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

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.

The CD24 surface antigen in neural development and disease.

A cell's surface molecular signature enables its reciprocal interactions with the associated microenvironments in development, tissue homeostasis and pathological processes. The CD24 surface antigen (heat-stable antigen, nectadrin; small cell lung cancer antigen cluster-4) represents a prime example of a neural surface molecule that has long been known, but whose diverse molecular functions in intercellular communication we have only begun to unravel. Here, we briefly summarize the molecular fundamentals of CD24 structure and provide a comprehensive review of CD24 expression and functional studies in mammalian neural developmental systems and disease models (rodent, human). Striving for an integrated view of the intracellular signaling processes involved, we discuss the most pertinent routes of CD24-mediated signaling pathways and functional networks in neurobiology (neural migration, neurite extension, neurogenesis) and pathology (tumorigenesis, multiple sclerosis).

Verteporfin-induced proteotoxicity impairs cell homeostasis and survival in neuroblastoma subtypes independent of YAP/TAZ expression.

Neuroblastoma (NB) is a highly aggressive extracranial solid tumor in children. Due to its heterogeneity, NB remains a therapeutic challenge. Several oncogenic factors, including the Hippo effectors YAP/TAZ, are associated with NB tumorigenesis. Verteporfin (VPF) is an FDA-approved drug shown to directly inhibit YAP/TAZ activity. Our study aimed to investigate VPF's potential as a therapeutic agent in NB. We show that VPF selectively and efficiently impairs the viability of YAP/TAZ-expressing NB GI-ME-N and SK-N-AS cells, but not of non-malignant fibroblasts. To investigate whether VPF-mediated NB cell killing is YAP-dependent, we tested VPF potency in CRISPR-mediated YAP/TAZ knock-out GI-ME-N cells, and BE(2)-M17 NB cells (a MYCN-amplified, predominantly YAP-negative NB subtype). Our data shows that VPF-mediated NB cell killing is not dependent on YAP expression. Moreover, we determined that the formation of higher molecular weight (HMW) complexes is an early and shared VPF-induced cytotoxic mechanism in both YAP-positive and YAP-negative NB models. The accumulation of HMW complexes, involving STAT3, GM130 and COX IV proteins, impaired cell homeostasis and triggered cell stress and cell death mechanisms. Altogether, our study shows significant in vitro and in vivo VPF-induced suppression of NB growth, making VPF a potential therapeutic candidate against NB.

Glycan Epitope and Integrin Expression Dynamics Characterize Neural Crest Epithelial-to-Mesenchymal Transition (EMT) in Human Pluripotent Stem Cell Differentiation.

The neural crest gives rise to progeny as diverse as peripheral neurons, myelinating cells, cranial muscle, bone and cartilage tissues, and melanocytes. Neural crest derivation encompasses complex morphological change, including epithelial-to-mesenchymal transition (EMT) and migration to the eventual target locations throughout the body. Neural crest cultures derived from stem cells provide an attractive source for developmental studies in human model systems, of immediate biomedical relevance for neurocristopathies, neural cancer biology and regenerative medicine, if only appropriate markers for lineage and cell type definition and quality control criteria were available. Implementing a defined, scalable protocol to generate neural crest cells from embryonic stem cells, we identify stage-defining cluster-of-differentiation (CD) surface markers during human neural crest development in vitro. Acquisition of increasingly mesenchymal phenotype was characterized by absence of neuroepithelial stemness markers (CD15, CD133, CD49f) and by decrease of CD57 and CD24. Increased per-cell-expression of CD29, CD44 and CD73 correlated with established EMT markers as determined by immunofluorescence and immunoblot analysis. The further development towards migratory neural crest was associated with decreased CD24, CD49f (ITGA6) and CD57 (HNK1) versus an enhanced CD49d (ITGA4), CD49e (ITGA5) and CD51/CD61 (ITGAV/ITGB3) expression. Notably, a shift from CD57 to CD51/CD61 was identified as a sensitive surrogate surface indicator of EMT in neural crest in vitro development. The reported changes in glycan epitope and integrin surface expression may prove useful for elucidating neural crest stemness, EMT progression and malignancies.

Surface marker profiling of SH-SY5Y cells enables small molecule screens identifying BMP4 as a modulator of neuroblastoma differentiation.

Neuroblastoma is the most common extra-cranial solid tumor in children. Its broad spectrum of clinical outcomes reflects the underlying inherent cellular heterogeneity. As current treatments often do not lead to tumor eradication, there is a need to better define therapy-resistant neuroblastoma and to identify new modulatory molecules. To this end, we performed the first comprehensive flow cytometric characterization of surface molecule expression in neuroblastoma cell lines. Exploiting an established clustering algorithm (SPADE) for unbiased visualization of cellular subsets, we conducted a multiwell screen for small molecule modulators of neuroblastoma phenotype. In addition to SH-SY5Y cells, the SH-EP, BE(2)-M17 and Kelly lines were included in follow-up analysis as in vitro models of neuroblastoma. A combinatorial detection of glycoprotein epitopes (CD15, CD24, CD44, CD57, TrkA) and the chemokine receptor CXCR4 (CD184) enabled the quantitative identification of SPADE-defined clusters differentially responding to small molecules. Exposure to bone morphogenetic protein (BMP)-4 was found to enhance a TrkAhigh/CD15-/CD184- neuroblastoma cellular subset, accompanied by a reduction in doublecortin-positive neuroblasts and of NMYC protein expression in SH-SY5Y cells. Beyond yielding novel marker candidates for studying neuroblastoma pathology, our approach may provide tools for improved pharmacological screens towards developing novel avenues of neuroblastoma diagnosis and treatment.

The Hippo pathway member YAP enhances human neural crest cell fate and migration.

The Hippo/YAP pathway serves as a major integrator of cell surface-mediated signals and regulates key processes during development and tumorigenesis. The neural crest is an embryonic tissue known to respond to multiple environmental cues in order to acquire appropriate cell fate and migration properties. Using multiple in vitro models of human neural development (pluripotent stem cell-derived neural stem cells; LUHMES, NTERA2 and SH-SY5Y cell lines), we investigated the role of Hippo/YAP signaling in neural differentiation and neural crest development. We report that the activity of YAP promotes an early neural crest phenotype and migration, and provide the first evidence for an interaction between Hippo/YAP and retinoic acid signaling in this system.

Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.