Transcriptional profiling of mESC-derived tendon and fibrocartilage cell fate switch

Abstract

The transcriptional regulators underlying induction and differentiation of dense connective tissues such as tendon and related fibrocartilaginous tissues (meniscus and annulus fibrosus) remain largely unknown. Using an iterative approach informed by developmental cues and single cell RNA sequencing (scRNA-seq), we establish directed differentiation models to generate tendon and fibrocartilage cells from mouse embryonic stem cells (mESCs) by activation of TGFβ and hedgehog pathways, achieving 90% induction efficiency. Transcriptional signatures of the mESC-derived cells recapitulate embryonic tendon and fibrocartilage signatures from the mouse tail. scRNA-seq further identify retinoic acid signaling as a critical regulator of cell fate switch between TGFβ-induced tendon and fibrocartilage lineages. Trajectory analysis by RNA sequencing define transcriptional modules underlying tendon and fibrocartilage fate induction and identify molecules associated with lineage-specific differentiation. Finally, we successfully generate 3-dimensional engineered tissues using these differentiation protocols and show activation of mechanotransduction markers with dynamic tensile loading. These findings provide a serum-free approach to generate tendon and fibrocartilage cells and tissues at high efficiency for modeling development and disease.

Tenogenic Induction From Induced Pluripotent Stem Cells Unveils the Trajectory Towards Tenocyte Differentiation

“…Interestingly, both tenogenic (Scx, Tnmd, and Mkx) and chondrogenic (Sox9, Acan, Col2a1, and Col11a1) markers were significantly upregulated when BMS493 and TGF-β2 were added (Figure 8C). Since ATRA has been reported to suppress fibrocartilage differentiation (Kaji et al, 2021) which is characterized by induction of cells simultaneously expressing Col1 and Col2 but negative for Tnmd (Benjamin and Evans, 1990;Sugimoto et al, 2013a), our results suggest that both tenogenic and fibrocartilaginous chondrogenic differentiation are negatively regulated by activation of RA signaling. We next investigated the relation between endogenous RA signaling and tenogenic/chondrogenic differentiation by analyzing the expression status of Scx and Aldh1a2 in our single cell analysis.…”

“…Tnmd is a widely accepted molecular marker of mature tenocytes ( Shukunami et al, 2006 ). Recent studies have also reported the induction of tenogenic cells from ScxGFP pluripotent cells by activation of TGF-β signaling using TGF-β1 ( Komura et al, 2020 ; Kaji et al, 2021 ). In the developing mouse limb, Tgfb2 was reported to show the highest level of expression compared to those of other Tgfb genes ( Havis et al, 2014 ).…”

“…Although in vitro tenogenic differentiation from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) has also been reported, these systems still have problems, including complicated procedures ( Nakajima et al, 2018 ) and poor tendon marker gene expression ( Chen et al, 2009 ; Chen et al, 2012 ; Komura et al, 2020 ). Recently, Kaji et al reported a system with high induction efficiency using ScxGFP ESCs, but this procedure is still relatively complicated because it is based on embryoid body (EB) formation ( Kaji et al, 2021 ).…”

Comparison of Tendon Development Versus Tendon Healing and Regeneration

“…Recent research explains this in more detail. It was demonstrated that RA initially contribute to the regulation of tendon progenitors, with mESCs undergoing neural differentiation when RA signaling is activated, while differentiating into tendon progenitors of paraxial mesoderm when RA signaling is inhibited ( Kaji et al, 2021 ). RA further regulates tendon progenitor cells, differentiating into the tenogenic lineage when RA signaling is activated, with reverse differentiation into fibrocartilage ( Kaji et al, 2021 ).…”

“…It was demonstrated that RA initially contribute to the regulation of tendon progenitors, with mESCs undergoing neural differentiation when RA signaling is activated, while differentiating into tendon progenitors of paraxial mesoderm when RA signaling is inhibited ( Kaji et al, 2021 ). RA further regulates tendon progenitor cells, differentiating into the tenogenic lineage when RA signaling is activated, with reverse differentiation into fibrocartilage ( Kaji et al, 2021 ). Besides, it was found that the variations of retinoids concentration in endogenous local region promoted the formation of tendon condensations and attachment sites within the extraocular area ( Comai et al, 2020 ).…”