Comprehensive Cell Surface Antigen Analysis Identifies Transferrin Receptor Protein-1 (CD71) as a Negative Selection Marker for Human Neuronal Cells

Abstract

Cell state‐, developmental stage‐, and lineage‐specific combinatorial expression of cluster of differentiation (CD) molecules enables the identification of cellular subsets via multicolor flow cytometry. We describe an exhaustive characterization of neural cell types by surface antigens, exploiting human pluripotent stem cell‐derived neural cell systems. Using multiwell screening approaches followed by detailed validation of expression patterns and dynamics, we exemplify a strategy for resolving cellular heterogeneity in stem cell paradigms. In addition to providing a catalog of surface antigens expressed in the neural lineage, we identified the transferrin receptor‐1 (CD71) to be differentially expressed in neural stem cells and differentiated neurons. In this context, we describe a role for N‐Myc proto‐oncogene (MYCN) in maintaining CD71 expression in proliferating neural cells. We report that in vitro human stem cell‐derived neurons lack CD71 surface expression and that the observed differential expression can be used to identify and enrich CD71− neuronal derivatives from heterogeneous cultures. stem cells 2019;37:1293–1306

Neural Stem Cell Transplantation for Neurodegenerative Diseases

“…In particular, noggin, a BMP inhibitor, and SB431542, a TGF-β inhibitor, efficiently block the BMP and activin/nodal pathways, promoting the neuralization of human pluripotent stem cells [14]. In-vitro NSC differentiation was tracked at various differential stages with intracellular (Table 1) [20][21][22][23][24][25][26][27][28][29][30][31] and cell-surface (Table 2) markers [15][16][17]28,[32][33][34][35]. The cells express paired box 6 (PAX6) and SRY-box transcription factors 1 (SOX1) and 2 (SOX2), transcription factors involved in the selfrenewal and maintenance of NSCs in an undifferentiated state [20,23].…”

“…Expression of the intermediate filament nestin signals the appearance of neural progenitors and correlates with distinctive properties of progenitor cells, such as multipotency, limited self-renewal and regenerative capacity [25]. In-vitro NSC differentiation was tracked at various differential stages with intracellular (Table 1) [20][21][22][23][24][25][26][27][28][29][30][31] and cell-surface (Table 2) markers [15][16][17]28,[32][33][34][35]. The cells express paired box 6 (PAX6) and SRY-box transcription factors 1 (SOX1) and 2 (SOX2), transcription factors involved in the self-renewal and maintenance of NSCs in an undifferentiated state [20,23].…”

“…Melanoma cell adhesion molecule (CD146) [16,32] P75 neurotrophin receptor (p75) CXCR4, C-X-C chemokine receptor type 4 (CD184) [28,32] Integrin β-1 (CD29)…”

“…This selection can be achieved through positive or negative selection for surface markers, taking advantage of fluorescence-activated or magnetic bead cell sorting. Several different analyses of neural clusters of differentiation (CD) antigen expression on NSCs and their derivatives have been performed [15][16][17]28,[32][33][34][35] and are summarized in Table 2. For example, negative selection for pluripotent markers, such as SSEA-3, SSEA-4, and the keratin sulfate-related antigens TRA-1-60 and TRA-1-81 [15], can be useful to eliminate undifferentiated cells, reducing the risk of teratoma formation after transplantation [16,17], whereas CD71 could be used to distinguish more mature neuronal derivatives from mixed cultures [32].…”

“…Several different analyses of neural clusters of differentiation (CD) antigen expression on NSCs and their derivatives have been performed [15][16][17]28,[32][33][34][35] and are summarized in Table 2. For example, negative selection for pluripotent markers, such as SSEA-3, SSEA-4, and the keratin sulfate-related antigens TRA-1-60 and TRA-1-81 [15], can be useful to eliminate undifferentiated cells, reducing the risk of teratoma formation after transplantation [16,17], whereas CD71 could be used to distinguish more mature neuronal derivatives from mixed cultures [32]. Early committed NSCs have been selected for the positive expression of prominin-1 (CD133), SSEA-1 (CD15), or FORSE-1 [32,33].…”