Loss of microenvironmental Ptn leads to enhanced stem cell maintenance in vitro. (A) Scheme of coculture experiment. In brief, Lin⁻ cells from Ly-5.2⁺ WT BM were cocultured with Ptn^KD and pLKO.1 stroma in contact. After 3 weeks, cultures were transplanted into irradiated WT mice. Recipients were killed 16 weeks after injection, and 5 × 10⁶ BM cells were again transplanted into irradiated WT recipients. (B) Total number of hematopoietic cells as percentage of the total cell number (hematopoietic + stromal cells) recovered from the culture. Statistics were performed with a paired Student t test. (C) Example of the flow cytometric analyses after coculture. (D) Hematopoietic subpopulations in stromal cocultures. Myeloid cells were gated as Gr1^low−high, CD11b⁺. LSKs and MMPs were gated as Lin⁻ Ly6a⁺ Kit⁺ cells and Lin⁻ Ly6a⁻ Kit⁺ cells, respectively. Statistics were performed with a paired Student t test. (E) Engraftment in PB 16 weeks after primary transplantation. Animals were counted positive with ≥ 1% engraftment in blood and with donor cells containing ≥ 1% myeloid and lymphoid cells, respectively. (F) Enumeration of Cd34⁻ LSK and MMP populations in BM 16 weeks after primary transplantation of cocultured cells. Shown are the results of mice with ≥ 1% engraftment in BM (n = 4, white bars, pLKO.1 stroma in cocultures; n = 8, black bars, Ptn^KD stroma in cocultures). (G) Frequency of lymphoid and myeloid cells within the donor cell population in PB of transplanted mice, 5, 10, and 16 weeks after transplantation (pLKO.1 = 6; Ptn^KD = 8). Secondary transplantation, donor engraftment levels in BM (H; n = 9), and in PB (I). (J) Secondary transplantation, total number of donor-derived Cd34⁻ LSKs in BM 16 weeks after the start of secondary transplantation, as well as (K) donor-derived MMPs in BM. All values are mean ± SEM; *P < .05.