Hematopoietic origin of microglial and perivascular cells in brain.
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.
Hess
David C
DC
Department of Neurology, Medical College of Georgia, Augusta, GA 30912, USA. dhess@mail.mcg.edu
Abe
Takanori
T
Hill
William D
WD
Studdard
Angeline Martin
AM
Carothers
Jo
J
Masuya
Masahiro
M
Fleming
Paul A
PA
Drake
Christopher J
CJ
Ogawa
Makio
M
D000818:Animals; D000941:Antigens / Q000378:metabolism; D001562:Benzimidazoles / Q000378:metabolism; D001921:Brain / Q000166:cytology* / Q000502:physiology; D002232:Carbocyanines / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D007839:Functional Laterality; D049452:Green Fluorescent Proteins; D006410:Hematopoiesis / Q000502:physiology*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D007150:Immunohistochemistry / Q000379:methods; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D017628:Microglia / Q000166:cytology*; D018613:Microscopy, Confocal / Q000379:methods; D009474:Neurons / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D037121:Plant Lectins / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D011828:Radiation Chimera; D013997:Time Factors; D014841:von Willebrand Factor / Q000276:immunology
D000941:Antigens; D001562:Benzimidazoles; D002232:Carbocyanines; D008164:Luminescent Proteins; D037121:Plant Lectins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C017707:Ricinus communis agglutinin-1; C029417:Von Willebrand antigen; C089187:cyanine dye 3; D014841:von Willebrand Factor; D049452:Green Fluorescent Proteins; C009712:bis-benzimidazole; D010751:Phosphopyruvate Hydratase