Correction: Characterization and Therapeutic Potential of Induced Pluripotent Stem Cell-Derived Cardiovascular Progenitor Cells.

<div><h3>Background</h3><p>Cardiovascular 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 <em>in vitro</em> expansion that maintain their multipotency.</p> <h3>Methodology/Principal Findings</h3><p>We 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 <em>in vitro</em> and transplantation <em>in vivo</em>. 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 <em>in vitro</em>. Flt1⁺/Flt4⁺ CPCs transplanted into left ventricles demonstrated robust engraftment and differentiation into mature cardiomyocytes (CMs).</p> <h3>Conclusion/Significance</h3><p>The 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 <em>in vitro</em> and robust ability for engraftment and differentiation into morphologically and electrophysiologically mature adult CMs <em>in vivo</em> post transplantation into adult hearts.</p> </div

Characterization and therapeutic potential of induced pluripotent stem cell-derived cardiovascular progenitor cells.

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

FACS analysis of cells dissociated from E15.5 mouse hearts.

<p>FACS analysis of E15.5 mouse embryonic hearts for different cell surface marker/s identified by microarray analysis of mouse ESC-derived Flk1⁺ cells. Flt1/Flt4 combination is the most specific to identify and enrich for Isl1⁺ cells in the E15.5 heart.</p

Flk1 is not a specific marker for endogenous and mouse ESC-derived Isl1⁺ CPCs.

<p>(A) Immunohistochemical staining of E15.5 mouse heart identifies Isl1-expressing CPCs (blue) located in niches in outflow tracts. (B) FACS analysis of mouse ESC-derived Flk1⁺ cells reveals a heterogeneous Flk1⁺ population with low enrichment for Isl1 (light blue bar) and Nkx2.5 (orange bar) cells (n = 3). (C & D) FACS analysis of differentiated mouse ESCs reveals that Flk1 represents <10% of Isl1⁺ cells (C; n = 3) and <5% of Nkx2.5⁺ cells (D; n = 3).</p

Transplanted iPSC-derived Flt1⁺/Flt4⁺ CPCs differentiate into morphologically and electrophysiologically mature CMs.

<p>(A) 10X phase contrast imaging of live, dissociated single CMs post-transplantation of GFP⁺ Flt1⁺/Flt4⁺ CPCs into the LV. (B) Immunofluorescence imaging of the same field of live cells confirms GFP⁺ mature CMs derived from transplanted CPCs. (C) 40X image of GFP⁺ CMs counterstained using DAPI. (D) FACS analysis of dissociated cells from transplanted hearts demonstrates that ∼6% of Troponin I⁺ CMs are GFP⁺ labeled using a GFP antibody. (E) Action potential and (F) fluorescence transient recorded simultaneously from a single GFP⁺ Flt1⁺/Flt4⁺-derived CM loaded with the Ca²⁺ indicator rhod-2-AM and paced at 0.2 Hz. (G) Confocal image of live single GFP⁺ Flt1⁺/Flt4⁺-derived CMs post-enzymatic digestion of mouse heart post-transplant (63X). (H) Live 2D Ca²⁺ imaging of the same GFP⁺ CM shown in G which was also loaded with Ca²⁺ indicator rhod-2-AM. The bright area on the left shows the beginning of a spontaneous Ca²⁺ wave. (I) Line scan image from the same CM during depolarization evoked by field stimulation. The synchronous onset of the Ca²⁺ transient and rapid increase in fluorescence indicate electrically triggered Ca²⁺ release. Fluorescence intensities are displayed in arbitrary units (a.u.) and action potential in millivolt (mV). Scale bars equal 25 µm.</p

Mouse iPSC-derived GFP⁺ Flt1⁺/Flt4⁺ CPCs can be clonally expanded while maintaining phenotype and multipotency.

<p>(Aa-c) Immunofluorescence imaging of live GFP⁺ Flt1⁺/Flt4⁺ single cell CPCs during clonal expansion: (a) single cell post FACS sort, day 1, (b) same colony on day 14 and (c) on day 30. (Ad-f) CPCs demonstrate trilineage cardiovascular differentiation potential post-clonal expansion. Immunofluorescence imaging of clonally expanded Flt1⁺/Flt4⁺ CPC colonies fourteen days post-differentiation demonstrating (d) TropC-expressing CMs, (e) α-SMA expressing smooth muscle cells, and (f) CD31 expressing endothelial cells. Cell nuclei are identified with DAPI. (B) FACS analysis thirty days post-clonal expansion confirms CPCs maintain their Flt1⁺/Flt4⁺ CPC phenotype.</p