Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human amniotic fluid stem cells

PLoS One. 2017 May 17;12(5):e0177824. doi: 10.1371/journal.pone.0177824. eCollection 2017.

Abstract

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and β-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.

MeSH terms

  • Amniotic Fluid / cytology*
  • Animals
  • Antigens, Surface / genetics
  • Cell Differentiation
  • Cells, Cultured
  • Cellular Reprogramming
  • Female
  • Fetal Stem Cells / cytology*
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Kruppel-Like Factor 4
  • Mice
  • Myocytes, Cardiac / cytology*
  • Nanog Homeobox Protein / genetics
  • Octamer Transcription Factor-3 / genetics
  • Pregnancy
  • Pregnancy Trimester, Second
  • Proteoglycans / genetics
  • Transfection

Substances

  • Antigens, Surface
  • KLF4 protein, human
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • NANOG protein, human
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • Proteoglycans
  • TRA-1-60 antigen, human
  • TRA-1-81 antigen, human

Grant support

This work was supported in part by grants from the American Heart Association (14BGIA18750004 to J. Jacot), the National Science Foundation (CBET-1547838 to J. Jacot), the National Institutes of Health (1R01HL130436-01 to J. Jacot), and Texas Children’s Hospital. K.D.B. is supported by the National Heart Lung and Blood Institute (NHLBI) grant R01HL134510, the Texas Hepatocellular Carcinoma Consortium (THCCC) (CPRIT #RP150587) and the Diana Helis Henry and Adrienne Helis Malvin Medical Research Foundations.