# Establishment and Molecular Characterization of a Human Stem Cell Line from a Primary Cell Culture Obtained from an Ectopic Calcified Lesion of a Tumoral Calcinosis Patient Carrying a Novel GALNT3 Mutation

## TL;DR

Researchers created a human stem cell line from a rare calcification disease patient, which could help understand the disease and develop new treatments.

## Contribution

A novel GALNT3 mutation and a stem cell line from a tumoral calcinosis patient were established and characterized.

## Key findings

- The TC1-SC line showed stem-like properties and could differentiate into adipogenic and osteogenic lineages.
- The cells expressed MSC and ESC markers and had a distinct miRNA and osteogenic gene expression signature.
- The cell line provides a new in vitro model for studying tumoral calcinosis pathogenesis.

## Abstract

Background/Objectives: Tumoral calcinosis (TC) is an extremely rare inherited disease characterized by multilobulated, dense ectopic calcified masses, usually in the periarticular soft tissue regions. In a previous study, we isolated a primary cell line from an ectopic lesion of a TC patient carrying a previously undescribed GALNT3 mutation. Here, we researched whether a stem cell (SC) subpopulation, which may play a critical role in TC progression, could be present within these lesions. Methods: A putative SC subpopulation was initially isolated by the sphere assay (marked as TC1-SC line) and characterized for its stem-like phenotype through several cellular and molecular assays, including colony forming unit assay, immunofluorescence staining for mesenchymal SC (MSC) markers, gene expression analyses for embryonic SC (ESC) marker genes, and multidifferentiation capacity. In addition, a preliminary expression pattern of osteogenesis-related pathways miRNAs and genes were assessed in the TC1-SC by quantitative Real-Time PCR (qPCR). Results: These cells were capable of differentiating into both the adipogenic and the osteogenic lineages. Moreover, they showed the presence of the MSC and ESC markers, confirmed respectively by using immunofluorescence and qualitative reverse transcriptase PCR (RT-PCR), and a good rate of clonogenic capacity. Finally, qPCR data revealed a signature of miRNAs (i.e., miR-21, miR-23a-3p, miR-26a, miR-27a-3p, miR-27b-3p, and miR-29b-3p) and osteogenic marker genes (i.e., ALP, RUNX2, COLIA1, OPG, OCN, and CCN2) characteristic for the established TC1-SC line. Conclusions: The establishment of this in vitro cell model system could advance the understanding of mechanisms underlying TC pathogenesis, thereby paving the way for the discovery of new diagnostic and novel gene-targeted therapeutic approaches for TC.