Hematopoietic origin of microglial and perivascular cells in brain

Abstract

Background: Bone marrow (BM)-derived cells differentiate into a wide variety of cell types. BM contains a heterogeneous population of stem and progenitor cells including hematopoietic stem cells, marrow stromal cells, and perhaps other progenitor cells. 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. Methods: We generated mice that exhibited a high level of hematopoietic reconstitution from a single enhanced green fluorescent protein (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 (Lin⁻, CD34⁻, c-kit⁺, and Sca-1⁺). Clonal populations of cells were then transplanted into lethally irradiated recipient mice. After 3–4 months of engraftment, some mice underwent middle cerebral artery (MCA) suture occlusion. EGFP immunocytochemistry and dual labeling was performed with cell-specific markers on tissue from various time points. Results: In all transplanted mice, EGFP⁺ highly ramified cells were seen in the brain parenchyma. These cells stained with RCA120 lectin and had the characteristics of parenchymal microglial cells. In brains without infarction and in uninfarcted brain regions of mice that underwent MCA occlusion, there were many EGFP⁺ cells in a perivascular distribution, associated with both small and larger blood vessels. The cells were tightly apposed to the vessel wall and some had long processes that enveloped the endothelial cells. After MCA occlusion, there was an influx of EGFP expressing cells in the ischemic tissue that colocalized with the “neovascularization.” These EGFP⁺ cells were wrapped around endothelial cells in an albuminal location and did not coexpress von Willebrand Factor or CD31. We detected rare dual-labeled EGFP and NeuN-expressing cells. We detected two staining patterns. The more frequent pattern was phagocytosis of NeuN cells by EGFP expressing cells. However, we also detected rarer cells where the EGFP and NeuN appeared to be colocalized by confocal microscopy. Conclusions: HSC differentiate into parenchymal microglial cells and perivascular cells in the brain. The numbers of these cells increase after cerebral ischemia. The HSC is therefore one source of parenchymal microglial cells and a source for perivascular cells. After a cerebral infarction, there are rare HSC-derived cells that stain with the neuronal marker, NeuN. However, the more common pattern appears to represent phagocytosis of damaged neurons by EGFP⁺ microglial cells.

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.