# Single-cell glycome and transcriptome profiling uncovers the glycan signature of each cell subpopulation of human iPSC-derived neurons

## TL;DR

A new method reveals hidden cell types in stem cell-derived neurons and identifies unique sugar markers to detect non-neural cells.

## Contribution

The study introduces glycan markers and detection probes for non-neuronal cells in iPSC-derived neurons using single-cell glycan-RNA sequencing.

## Key findings

- Four subpopulations of iPSC-derived neurons were identified, including non-neural undiffNPCs and mesenchymal cells.
- Glycan markers like α1,3-fucose and poly-LacNAc were found to distinguish undiffNPCs and mesenchymal cells.
- Pseudotime analysis showed NPCs can transition into mesenchymal cells during differentiation.

## Abstract

Human induced pluripotent stem cell (iPSC)-derived neurons are often heterogeneous, posing challenges for disease modeling and cell therapy. We previously developed single-cell glycan and RNA sequencing (scGR-seq) to analyze the glycome and transcriptome simultaneously. Here, we applied scGR-seq to examine heterogeneous populations of human iPSC-derived neurons. We identified four subpopulations: mature neurons, immature neurons, undifferentiated neural progenitor cells (undiffNPCs), and mesenchymal cells (MCs). Lectin-binding patterns indicated high α1,3-fucose expression in undiffNPCs. MCs exhibited strong binding of a poly-LacNAc-recognizing lectin (rLSLN) and high expression of B3GNT2, a poly-LacNAc synthetic enzyme. Pseudotime analysis revealed that a subpopulation of NPCs acquired mesenchymal features and differentiated into MCs. Immunocytochemistry confirmed the specific detection of undiffNPCs and MCs using anti-Lewis X (α1,3-fucosylated glycan) antibodies and rLSLN. Beyond identifying cell heterogeneity, scGR-seq enables the discovery of glycan markers and detection probes for iPSC-derived cells, aiding in their further cell processing and manipulation.

•Single-cell glycan and RNA sequencing revealed heterogeneity in iPSC-derived neurons•Undifferentiated NPCs and mesenchymal cells (MCs) were identified as non-neural cells•Glycan markers of undifferentiated NPCs and MCs were identified•Glycan markers enabled the detection of non-neural cells in neural cultures

Single-cell glycan and RNA sequencing revealed heterogeneity in iPSC-derived neurons

Undifferentiated NPCs and mesenchymal cells (MCs) were identified as non-neural cells

Glycan markers of undifferentiated NPCs and MCs were identified

Glycan markers enabled the detection of non-neural cells in neural cultures

Tateno and colleagues applied single-cell glycan-RNA sequencing (scGR-seq) to induced pluripotent stem cell (iPSC)-derived neurons, revealing heterogeneity and identifying glycan signatures for each cell type. In addition, we identified glycan markers of non-neuronal cells and their detection probes, lectins. These results demonstrate the value of scGR-seq in developing technologies to detect and remove undesirable cells in regenerative medicine.