# Protocol for differentiation of vascular smooth muscle cells from human iPSCs and their application in CRISPRa-mediated gene regulation

## TL;DR

This paper provides a detailed protocol for generating vascular smooth muscle cells from human iPSCs and using them for gene regulation studies.

## Contribution

A reproducible protocol for differentiating hiPSCs into vSMCs under chemically defined conditions, with CRISPRa-ready applications.

## Key findings

- A step-by-step protocol for hiPSC-derived vSMC differentiation is described.
- Functional and molecular analysis methods for contractile vSMCs are detailed.
- CRISPRa-mediated gene regulation is enabled in the derived vSMCs.

## Abstract

Directed differentiation of human induced pluripotent stem cells (hiPSCs) holds major promise for the development of disease models, drug screening platforms, and regenerative medicine. Here, we provide a step-by-step highly reproducible protocol for differentiating vascular smooth muscle cells (vSMCs) from hiPSCs, including hiPSC culture, hiPSC differentiation, and vSMC passaging under chemically defined conditions. We also detail molecular and functional analysis procedures for hiPSC-derived contractile vSMCs along with endogenous transcriptional activation modulation ready for any downstream application.

•Steps for generating vSMCs from hiPSCs in chemically defined conditions•Procedures for differentiating epicardial-like intermediates into vSMCs•Guidance on assessing contractility using collagen gel contraction assays•Instructions for molecular characterization and CRISPRa in vSMCs

Steps for generating vSMCs from hiPSCs in chemically defined conditions

Procedures for differentiating epicardial-like intermediates into vSMCs

Guidance on assessing contractility using collagen gel contraction assays

Instructions for molecular characterization and CRISPRa in vSMCs

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

Directed differentiation of human induced pluripotent stem cells (hiPSCs) holds major promise for the development of disease models, drug screening platforms, and regenerative medicine. Here, we provide a step-by-step highly reproducible protocol for differentiating vascular smooth muscle cells (vSMCs) from hiPSCs, including hiPSC culture, hiPSC differentiation, and vSMC passaging under chemically defined conditions. We also detail molecular and functional analysis procedures for hiPSC-derived contractile vSMCs along with endogenous transcriptional activation modulation ready for any downstream application.