Growth factors mobilize CXCR4 low/negative primitive hematopoietic stem/progenitor cells from the bone marrow of nonhuman primates Nadim Mahmud, Hetal.

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Growth factors mobilize CXCR4 low/negative primitive hematopoietic stem/progenitor cells from the bone marrow of nonhuman primates Nadim Mahmud, Hetal Patel, Ronald Hoffman Biology of Blood and Marrow Transplantation Volume 10, Issue 10, Pages 681-690 (October 2004) DOI: 10.1016/j.bbmt.2004.07.002 Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Expression of CXCR4 by PB MNC is inversely related to the number of PB CFC after growth factor-induced mobilization. During administration of recombinant human stem cell factor and rhG-CSF to juvenile baboons, PB MNC were analyzed at different time points to determine the kinetics of the appearance of CXCR4+ cells by flow cytometry (A), as well as assayable progenitor cells in PB MNC (B). The kinetics of CXCR4+ cells and the number of CFC are plotted from 2 different baboons (PA6660 and PA5909). Biology of Blood and Marrow Transplantation 2004 10, 681-690DOI: (10.1016/j.bbmt.2004.07.002) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 The absolute number of CD34+ cells in PB and BM during steady state (day 0) and after growth factor administration (day 7). The data were obtained from 2 different baboons: PA6660 (A) and PA5909 (B). Each bar represents the number of CD34+ cells per microliter of bone marrow or peripheral blood (mean ± SE) obtained from 2 separate animals. Biology of Blood and Marrow Transplantation 2004 10, 681-690DOI: (10.1016/j.bbmt.2004.07.002) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Subpopulations of CD34+ bone marrow cells were sorted on the basis of Hoechst/rhodamine (Ho/Rho) staining as described previously [16]. Forward and side scatter profiles of CD34+HoLow/RhoLow cells (A) and gating on propidium iodide-negative live cells (B) are shown. Gated live cells on the basis of the gate shown in (A) and (B) are presented as histograms (C). The dark filled histogram represents the isotype control, the dotted line histogram is an overlay representing expression of CXCR4 by CD34+ cells before sorting, and the continuous line histogram is a overlay representing expression of CXCR4 by CD34+ cells sorted flow cytometrically on the basis of HoLow/RhoLow, the most quiescent and primitive population in baboons [16]. M1 was set on the basis of isotype control. Biology of Blood and Marrow Transplantation 2004 10, 681-690DOI: (10.1016/j.bbmt.2004.07.002) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Bone marrow CD34+ cells were sorted flow cytometrically on the basis of CXCR4 expression. The isotype for CXCR4 phycoerythrin is shown in (A), and the cursor lines in the dot plots are set according to the isotype control. The sort regions for CD34+CXCR4+ and CD34+CXCR4− cells are shown in (B). Biology of Blood and Marrow Transplantation 2004 10, 681-690DOI: (10.1016/j.bbmt.2004.07.002) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Content of hematopoietic progenitor cells within CD34+CXCR4+ and CD34+CXCR4− cells obtained from steady-state baboon bone marrow. The bar graph represents the mean number of colonies ± SEM assayed in triplicate from 2 different baboons. Biology of Blood and Marrow Transplantation 2004 10, 681-690DOI: (10.1016/j.bbmt.2004.07.002) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 Distribution of CAFC frequency among different subpopulations of marrow CD34+ cells based on CXCR4 expression from steady-state baboon bone marrow. The bar graph represents the mean ± SEM of the frequency of CAFC obtained from 2 different animals. Biology of Blood and Marrow Transplantation 2004 10, 681-690DOI: (10.1016/j.bbmt.2004.07.002) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 7 CD34+ MPB and BM cells were cultured ex vivo in media supplemented with FBS containing G-CSF and SCF for 40 hours and examined flow cytometrically for expression of CXCR4 on CD34+ cells. MPB cells are shown before (A) and after (B) culture. (C) BM before culture; (D) BM CD34+ cells after culture. The cursor line on the dot plot was placed on the basis of matched isotype control. The percentage of each subpopulation of cells is shown in each quadrant. Biology of Blood and Marrow Transplantation 2004 10, 681-690DOI: (10.1016/j.bbmt.2004.07.002) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions