DPT is essential for ex vivo proliferation and maintenance of long-term repopulating cells. (A) Fold difference in the expression of membrane-bound or extracellular matrix genes between nonirradiated or irradiated AFT024 and 2018 stroma at the RNA level based on qRT-PCR (ΔΔ cycle threshold [ΔΔCt] method). (B) Comparison of proliferation rates of founder HSCs cultured on different knockdown AFT024 lines (gray bars) or wild-type (wt) stroma (AFT024, dark gray bar; 2018, white bar). AFT024 knockdown lines included scrambled shRNA control (n = 3 independent experiments, 103 trees), single DLK1^KD (20% knockdown efficiency, n = 3 independent experiments, 133 trees), single DLK1^KD (90% knockdown by fluorescence-activated cell sorting, n = 4 independent experiments, 163 trees), single DPT^KD (99% knockdown, RNA level, n = 6 independent experiments, 211 trees), single FAP^KD (95% knockdown, RNA level, n = 4 independent experiments, 109 trees), double DLK1DPT^KD (n = 3 independent experiments, 111 trees), double DLK1FAP^KD (n = 4 independent experiments, 150 trees), double DPTFAP^KD (n = 3 independent experiments, 120 trees), and triple DLK1DPTFAP^KD (n = 5 independent experiments, 196 trees). (C) Proliferation rates of founder HSCs or early MPPs upon coculture with wild-type, DLK1^KD (n = 3 independent experiments, early MPP 108 trees), DPT^KD (n = 3 independent experiments, 91 early MPP trees), or FAP^KD stroma (n = 4 independent experiments, 184 early MPP trees). (D) Experimental approach for in vivo transplantation of sorted HSCs cultured on knockdown cell lines prior to injection into sublethally irradiated recipients. (E) CD45.1 HSCs (1250) were sorted and cocultured with different stroma cell lines. After 7 days of coculture, the content of each well was transplanted into a CD45.2 sublethally irradiated recipient. The peripheral blood (PB) contribution of donor CD45.1 cells was analyzed at several time points up to 32 weeks posttransplantation.