The immunogenicity of transplanted allogeneic cells and tissues is a major hurdle to the advancement of cell therapies. Here we show that the overexpression of eight immunomodulatory transgenes (Pdl1, Cd200, Cd47, H2-M3, Fasl, Serpinb9, Ccl21 and Mfge8) in mouse embryonic stem cells (mESCs) is sufficient to immunologically 'cloak' the cells as well as tissues derived from them, allowing their survival for months in outbred and allogeneic inbred recipients. Overexpression of the human orthologues of these genes in human ESCs abolished the activation of allogeneic human peripheral blood mononuclear cells and their inflammatory responses. Moreover, by using the previously reported FailSafe transgene system, which transcriptionally links a gene essential for cell division with an inducible and cell-proliferation-dependent kill switch, we generated cloaked tissues from mESCs that served as immune-privileged subcutaneous sites that protected uncloaked allogeneic and xenogeneic cells from rejection in immune-competent hosts. The combination of cloaking and FailSafe technologies may allow for the generation of safe and allogeneically accepted cell lines and off-the-shelf cell products.
© 2023. The Author(s).

h4>ABSTRACT/h4> A vast number of diseases could be treated with therapeutic cells derived from pluripotent stem cells (PSCs). However, cell products that come from non-autologous sources can be immune rejected by the recipient’s immune system. Here, we show that forced expression of eight immunomodulatory transgenes, including Ccl21, Pdl1, Fasl, Serpinb9, H2-M3, Cd47, Cd200 , and Mfge8 , allows mouse embryonic stem cells (mESCs) and their derivatives to escape immune rejection in fully immunocompetent, allogeneic recipients. Despite no genetic alterations to major histocompatibility complex (MHC) genes, immune-modified C57BL/6 mESCs could generate long-term, allogeneic tissues in inbred FVB/N, C3H, and BALB/c, as well as outbred CD-1 recipients. Due to the tandem incorporation of our safe-cell suicide system, which allows tight and drug-inducible control over proliferation in vivo , these allotolerated cells can generate safe and dormant ectopic tissues in the host. We show that these ectopic tissues maintain high expression of all eight immunomodulatory transgenes and are immune-privileged sites that can host and protect unmodified mouse and human cells from rejection in allogeneic and xenogeneic settings, respectively. If translated to human clinical settings, we envision the development of a single pluripotent cell line that can be used to generate allo-tolerated, off-the-shelf cell products to serve all humankind, as well as immune-privileged ectopic tissues to host and immune-protect any kind of therapeutic cell product.