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Single Cell Transcriptomics-Informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineag

ABSTRACT: Single Cell Transcriptomics-Informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineag

PROVIDER: PRJNA1202147 | ENA |

REPOSITORIES: ENA

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	SRR33830076.fastq.gz	Fastqsanger.gz
	SRR33830079.fastq.gz	Fastqsanger.gz
	SRR33830081.fastq.gz	Fastqsanger.gz
	SRR33830082.fastq.gz	Fastqsanger.gz

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Single Cell Transcriptomics-Informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineag

Project description:During vertebrate embryogenesis, axial tendons develop from the paraxial mesoderm and differentiate through specific developmental stages to reach the syndetome stage. While the main roles of signaling pathways in the earlier stages of the differentiation have been well established, pathway nuances in syndetome specification from the sclerotome stage have yet to be explored. Here, we show stepwise differentiation of human iPSCs to the syndetome stage using chemically defined media and small molecules that were modified based on single cell RNA-sequencing and pathway analysis. We identified a significant population of branching off-target cells differentiating towards a neural phenotype overexpressing Wnt. Further transcriptomics post-addition of a WNT inhibitor at the somite stage and onwards revealed not only total removal of the neural off-target cells, but also increased syndetome induction efficiency. Fine-tuning tendon differentiation in vitro is essential to address the current challenges in developing a successful cell-based tendon therapy.

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Project description:The musculoskeletal system is integrated by tendons that are characterized by expression of a functionally important transcription factor Scleraxis (Scx). To date, there has been no solid culture system for tenogenic differentiation. Here we developed the tenocyte induction method using induced pluripotent stem cells established from ScxGFP transgenic mice by monitoring fluorescence that reflects a series of dynamic differentiation process. Among several developmentally relevant factors, TGF-2 was the most potent inducer for differentiation of tenocytes expressing Tenomodulin, a mature differentiation marker. Single-cell RNA sequencing (scRNA-seq) revealed 11 distinct clusters including mature tenocyte population and tenogenic differentiation trajectory, which well recapitulates in vivo developmental process. Dataset of scRNA-seq and transcriptome of Scx deficient tendon enabled identification of Scx-related genes in which tendon-related genes are enriched. Collectively, this study leads to a new opportunity for tendon research and provides further insight into mechanistic understanding of the differentiation pathway to a tenogenic fate.

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