Figure 2
Loss of microenvironmental Ptn leads to enhanced stem cell maintenance in vivo. (A) Experimental design of serial transplantation experiments. WT Ly5.1 BM cells (2 × 105) were transplanted into Ptn−/− and Ptn+/+ littermate (Ly-5.2+) recipients irradiated with 9 Gy. Sixteen weeks after transplantation, recipients were killed, and 1 × 106 BM cells were serially transplanted into WT secondary recipients. Again, mice were killed 16 weeks after transplantation, and 2 × 106 BM cells were serially transplanted into tertiary irradiated WT mice. Finally, 5 × 106 BM cells of these tertiary recipients were again transplanted into irradiated quaternary WT recipients. (B) Engraftment levels as percentage of Ly-5.1+ donor cells in BM of first-degree recipients, n = 5 (+/+), n = 10 (−/−). (C) Primary transplantation, engraftment levels in PB 16 weeks after transplantation. (D) Secondary transplantation, engraftment levels in BM 16 weeks after transplantation, percentage of donor cells in total cell count of 4 long bones, n = 10 (Ptn+/+), n = 8 (Ptn−/−). (E) Secondary transplantation, engraftment levels in PB 16 weeks after transplantation. (F) Tertiary transplantation, engraftment levels in BM 16 weeks after transplantation as percentage of donor cells; n = 7 (Ptn+/+), n = 5 (Ptn−/−). (G) Tertiary transplantation, engraftment levels in PB 16 weeks after transplantation. (H) Quaternary transplantation, engraftment levels in BM 16 weeks after transplantation, as percentage of donor cells; n = 5 (Ptn+/+), n = 6 (Ptn−/−). (I) Quaternary transplantation, engraftment levels in PB 16 weeks after transplantation. (J) Calculated absolute numbers of Cd34−LSK and MMP cells per 4 long bones in the serial transplantations, based on the number of transplanted BM cells. (K) Flow cytometric analysis of BM of quaternary transplants. LSK and MMP are gated in Lin− cells, Cd34−LSKs were gated in the LSK cells. All values are mean ± SEM; *P < .05.

Loss of microenvironmental Ptn leads to enhanced stem cell maintenance in vivo. (A) Experimental design of serial transplantation experiments. WT Ly5.1 BM cells (2 × 105) were transplanted into Ptn−/− and Ptn+/+ littermate (Ly-5.2+) recipients irradiated with 9 Gy. Sixteen weeks after transplantation, recipients were killed, and 1 × 106 BM cells were serially transplanted into WT secondary recipients. Again, mice were killed 16 weeks after transplantation, and 2 × 106 BM cells were serially transplanted into tertiary irradiated WT mice. Finally, 5 × 106 BM cells of these tertiary recipients were again transplanted into irradiated quaternary WT recipients. (B) Engraftment levels as percentage of Ly-5.1+ donor cells in BM of first-degree recipients, n = 5 (+/+), n = 10 (−/−). (C) Primary transplantation, engraftment levels in PB 16 weeks after transplantation. (D) Secondary transplantation, engraftment levels in BM 16 weeks after transplantation, percentage of donor cells in total cell count of 4 long bones, n = 10 (Ptn+/+), n = 8 (Ptn−/−). (E) Secondary transplantation, engraftment levels in PB 16 weeks after transplantation. (F) Tertiary transplantation, engraftment levels in BM 16 weeks after transplantation as percentage of donor cells; n = 7 (Ptn+/+), n = 5 (Ptn−/−). (G) Tertiary transplantation, engraftment levels in PB 16 weeks after transplantation. (H) Quaternary transplantation, engraftment levels in BM 16 weeks after transplantation, as percentage of donor cells; n = 5 (Ptn+/+), n = 6 (Ptn−/−). (I) Quaternary transplantation, engraftment levels in PB 16 weeks after transplantation. (J) Calculated absolute numbers of Cd34LSK and MMP cells per 4 long bones in the serial transplantations, based on the number of transplanted BM cells. (K) Flow cytometric analysis of BM of quaternary transplants. LSK and MMP are gated in Lin cells, Cd34LSKs were gated in the LSK cells. All values are mean ± SEM; *P < .05.

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