Vignesh Subramanian ’24

Neural stem cells (NSCs) are multipotent cells capable of differentiating into the major types of mature cells in the central nervous system (CNS) – neurons and glia – and giving rise to unlimited generations of those cells. Alongside progenitor cells, which are descended from NSCs and differentiate into specific target cells, NSCs are critical in advancing neurogenesis, the development of neurons from an organism’s embryonic stage onward. Investigative examinations of neural progenitor cell (NPC) populations have been hindered by the fact that current localization markers like nestin and Sox-2 are not selective for NPCs, and as such cannot tell NPC populations apart from astroglial and other lineages not of interest. One recently explored approach of identifying such novel markers for NPCs involves identifying the epitopes, or determinant binding regions, of antigens to which antibodies released by originating NSCs bind. To determine whether this approach could distinguish surface antigen proteins that bind to NSCs, Stony Brook researcher Dr. Manganas and his team examined the extent of immunolabeling by several antibodies that responsively bind to expressed antigen epitopes.

Researchers first immunocytochemically screened the tail bleeds and whole brain extracts of wild-type C57/BL6 and BALB/C mice to establish whether staining of various cultures by NSC-derived antibodies was visibly apparent. The cultures – subcloned hybridomas (fusions of antibody-producing lymphocytes and myeloma cells), human neuroprogenitor cells, and GFP marker-containing neurospheres – underwent spot excisions, RT-PCR, and LC-MS analyses to narrowly characterize expression of the antigen bound by the specific antibody NSC-6. Researchers then deployed double- and triple-stain immunoblotting protocols to determine whether and where NSC-6 could localize the antigen epitope and thereby establish NSC presence.

Researchers found that the NSC-6 antibody successfully stained both mouse and human NPCs and that NSC-6 could recognize the antigen’s epitope, identified to be brain acid-soluble protein 1 (BASP1). The antibody immunolabeled BASP1 among radial NSCs in the embryonic mouse brain and neurogenic regions like the corpus callosum, anterior commissure, and cerebellum of the postnatal mouse brain, as well as label NSCs in human brain organoids. NSC-6 also localized BASP1 directly to NSCs to the hippocampus, subventricular zone, and spinal cord of the postnatal mouse brain while avoiding most other CNS cells. Researchers could ultimately characterize the temporal regulation of expressed BASP1, suggesting NSC-6 could viably serve as a selective marker. Future research may reaffirm that such markers could be used to track the prognosis of faltering neurogenesis with maximal accuracy.

Works Cited:

[1] L. Manganas, et al., BASP1 labels neural stem cells in the neurogenic niches of the mammalian brain. Nature Scientific Reports 11, 5546 (2021). DOI: 10.1038/s41598-021-85129-1

[2] Image retrieved from: https://directorsblog.nih.gov/2017/04/13/snapshots-of-life-healing-spinal-cord-injuries/