Hess David C, Abe Takanori, Hill William D, Studdard Angeline Martin, Carothers Jo, Masuya Masahiro, Fleming Paul A, Drake Christopher J, Ogawa Makio
Department of Neurology, Medical College of Georgia, Augusta, GA 30912, USA.
Exp Neurol. 2004 Apr;186(2):134-44. doi: 10.1016/j.expneurol.2003.11.005.
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.
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.
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.
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.
骨髓来源的细胞可分化为多种细胞类型。骨髓包含异质性的干细胞和祖细胞群体，包括造血干细胞、骨髓基质细胞以及其他可能的祖细胞。为明确造血细胞向非造血细胞（内皮细胞、神经元和神经胶质细胞）转分化的能力，必须证明单个细胞或该单个细胞的克隆（克隆分析）能分化为构成血管或脑内其他细胞的细胞。
我们培育出了从单个增强型绿色荧光蛋白（EGFP）干细胞实现高水平造血重建的小鼠。为此，我们结合荧光激活细胞分选（FACS）和细胞培养，以产生源自单个造血干细胞（Lin-、CD34-、c-kit+和Sca-1+）的细胞群体。然后将细胞克隆群体移植到经致死性照射的受体小鼠体内。移植后3 - 4个月，部分小鼠接受大脑中动脉（MCA）缝合闭塞手术。在不同时间点对组织进行EGFP免疫细胞化学检测以及用细胞特异性标志物进行双重标记。
在所有移植小鼠的脑实质中均可见EGFP+高度分支状细胞。这些细胞用RCA120凝集素染色，具有实质小胶质细胞的特征。在未发生梗死的小鼠脑内以及接受MCA闭塞手术小鼠的未梗死脑区，有许多EGFP+细胞呈血管周围分布，与大小血管均有关联。这些细胞紧密贴附于血管壁，有些细胞具有包裹内皮细胞的长突起。MCA闭塞后，缺血组织中有表达EGFP的细胞流入，与“新生血管形成”共定位。这些EGFP+细胞在内皮细胞周围呈白蛋白样位置包裹，不共表达血管性血友病因子或CD31。我们检测到罕见的EGFP和NeuN双标记细胞。我们检测到两种染色模式。较常见的模式是EGFP表达细胞吞噬NeuN细胞。然而，我们也检测到更罕见的细胞，通过共聚焦显微镜观察，EGFP和NeuN似乎共定位。
造血干细胞可分化为脑实质小胶质细胞和血管周围细胞。脑缺血后这些细胞数量增加。因此，造血干细胞是脑实质小胶质细胞的一个来源以及血管周围细胞的一个来源。脑梗死后，有罕见的造血干细胞来源细胞用神经元标志物NeuN染色。然而，更常见的模式似乎是EGFP+小胶质细胞吞噬受损神经元。
