Mechanical and molecular parameters that influence the tendon differentiation potential of C3H10T1/2 cells in 2D- and 3D-culture systems.

Ludovic Gaut,Mathias Mericskay,Marie-Ange Bonnin,Monika Orpel,Isabelle Cacciapuoti,Delphine Duprez,Cédrine Blavet

doi:10.1242/bio.047928

Abstract

ABSTRACTOne of the main challenges relating to tendons is to understand the regulators of the tendon differentiation program. The optimum culture conditions that favor tendon cell differentiation have not been identified. Mesenchymal stem cells present the ability to differentiate into multiple lineages in cultures under different cues ranging from chemical treatment to physical constraints. We analyzed the tendon differentiation potential of C3H10T1/2 cells, a murine cell line of mesenchymal stem cells, upon different 2D- and 3D-culture conditions. We observed that C3H10T1/2 cells cultured in 2D conditions on silicone substrate were more prone to tendon differentiation, assessed with the expression of the tendon markers Scx, Col1a1 and Tnmd as compared to cells cultured on plastic substrate. The 3D-fibrin environment was more favorable for Scx and Col1a1 expression compared to 2D cultures. We also identified TGFβ2 as a negative regulator of Tnmd expression in C3H10T1/2 cells in 2D and 3D cultures. Altogether, our results provide us with a better understanding of the culture conditions that promote tendon gene expression and identify mechanical and molecular parameters upon which we could act to define the optimum culture conditions that favor tenogenic differentiation in mesenchymal stem cells.

Highlights

Mesenchymal stem cells (MSCs) are multipotent cells that can be induced to differentiate in various tissue lineages upon specific molecular or mechanical cues
We used tendon genes identified in the transcriptomic analysis of mouse tendon cells during development (Havis et al, 2014), such as aquaporin1 (Aqp1) gene coding for a water channel protein and thrombospondin 2 (Thsb2) coding for an adhesive glycoprotein with antiangiogenic properties, both expressed in developing limb tendons
Differentiation potential of C3H10T1/2 cells cultured on plastic substrate over time We investigated the tendon differentiation potential of C3H10T1/2 cells cultured on plastic substrate over time

Summary

Introduction

Mesenchymal stem cells (MSCs) are multipotent cells that can be induced to differentiate in various tissue lineages upon specific molecular or mechanical cues. Based on specific lineage markers and identified master genes, established protocols are recognized to drive differentiation towards osteocytes, chondrocytes and adipocytes (Caplan, 1991; Pittenger et al, 1999; Prockop, 1997). Received 15 September 2019; Accepted 6 January 2020 differentiation upon molecular and mechanical cues from MSCs (reviewed in Nourissat et al, 2015; Zhang et al, 2018), tendon lineage is less studied than other tissue-specific lineages. There is no recognized/established protocol with external inducers to differentiate MSCs towards a tendon phenotype. There is no identified master gene that initiates the tenogenic program in cell cultures as for the cartilage (Sox9), bone (Runx2) and muscle (muscle regulatory factors) programs (Buckingham, 2017; Karsenty et al, 2009; Liu et al, 2017)

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