DPT is sufficient to improve ex vivo maintenance of long-term repopulating cells. (A) Comparison of proliferation rates of founder HSCs cultured on wt (2018: n = 5 independent experiments, 264 trees; AFT024: n = 7 independent experiments, 290 trees) or virally transduced 2018 stroma overexpressing tdTOMATO − 2018^tdTOMATO (mock, n = 3 independent experiments, 120 trees), DLK1 − 2018^DLK1 (n = 4 independent experiments, 162 trees), DPT − 2018^DPT (n = 4 independent experiments, 202 trees), FAP − 2018^FAP (n = 4 independent experiments, 138 trees) or combinations, such as DLK1 and DPT − 2018^DLK1DPT (n = 4 independent experiments, 132 trees), DLK1 and FAP − 2018^DLK1FAP (n = 3 independent experiments, 103 trees), DPT and FAP – 2018^DPTFAP (n = 4 independent experiments, 150 trees) or all 3 − 2018^DLK1DPTFAP (n = 4 independent experiments, 216 trees). (B) Generation-based analysis of HSC and their progeny cultured on the same conditions as in panel A. (C) Similar analysis for early MPPs on wild-type (2018: n = 3 independent experiments, 270 trees; AFT024: n = 5 independent experiments, 279 trees) or virally transduced 2018 stroma overexpressing DLK1 (n = 5 independent experiments, 150 trees), DPT (n = 3 independent experiments, 194 trees), FAP (n = 3 independent experiments, 90 trees), DLK1 and DPT (n = 5 independent experiments, 169 trees), DLK1 and FAP (n = 3 independent experiments, 90 trees), DPT and FAP (n = 3 independent experiments, 93 trees), or all 3 (n = 3 independent experiments, 90 trees). (D) CD45.1 HSCs (125) were sorted and cocultured with wild-type lines (AFT024, 2018) or DPT expressing 2018 stroma for 7 days before being transplanted into sublethally irradiated W41 recipients. Donor contribution was calculated in the peripheral blood (PB) and bone marrow (BM) 20 weeks posttransplantation. (E) Cell-type–specific contribution of donor cells in recipient’s bone marrow. (F) Lineage-specific donor contribution in recipients’ peripheral blood 20 weeks posttransplant.