title: Directing cardiomyogenic differentiation of human pluripotent stem cells by plasmid-based transient overexpression of cardiac transcription factors.
abstract: Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) possess a high potential for regenerative medicine. Previous publications suggested that viral transduction of a defined set of transcription factors (TFs) known to play pivotal roles in heart development also increases cardiomyogenesis in vitro upon overexpression in mouse or human ES cells. To circumvent issues associated with viral approaches such as insertional mutagenesis, we have established a transient transfection system for straightforward testing of TF combinations. Applying this method, the transfection efficiency and the temporal pattern of transgene expression were extensively assessed in hPSCs by quantitative real time-polymerase chain reaction (qRT-PCR), TF-specific immunofluorescence analysis, and flow cytometry. Testing TF combinations in our approach revealed that BAF60C, GATA4, and MESP1 (BGM) were most effective for cardiac forward programming in human induced pluripotent stem cell lines and human ES cells as well. Removal of BAF60C slightly diminished formation of CM-like cells, whereas depletion of GATA4 or MESP1 abolished cardiomyogenesis. Each of these TFs alone had no inductive effect. In addition, we have noted sensitivity of CM formation to cell density effects, which highlights the necessity for cautious analysis when interpreting TF-directed lineage induction. In summary, this is the first report on TF-induced cardiomyogenesis of hPSCs applying a transient, nonintegrating method of cell transfection.
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs, Department of Cardiac, Thoracic-, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Hartung
authors.forename: Kristin
authors.initials: K
authors.lastname: Schwanke
authors.forename: Alexandra
authors.initials: A
authors.lastname: Haase
authors.forename: Robert
authors.initials: R
authors.lastname: David
authors.forename: Wolfgang-Michael
authors.initials: WM
authors.lastname: Franz
authors.forename: Ulrich
authors.initials: U
authors.lastname: Martin
authors.forename: Robert
authors.initials: R
authors.lastname: Zweigerdt
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation; D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone; D050991:GATA4 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006321:Heart / Q000196:embryology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D002868:Chromosomal Proteins, Non-Histone; D050991:GATA4 Transcription Factor; D014157:Transcription Factors; C484170:GATA4 protein, human; C491649:SMARCD3 protein, human