Genetically Engineered Hypoimmune Human Muscle Progenitor Cells Can Reduce Immune Rejection

Abstract

Cells face two challenges after transplantation: recognition and killing by lymphocytes, and cell apoptosis induced by the transplantation environment. Our hypoimmune cells aim to address these two challenges through editing of immunomodulatory proteins and overexpression of anti-apoptotic proteins.

FIGURE 1

9G2X enhances the immune‐evasive abilities of mESCs/adult muscle progenitor. a. Candidate immunomodulatory factors and their role in innate and adaptive immune pathways. b. Schematic diagram of PB‐9G2X transposon plasmid construction, electroporation and hypoimmune cell clones screening. c. mESCs (luc vs. 9G2X) were transplanted subcutaneously into the back of allogeneic female C3H mice, and the survival of the transplanted cells in vivo was recorded by in vivo bioluminescence imaging (BLI). d. Statistical analysis of the data in (a) showed that the fluorescence signal of 9G2X mESCs could be maintained longer, relative to those co‐cultured with mESCs controls, n = 3, “ns” represents no significant difference, and “*” represents p < 0.05. e. Representative illustration of the remaining transplant grafts after dissection of allogeneic C3H mice on day 21. f. Identify MHCI‐MHCII‐ double negative cell populations and generate hypoimmune clones by flow cytometry. g. Western blot was used to verify the knockout of B2M and CIITA at the protein level in these hypoimmune cells. h. The expression levels of representative immunosuppressive factors in these clones were detected by qPCR and displayed as radar graphs. i. After mouse splenocytes were co‐cultured with LTS or hypoimmune cells for 5 days, the secretion level of TNFα in the culture supernatant was detected. The secretion of TNFα in groups 2#64, 2#108, 3#78 and 3#86 was significantly reduced, while that in group 2#54 was not significantly changed, relative to those co‐cultured with LTS controls, “ns” represents no significant difference, and “***” represents p < 0.001. j. Mouse splenocytes proliferation incubated with LTS or hypoimmune cells was measured by CFSE staining. Groups 2#64, 2#108, 3#78, and 3#86 reduced the potential for lymphocyte proliferation, while group 2#54 showed no significant change, relative to those co‐cultured with LTS controls, n = 3, “ns” represents no significant difference, “*” represents p < 0.05, and “**” represents p < 0.01. It is possible that clone 2#52 may have accumulated mutations that rendered its epitopes immunogenic.

FIGURE 2

Hypoimmune muscle progenitors have stronger survival potential in vivo. a. Representative images of LTS‐GFP or hypoimmune clone 3#78‐GFP co‐cultured with mouse splenocytes. Statistical analysis of the corresponding proliferation capacity of target cells, n = 3, p < 0.05. b. Detection of CD107a positive rate of mouse splenocytes co‐cultured with LTS or hypoimmune clone 3#78, n = 3, p < 0.001. c. After mouse splenocytes were co‐cultured with LTS or hypoimmune clone 3#78 for 48 h. Annexin‐v positive rate of lymphocytes was detected, n = 3, p < 0.001. d. Changes in the number of cells per unit area during co‐culture of mouse splenocytes with LTS or hypoimmune clone 3#78, n = 3, p < 0.05. e. LTS‐luc and hypoimmune clone 3#78 in vivo validation design and representative images of BLI. f. Representative diagram of mouse dissection and sampling 11 days after LTS and hypoimmune cell transplantation. g. Determination of LTS and hypoimmune cell graft volume. The hypoimmune cell graft volume was significantly larger than that of the LTS control group, n = 3, p < 0.01. In addition, the residual graft volume of 2#62 and 3#86 is also significantly larger than that of 3#78 (not shown in the figure, 2#62 vs. 3#78 (p < 0.01), 3#86 vs. 3#78 (p < 0.05)). h. Detection of inflammation levels in graft. The inflammation levels of hypoimmune clones 2#62 and 3#86 were significantly reduced, n = 3, “ns” represents no significant difference, “*” represents p < 0.05, “**” represents p < 0.01, and “***” represents p < 0.001. i. Pro‐apoptotic factor expression detection, and they showed a significantly down‐regulation trend in 2#62 and 3#86, n = 3, “ns” represents no significant difference, “*” represents p < 0.05, “**” represents p < 0.01, and “***” represents p < 0.001. j. Detection of protein expression levels of immunosuppressive factors in hypoimmune cells.