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.
Highlights
Congenital heart defects (CHD) are the most common birth defects and the leading cause of infant death in the United States [1]
The objectives of this study were to test whether Amniotic fluid stem cells (AFSC) can be reprogrammed to induced pluripotent stem cells (iPSC) by mRNA delivery and whether non-virally attained AFSC-iPSC are capable of cardiac differentiation
MRNA reprogramming of Passage 3 AFSC seeded at 2.6 x 104 cells per cm2 yielded transfected and highly proliferative cells
Summary
Congenital heart defects (CHD) are the most common birth defects and the leading cause of infant death in the United States [1]. Derived contractile cardiac cells can be applied to patches for structural defect repair [2], engineered heart tissue[3], cells for cardiomyoplasty [4], and gene editing correction of specific defects[5]. With 80% of CHDs diagnosed in the second trimester [6], amniotic fluid presents an ideal source for autologous cells for use in neonatal CHD treatment [4, 7]. Amniotic fluid stem cells (AFSC) are broadly multipotent, but do not directly differentiate into contractile cardiomyocytes (CM). AFSC express mesenchymal stem cell markers (CD29, CD44, CD90, and CD105), certain pluripotent markers (SOX2), and are capable of differentiating into all three germ layers[8]. While attempts at direct cardiac differentiation have shown gene and protein level similarities (GATA4, Nkx2.5, α-actinin, cTnT), resulting cells lack contractility[8, 9]
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