Contribution of Hematopoietically-Derived Progenitors to the Central Nervous System in Mixed Chimeric Mice

Abstract

Introduction: Microglia are resident immune cells of the central nervous system (CNS) and work to maintain a homeostatic environment to allow for proper neuronal functioning. Previous studies suggest microglia originate both from a neural progenitor cell population and possibly a myeloid progenitor. This study aims to establish a working model to both quantitatively and qualitatively determine the contribution of hematopoietically-derived myeloid precursors to the CNS microglia population in a non-disease state. Methods: Normal C57BL/6 mice were treated with Busulfan (150mg/10g weight; days -2 & -1) followed by administration of syngeneic green fluorescent protein (GFP+) positive bone marrow cells (1× and 20×106) to create hematopoietic chimeras. Peripheral chimerism in the blood was assessed at 24 hours, 48 hours, and biweekly post bone marrow transplant (BMT) using flow cytometric analysis. Mice were saline perfused, brains extracted and split along the midsagittal plane to allow for both flow cytometic analysis and immunoflourescent imaging of each animal at 48 hours, and one, seven, eighteen and twenty-four weeks post BMT. 30μm tissue sections were obtained with a freezing microtome and microglia were identified with anti-mouse Iba-1 primary and Alexa Fluor 594 fluorescent secondary antibodies. Results: By four weeks post-BMT, stable peripheral chimerism was established across multiple cell lineages, including greater than 90% GFP+ donor cells in the myeloid lineages. Both flow cytometric and immunoflourescent data demonstrate that GFP+ donor cells contribute to the CNS by seven weeks post-BMT, as seen by the appearance of GFP+ cells within the Mac1+ CD45mid population (˜3-5%). Figure 1 presents immunofluorescent staining of week 7 brain tissue within the left prefrontal cortex of a chimeric mouse: Panel A represents Iba-1+ labeled microglia cells; Panel B represents GFP+ cells; and Panel C represents a merged image showing double positive cells in yellow as the newly infiltrated donor-derived microglia. White scale bars represent 50μm.Figure: [Iba1+ GFP+ in mixed chimera]Conclusion: This study demonstrates a novel experimental model that utilizes a non-irradiation conditioning protocol to establish chimerism and allows for simultaneous quantitative and qualitative analysis of the microglia population within the CNS tissue. These data demonstrate by both flow cytometric and immunofluorescent analysis that hematopoietically-derived progenitors can contribute to the CNS, and may appear as early as seven weeks post-BMT.