Optogenetic activation of TGFβ signaling drives ligand-free chondrogenesis in hESC-derived MSCs

Jeongmin Lee; Gabsang Lee; Yohan Oh

doi:10.1093/stmcls/sxaf083

Optogenetic activation of TGFβ signaling drives ligand-free chondrogenesis in hESC-derived MSCs

Stem Cells. 2025 Dec 26:sxaf083. doi: 10.1093/stmcls/sxaf083. Online ahead of print.

Authors

Jeongmin Lee¹, Gabsang Lee^{2

3

4}, Yohan Oh^{1

5

6

7}

Affiliations

¹ Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, 04763, Republic of Korea.
² Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
³ Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
⁴ The Solomon Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
⁵ Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, 04763, Republic of Korea.
⁶ Hanyang Institute of Bioscience and Biotechnology, Hanyang University, Seoul, 04763, Republic of Korea.
⁷ Hanyang Institute of Advanced BioConvergence, Hanyang University, Seoul, 04763, Republic of Korea.

PMID: 41452756
DOI: 10.1093/stmcls/sxaf083

Abstract

Optogenetics holds great potential for diverse biological applications, including fundamental research, tissue engineering, and regenerative medicine, by enabling the precise spatial and temporal control of cellular signaling pathways. Transforming growth factor-beta (TGFβ), a multifunctional cytokine, is a critical regulator of cell proliferation, differentiation, and particularly chondrogenesis. Although TGFβ signaling is necessary for effective chondrogenic differentiation, previous studies have primarily relied on recombinant TGFβ ligand supplementation. In this study, we established an advanced optogenetic platform by knocking-in opto-TGFβ receptors in the AAVS1 locus of human embryonic stem cells (hESCs), enabling precise optogenetic activation of endogenous TGFβ signaling. Blue light illumination specifically activated TGFβ signaling, indicated by enhanced SMAD2 phosphorylation. Employing a three-dimensional pellet culture system, we demonstrated that direct optogenetic activation of TGFβ receptors, without exogenous ligand supplementation, is sufficient for robust chondrogenic differentiation of hESC-derived mesenchymal stem cells. The efficiency of optogenetic differentiation was comparable to conventional recombinant TGFβ protein treatment, evidenced by the expression of chondrogenic markers and deposition of cartilage-specific extracellular matrix components, including aggrecan and type II collagen. Our findings directly confirm the sufficiency and critical role of TGFβ receptor activation itself in chondrogenesis. Furthermore, this optogenetic approach provides a theoretical advantage by enabling non-invasive external modulation of TGFβ signaling post-transplantation, potentially facilitating further maturation and functional integration of transplanted chondrocytes. Thus, our results highlight a promising recombinant-protein-free strategy for use in cartilage tissue engineering and regenerative medicine.

Keywords: cell signaling; chondrocyte; human embryonic stem cell (hESC); optogenetics; transforming growth factor-beta (TGFβ).

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