RT Journal Article SR Electronic T1 Engineering Human Pluripotent Stem Cell Lines to Evade Xenogeneic Transplantation Barriers JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.06.27.546594 DO 10.1101/2023.06.27.546594 A1 Pizzato, Hannah A. A1 Alonso-Guallart, Paula A1 Woods, James A1 Johannesson, Bjarki A1 Connelly, Jon P. A1 Fehniger, Todd A. A1 Atkinson, John P. A1 Pruett-Miller, Shondra M. A1 Monsma, Frederick J. A1 Bhattacharya, Deepta YR 2023 UL http://biorxiv.org/content/early/2023/06/29/2023.06.27.546594.abstract AB Allogeneic human pluripotent stem cell (hPSC)-derived cells and tissues for therapeutic transplantation must necessarily overcome immunological rejection by the recipient. To define these barriers and to create cells capable of evading rejection for preclinical testing in immunocompetent mouse models, we genetically ablated β2m, Tap1, Ciita, Cd74, Mica, and Micb to limit expression of HLA-I, HLA-II, and natural killer cell activating ligands in hPSCs. Though these and even unedited hPSCs readily formed teratomas in cord blood-humanized immunodeficient mice, grafts were rapidly rejected by immunocompetent wild-type mice. Transplantation of these cells that also expressed covalent single chain trimers of Qa1 and H2-Kb to inhibit natural killer cells and CD55, Crry, and CD59 to inhibit complement deposition led to persistent teratomas in wild-type mice. Expression of additional inhibitory factors such as CD24, CD47, and/or PD-L1 had no discernible impact on teratoma growth or persistence. Transplantation of HLA-deficient hPSCs into mice genetically deficient in complement and depleted of natural killer cells also led to persistent teratomas. Thus, T cell, NK cell, and complement evasion are necessary to prevent immunological rejection of hPSCs and their progeny. These cells and versions expressing human orthologs of immune evasion factors can be used to refine tissue- and cell type-specific immune barriers, and to conduct preclinical testing in immunocompetent mouse models.Competing Interest StatementSana Biotechnology has licensed intellectual property of H.A.P., D.B., and Washington University in St. Louis. Gilead Biosciences has licensed intellectual property of D.B. and Stanford University. Clade Therapeutics has licensed intellectual property of D.B., H.A.P., and The University of Arizona. D.B. is a co-founder of Clade Therapeutics. D.B. served on an advisory panel for GlaxoSmithKline. T.A.F. has licensed patents, equity, consulting, and potential royalty interests in Wugen; equity interests in Indapta Therapeutics and Orca Bio; and serves as a consultant for Affimed, AI Proteins, Smart Immune, Simcha. B.J. is an employee of AstraZeneca. The authors have no additional financial interests.