Quantitation of Human Cells that Produce Neutrophils and Platelets in Vivo Obtained from Normal Donors Treated with Granulocyte Colony–Stimulating Factor.

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Quantitation of Human Cells that Produce Neutrophils and Platelets in Vivo Obtained from Normal Donors Treated with Granulocyte Colony–Stimulating Factor and/or Plerixafor Paul H. Miller, Naoto Nakamichi, David J.H.F. Knapp, Gabrielle Rabu, Kirk R. Schultz, David M. Jones, Stephen Couban, Connie J. Eaves Biology of Blood and Marrow Transplantation Volume 22, Issue 11, Pages 1945-1952 (November 2016) DOI: 10.1016/j.bbmt.2016.07.023 Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Experimental design. (A) Five healthy adult donors received a single subcutaneous injection of P. (B) Another 5 donors received a single injection of P 24 hours after 4 daily doses of G. Pretreatment (D0), post-G/pre-P (post-G), 4 hours after P (P), and 24 hours after P (P+24) PB samples were collected as shown. (C) Assessments of CD34+ cells, CFCs, and CFC output activity in 3-week and 6-week LTCs performed on separately pooled PB and BM samples for each time point for both protocols (prepared from equal aliquots by volume [PB] or cell number [BM]). (D) Assessments of multiple measures of hematopoietic activity in matching individual day 0 and post-G and/or P PB samples. *- indicates a time point when only PB was collected. Biology of Blood and Marrow Transplantation 2016 22, 1945-1952DOI: (10.1016/j.bbmt.2016.07.023) Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Hematopoietic progenitors are maximally elevated in the PB of normal donors 4 hours after P administration, regardless of a prior course of G. Kinetics of changes in progenitor levels in matched PB (A-D) and BM (E-H) samples from donors treated with P only (red), just after G (blue), and after G + P (purple). Pooled samples were assessed for CD34+ cells (Figures 2A,E), CFCs (Figures 2B,F), CFC outputs in 3-week LTCs (Figures 2C,G), and in 6-week LTCs (Figures 2D,H). PB and BM sample analyses were based on initial blood volume and TNC counts, respectively. (This figure is available in color online at www.bbmt.org.) Biology of Blood and Marrow Transplantation 2016 22, 1945-1952DOI: (10.1016/j.bbmt.2016.07.023) Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 G + P produced the greatest increases in PB content of multiple progenitor types. (A) CFC assays showed significantly greater increases in G + P (purple) versus day 0 as compared to donors treated with G (blue) or P only (red). Three-week LTCs (B) and 6-week LTCs (C) showed consistently greater increases in outputs of both mature cells (TNCs and CD33/15+ cells) and precursors (CD34+ cells and CFCs). LTCs were initiated with volume-matched aliquots of PB cells from each donor. Horizontal bars with asterisks indicate statistical significance (* = P ≤ .05; ** = P ≤ .01; *** = P ≤ .001). (This figure is available in color online at www.bbmt.org.) Biology of Blood and Marrow Transplantation 2016 22, 1945-1952DOI: (10.1016/j.bbmt.2016.07.023) Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 In vivo xenograft assays show greatest activity elicited by the G + P mobilization protocol. Individual day 0 and post-treatment PB samples were transplanted into irradiated NRG-3GS mice, and 3, 6, and 12 weeks later, total human CD45+, CD45+CD33/15+ (myeloid), CD45+CD19+ (B-lineage), and CD41+CD61+ platelets were assessed in the PB of these mice. Blue, red, and purple bars indicate results for G-only, P only, and G + P samples, respectively. Horizontal bars with asterisks indicate statistical significance (* = P ≤ .05; ** = P ≤ .01; *** = P ≤ .001). (This figure is available in color online at www.bbmt.org.) Biology of Blood and Marrow Transplantation 2016 22, 1945-1952DOI: (10.1016/j.bbmt.2016.07.023) Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Hierarchical clustering reveals a high concordance between human myeloid cell outputs in 3-week LTC and 3-weeks after transplantation in NRG-3GS mice. Dendrograms show the results of an unsupervised analysis of the relatedness of the rank-ordered activities measured in each of the 23 in vitro and in vivo assays performed on individual PB samples. Separate dendrograms are shown for the activities measured from donors treated only with G, only with P, and with G + P, as indicated. All results were derived from post-thaw samples except for prefreeze CD34 counts. Brown, orange, and green letters identify phenotypic-, culture-based, and in vivo assays, respectively. Colored branches identify assay clusters. (This figure is available in color online at www.bbmt.org.) Biology of Blood and Marrow Transplantation 2016 22, 1945-1952DOI: (10.1016/j.bbmt.2016.07.023) Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions