Abstract
BackgroundCardiovascular progenitor cells (CPCs) have been identified within the developing mouse heart and differentiating pluripotent stem cells by intracellular transcription factors Nkx2.5 and Islet 1 (Isl1). Study of endogenous and induced pluripotent stem cell (iPSC)-derived CPCs has been limited due to the lack of specific cell surface markers to isolate them and conditions for their in vitro expansion that maintain their multipotency.Methodology/Principal FindingsWe sought to identify specific cell surface markers that label endogenous embryonic CPCs and validated these markers in iPSC-derived Isl1+/Nkx2.5+ CPCs. We developed conditions that allow propagation and characterization of endogenous and iPSC-derived Isl1+/Nkx2.5+ CPCs and protocols for their clonal expansion in vitro and transplantation in vivo. Transcriptome analysis of CPCs from differentiating mouse embryonic stem cells identified a panel of surface markers. Comparison of these markers as well as previously described surface markers revealed the combination of Flt1+/Flt4+ best identified and facilitated enrichment for Isl1+/Nkx2.5+ CPCs from embryonic hearts and differentiating iPSCs. Endogenous mouse and iPSC-derived Flt1+/Flt4+ CPCs differentiated into all three cardiovascular lineages in vitro. Flt1+/Flt4+ CPCs transplanted into left ventricles demonstrated robust engraftment and differentiation into mature cardiomyocytes (CMs).Conclusion/SignificanceThe cell surface marker combination of Flt1 and Flt4 specifically identify and enrich for an endogenous and iPSC-derived Isl1+/Nkx2.5+ CPC with trilineage cardiovascular potential in vitro and robust ability for engraftment and differentiation into morphologically and electrophysiologically mature adult CMs in vivo post transplantation into adult hearts.
Highlights
Despite therapeutic advancements, cardiovascular disease remains a major cause of morbidity and mortality worldwide
We believe that the optimal cell type would be lineage-committed, multipotent Cardiovascular progenitor cells (CPCs) that satisfy the need for multilineage differentiation while limiting the oncogenic risk of injecting undifferentiated induced pluripotent stem cell (iPSC) or Embryonic stem cells (ESCs)
Cardiovascular progenitors that contribute to all three cardiovascular lineages identified by the transcription factor Islet 1 (Isl1) have been described in the developing human and mouse fetal heart [16]
Summary
Cardiovascular disease remains a major cause of morbidity and mortality worldwide. Since most preclinical studies have demonstrated very low rates of cardiac differentiation when using these cells [5], there is increasing consensus that transplanted adult stem cells may have a limited capacity for true cardiac regeneration and their beneficial effects are more likely related to paracrine mechanisms [6]. This highlights the need for cell types that can provide long-lasting engraftment and myogenesis either alone or in combination with existing cell types. Study of endogenous and induced pluripotent stem cell (iPSC)-derived CPCs has been limited due to the lack of specific cell surface markers to isolate them and conditions for their in vitro expansion that maintain their multipotency
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