Abstract

RationalePluripotent stem cell–derived cardiac progenitor cells (CPCs) have emerged as a powerful tool to study cardiogenesis in vitro and a potential cell source for cardiac regenerative medicine. However, available methods to induce CPCs are not efficient or require high-cost cytokines with extensive optimization due to cell line variations.ObjectiveBased on our in-vivo observation that early endodermal cells maintain contact with nascent pre-cardiac mesoderm, we hypothesized that direct physical contact with endoderm promotes induction of CPCs from pluripotent cells.Method and ResultTo test the hypothesis, we cocultured mouse embryonic stem (ES) cells with the endodermal cell line End2 by co-aggregation or End2-conditioned medium. Co-aggregation resulted in strong induction of Flk1+ PDGFRa+ CPCs in a dose-dependent manner, but the conditioned medium did not, indicating that direct contact is necessary for this process. To determine if direct contact with End2 cells also promotes the induction of committed cardiac progenitors, we utilized several mouse ES and induced pluripotent (iPS) cell lines expressing fluorescent proteins under regulation of the CPC lineage markers Nkx2.5 or Isl1. In agreement with earlier data, co-aggregation with End2 cells potently induces both Nkx2.5+ and Isl1+ CPCs, leading to a sheet of beating cardiomyocytes. Furthermore, co-aggregation with End2 cells greatly promotes the induction of KDR+ PDGFRa+ CPCs from human ES cells.ConclusionsOur co-aggregation method provides an efficient, simple and cost-effective way to induce CPCs from mouse and human pluripotent cells.

Highlights

  • The availability of embryonic stem (ES) cells and induced pluripotent stem cells has opened new fields of research and medicine[1,2,3]

  • Isl1Cre; RosaYFP or RosaRFP were derived on irradiated mouse embryonic fibroblasts (MEF) in knockout (KO) DMEM supplemented with 15% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 2 mM GlutaMAX (Invitogen), 0.1 mM sodium pyruvate (Invitogen), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), and 100 U/ml leukemia inhibitory factor (LIF, Millipore), 3 mM CHIR99021 and 1 mM PD0325901 by standard procedures

  • The basic helix-loop-helix transcription factor Mesp1 is transiently expressed in the earliest cardiovascular progenitor cell population that gives rise to the entire heart and vasculature [15]

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Summary

Introduction

The availability of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells has opened new fields of research and medicine[1,2,3]. ES/iPS cells have the potential to become all types of somatic cells, and they are potential cell sources for disease modeling, drug discovery and regenerative medicine. Heart malformation is the most frequent form of human birth defects, and heart disease is the number one killer of adults worldwide [4]. Recent advances point to the potential of therapies based on cardiac progenitor cells (CPCs). CPCs can be purified from ES/iPS cells and manipulated in culture to expand and differentiate into various types of cardiac cells including cardiomyocytes, vascular endothelial cells and smooth muscle cells [5,6]. CPCs might be potential cell types for cardiac regenerative therapy

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