Figure 4.
Evidence for a critical role of local BM KITL production for erythropoiesis but not HSCs. (A) Representative FACS profiles and mean (± SEM) percentage recipient CD45.1 reconstitution of LSK and LSK CD150+CD48– (HSCs) in kidney capsule–transplanted (CD45.2) E15.5 wild-type (control; n = 8) and Sl/Sl (n = 4) bones 3 weeks after transplantation. (B) Representative FACS plots showing the distribution of LEPR+ PVCs and CD31+ ECs after first gating as negative for the hematopoietic antigens CD45 and TER119 (CD45–TER119–) in the BM of a nontransplanted femur from 2.5-week-old wild-type mice (n = 5) and in E15.5 femur from wild-type (n = 8) and Sl/Sl (n = 4) embryos analyzed 3 to 4 weeks after transplantation under the kidney capsule of wild-type adult recipient mice. (C) Mean (± SEM) expression of total Kitl (Kitl exon 2-3 amplicon) relative to housekeeping genes (Hprt, B2m, and Gapdh) in ECs and LEPR+ PVCs isolated from femurs of 2.5-week-old wild-type mice (n = 5), and E15.5 femurs from wild-type (n = 5) and Sl/Sl (n = 4) embryos 3 to 4 weeks after kidney capsule transplantation (KCT). (D) Experimental design for evaluating impact on hematopoietic stem and progenitor cell (HSPC) numbers within E15.5 wild-type or Sl/Sl (Kitl-deficient) bones transplanted into recipients with normal levels of KITL. (E) Mean (± SEM) BM cellularity of E15.5 femurs derived from CD45.2 wild-type (control) and Sl/Sl mice 3 weeks after transplantation under the kidney capsule of adult CD45.2 wild-type mice (n = 8 of each genotype). (F-G) Representative FACS profiles with gated cell populations as percentages of total BM cells (F) and mean (± SEM) LSK CD150+CD48– (HSC) numbers (G) per femur (n = 5 of each genotype). (H) Mean (± SEM) PB reconstitution 4 and 16 weeks after competitive transplantation of whole BM cells (CD45.2) from transplanted femurs from (E-G) in competition with wild-type CD45.1 BM cells into irradiated CD45.1 recipients. (I) Mean (± SEM) number of Lin–SCA1–KIT+ (LK) cells (including all committed progenitor cells) in transplanted femurs from panels D and E (n = 8 of each genotype). (J-K) Representative FACS profiles (J) and mean (± SEM) numbers (K, top row) and frequency (K, bottom) of distinct erythroid progenitor subsets in the BM of transplanted femurs from panels D and E (n = 8). All data represent mean ± SEM, and the nonparametric Mann-Whitney test was used to assess statistical significance. ∗P < .05; ∗∗P < .01.

Evidence for a critical role of local BM KITL production for erythropoiesis but not HSCs. (A) Representative FACS profiles and mean (± SEM) percentage recipient CD45.1 reconstitution of LSK and LSK CD150+CD48– (HSCs) in kidney capsule–transplanted (CD45.2) E15.5 wild-type (control; n = 8) and Sl/Sl (n = 4) bones 3 weeks after transplantation. (B) Representative FACS plots showing the distribution of LEPR+ PVCs and CD31+ ECs after first gating as negative for the hematopoietic antigens CD45 and TER119 (CD45–TER119–) in the BM of a nontransplanted femur from 2.5-week-old wild-type mice (n = 5) and in E15.5 femur from wild-type (n = 8) and Sl/Sl (n = 4) embryos analyzed 3 to 4 weeks after transplantation under the kidney capsule of wild-type adult recipient mice. (C) Mean (± SEM) expression of total Kitl (Kitl exon 2-3 amplicon) relative to housekeeping genes (Hprt, B2m, and Gapdh) in ECs and LEPR+ PVCs isolated from femurs of 2.5-week-old wild-type mice (n = 5), and E15.5 femurs from wild-type (n = 5) and Sl/Sl (n = 4) embryos 3 to 4 weeks after kidney capsule transplantation (KCT). (D) Experimental design for evaluating impact on hematopoietic stem and progenitor cell (HSPC) numbers within E15.5 wild-type or Sl/Sl (Kitl-deficient) bones transplanted into recipients with normal levels of KITL. (E) Mean (± SEM) BM cellularity of E15.5 femurs derived from CD45.2 wild-type (control) and Sl/Sl mice 3 weeks after transplantation under the kidney capsule of adult CD45.2 wild-type mice (n = 8 of each genotype). (F-G) Representative FACS profiles with gated cell populations as percentages of total BM cells (F) and mean (± SEM) LSK CD150+CD48– (HSC) numbers (G) per femur (n = 5 of each genotype). (H) Mean (± SEM) PB reconstitution 4 and 16 weeks after competitive transplantation of whole BM cells (CD45.2) from transplanted femurs from (E-G) in competition with wild-type CD45.1 BM cells into irradiated CD45.1 recipients. (I) Mean (± SEM) number of Lin–SCA1–KIT+ (LK) cells (including all committed progenitor cells) in transplanted femurs from panels D and E (n = 8 of each genotype). (J-K) Representative FACS profiles (J) and mean (± SEM) numbers (K, top row) and frequency (K, bottom) of distinct erythroid progenitor subsets in the BM of transplanted femurs from panels D and E (n = 8). All data represent mean ± SEM, and the nonparametric Mann-Whitney test was used to assess statistical significance. ∗P < .05; ∗∗P < .01.

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