Hematopoietic origin of microglial and perivascular cells in brain
Introduction
Bone marrow (BM)-derived progenitor and stem cells show surprising plasticity and are capable of multilineage engraftment. Recent studies reveal that BM cells differentiate into hepatocytes, muscle cells, endothelial cells, glial cells, and neurons in vivo Brazelton et al., 2000, Eglitis and Mezey, 1997, Ferrari et al., 1998, Jackson et al., 2001, Krause et al., 2001, Lagasse et al., 2000, Mezey et al., 2000, Priller et al., 2001a, Priller et al., 2001b. BM cells also generate new neurons in humans (Mezey et al., 2003). This engraftment of BM cells into nonhematopoietic tissues is enhanced by injury to the organ Hess et al., 2002, Krause et al., 2001, Lagasse et al., 2000. Since the brain has limited repair capacity after injury, the findings that BM cells generate neurons has potential clinical and therapeutic relevance. In a brain injury such as stroke, where multiple cell types are injured and die, it will be important to repair not only neurons but also the blood vessels and glial cells, which support these neurons.
One of the limitations of the studies demonstrating BM plasticity is the heterogeneity of BM cells transplanted. Crude or unfractionated BM contains at least two populations of stem cells: hematopoietic stem cells (HSC) and mesenchymal stromal cells (Bianco et al., 2001). Crude BM may also contain endothelial stem cells (angioblasts) or even neural and other progenitor cells. Most studies to date showing differentiation of BM cells into microglia, neurons, or astrocytes have utilized crude, unfractionated BM cell transplantation. There is less known about the differentiation potential of purer populations such as clonal HSC. HSC differentiate into epithelial cells of the lung, GI tract, and skin and into hepatocytes (Krause et al., 2001). In another study that transplanted single HSC, there was little engraftment in noninjured organs, although a single Purkinje cell in the cerebellum was HSC derived (Wagers et al., 2002).
To establish unequivocally the transdifferentiation capability of a hematopoietic cell to a nonhematopoietic cell (endothelial cells, neurons, and glial cells), it is imperative to demonstrate that a single cell or clone of that single cell (clonal analysis) differentiates into cells comprising vessels or other cells in the brain. We demonstrate that parenchymal microglial cells and perivascular cells are derived from the HSC. After temporary middle cerebral artery (MCA) occlusion, there is dramatic increase in these cell types.
Section snippets
Mice
C57BL/6-Ly5.1 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Transgenic enhanced green fluorescent protein (EGFP) mice (C57BL/6-Ly5.2 background) were kindly provided by M. Okabe (Osaka University, Japan). These two strains of mice were bred and maintained at the Animal Research Facility of the Veterans Affairs Medical Center. All aspects of the animal research have been conducted in accordance with the guidelines set by the Institutional Animal Care and Use Committee of the
Immunocytochemistry
EGFP epifluorescence was detected directly simply using an FITC or GFP filter set, and EGFP immunofluorescence was examined using anti-GFP antibodies (rabbit anti-GFP, Molecular Probes) with detection of the anti-GFP antibody via anti-rabbit IgG-FITC-conjugated secondary antibodies (Jackson ImmunoResearch). Double labeling was done with antibodies to specific cell types: endothelial: vWF (rabbit, DAKO); CD31/PECAM (goat, Santa Cruz); microglial: RCA 120 lectin (Vector); F4/80 (rat, Serotec);
Results
We generated mice that exhibit a high level of hematopoietic reconstitution from a single EGFP+ stem cell. To achieve this, we combined FACS sorting and cell culture to generate a population of cells derived from a single hematopoietic stem cell. Clonal populations of cells were then transplanted into lethally irradiated recipient mice. Two months later, analysis of the nucleated blood cells from these mice revealed variable levels of hematopoietic chimerism and lineage combinations. Fig. 1 is
Discussion
We were able to generate and study mice that exhibit a high level of hematopoietic reconstitution from a single EGFP stem cell. The advantage of this approach over transplanting unfractionated BM is the ability to trace the lineage of an EGFP-labeled cell in the brain back to a single HSC. This is important because BM contains a heterogeneous population of stem and progenitor cells. This approach allowed us to examine the in vivo differentiation potential of the HSC as opposed to mesenchymal
Acknowledgements
Supported by NIH 1R21 NS43487-01, VA Medical Research Service, NIH grants PO1-CA78582, RO1-DK54197, RO1-HL69123, HL 57375, HL 52813, and DAMD 17-00-1-0038 and the Cardiovascular Development Biology Center Medical University of South Carolina (MUSC). We would also like to acknowledge assistance of Dr. Haiqun Zeng in FACS sorting and staffs of the Department of Radiation Oncology at MUSC in irradiation of mice.
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