Figure 3
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 PtnKD 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 Gr1low−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, PtnKD 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; PtnKD = 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.

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 PtnKD 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 Gr1low−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, PtnKD 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; PtnKD = 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.

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