title: Activin-A and Bmp4 levels modulate cell type specification during CHIR-induced cardiomyogenesis.
abstract: The use of human pluripotent cell progeny for cardiac disease modeling, drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyogenic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success, recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR, which in turn induces the Activin-A and Bmp pathways, is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis, and to ultimately promote myocyte maturation, we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq, induction with CHIR during Day 1 (Days 0-1) was followed by immediate expression of Nodal ligands and receptors, followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic Activin-A (50-100 ng/ml) during Day 0-1 efficiently induced definitive endoderm, whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency, even when CHIR alone was ineffective. Moreover, co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis, as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast, co-induction with CHIR plus low levels (3-10 ng/ml) of Bmp4 during Day 0-1 consistently and strongly inhibited cardiomyogenesis. These findings, which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression, are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages, while Bmp levels regulate subsequent allocation into mesodermal cell types.
authors.affiliation: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Min-Su
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Audrey
authors.initials: A
authors.lastname: Horst
authors.affiliation: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Steven
authors.initials: S
authors.lastname: Blinka
authors.affiliation: Department of Surgery, Division of Cardiothoracic Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Karl
authors.initials: K
authors.lastname: Stamm
authors.affiliation: Department of Surgery, Division of Cardiothoracic Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Donna
authors.initials: D
authors.lastname: Mahnke
authors.affiliation: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: James
authors.initials: J
authors.lastname: Schuman
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Rebekah
authors.initials: R
authors.lastname: Gundry
authors.affiliation: Department of Surgery, Division of Cardiothoracic Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; The Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Aoy
authors.initials: A
authors.lastname: Tomita-Mitchell
authors.affiliation: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; The Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Lough
mesh_terms: D028341:Activins / Q000502:physiology*; D055415:Bone Morphogenetic Protein 4 / Q000502:physiology*; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology*; D017423:Sequence Analysis, RNA
chemical_list: D055415:Bone Morphogenetic Protein 4; C422405:activin A; D028341:Activins