A multi-omics study to characterize the transdifferentiation of human dermal fibroblasts to osteoblast-like cells

Sandra Pihlström; Kirsi Määttä; Tiina Öhman; Riikka E Mäkitie; Mira Aronen; Markku Varjosalo; Outi Mäkitie; Minna Pekkinen

doi:10.3389/fmolb.2022.1032026

A multi-omics study to characterize the transdifferentiation of human dermal fibroblasts to osteoblast-like cells

Front Mol Biosci. 2022 Nov 17:9:1032026. doi: 10.3389/fmolb.2022.1032026. eCollection 2022.

Authors

Sandra Pihlström^{1

2}, Kirsi Määttä^{1

2}, Tiina Öhman³, Riikka E Mäkitie^{1

2

4}, Mira Aronen¹, Markku Varjosalo³, Outi Mäkitie^{1

2

5

6}, Minna Pekkinen^{1

2

5}

Affiliations

¹ Institute of Genetics, Folkhälsan Research Center, Helsinki, Finland.
² Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
³ Institute of Biotechnology and Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.
⁴ Department of Otorhinolaryngology-Head and Neck Surgery, Helsinki University Hospital and University of Helsinki, Helsinki, Finland.
⁵ Children's Hospital, Helsinki University Hospital and University of Helsinki, Helsinki, Finland.
⁶ Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.

Abstract

Background: Various skeletal disorders display defects in osteoblast development and function. An in vitro model can help to understand underlying disease mechanisms. Currently, access to appropriate starting material for in vitro osteoblastic studies is limited. Native osteoblasts and their progenitors, the bone marrow mesenchymal stem cells, (MSCs) are problematic to isolate from affected patients and challenging to expand in vitro. Human dermal fibroblasts in vitro are a promising substitute source of cells. Method: We developed an in vitro culturing technique to transdifferentiate fibroblasts into osteoblast-like cells. We obtained human fibroblasts from forearm skin biopsy and differentiated them into osteoblast-like cells with ß-glycerophosphate, ascorbic acid, and dexamethasone treatment. Osteoblastic phenotype was confirmed by staining for alkaline phosphatase (ALP), calcium and phosphate deposits (Alizarin Red, Von Kossa) and by a multi-omics approach (transcriptomic, proteomic, and phosphoproteomic analyses). Result: After 14 days of treatment, both fibroblasts and MSCs (reference cells) stained positive for ALP together with a significant increase in bone specific ALP (p = 0.04 and 0.004, respectively) compared to untreated cells. At a later time point, both cell types deposited minerals, indicating mineralization. In addition, fibroblasts and MSCs showed elevated expression of several osteogenic genes (e.g. ALPL, RUNX2, BMPs and SMADs), and decreased expression of SOX9. Ingenuity Pathways Analysis of RNA sequencing data from fibroblasts and MSCs showed that the osteoarthritis pathway was activated in both cell types (p_adj. = 0.003 and 0.004, respectively). Discussion: These data indicate that our in vitro treatment induces osteoblast-like differentiation in fibroblasts and MSCs, producing an in vitro osteoblastic cell system. This culturing system provides an alternative tool for bone biology research and skeletal tissue engineering.

Keywords: fibroblasts; in vitro method; osteoblast-like cells; osteoblastic differentiation treatment; transdifferentiation.