Delayed Marrow Infusion in Mice Enhances Hematopoietic and Osteopoietic Engraftment by Facilitating Transient Expansion of the Osteoblastic Niche Roberta.

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Delayed Marrow Infusion in Mice Enhances Hematopoietic and Osteopoietic Engraftment by Facilitating Transient Expansion of the Osteoblastic Niche Roberta Marino, Satoru Otsuru, Ted J. Hofmann, Timothy S. Olson, Valeria Rasini, Elena Veronesi, Kelli Boyd, Mostafa Waleed Gaber, Caridad Martinez, Paolo Paolucci, Massimo Dominici, Edwin M. Horwitz Biology of Blood and Marrow Transplantation Volume 19, Issue 11, Pages 1566-1573 (November 2013) DOI: 10.1016/j.bbmt.2013.07.025 Copyright © 2013 Terms and Conditions

Figure 1 Kinetics of osteoblast proliferation and osteopoietic/hematopoietic chimerism. (A) Representative photomicrographs from 6 hours (left) and 48 hours (right) after TBI depicting the proliferation of the endosteal osteoblasts (arrows). (B) Time course of endosteal osteoblast proliferation in the metaphysis after TBI. Proliferation index as described in the text. Data points represent mean ± standard deviation. (C) Bone chimerism by time to cell infusion. Chimerism was defined as the fraction of GFP+ osteoblasts and osteocytes in the metaphysis and epiphysis, determined by immunohistochemical staining and microscopic evaluation. (D) Chimerism within the KLS subpopulation of marrow cells according to the intervals from TBI to cell infusion, as determined by multicolor flow cytometric analysis of marrow isolated from transplanted mice. (E) Bone chimerism, determined as in (C), after different marrow dosing schedules. Bars represent mean ± standard deviation values. Biology of Blood and Marrow Transplantation 2013 19, 1566-1573DOI: (10.1016/j.bbmt.2013.07.025) Copyright © 2013 Terms and Conditions

Figure 2 Comparison of osteopoietic and hematopoietic chimerism in irradiated versus shielded (nonirradiated) limbs according to time from TBI to marrow infusion. Total marrow cell, KLS marrow cell, and bone chimerism were determined in shielded versus irradiated legs 3 weeks post-transplantation, when donor cells were transplanted at (A) 6 hours or (B) 24 hours. Histograms are representative of the flow cytometric analyses for the bone marrow and KLS chimerism in shielded (- - -) and unshielded (—) legs. Total marrow cell and bone chimerism were determined in left and right legs (both unshielded during irradiation) of control mice 3 weeks post-transplantation, when donor cells were transplanted at (C) 6 hours or (D) 24 hours. In all graphs, bars represent the mean ± standard deviation (n = 10). NS, not statistically significant. Biology of Blood and Marrow Transplantation 2013 19, 1566-1573DOI: (10.1016/j.bbmt.2013.07.025) Copyright © 2013 Terms and Conditions

Figure 3 Independent analysis of irradiated and shielded (nonirradiated) limbs for osteopoietic and hematopoietic chimerism. Total marrow cell, KLS marrow cell, and bone chimerism were assessed in the irradiated (A) and shielded (nonirradiated) (B) legs of mice infused with donor cells 6 hours versus 24 hours after TBI. These data are derived from the same experiment as in Figure 2. Bars represent mean ± standard deviation values. NS, not statistically significant. Biology of Blood and Marrow Transplantation 2013 19, 1566-1573DOI: (10.1016/j.bbmt.2013.07.025) Copyright © 2013 Terms and Conditions

Figure 4 Engraftment of CD150+ CD48− CD41− (SLAM) cells in irradiated and shielded (nonirradiated) limbs. SLAM cells were measured in the bone marrow from the shielded or irradiated legs of mice transplanted at (A) 6 hours or (B) 24 hours after TBI and analyzed for chimerism and other engraftment parameters. The absolute SLAM count represents the number of SLAM cells per 106 viable marrow cells as determined by flow cytometry. Chimerism is the percentage of donor-derived (GFP+) SLAM cells. The absolute GFP+ SLAM count represents the number of GFP+ SLAM cells per 106 viable marrow cells. (C) Comparison of the absolute GFP+ SLAM count in the irradiated limb when marrow was infused 6 hours versus 24 hours after TBI. (D) Comparison of the absolute GFP+ SLAM count in the shielded (nonirradiated) limb when marrow was infused 6 hours versus 24 hours after TBI. In all panels, bars represent the mean ± standard deviation values. NS, not statistically significant. Biology of Blood and Marrow Transplantation 2013 19, 1566-1573DOI: (10.1016/j.bbmt.2013.07.025) Copyright © 2013 Terms and Conditions

Figure 5 Endosteal osteoblast proliferation in the irradiated versus shielded (nonirradiated) limbs. The photomicrographs depict H & E staining of femora taken from mice 24 hours after TBI. (A) Femur section from an irradiated limb; arrows indicate osteoblasts. (B) Femur section from shielded limb depicting regions of proliferating osteoblasts (multiple cell layers, left) and quiescent osteoblasts (single cell layer, right). (C) Femur section from a control (nonirradiated) mouse included for comparison; arrows indicate osteoblasts. (D) Selected region of a femur from a shielded limb demonstrating so-called plump osteoblasts in contrast to the more flattened cells seen elsewhere in the shielded limb (B) or during homeostasis (C). (E) Comparison of the magnitude of osteoblast proliferation, scored according to the proliferation index (see Methods) among femora from nonirradiated mice (controls), shielded limbs from irradiated mice, and irradiated limbs. Data were analyzed using a 1-way analysis of variance with Tukey's multiple comparison test comparing each experimental group with the controls. The bars represent mean ± standard deviation. Biology of Blood and Marrow Transplantation 2013 19, 1566-1573DOI: (10.1016/j.bbmt.2013.07.025) Copyright © 2013 Terms and Conditions