Transplantation of mESC-derived TEPs improves thymic reconstitution after allogeneic BMT. (A) Lethally irradiated C57BL/6 mice were injected intrathymically with 1 × 10⁴, 5 × 10⁴, and 10 × 10⁴ TC-1 mESC-derived EpCAM1⁺, EpCAM1⁻ cells (or PBS) and intravenously with 5 × 10⁶ TCD BM from BALB/c mice. Thirty days after BMT, thymic cellularity was analyzed. (B-D) Lethally irradiated C57BL/6 mice were injected intrathymically with 5 × 10⁴ mESC-derived EpCAM1⁺, EpCAM1⁻ cells, or PBS, and intravenously with 5 × 10⁶ TCD BM from BALB/c mice. Thirty days after BMT, the numbers of (B) thymocyte subsets, including CD4 and CD8 double-negative (DN), double-positive (DP), and CD4 or CD8 single-positive (SP) cells; (C) donor-origin ETPs (lin⁻IL-7Rα ⁻c-Kit⁺CD44⁺CD25⁻), and (D) total TECs (CD45⁻EpCAM1⁺MHC II⁺), cTECs (CD45⁻EpCAM1⁺MHC II⁺Ly51⁺), and mTECs (CD45⁻EpCAM1⁺MHC II⁺Ly51⁻) were quantified by flow cytometry. Mean percentages of donor-origin thymocytes in 5 × 10⁴ mESC-derived EpCAM1⁺ cell–, EpCAM1⁻ cell–, or PBS-treated BM transplant recipients are 89.6%, 88.7%, and 88.6%, respectively. Means ± SDs are presented (n = 5-6). *P < .05, **P < .01, and ***P < .001. The data are representative of 3 independent experiments.