Figure 2
Figure 2. VDR loss alters BM hematopoiesis but not PB hematopoiesis. (A) No change in BM-resident colony number. VDR−/− and VDR+/+ animals were fed a normal diet immediately after weaning. At 8 weeks of age, animals were killed, and the bones were removed and crushed with a mortar and pestle. Total femur MNC numbers were obtained with a hemocytometer (trypan blue exclusion). MNCs were plated in M3434 methylcellulose (Stem Cell Technologies) and scored 12 days later for total colony (defined by a cluster of ≥ 20 cells) number. Colony number was then corrected for total BM MNC number per femur. Bars represent averages. Individual mice are represented by individual data points (n = 8). P value was calculated via an unpaired Student t test. (B) No change in BM HSC frequency. VDR−/− and VDR+/+ animals were fed a normal diet immediately after weaning. At 8 weeks of age, animals were killed, and the bones removed and crushed with a mortar and pestle. BM MNCs from 3 pooled VDR+/+ animals or 3 pooled VDR−/− animals (WT and KO were both CD45.2+) were competed at various doses (2 × 106, 2 × 105, 5 × 104, and 1 × 104) with a constant (2 × 105) number of CD45.1+ WT BM MNCs and transplanted into lethally irradiated (9.5 Gy) hosts (8-10 recipients per cell dose). Sixteen weeks after transplantation, multilineage reconstitution (B220; Gr-1 or CD11b; CD4 or CD8). All antibodies were purchased from BioLegend, BD Biosciences PharMingen, and eBioscience. Antibody clones used are as follows: GK1.5 (anti-CD4), 53-6.7 (anti-CD8a), RA3-6B2 (anti-B220), RB6-865 (anti–Gr-1), and M1/70 (anti-CD11b) in the PB obtained by tail vein nicking by VDR+/+ and VDR−/− donor cells was assessed, and recipient mice, which possessed less than 1% VDR donor cells in any one of the 3 lineages, were scored as nonresponders. Each one of the dosage groups was evaluated for nonresponders. The frequency of multilineage reconstituting HSCs in the original donor marrow was calculated from these data using Poisson statistics in the L-Calc software package (StemCell Technologies). For ease of graphing on a logarithmic scale, groups in which zero nonresponders were present were arbitrarily assigned a “1%” value in panel B (□ represents WT data points; ▵, KO data points). Broken line represents WT trend line; and solid line, KO trend line. P value for the comparison of VDR−/− and VDR+/+ HSC frequency was also calculated in L-Calc using Poisson statistics. (C) Decreased BM cellularity in VDR−/− animals. VDR−/− and VDR+/+ animals were fed a normal diet immediately after weaning. At 8 weeks of age, animals were killed, and the bones removed and crushed with a mortar and pestle. Total femur MNC numbers were obtained with a hemocytometer (trypan blue exclusion). Bars represent averages. Individual mice are represented by individual data points (n = 8). P value was calculated via an unpaired Student t test. (D) No change in PB HPC number. VDR−/− and VDR+/+ animals were fed a normal diet immediately after weaning. At 8 weeks of age, PB was obtained by tail vein nicking. The red blood cells (RBCs) in a defined volume of blood were lysed in ammonium chloride lysis buffer, and the resultant PB MNCs were plated in M3434 methylcellulose (StemCell Technologies) and scored 12 days later for total colony (defined by a cluster of ≥ 20 cells) number. Bars represent averages. Individual mice are represented by individual data points (n = 6). P value was calculated via an unpaired Student t test. (E) No change in PB white blood cell (WBC) count. VDR−/− and VDR+/+ animals (n = 3) were fed a normal diet immediately after weaning. At 8 weeks of age, PB was obtained by tail vein nicking. Complete blood counts (CBCs) were obtained using a Hemavet 850 (Drew Scientific). (F) No change in PB RBC count. VDR−/− and VDR+/+ animals (n = 3) were fed a normal diet immediately after weaning. At 8 weeks of age, PB was obtained by tail vein nicking. CBCs were obtained using a Hemavet 850 (Drew Scientific). (G) No change in PB platelet count. VDR−/− and VDR+/+ animals (n = 3) were fed a normal diet immediately after weaning. At 8 weeks of age, PB was obtained by tail vein nicking. CBCs were obtained using a Hemavet 850 (Drew Scientific).

VDR loss alters BM hematopoiesis but not PB hematopoiesis. (A) No change in BM-resident colony number. VDR−/− and VDR+/+ animals were fed a normal diet immediately after weaning. At 8 weeks of age, animals were killed, and the bones were removed and crushed with a mortar and pestle. Total femur MNC numbers were obtained with a hemocytometer (trypan blue exclusion). MNCs were plated in M3434 methylcellulose (Stem Cell Technologies) and scored 12 days later for total colony (defined by a cluster of ≥ 20 cells) number. Colony number was then corrected for total BM MNC number per femur. Bars represent averages. Individual mice are represented by individual data points (n = 8). P value was calculated via an unpaired Student t test. (B) No change in BM HSC frequency. VDR−/− and VDR+/+ animals were fed a normal diet immediately after weaning. At 8 weeks of age, animals were killed, and the bones removed and crushed with a mortar and pestle. BM MNCs from 3 pooled VDR+/+ animals or 3 pooled VDR−/− animals (WT and KO were both CD45.2+) were competed at various doses (2 × 106, 2 × 105, 5 × 104, and 1 × 104) with a constant (2 × 105) number of CD45.1+ WT BM MNCs and transplanted into lethally irradiated (9.5 Gy) hosts (8-10 recipients per cell dose). Sixteen weeks after transplantation, multilineage reconstitution (B220; Gr-1 or CD11b; CD4 or CD8). All antibodies were purchased from BioLegend, BD Biosciences PharMingen, and eBioscience. Antibody clones used are as follows: GK1.5 (anti-CD4), 53-6.7 (anti-CD8a), RA3-6B2 (anti-B220), RB6-865 (anti–Gr-1), and M1/70 (anti-CD11b) in the PB obtained by tail vein nicking by VDR+/+ and VDR−/− donor cells was assessed, and recipient mice, which possessed less than 1% VDR donor cells in any one of the 3 lineages, were scored as nonresponders. Each one of the dosage groups was evaluated for nonresponders. The frequency of multilineage reconstituting HSCs in the original donor marrow was calculated from these data using Poisson statistics in the L-Calc software package (StemCell Technologies). For ease of graphing on a logarithmic scale, groups in which zero nonresponders were present were arbitrarily assigned a “1%” value in panel B (□ represents WT data points; ▵, KO data points). Broken line represents WT trend line; and solid line, KO trend line. P value for the comparison of VDR−/− and VDR+/+ HSC frequency was also calculated in L-Calc using Poisson statistics. (C) Decreased BM cellularity in VDR−/− animals. VDR−/− and VDR+/+ animals were fed a normal diet immediately after weaning. At 8 weeks of age, animals were killed, and the bones removed and crushed with a mortar and pestle. Total femur MNC numbers were obtained with a hemocytometer (trypan blue exclusion). Bars represent averages. Individual mice are represented by individual data points (n = 8). P value was calculated via an unpaired Student t test. (D) No change in PB HPC number. VDR−/− and VDR+/+ animals were fed a normal diet immediately after weaning. At 8 weeks of age, PB was obtained by tail vein nicking. The red blood cells (RBCs) in a defined volume of blood were lysed in ammonium chloride lysis buffer, and the resultant PB MNCs were plated in M3434 methylcellulose (StemCell Technologies) and scored 12 days later for total colony (defined by a cluster of ≥ 20 cells) number. Bars represent averages. Individual mice are represented by individual data points (n = 6). P value was calculated via an unpaired Student t test. (E) No change in PB white blood cell (WBC) count. VDR−/− and VDR+/+ animals (n = 3) were fed a normal diet immediately after weaning. At 8 weeks of age, PB was obtained by tail vein nicking. Complete blood counts (CBCs) were obtained using a Hemavet 850 (Drew Scientific). (F) No change in PB RBC count. VDR−/− and VDR+/+ animals (n = 3) were fed a normal diet immediately after weaning. At 8 weeks of age, PB was obtained by tail vein nicking. CBCs were obtained using a Hemavet 850 (Drew Scientific). (G) No change in PB platelet count. VDR−/− and VDR+/+ animals (n = 3) were fed a normal diet immediately after weaning. At 8 weeks of age, PB was obtained by tail vein nicking. CBCs were obtained using a Hemavet 850 (Drew Scientific).

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