# Efficient Generation of Induced Pluripotent Stem Cell-Derived Definitive Endoderm Cells with Growth Factors and Small Molecules

## TL;DR

This study compares two methods for generating definitive endoderm cells from stem cells and finds that both work similarly but differ in liver cell development.

## Contribution

The study identifies protocol-specific differences in hepatic specification during definitive endoderm differentiation.

## Key findings

- Both GF and SM protocols produce similar definitive endoderm cells in morphology and gene expression.
- GF protocol leads to better hepatic specification with distinct metabolic protein profiles in hepatoblasts.
- Divergence between protocols is observed during liver-specific development stages.

## Abstract

Definitive endoderm (DE) differentiation leads to the development of the major internal organs including the liver, intestines, pancreas, gall bladder, prostate, bladder, thyroid, and lungs. The two primary methods utilized for in vitro differentiation of induced pluripotent stem cells (iPSCs) into DE cells are the growth factor (GF) and the small molecule (SM) approaches. The GSK-3 inhibitor (CHIR99021) is a key factor for the SM approach. Activin A and Wnt3a are utilized in the GF approach. In this study, both the GF and SM protocols were compared to each other. The results show that both the GF and SM protocol produce DE with a similar morphological phenotype, gene and protein expression, and a similar level of homogeneity and functionality. However, on both the gene expression and proteomic level, there is a divergence between the two protocols during hepatic specification. Proteomic analysis shows that hepatoblasts from the GF protocol have significantly differentially expressed proteins (DEPs) involved in liver metabolic pathways compared to the SM protocol. Well-validated DE differentiation protocols are needed to fully unlock the clinical potential of iPSCs. In the first step of generating DE-derived tissue, either protocol can be utilized. However, for hepatic specification, the GF protocol is more effective.