Single Cell Transcriptomics-Informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineage.
Publication Year:
2024
PubMed ID:
37090543
Funding Grants:
Public Summary:
This study focuses on improving methods to generate tendon-forming cells from human induced pluripotent stem cells (iPSCs). By studying gene activity in single cells during differentiation, the researchers mapped out the developmental steps that mimic how tendons naturally form in the embryo. They discovered that many cells were accidentally turning into neuron-like cells due to increased Wnt signaling. By adding a Wnt inhibitor at a key stage, they prevented this off-target neural development and greatly improved the efficiency of producing the desired tendon lineage (syndetome). These findings show that precisely adjusting developmental signals can enhance tendon cell production, helping advance future cell-based therapies for tendon injuries.
Scientific Abstract:
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.