# Pyrophosphate Regulates Multilineage Differentiation in Stem Cells From Human Exfoliated Deciduous Teeth

## TL;DR

This study shows that inorganic pyrophosphate affects how stem cells from baby teeth develop into different cell types, such as bone and fat cells.

## Contribution

The study reveals a novel regulatory role of pyrophosphate in multilineage differentiation of SHED cells.

## Key findings

- PPi inhibited osteogenic and adipogenic differentiation of SHED cells.
- PPi reduced TRAP-positive osteoclast formation and altered RANKL/OPG expression.
- PPi modulated lipid metabolism and signaling pathways like TGF-β1 and NOTCH.

## Abstract

To explore the cellular behavior of stem cells derived from human exfoliated deciduous teeth (SHED) in response to inorganic pyrophosphate (PPi).

SHED cells were isolated from the dental pulp tissues of human primary exfoliated teeth. Cell proliferation was examined using the MTT assay, colony‐forming unit assay, and cell cycle analysis. Cell migration was evaluated using the scratch assay. Osteogenic differentiation was assessed by the expression of osteogenic marker genes and in vitro mineral deposition. Oil Red O staining was employed to determine intracellular lipid accumulation under adipogenic differentiation. For osteoclast differentiation, TRAP staining was used. The global gene expression profile was examined by RNA sequencing analysis.

PPi reduced early cell apoptosis and enhanced cell migration. PPi inhibited mineral deposition dose‐dependently and significantly reduced DSPP and BGLAP expression. The higher dose of 10 μM PPi decreased RANKL mRNA expression, while it did not influence OPG mRNA levels, resulting in the reduction of the RANKL/OPG expression ratio. Culture medium from PPi‐treated SHED reduced the number of TRAP‐positive multinucleated cells. Further, PPi inhibited CEBPA but not PPARG and LPL mRNA expression under adipogenic induction. The intracellular lipid accumulation tended to decrease in PPi‐treated conditions (10 μM). The transcriptomic profiles illustrated that PPi potentially modulated several pathways, including the metabolism of lipids, interleukin‐6, TGF‐β1, and NOTCH signaling.

PPi inhibited osteo/odontogenic, adipogenic and indirectly attenuated osteoclast differentiation by SHED. This study implicated that PPi can modulate the cellular responses of SHED.