# A Xeno-Free Protocol for Rapid Differentiation of Human iPSC-Derived Microglia from the KOLF2.1J Reference Line

## TL;DR

This paper introduces a xeno-free method to rapidly generate human microglia from a standard stem cell line for use in disease modeling and research.

## Contribution

A xeno-free, rapid, and reproducible protocol for generating functional human microglia from the KOLF2.1J hiPSC line.

## Key findings

- The protocol enables uniform microglial differentiation within two weeks using doxycycline-inducible transcription factors.
- Generated i-Microglia display mature features like phagocytic activity and immune marker upregulation upon LPS stimulation.
- The system is compatible with co-culture with neurons and includes a fluorescent reporter for tracking microglia.

## Abstract

We present a detailed, xeno-free protocol for the rapid differentiation of human induced pluripotent stem cells (hiPSCs) into microglia using the well-characterized KOLF2.1J reference line. This system employs doxycycline-inducible expression of six transcription factors (6-TF), stably integrated into the CLYBL safe harbor locus, to drive uniform microglial differentiation within two weeks. Building upon an established transcription factor-driven approach, our protocol includes key optimizations for KOLF2.1J, including culture on Laminin-521 to support xeno-free conditions. The resulting i-Microglia exhibit hallmark features of mature microglia, including expression of P2RY12, loss of the pluripotency marker SSEA4, phagocytic activity, and upregulation of immune markers (e.g., CD80, CD83) upon LPS stimulation. We also demonstrate compatibility with co-culture systems using iPSC-derived neurons. Additionally, we describe a modification of the line to include a constitutive mCherry reporter integrated into the SH4-2 safe harbor locus, enabling fluorescent tracking of microglia in mixed cultures or in vivo. This protocol provides a reproducible and scalable platform for generating functional human microglia from a widely used hiPSC line, supporting applications in brain tumors and disease modeling, neuroinflammation research, and therapeutic screening.