Abstract 394: Transient Bone Morphogenic Protein Antagonism Directs Differentiation of iPSCs into the Cardiac Neural Crest and cKit+ Myocardial Progenitor Lineages
Introduction: The capability of cKit+ cardiac progenitor cells (CPCs) to participate in cardiomyocyte regeneration remains controversial, despite basic and clinical studies supporting such a role.
Hypothesis: A non-permissive cardiac milieu minimizes the generation of cardiomyocytes from CPCs.
Methods: We lineage-traced CPCs using novel dual-recombinase responsive indicator mice (cKitCreERT2;Wnt1::Flpe;RC::Fela) and iPSCs derived from cKitCreERT2;IRG (iPSCKit) mice.
Results: Intersectional genetic fate-mapping of cKitCreERT2;Wnt1:: Flpe;RC::Fela embryos supported that cKit marks Wnt1-expressing cardiac neural crest (CNC) progenitors, emerging at ~E9.5 and contributing a limited number of cardiomyocytes. To decipher the mechanisms underlying cardiomyocyte differentiation of CPCs, we lineage-traced CPCs during stage-specific cardiogenic differentiation of iPSCKit. Ascorbate treatment promoted differentiation of cKit+ iPSC-derived embryoid bodies (EBs) into Nkx2.5+ myocardium, 45.5%±6.7% of which co-expressed the Cre-reporter EGFP (n=154 EBs; 12 preparations), suggesting that CPCs encompass fully competent cardiomyogenic progenitors. Noggin (or Dorsomorphin), a BMP antagonist transiently expressed in the heart at E7.5-E8.5 but not during CNC invasion, directed the differentiation of iPSCkit-EBs into Mesp1+/Isl1+/Nkx2.5+ cardiac mesoderm progenitors (p≤0.0001). Remarkably, the same signaling pathway subsequently directed EBs into the cKit+/Wnt1+/Pax3+/Mitf-H+/Isl1+/Nkx2.5+ CNC lineage (p≤0.0001), while suppressing the generation of WT1+/Tbx18+ epicardium (p<0.05). Stage-specific induction of Cre-recombination delineated that iPSCkit-derived CPCs encompass Mesp1–/cKit+/Nkx2.5+ CNC progenitors which contributed EGFP+ CNC derivatives, including Nkx2-5+ cardiomyocytes, to 60.7%±7.3% of spontaneously beating EBs (n=147 EBs; 12 preparations).
Conclusions: Collectively, our data show that CPCkit are fully competent CNC-derived cardiomyogenic progenitors, whose differentiation to cardiomyocytes is minimized by a latent Noggin-mediated signaling pathway. Therefore exploiting CPCkit therapeutically, provides an important strategy for maximizing myocardial regeneration.
Author Disclosures: K.E. Hatzistergos: 7. Ownership Interest; Modest; Vestion Inc.. 8. Consultant/Advisory Board; Modest; Vestion Inc.. L.M. Takeuchi: None. D. Saur: None. B. Seidler: None. S.M. Dymecki: None. J. Mai: None. R. Kanashiro-Takeuchi: None. W. Balkan: None. J.M. Hare: 7. Ownership Interest; Modest; Vestion Inc. 8. Consultant/Advisory Board; Modest; Vestion Inc.
- © 2015 by American Heart Association, Inc.