Analysis of engraftment and differentiation of in vitro–derived progenitor T cells in immunodeficient mice. Human UCB CD34⁺CD38^−/lo cells were differentiated on OP9-DL1 cells for 10 to 12 days, and CD34⁺CD7⁺⁺ (proT) cells were sorted by flow cytometry. Neonatal NOD/SCID/γc^null or BALB/c Rag2^−/−γc^−/− mice were injected intrahepatically with 2.5 to 5.0 × 10⁵ proT cells or 1.5 to 2.5 × 10⁵ UCB-derived CD34⁺ cells. (A) Thymuses were harvested 4 to 6 weeks after transplantation, single-cell suspensions were obtained, and total cellularity was determined. The cellularity shown is for NOD/SCID/γc^null mice that received a transplant of proT cells and that displayed higher than 70% human CD45⁺ chimerism. (B) The percentage of human CD45⁺ cells present in the thymus of individual BALB/c Rag2^−/−γc^−/− and NOD/SCID/γc^null mice that received a transplant of UCB CD34⁺ or in vitro–generated proT cells is shown, with the mean percentage indicated by a horizontal bar. (C) Flow cytometric analysis for cell surface expression of human CD45 of thymocytes from a representative UCB CD34⁺- and proT-injected mouse (top row); middle row panels show human CD45⁺-gated cells analyzed for CD4 and CD8 cell surface expression, and CD3 expression is shown for either DP-gated cells (bottom row) or SP-gated cells (middle and bottom rows), as indicated by arrows. (D) Flow cytometric analysis of human CD45⁺-gated thymocytes from a mouse injected with in vitro–generated proT cells is shown for CD3 and TCRαβ expression (left); and CD3 and TCRγδ expression on CD3⁺TCRαβ⁻-gated cells, as indicated by arrow (right).