PMC

Outline of the process of reprogramming hAFSCs into transient universal (hypoimmunogenic) hiPSCs prepared from multiple AF donors (A) and conventional hiPSCs prepared from a single AF donor (B). The immunotolerance of transient universal (hypoimmunogenic) hiPSCs and their differentiated cardiomyocytes or embryoid bodies (EBs) is evaluated after treatment with mononuclear cells donated by different AF donors

Expression of HLA class I and class II of hAFSCs, several hPSCs and their differentiated cells. A, HLA class Ia and class II expression of hESC (H9)‐derived cardiomyocytes (a) and hiPSC (HPS0077)‐derived cardiomyocytes (b) on day 14 of induction. B, HLA class Ia and class II expression of hAFSCs derived from a single AF donor (hAFSCs (single)) (a), hiPSCs (single) reprogrammed from hAFSCs (single) at passage 20 (b), and cardiomyocytes derived from hiPSCs (single) of passage 20 (c). C, HLA class Ia and class II expression of hAFSCs derived from mixing AF from 2 donors (hAFSCs (mix)) (a), universal hiPSCs (mix‐2) reprogrammed from hAFSCs (mix) at passage 21 (b), and cardiomyocytes derived from universal hiPSCs (mix‐2) of passage 21 (c). D, HLA class Ia and class II expression of hAFSCs derived from mixing AF from 5 donors (hAFSCs (mix)) (a), transient universal hiPSCs (mix‐5) reprogrammed from hAFSCs (mix) at passage 22 (b), and cardiomyocytes derived from transient universal hiPSCs (mix‐5) of passage 22 (c)

Immunogenic reaction of several hPSCs treated with mononuclear cells. A, (a) Survival rates of hESCs (H9), hiPSCs (HPS0077), hiPSCs (single) at passage 20, transient universal hiPSCs (mix‐2) at passage 20, and transient universal hiPSCs (mix‐5) at passage 20, which were calculated before (left red bar) and after (right blue bar) mononuclear cell treatment for 2 days at 37°C. (b) Survival rates of cardiomyocytes derived from hESCs (H9), hiPSCs (HPS0077), hiPSCs (single) at passage 20, transient universal hiPSCs (mix‐2) at passage 21 and transient universal hiPSCs (mix‐5) at passage 22, which were evaluated using flow cytometry before (left red bar) and after (right blue bar) mononuclear cell treatment for 2 days at 37°C. ^* P < .05, ^** P > .05. B, Production of interferon‐γ (IFN‐γ) by (a) hPSCs (hESCs (H9), hiPSCs (HPS0077), hiPSCs (single) at passage 20, transient universal hiPSCs (mix‐2) at passage 20 and transient universal hiPSCs (mix‐5) at passage 20) and (b) cardiomyocytes derived from these hPSCs before (left red bar) and after (right blue bar) mononuclear cell treatment for 2 days at 37°C. ^* P < .05. ^** P > .05. C, Production of interleukin‐6 (IL‐6) by (a) hPSCs (hESCs (H9), hiPSCs (HPS0077), hiPSCs (single) at passage 20, transient universal hiPSCs (mix‐2) at passage 20 and transient universal hiPSCs (mix‐5) at passage 20) and by (b) cardiomyocytes derived from these hPSCs before (left red bar) and after (right blue bar) mononuclear cell treatment for 2 days at 37°C. ^* P < .05, ^** P > .05

Evaluation of the immunogenic reaction of several hPSCs treated with mononuclear cells. A, Outline of immunogenic reaction experiments of hPSCs after contact with mononuclear cells. B, Live (green) and dead (red) staining of (i, v) hESCs (H9), (ii, vi) hiPSCs (HPS0077), (iii, vii) transient universal hiPSCs (mix‐2) at passage 20, which were reprogrammed from hAFSCs derived from multiple AF donors (2 donors), and (iv, viii) transient universal hiPSCs (mix‐5) at passage 20, which were reprogrammed from hAFSCs derived from multiple AF donors (5 donors), where the cells were treated with mononuclear cells for 2 days. The bar indicates 500 μm. C, Live (green) and dead (red) staining of cardiomyocytes derived from (i, v) hESCs (H9), (ii, vi) hiPSCs (HPS0077), (iii, vii) transient universal hiPSCs (mix‐2) at passage 21, and (iv, viii) transient universal hiPSCs (mix‐5) at passage 22, where the cells were treated with mononuclear cells for 2 days. The bar indicates 500 μm. D, E, Live (green, iii, vii) and dead (red, iv, viii) staining of (D) hiPSCs (single) and (E) cardiomyocytes derived from hiPSCs (single) at passage 20 before (i‐iv) and after (v‐viii) treatment of mononuclear cells for 2 days. (i, v) phase contrast images. Photograph (ii) was generated by merging (iii) and (iv). Photograph (vi) was generated by merging (vii) and (viii). The bar indicates 500 μm. F, Live (green) and dead (red) staining of EBs derived from (i, v) hESCs (H9), (ii, vi) hiPSCs (HPS0077), (iii, vii) transient universal hiPSCs (mix‐2) at passage 21, and (iv, viii) transient universal hiPSCs (mix‐5) at passage 21, where the cells were treated with mononuclear cells for 2 days. The bar indicates 500 μm