Transplantation of hypoantigeneic hESC. (a) HLA I knockdown in hESC^KD was followed by flow cytometry and showed levels between 1% and 12% of those of naïve hESC between days 7 and 42 (means ± s.e.m.). (b) A total of 10⁶ hESCs or hESC^KD were transplanted into the gastrocnemius muscle of either immunocompetent Balb/c or severely immunocompromised SCID-beige mice. All Balb/c mice rapidly rejected the hESC transplants, whereas the cells survived in SCID-beige recipients. Rejection of hESC^KD was markedly attenuated and four out of ten hESC^KD grafts achieved long-term survival. (c) On day 5, BLI signals from hESCs in SCID-beige and hESC^KD in Balb/c were similarly strong, whereas signals from hESCs in Balb/c were negligible. (d) Balb/c cellular immune activation on the same day was significantly stronger after hESC rather than hESC^KD transplantation. *P<0.05 compared with hESCs; ^†P<0.001 compared with hESCs. (e) The percentage of Treg cells (CD25+ Foxp3+ cells) among the CD4+ population in inguinal lymph nodes was monitored over time. The Treg fraction increased after both hESC and hESC^KD transplantation (*P<0.05 compared with native Balb/c) but remained elevated only in the hESC^KD group (^§P<0.01 compared with hESC after 14 days).

Histology of hESC transplants. Intramuscularly-injected hESCs were identified according to their morphology in hematoxylin and eosin (a,b) and their Fluc-positivity (c). At 5 days after transplantation, hESC grafts showed markedly more dense infiltrates of MAC2+ macrophages and CD3+ lymphocytes than hESC^KD grafts (d). Only a few KLRA1+ NK cells were found after hESC or hESC^KD transplantation. (e) Cell numbers of all three cell populations were significantly lower within hESC^KD rather than hESC grafts. ^†P<0.001 compared with hESCs.

Antibody response after hESC transplantation. (a) At 5 days after transplantation into Balb/mice, flow cytometry showed a significantly stronger hESC-specific antibody production for hESCs compared with hESC^KD. *P<0.05 compared with both before transplantation and with hESC^KD transplantation. (b) Luminex single-antigen assays revealed HLA class I-specific antibody induction by hESCs but not hESC^KD, which was massively boosted by hESC re-injection. *P<0.05 compared with native Balb/c; ^§P<0.05 compared with hESCs. HLA class II antibody induction was not observed. (c,d) In recipients of hESC grafts, the mean Z-score of hESC-specific class I antibodies was significantly higher than that of non-hESC-specific antibodies and all of the six hESC-specific HLA-A, -B and -C antibody Z-scores were above a threshold of 3 s.d. #P<0.05. The recipients of hESC^KD did not significantly induce hESC-specific class I antibody production, and recipients of both groups did not develop relevant hESC-specific class II antibodies.