# PPDPF is not a key regulator of human pancreas development

## TL;DR

This study shows that PPDPF is not a key regulator of human pancreas development, unlike its zebrafish counterpart.

## Contribution

The study reveals the limited role of PPDPF in human pancreatic development using CRISPR/Cas9 in hPSCs.

## Key findings

- PPDPF knockout in hPSCs had a modest effect on pancreatic progenitor development in vitro.
- PPDPF knockout did not affect lineage specification in vivo after orthotopic transplantation.
- PPDPF genetic variants are linked to glucose levels but not to type 2 diabetes risk in humans.

## Abstract

Given their capability to differentiate into each cell type of the human body, human pluripotent stem cells (hPSCs) provide a unique platform for developmental studies. In the current study, we employed this cell system to understand the role of pancreatic progenitor differentiation and proliferation factor (PPDPF), a protein that has been little explored so far. While the zebrafish orthologue exdpf is essential for exocrine pancreas specification, its importance for mammalian and human development has not been studied yet. We implemented a four times CRISPR/Cas9 nicking approach to knockout PPDPF in human embryonic stem cells (hESCs) and differentiated PPDPFKO/KO and PPDPFWT/WT cells towards the pancreatic lineage. In contrast to data obtained from zebrafish, a very modest effect of the knockout was observed in the development of pancreatic progenitors in vitro, not affecting lineage specification upon orthotopic transplantation in vivo. The modest effect is in line with the finding that genetic variants near PPDPF are associated with random glucose levels in humans, but not with type 2 diabetes risk, supporting that dysregulation of this gene may only result in minor alterations of glycaemic balance in humans. In addition, PPDPF is less organ- and cell type specifically expressed in higher vertebrates and its so far reported functions appear highly context-dependent.

Human pluripotent stem cells (hPSCs) care capable of differentiating into nearly all cells of the human body and thereby serve in research as a powerful tool to study developmental processes in humans. In this study, we explored the role of PPDPF, a poorly understood gene linked to pancreas development. While the zebrafish equivalent of this gene, exdpf, plays a critical role in forming the exocrine pancreas, its developmental function in mammals, including humans, had not been studied in detail. By using CRISPR/Cas9 technology, we “knocked out” the PPDPF gene in hPSCs and directed these cells to form pancreas-specific cells. Interestingly, unlike zebrafish, the “knock-out” of PPDPF was not essential for proper pancreatic cell type specification in human cells. Our findings underscore the importance of studying genes across different species to understand their evolutionary and functional nuances. The role of PPDPF appears to be more specific and critical in zebrafish, while in humans, it may rather act as a subtle regulator rather than a key driver. This less important role aligned with genetic data suggesting that variations in the PPDPF gene influence glucose levels but are not significantly associated with the risk to develop diabetes.