# Protocol for morphogen-guided differentiation of brain cell types using human induced pluripotent stem cells

## TL;DR

This paper provides a detailed protocol for differentiating human stem cells into brain cell types to study neurological disorders.

## Contribution

A novel protocol for morphogen-guided differentiation of hiPSCs into microglia, astrocytes, and mixed cortical cultures is introduced.

## Key findings

- Hypoxia enhances microglial production from hiPSCs.
- Serum-free astrocyte differentiation is achieved using knockout serum replacement.
- Quality-control measures improve differentiation outcomes for astrocytes and mixed cortical cultures.

## Abstract

Modeling neurological disorders is challenging due to differing functional genomics and phenotypes among species. Here, we present a protocol for morphogen-guided differentiation of human induced pluripotent stem cells (hiPSCs) into microglia, astrocytes, and mixed cortical cultures (MCCs) for studying human brain disorders. We describe steps for enhancing microglial production using hypoxia and implementing quality-control measures for astrocyte and MCC differentiations. We detail knockout serum replacement procedures for serum-free astrocytes. This protocol enables cell-type-specific investigation of disease mechanisms and drug screening.

•Guidance for morphogen- and small-molecule differentiation of human iPSC neural cultures•Steps for hypoxia-facilitated differentiation of hematopoietic progenitors at high yield•Instructions for serum-free astrocyte differentiation•Procedures for troubleshooting neural differentiation with contaminant cell types

Guidance for morphogen- and small-molecule differentiation of human iPSC neural cultures

Steps for hypoxia-facilitated differentiation of hematopoietic progenitors at high yield

Instructions for serum-free astrocyte differentiation

Procedures for troubleshooting neural differentiation with contaminant cell types

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Modeling neurological disorders is challenging due to differing functional genomics and phenotypes among species. Here, we present a protocol for morphogen-guided differentiation of human induced pluripotent stem cells (hiPSCs) into microglia, astrocytes, and mixed cortical cultures (MCCs) for studying human brain disorders. We describe steps for enhancing microglial production using hypoxia and implementing quality-control measures for astrocyte and MCC differentiation. We detail knockout serum replacement procedures for serum-free astrocytes. This protocol enables cell-type-specific investigation of disease mechanisms and drug screening.