Fig. 3

Assessment of T potential of single iHECs from iR9-ESC. a The strategy of T cell induction from iR9-ESC-derived single iHECs. Single iHECs were sorted into individual wells (24-well plates) pre-seeded with OP9-DL1 feeder cells (10,000 cells/well) 12 h prior maturation in EM medium with doxycycline (1 μg/mL). Doxycycline was sustained for 10 days during the maturation step. After maturation, the bulk blood cells were assessed for T lineage generation potential. For in vivo T cell regeneration, the single iHEC-derived bulk hematopoietic cells (day 10) were transplanted into individual B-NDG recipients. For in vitro T cell induction, the medium was changed to T cell induction medium (TIM, α-MEM, 20% DFBS, and 1% GlutaMAX) supplemented with 2% conditioned medium derived from supernatants of AFT024-hFlt3L and AFT024-hIL7 cell culture for sustaining 12 days. b Single iHECs efficiently gave rise to T cells. Three hundred and eighty-four single iHECs at day 11 were sorted into individual wells (24 well plates). Thirty single-iHEC-formed blood colonies were induced for T cell generation in vitro. Cell collections of Twenty-five single-iHEC-formed blood colonies were transplanted into 25 individual B-NDG mice for the assessment of T lymphopoiesis in vivo. c Flow cytometry analysis of induced T cells from in vitro induction of single iHECs. iT cells from single iHEC culture product (day 22) were analyzed. Plots of iT cells induced from one representative colony are shown. d Single iHEC-derived hematopoietic cells gave rise to mature iT cells in PB of B-NDG recipient mice 4 weeks after transplantation. Plots of one representative mouse are shown