Induced Pluripotent Stem Cell-Derived Fibroblasts Efficiently Engage Senescence Pathways but Show Increased Sensitivity to Stress Inducers.

The risk of aberrant growth of induced pluripotent stem cell (iPSC)-derived cells in response to DNA damage is a potential concern as the tumor suppressor genes TP53 and CDKN2A are transiently inactivated during reprogramming. Herein, we evaluate the integrity of cellular senescence pathways and DNA double-strand break (DSB) repair in Sendai virus reprogrammed iPSC-derived human fibroblasts (i-HF) compared to their parental skin fibroblasts (HF). Using transcriptomics analysis and a variety of functional assays, we show that the capacity of i-HF to enter senescence and repair DSB is not compromised after damage induced by ionizing radiation (IR) or the overexpression of H-RASV12. Still, i-HF lines are transcriptionally different from their parental lines, showing enhanced metabolic activity and higher expression of p53-related effector genes. As a result, i-HF lines generally exhibit increased sensitivity to various stresses, have an elevated senescence-associated secretory phenotype (SASP), and cannot be immortalized unless p53 expression is knocked down. In conclusion, while our results suggest that i-HF are not at a greater risk of transformation, their overall hyperactivation of senescence pathways may impede their function as a cell therapy product.

Myogenic progenitor cells derived from human induced pluripotent stem cell are immune-tolerated in humanized mice.

It is still unclear if immune responses will compromise the large-scale utilization of human induced pluripotent stem cells (hiPSCs)-derived cell therapies. To answer this question, we used humanized mouse models generated by the adoptive transfer of peripheral blood mononuclear cells or the cotransplantation of hematopoietic stem cells and human thymic tissue. Using these mice, we evaluated the engraftment in skeletal muscle of myoblasts derived either directly from a muscle biopsy or differentiated from hiPSCs or fibroblasts. Our results showed that while allogeneic grafts were mostly rejected and highly infiltrated with human T cells, engraftment of autologous cells was tolerated. We also observed that hiPSC-derived myogenic progenitor cells (MPCs) are not targeted by autologous T cells and natural killer cells in vitro. These findings suggest that the reprogramming and differentiation procedures we used are not immunogenic and that hiPSC-derived MPCs will be tolerated in the presence of a competent human immune system.

Natural Killer Cells Prevent the Formation of Teratomas Derived From Human Induced Pluripotent Stem Cells.

The safe utilization of induced pluripotent stem cell (iPSC) derivatives in clinical use is attributed to the complete elimination of the risk of forming teratomas after transplantation. The extent by which such a risk exists in immune-competent hosts is mostly unknown. Here, using humanized mice reconstituted with fetal hematopoietic stem cells and autologous thymus tissue (bone-liver-thymus humanized mice [Hu-BLT]) or following the adoptive transfer of peripheral blood mononuclear cells(PBMCs) (Hu-AT), we evaluated the capacity of immune cells to prevent or eliminate teratomas derived from human iPSCs (hiPSCs). Our results showed that the injection of hiPSCs failed to form teratomas in Hu-AT mice reconstituted with allogeneic or autologous PBMCs or purified natural killer (NK) cells alone. However, teratomas were observed in Hu-AT mice reconstituted with autologous PBMCs depleted from NK cells. In line with these results, Hu-BLT, which do not have functional NK cells, could not prevent the growth of teratomas. Finally, we found that established teratomas were not targeted by NK cells and instead were efficiently rejected by allogeneic but not autologous T cells in Hu-AT mice. Overall, our findings suggest that autologous hiPSC-derived therapies are unlikely to form teratomas in the presence of NK cells.