Abstract 15441: A Novel and Efficient Combinatorial Method of Using DNAs and mRNAs to Generate Integration-free Induced Pluripotent Stem Cells and Functional Cardiomyocytes
Introduction: Efficient generation of viral integration-free induced pluripotent cells (iPSCs) from human somatic cells and differentiation into cardiomyocytes remain major hurdles in regenerative cardiology.
Hypothesis: Transfection with a combination of DNAs and mRNAs will generate iPSCs from any human somatic cell with subsequent differentiation into cardiomyocytes.
Methods: In a series of experiments, adult human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs) were treated with combination of DNAs containing Oct4, Nanog, Sox2, and Lin28, followed by a cocktail of mRNAs containing Oct4, Sox2, Klf4, cMyc, and Lin28 daily for 11 days in culture.
Results: We made the following observations: 1) this treatment regimen efficiently and reliably generated iPSC colonies from HSF (referred as hf-iPSCs) and HUVECs (referred as he-iPSCs) by day 17. 2) Our flow cytometry data showed that 4% of hf-iPSCs at passage 0 (P0) were positive for Oct4 and Sox2, which is higher than currently available methods. However, plasmid DNA alone or cocktail of mRNA alone were not able to produce any colony. 3) Our PCR array data revealed that a total of 44 and 49 pluripotent genes were significantly expressed in hf-iPSCs and he-iPSCs respectively. Among the mRNA transcripts, Oct4, Nanog, Sox2, Lefty2, Lin28, TDGF1, and DNMT3b were strongly (>100 fold) upregulated. These pluripotent mRNA expressions were further corroborated by immunofluorescence staining analysis. 4) These iPSCs were further differentiated into all three types of germ layer cells, including cardiomyocytes with >88% of cells positive for troponin T and Gata4 by flow cytometry (Figure). Sustained beating was observed in differentiated cells.
Conclusion: This select combination of DNAs and mRNAs can efficiently generate iPSCs, and subsequently functional cardiomyocytes, from human somatic cells. This novel method may be used to produce cells for human therapy.
Author Disclosures: J. Rajasingh: None. S. Rajasingh: None. J. Thangavel: None. B. Barani: None. B. Dawn: None.
This research has received full or partial funding support from the American Heart Association.
- © 2014 by American Heart Association, Inc.