Astrocyte cell surface marker phenotyping: Identification of multipotent ACSA-2-/GLAST+ cerebellar progenitor cells

Astrocytes, a member of the glial cell family, are the most abundant neural cell type of the central nervous system (CNS) and present functional and morphological heterogeneity. Among other characteristics, astrocytes provide trophic functions, are essential for the formation of the blood-brain barrier, regulate ion homeostasis, synaptogenesis and synaptic plasticity. Despite their diversity astrocyte subpopulations are not well characterized mainly due to the lack of markers that could classify functional subclasses. This study aimed at the phenotyping of astrocyte subpopulations based on cell surface marker expression, their prospective isolation and subsequent molecular and cellular characterization of the identified subsets. The glutamate aspartate transporter (GLAST) is a common astrocyte marker expressed by astrocytes in the CNS. As the GLAST antibody allows for the isolation of astrocytes it was chosen as principal component for the identification of astrocyte subpopulations. One approach, which was not successful, was the generation of novel monoclonal astrocyte subpopulation specific antibodies using GLAST isolated astrocytes and hybridoma techniques. Another approach was the identification of novel astrocyte markers by flow cytometry. 232 cell surface markers were tested for their expression on astrocytes. In total, 15 candidates were identified and subsequently validated by immunohistochemistry (IHC) and the recently established in house technology: Multidimensional In Situ Cytometry Survey (MICS). The expression profile and the nature of the target of the novel Anti-ACSA-2 (ACSA: astrocyte cell surface antigen) antibody were addressed. In co-operation with the group of Dr. Harold Cremer at the Institut de Biologie du Développement de Marseille, the expression profile of the ACSA-2 antigen was analyzed systematically using IHC, immunocytochemistry (ICC), MICS and flow cytometry. Co-expression of ACSA-2 with common astrocyte markers such as GFAP, S100β and GLAST and missing expression on neurons, oligodendrocytes validated ACSA-2 as an astrocyte specific marker. Additional Western Blot analysis, immunoprecipitation and deglycosylation assays pointed out the ACSA-2 antibody addresses a glycosylation structure expressed by astrocytes. In the next step, the expression profile of ACSA-2 and GLAST was investigated in comprehensive analyses of embryonal, neonatal and adult mouse brain using IHC, ICC, MICS, and flow cytometry. ACSA-2 and GLAST demonstrated differential marker expression in distinct brain regions, retina and spinal cord of neonatal mice. Additional studies addressed ACSA-2 and GLAST expression on neural stem cells. The expression of ACSA-2 in the subventricular zone (SVZ) was analyzed using IHC, in vivo electroporation and flow cytometry. ACSA-2 was demonstrated to be co-expressed with GLAST in the adult SVZ as shown by IHC and flow cytometry. Differences between ACSA-2 and GLAST expression were also identified in the rostral migratory stream (RMS) and hippocampus of the adult mouse brain. However, these differences could not be confirmed on the single cell level by flow cytometry. Cerebellar glia revealed significant differences in GLAST and ACSA-2 expression in the adult mouse brain. To address the origin of these potential subpopulations GLAST and ACSA-2 expression was monitored between E13 and P12 in the developing cerebellum. Thereby, ACSA-2-/GLAST+ and ACSA-2+/GLAST+/- cells were identified. Populations were most discriminative between E17 to P3 and frequencies of ACSA-2-/GLAST+ cells decreased with the onset of development. Both fractions were isolated to high purities by magnetic cell sorting. Cell characteristics were addressed by ICC, neurosphere assay, gene expression profiling and transplantation assay. Cell differentiation in vivo was investigated in collaboration with the group of Dr. Annalisa Buffo at the Neuroscienze Institute Cavalieri Ottolenghi in Turin. ACSA-2-/GLAST+ and ACSA-2+/GLAST+/- cells were isolated from the cerebellum of P0-P3 β-actin GFP mice and grafted into cerebellum of homochronic wild-type mice. ACSA-2-/GLAST+ cells were able to differentiate into interneurons (stellate and basket cells), Bergmann glia, astrocytes and oligodendrocytes. In contrast, ACSA-2+/GLAST+/- cells only differentiated into astrocytes and oligodendrocytes. Gene expression profiling was performed to address differences on the transcriptome level. Genes related to a multipotent and GABAergic phenotyp were enriched in the ACSA-2-/GLAST+ sample set (Gli1, Wif1, Nestin, Ptf1a, Ascl-1). Genes involved in cell-cell interactions as well as cell matrix proteins (Gjb2, Gjb6, Gja1, Vitronectin) were enriched in the ACSA-2+/GLAST+/- sample set. In conclusion, this study presents a regional, functional and molecular discrimination of astrocyte subpopulations based on specific cell surface markers. As a result it describes the identification of a novel ACSA-2-/GLAST+ subpopulation of astrocytes which act as multipotent progenitors in the cerebellum

Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes

International audienceAstrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2+ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages