A Guide to Neural Stem Cell Markers

Originating from the embryonic neuroectoderm as neuroepithelial cells, neural stem cells generate neurons, astrocytes, and oligodendrocytes—the majority of the cells in the central nervous system. Neural stem cells have two primary functions: self-renewal and differentiation. This guide summarizes the most commonly used markers of neural stem cells and their immediate progeny.

Markers of Embryonic Neural Stem Cell Self-Renewal

Although the ultimate goal of any stem cell is to differentiate, all stem cell populations, including neural stem cells, must balance this fate with self-renewal. The process of self-renewal allows neural stem cell populations to expand and maintain their stemness, providing a continual source of new cells for differentiation when needed. The balance between differentiation and self-renewal is regulated by opposing transcriptional effectors.

The Notch signaling pathway is integral to this balance. Activation of the Notch 1 receptor leads to the expression of Hes1, Hes3, and Hes5—transcriptional repressors that inhibit proneural gene expression and promote pluripotency and self-renewal. With Notch activation, neural stem cells differentiate. The Hes family of transcription factors is not the only one touched by the Notch signaling pathway, however. The Gli (Gli1, Gli2, Gli3) and SoxB1 (Sox1, Sox2, Sox3) families of transcription factors also help foster pluripotency and self-renewal. Gli transcription factors do so by regulating the expression of Hes transcription factors downstream of Notch activation, whereas SoxB1 transcription factors do so by antagonizing proneural transcriptional effectors such as Ngn2 and ASCL1. SOX2 in particular is among the earliest markers of neural stem cell identity.

Other transcriptional regulators that regulate neural stem cell self-renewal include ID4 and HesR1/HesR2. The former promotes proliferation (but not pluripotency) and the latter represses neural differentiation.

Outside of transcriptional regulators, the filament protein neuroepithelial stem cell protein—or nestin, as it’s more commonly known—participates in neural stem cell survival and self-renewal.

Finally, the nonspecific proliferation markers BrdU (a thymidine analog that is incorporated into proliferating cells and can be detected by antibodies) and Ki67 (a nuclear protein universally associated with self-renewal) can also be used to triangulate neural stem cells, but only in combination with neural stem cell-specific markers.

Markers of Embryonic Neural Stem Cell Differentiation

Neural stem cell differentiation involves the downregulation of many of the self-renewal factors described above. This downregulation of self-renewal factors is accompanied by upregulation of proneural or proglial factors that drive the differentiation of neural stem cells into neurons, astrocytes, and oligodendrocytes.

The switch from radial glial cell to intermediate progenitor cell marks the first step in the process of differentiation. This switch occurs with the upregulation of transcriptional regulators Tbr1/Tbr2, Svet1, Cux1/Cux2, EMX2, FEZF2, and Ctip2. Tbr2 in particular is an exquisite marker of the intermediate progenitor cell stage of neural stem cell differentiation. Its expression is turned off in the next stage, and its expression in earlier stages (the radial glial cell stage) specifies intermediate progenitor cell identity.

Intermediate progenitor cells differentiate into neural progenitor cells, after which these cells differentiate into neurons and glia. These fates and the markers associated with them are outlined below.

Neuronal Differentiation

Neuronal differentiation is induced by neurogenic basic loop-helix-loop (bHLH) transcription factors, such as Ngn2 and Ascl1. As mentioned above, these transcription factors directly oppose transcriptional regulators of neural stem cell proliferation and self-renewal, such as SoxB1 family members.

The transcription factor Pax6 promotes neurogenesis and antagonizes self-renewal whether it’s expressed at high levels or not at all. High levels of Pax6 lead to increased interactions between Pax6 and proneural factors (such as Ascl1), whereas loss of Pax6 lowers the expression of cell cycle regulators, thereby promoting neurogenesis. Pax6 also controls the expression of Notch ligands and Hes5.

In the later stages of neuronal differentiation, neural stem cells begin to express the classical neuronal markers MAP2 and TUJ1. They also express vimentin, but this marker is also expressed by glia.

A number of cell surface markers of early neuronal commitment have been identified in vitro, including CD133, CD146, CD15, CD171, CD184, CD24, CD29, CD326, CD49d, and CDH1/CDH2. However, cell culture conditions, cell cycle phases, and cell sources (i.e. embryonic stem cells versus induced pluripotent stem cells) can influence the expression of these markers. These markers are more thoroughly discussed by De Gioia and colleagues (2020).

Astrocyte Differentiation

Gliogenesis is inhibited during neurogenesis by proneural bHLH transcription factors (e.g. Ngn2). Yet, during the process of neurogenesis, neural progenitor cells become receptive to gliogenic signals, enabling a gliogenic switch toward an astrocyte or oligodendrocyte fate. This switch is driven by gliogenic transcription factors Sox9, Olig1, and Olig2—all three of which support both astrocyte and oligodendrocyte differentiation. The neurogenic transcription factor Pax6 also plays a role in the maturation of astrocytes. The A2B5 antibody has been used to identify glial precursors.

A few markers can be used to infer astrocytic commitment in neural stem cells. GFAP is a commonly used marker of astrocytes, although it is not highly specific to these cells. Further, CD184, CD44, and CD49d have been identified as markers of astrocyte precursors in vitro.

Oligodendrocyte Differentiation

Markers of oligodendrocyte differentiation are better defined than those of astrocytic differentiation. In addition to the markers shared by astrocyte and oligodendrocyte precursors (discussed above), the latter also express Nkx2.2, Nkx6.2, PDGFA, and Sox10. PDGFA is a key determinant of oligodendrocyte fate as well as one of the most useful markers of oligodendrocyte progenitor cells.

Markers of Adult Neural Stem Cells

Neurogenesis persists into adulthood, although it is limited to the subgranular zone of the dentate gyrus of the hippocampus and the subventricular zone bordering the lateral ventricles. The transcriptional programs and markers that typify embryonic neurogenesis largely mirror those of adult neurogenesis. For example, Pax6, Hes family members, SoxB1 family members, neurogenic bHLH transcription factors, and Tbr2 all participate in both developmental phases.

Although the players are largely the same, the timing of expression differs slightly for some markers between embryonic and adult neurogenesis, as discussed by Christie and colleagues (2013).

Table of Neural Stem Cell Markers

The table below lists characteristic neural stem cell proteins as described by review literature. The list includes a variety of marker types, including transcription factors, cell surface proteins, and structural proteins. Accompanying each marker are links to relevant antibodies and ELISA kits that can be used to detect neural stem cells in vitro and in vivo. The associated products are offered by a variety of manufacturers and can serve as a useful reference for neural stem cell characterization.

Note: *A2B5 and FORSE-1 are antibody clones, not genes/proteins. **BRDU is a thymidine analog used to mark proliferating cells. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only). 

References

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2. Yuan SH, Martin J, Elia J, et al. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One. 2011;6(3):e17540. doi:10.1371/journal.pone.0017540

3. Christie KJ, Emery B, Denham M, Bujalka H, Cate HS, Turnley AM. Transcriptional regulation and specification of neural stem cells. Adv Exp Med Biol. 2013;786:129-155. doi:10.1007/978-94-007-6621-1_8

4. Zhang J, Jiao J. Molecular Biomarkers for Embryonic and Adult Neural Stem Cell and Neurogenesis. Biomed Res Int. 2015;2015:727542. doi:10.1155/2015/727542

5. Vinci L, Ravarino A, Fanos V, et al. Immunohistochemical markers of neural progenitor cells in the early embryonic human cerebral cortex. Eur J Histochem. 2016;60(1):2563. doi:10.4081/ejh.2016.2563

6. De Gioia R, Biella F, Citterio G, et al. Neural Stem Cell Transplantation for Neurodegenerative Diseases. Int J Mol Sci. 2020;21(9):3103. doi:10.3390/ijms21093103