IS - 2157-6564 (Print) IS - 2157-6580 (Electronic) IS - 2157-6564 (Linking) VI - 4 IP - 10 DP - 2015 Oct TI - Targeted Disruption of the beta2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells. PG - 1234-45 LID - 10.5966/sctm.2015-0049 [doi] AB - Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of beta2-microglobulin on both gene alleles in hESCs. Because beta2-microglobulin serves as the HLA-I light chain, disruption of the beta2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-gamma (IFN-gamma) treatment significantly induced beta2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with beta2-microglobulin-null hESCs and their derivatives treated with IFN-gamma. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, beta2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that beta2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. SIGNIFICANCE: This study reports the generation of a novel beta2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-gamma stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M-/- hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M-/- hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M-/- hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells. CI - (c)AlphaMed Press. FAU - Wang, Dachun AU - Wang D AD - The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases and Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas, USA rick.a.wetsel@uth.tmc.edu dachun.wang@uth.tmc.edu. FAU - Quan, Yuan AU - Quan Y AD - The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases and Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas, USA. FAU - Yan, Qing AU - Yan Q AD - The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases and Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas, USA. FAU - Morales, John E AU - Morales JE AD - The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases and Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas, USA. FAU - Wetsel, Rick A AU - Wetsel RA AD - The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases and Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas, USA rick.a.wetsel@uth.tmc.edu dachun.wang@uth.tmc.edu. LA - eng GR - R21 HL102833-01/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20150818 PL - England TA - Stem Cells Transl Med JT - Stem cells translational medicine JID - 101578022 RN - 0 (HLA Antigens) RN - 0 (beta 2-Microglobulin) RN - 82115-62-6 (Interferon-gamma) SB - IM MH - Alleles MH - Animals MH - CD8-Positive T-Lymphocytes/immunology MH - Cell Self Renewal MH - Cell Survival MH - Cell Transplantation/adverse effects MH - Cytotoxicity, Immunologic MH - Exons/genetics MH - Gene Expression Profiling MH - *Gene Knockdown Techniques MH - Genetic Vectors MH - Genomic Instability MH - Graft Rejection/prevention & control MH - HLA Antigens MH - Heterografts MH - Histocompatibility MH - Human Embryonic Stem Cells/cytology/*immunology/transplantation MH - Humans MH - Interferon-gamma/pharmacology MH - Killer Cells, Natural/immunology MH - Mice MH - Mice, SCID MH - Pluripotent Stem Cells/cytology/immunology/transplantation MH - Sequence Deletion MH - Teratoma/etiology/immunology MH - beta 2-Microglobulin/*genetics/physiology PMC - PMC4572902 OTO - NOTNLM OT - Differentiation and characterization OT - Human leukocyte antigen class I OT - Immunogenicity of human embryonic stem cells OT - beta2-Microglobulin gene targeting strategy EDAT- 2015/08/20 06:00 MHDA- 2015/12/19 06:00 PMCR- 2016/04/01 CRDT- 2015/08/20 06:00 PHST- 2015/03/16 00:00 [received] PHST- 2015/06/22 00:00 [accepted] PHST- 2015/08/20 06:00 [entrez] PHST- 2015/08/20 06:00 [pubmed] PHST- 2015/12/19 06:00 [medline] PHST- 2016/04/01 00:00 [pmc-release] AID - sctm.2015-0049 [pii] AID - 20150049 [pii] AID - 10.5966/sctm.2015-0049 [doi] PST - ppublish SO - Stem Cells Transl Med. 2015 Oct;4(10):1234-45. doi: 10.5966/sctm.2015-0049. Epub 2015 Aug 18.