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

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.

The Scleraxis Transcription Factor Directly Regulates Multiple Distinct Molecular and Cellular Processes During Early Tendon Cell Differentiation

Proper development of tendons is crucial for the integration and function of the musculoskeletal system. Currently little is known about the molecular mechanisms controlling tendon development and tendon cell differentiation. The transcription factor Scleraxis (Scx) is expressed throughout tendon development and plays essential roles in both embryonic tendon development and adult tendon healing, but few direct target genes of Scx in tendon development have been reported and genome-wide identification of Scx direct target genes in vivo has been lacking. In this study, we have generated a ScxFlag knockin mouse strain, which produces fully functional endogenous Scx proteins containing a 2xFLAG epitope tag at the carboxy terminus. We mapped the genome-wide Scx binding sites in the developing limb tendon tissues, identifying 12,097 high quality Scx regulatory cis-elements in-around 7,520 genes. Comparative analysis with previously reported embryonic tendon cell RNA-seq data identified 490 candidate Scx direct target genes in early tendon development. Furthermore, we characterized a new Scx gene-knockout mouse line and performed whole transcriptome RNA sequencing analysis of E15.5 forelimb tendon cells from Scx–/– embryos and control littermates, identifying 68 genes whose expression in the developing tendon tissues significantly depended on Scx function. Combined analysis of the ChIP-seq and RNA-seq data yielded 32 direct target genes that required Scx for activation and an additional 17 target genes whose expression was suppressed by Scx during early tendon development. We further analyzed and validated Scx-dependent tendon-specific expression patterns of a subset of the target genes, including Fmod, Kera, Htra3, Ssc5d, Tnmd, and Zfp185, by in situ hybridization and real-time quantitative polymerase chain reaction assays. These results provide novel insights into the molecular mechanisms mediating Scx function in tendon development and homeostasis. The ChIP-seq and RNA-seq data provide a rich resource for aiding design of further studies of the mechanisms regulating tendon cell differentiation and tendon tissue regeneration. The ScxFlag mice provide a valuable new tool for unraveling the molecular mechanisms involving Scx in the protein interaction and gene-regulatory networks underlying many developmental and disease processes.

Scleraxis is required for the growth of adult tendons in response to mechanical loading

Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading, and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (ScxΔ), and then induced tendon growth in Scx+ and ScxΔ adult mice via plantaris tendon mechanical overload. Compared to the wild type Scx+ group, ScxΔ mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically-stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage, and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.

Synchronized mechanical oscillations at the cell–matrix interface in the formation of tensile tissue

The formation of uniaxial fibrous tissues with defined viscoelastic properties implies the existence of an orchestrated mechanical interaction between the cytoskeleton and the extracellular matrix. This study addresses the nature of this interaction. The hypothesis is that this mechanical interplay underpins the mechanical development of the tissue. In embryonic tendon tissue, an early event in the development of a mechanically robust tissue is the interaction of the pointed tips of extracellular collagen fibrils with the fibroblast plasma membrane to form stable interface structures (fibripositors). Here, we used a fibroblast-generated tissue that is structurally and mechanically matched to embryonic tendon to demonstrate homeostasis of cell-derived and external strain-derived tension over repeated cycles of strain and relaxation. A cell-derived oscillatory tension component is evident in this matrix construct. This oscillatory tension involves synchronization of individual cell forces across the construct and is induced in each strain cycle by transient relaxation and transient tensioning of the tissue. The cell-derived tension along with the oscillatory component is absent in the presence of blebbistatin, which disrupts actinomyosin force generation of the cell. The time period of this oscillation (60–90 s) is well-defined in each tissue sample and matches a primary viscoelastic relaxation time. We hypothesize that this mechanical oscillation of fibroblasts with plasma membrane anchored collagen fibrils is a key factor in mechanical sensing and feedback regulation in the formation of tensile tissues.

The Role of Scleraxis in Fate Determination of Mesenchymal Stem Cells for Tenocyte Differentiation

Mesenchymal stem cells (MSCs) are pluripotent cells that primarily differentiate into osteocytes, chondrocytes and adipocytes. Recent studies indicate that MSCs can also be induced to generate tenocyte-like cells; moreover, MSCs have been suggested to have great therapeutic potential for tendon pathologies. Yet the precise molecular cascades governing tenogenic differentiation of MSCs remain unclear. We demonstrate scleraxis, a transcription factor critically involved in embryonic tendon development and formation, plays a pivotal role in the fate determination of MSC towards tenocyte differentiation. Using murine C3H10T1/2 pluripotent stem cells as a model system, we show scleraxis is extensively expressed in the early phase of bone morphogenetic protein (BMP)-12-triggered tenocytic differentiation. Once induced, scleraxis directly transactivates tendon lineage-related genes such as tenomodulin and suppresses osteogenic, chondrogenic and adipogenic capabilities, thus committing C3H10T1/2 cells to differentiate into the specific tenocyte-like lineage, while eliminating plasticity for other lineages. We also reveal that mechanical loading-mediated tenocytic differentiation follows a similar pathway and that BMP-12 and cyclic uniaxial strain act in an additive fashion to augment the maximal response by activating signal transducer Smad8. These results provide critical insights into the determination of multipotent stem cells to the tenocyte lineage induced by both chemical and physical signals.