Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Nat Commun. 2021 Jul 9;12(1):4208. doi: 10.1038/s41467-021-24535-5.
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.
目前尚不清楚诱导和分化致密结缔组织（如肌腱和相关纤维软骨组织（半月板和纤维环））的转录调控因子。我们采用基于发育线索和单细胞 RNA 测序（scRNA-seq）的迭代方法，通过激活 TGFβ 和 hedgehog 途径，从小鼠胚胎干细胞（mESCs）中建立定向分化模型来生成肌腱和纤维软骨细胞，实现了 90%的诱导效率。mESC 衍生细胞的转录特征再现了来自小鼠尾巴的胚胎肌腱和纤维软骨特征。scRNA-seq 进一步表明视黄酸信号是 TGFβ 诱导的肌腱和纤维软骨谱系之间细胞命运转换的关键调节因子。通过 RNA 测序进行轨迹分析确定了肌腱和纤维软骨命运诱导的转录模块，并鉴定了与谱系特异性分化相关的分子。最后，我们使用这些分化方案成功地生成了 3 维工程组织，并显示出在动态拉伸加载下机械转导标志物的激活。这些发现为高效地生成肌腱和纤维软骨细胞和组织提供了无血清方法，可用于发育和疾病的建模。
