title: Comparative analysis of cardiomyocyte differentiation from human embryonic stem cells under 3-D and 2-D culture conditions.
abstract: Post-myocardial infarction cardiomyocytes are the most important target cell types for cardiac repair. Many of the applications envisaged for human embryonic stem cells (hESC)-derived cardiomyocytes demand that the differentiation procedure be robust, cost effective and high yielding. Various lines of evidence including our earlier study suggest that hESCs have distinct preferences to become heart cells. However, a direct comparison between different protocols has not yet been reported to date. Here, we performed a logical and systematic comparison of cardiomyocytes obtained from hESCs via embryoid bodies (EBs) in suspension versus adherent static cultures of feeder-free hES colonies representing three-dimensional (3-D) and two-dimensional (2-D) culture systems, respectively. An in-depth characterization of the beating cells revealed appropriate cardiac marker expression both at gene and protein levels. Despite using similar media, 3-D and 2-D cultures showed significant variation in growth and ability to form beating areas. While the expression of pre-cardiac mesoderm markers like GATA-4, HAND1, Myf5, Msx1, and BMP-IIR remained unaltered; levels of functional heart-specific markers such as MLC-2A/2V, cTnT, ANP, Phospholamban, α-MHC and KV4.3 were substantially up-regulated in 3-D compared to 2-D cultures. Concurrently we observed a sharp decline in the expression of ESC, ectoderm and endoderm markers including Oct-4, Sox-2, NFH, Sox-1, Sox-17 and AFP. Further immunocytochemistry and flow cytometry demonstrated a higher percentage of cells positive for Brachyury, desmin and cardiac troponin in 3-D cultures. Our results underscore the higher efficiency of cardiomyocytes derived via 3-D cultures. This finding enriches our basic understanding of the differentiation pattern in hESC-derived cardiomyocytes.
authors.affiliation: Manipal Institute of Regenerative Medicine, Manipal University Branch Campus, Yelahanka, Bangalore 560 065, India. rajarshi.pal@manipal.edu
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Pal
authors.forename: Murali Krishna
authors.initials: MK
authors.lastname: Mamidi
authors.forename: Anjan Kumar
authors.initials: AK
authors.lastname: Das
authors.forename: Ramesh
authors.initials: R
authors.lastname: Bhonde
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D004475:Ectoderm / Q000378:metabolism; D058732:Embryoid Bodies / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004707:Endoderm / Q000378:metabolism; D005326:Fetal Proteins / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D008648:Mesoderm / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D015203:Reproducibility of Results; D055747:SOX Transcription Factors / Q000378:metabolism; D020825:T-Box Domain Proteins / Q000378:metabolism; D013997:Time Factors; D000098908:Brachyury Protein
chemical_list: D015415:Biomarkers; D005326:Fetal Proteins; D050814:Octamer Transcription Factor-3; D055747:SOX Transcription Factors; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein