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Direct lineage conversion can generate new cells for regeneration. Single-cell RNA sequencing revealed that suppressing the splicing factor Ptbp1 enhances the efficiency of reprogramming fibroblasts into induced cardiomyocytes (iCMs).

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Area of Science:

  • Cell biology
  • Regenerative medicine
  • Genomics

Background:

  • Direct lineage conversion is a promising strategy for tissue regeneration and disease modeling.
  • Studying the dynamic process of fibroblast reprogramming to target cell fates is challenging due to cellular heterogeneity and asynchronous progression.
  • Bulk genomic techniques are insufficient to capture the nuances of these complex cellular transitions.

Purpose of the Study:

  • To investigate the dynamic transcriptome changes during the direct reprogramming of mouse fibroblasts into induced cardiomyocytes (iCMs) using single-cell RNA sequencing.
  • To identify distinct cell subpopulations, understand the reprogramming trajectory, and uncover key regulatory factors.
  • To explore the role of mRNA processing and splicing factors in the efficiency of iCM induction.

Main Methods:

  • Single-cell RNA sequencing (scRNA-seq) was employed to analyze global transcriptome changes at early stages of fibroblast reprogramming.
  • Unsupervised dimensionality reduction and clustering algorithms were used to identify cell subpopulations and map reprogramming routes.
  • Functional analysis, including depletion studies, was performed on candidate genes, such as Ptbp1, to assess their role in iCM induction.

Main Results:

  • scRNA-seq identified distinct molecularly defined subpopulations during fibroblast reprogramming.
  • The study delineated the routes of induced cardiomyocyte (iCM) formation and correlated cell proliferation with iCM induction.
  • Unexpected downregulation of mRNA processing and splicing factors was observed, with Ptbp1 identified as a critical barrier to cardiomyocyte-specific splicing and iCM reprogramming.

Conclusions:

  • Single-cell transcriptomics provides a powerful approach to reconstruct reprogramming trajectories and identify intermediate cell states.
  • Ptbp1 acts as a significant barrier to iCM reprogramming, and its depletion enhances cardiac gene expression and reprogramming efficiency.
  • The study identified novel surface markers for iCM enrichment and revealed correlations between reprogramming factor expression and cellular reprogramming progress.