1. Low c-Kit Expression Level Induced by Stem Cell Factor Does Not Compromise Transplantation of Hematopoietic Stem Cells 
Chia-Ling Chen, Katerina Faltusova, Martin Molik, Filipp Savvulidi, Ko-Tung Chang, Emanuel Necas 
Biology of Blood and Marrow Transplantation 
Volume 22, Issue 7, Pages (July 2016)
DOI: /j.bbmt
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

2. Biology of Blood and Marrow Transplantation 2016 22, 1167-1172DOI: (10
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 1
Gating strategy to analyze immature hematopoietic cells in the bone marrow with depressed c-Kit expression level. c-Kit fluorescence-minus-one samples were used to delimit c-Kit+ cells. c-Kit+ cells lacking lineage markers (Lin−) were further characterized according to the expression of Sca-1 antigen and CD150 and CD48 markers. The mean values ± standard deviation of the percentage of CD150/CD48 LSK subtypes are from 10 independent determinations.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 2
Spleen colonies and blood cell production derived from transplanted normal and regenerating bone marrow. (A) Lethally irradiated mice were transplanted with 105 normal BMCs or with 5 × 105 BMCs obtained 16 days after sublethal irradiation with 6 Gy. c-Kit expression profiles of Lin−c-Kit+ cells in the donor bone marrows was determined and the results presented. Spleens were collected 12 days after transplantation. (B) Equal numbers (20,000 per mouse) of Lin−c-Kit+ cells from normal bone marrow (CD45.1) and from bone marrow collected 14 days after irradiation of mice (CD45.2) with 6 Gy were co-transplanted 1:1 to lethally irradiated CD45.1/CD45.2 F1 hybrid mice. The ratio of CD45.1-to-CD45.2 nucleated BMCs was determined in peripheral blood 1 month and 6 months after transplantation. c-Kit expression profiles of Lin−c-Kit+ cells in the donor bone marrows was determined and the results are presented. ∗∗∗ P <.001.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 3
Exposure to SCF in vitro decreased c-Kit receptors in Lin−c-Kit+ cells proportionally to the SCF concentration. Aliquots of normal BMCs were incubated for 60 minutes in DMEM without (Control) or with various concentrations of SCF. After incubation the cells were washed and stained with lineage cocktail antibodies and anti-Sca-1 and anti c-Kit antibodies. In experiments that tested the transplantability of HSPCs with down-regulated c-Kit receptors (see Figures 5 and 6) the concentrations of SCF concentrations 100 to 1000 ng/mL were used.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 4
c-Kit receptors are fully reconstituted on HSPCs within 12 to 14 hours after their consumption induced by SCF. Bone marrow was collected 1 to 14 hours after 1000 ng SCF given i.p. (A) or 1 to 46 hours after 1000 ng SCF given i.v. (B). Control mice were given PBS and were included in each experiment. The MFI was related to the values in control bone marrow in various cell types in each experiment. Data pooled from 6 independent experiments are presented, and each time point shows the results from a single mice.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 5
Down-regulation of c-Kit by SCF did not influence the homing of transplanted LS−K and LSK cells in the bone marrow of sublethally irradiated mice. Bone marrow was split into 2 aliquots, incubated with or without SCF and transplanted, 11 million CD45.2 (A) or 40 million UBC-GFP cells (B), to congenic CD45.1 mice irradiated 3 days earlier with a dose of 4 Gy. After 17 hours in the CD45.2 experiment (A) and 20 hours in the UBC-GFP experiment (B) the bone marrow of recipient mice was analyzed for the proportion of donor LS−K or LSK cells in all cells of the particular phenotype.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

8. Figure 6
Down-regulation of c-Kit by SCF did not influence the capacity of BMCs or separated HSPCs to be transplanted. (A) Colony-forming units-spleen assay. BMCs incubated with or without SCF were transplanted (60,000 cells per mouse) to 9-Gy-irradiated mice. Spleen colonies were counted after 8 or 10 days. Ten million (B) or 8 million (C) BMCs incubated with or without SCF were transplanted to congenic sublethally irradiated (6 or 6.5 Gy) mice. The down-regulation of c-Kit (not shown) did not decrease the engraftment of transplanted cells. (D) BMCs from CD45.1 and CD45.2 mice were split into 2 aliquots and incubated with or without SCF. After washing, congenic samples were mutually mixed in a 1:1 ratio, analyzed for c-Kit expression level, and co-transplanted to irradiated (6.5 Gy) dual CD45.2/CD45.1 F1 mice. CD45.2 cells were more represented independently whether they were or were not exposed to SCF. Therefore, no effect of the markedly different density of c-Kit receptors on the engraftment of transplanted HSPCs was demonstrated. (E) A similar experiment used double-sorted Lin−c-Kit+ CD45.1 and CD45.2 cells (96% and 98% purity). The cells were split into 2 aliquots and incubated with or without SCF, washed, mixed in a 1:1 ratio, and co-transplanted to irradiated (6.5 Gy) dual CD45.2/CD45.1 F1 mice. Markedly different c-Kit expression level (not shown) had no significant effect on competing of the repopulating cells. (F and G) BMCs of CD45.1 mice were incubated with or without SCF and transplanted, 26 million cells to each normal (F) or 15 million cells to each 1 Gy irradiated (G) UBC-GFP mouse. No effect of exposure to SCF was demonstrated. Data from individual mice and their means are presented throughout the figure.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions