Marianne E Yassa1, Iman A Mansour2, Nadia I Sewelam2, Hala Hamza3, Taghrid Gaafar2. 1. Department of Clinical and Chemical Pathology, Kasr Al-Ainy School of Medicine, Cairo University, Kasr Al-Ainy St., 11562, Cairo, Egypt. Electronic address: marianne.yassa@kasralainy.edu.eg. 2. Department of Clinical and Chemical Pathology, Kasr Al-Ainy School of Medicine, Cairo University, Kasr Al-Ainy St., 11562, Cairo, Egypt. 3. Department of Paediatric Cardiology, Kasr Al-Ainy School of Medicine, Cairo University, Egypt.
Abstract
Human induced pluripotent stem cells (hiPSCs) act as a promising therapeutic alternative for cardiovascular diseases. They yield a large number of functional cardiomyocytes (CMs) from autologous cell sources without ethical or immunological problems. However, significant limitations still remain in terms of line-to-line variability in CM yield and reproducibility. AIM: To efficiently enhance NP0040 hiPSCs differentiation into CMs. MAIN METHODS: Following a standard cardiac differentiation protocol using small molecules targeting the canonical Wnt signaling, growth factors (BMP4 and FGF2) and ascorbic acid were added further in order to increase the cardiac differentiation efficiency. All cultures were conducted in serum-free, feeder-free monolayer system followed by lactate purification. KEY FINDINGS: Using NP0040 hiPSCs, the CM yield resulting from modulation of the Wnt signaling pathway alone was inefficient compared to previous studies while the addition of BMP4, FGF2 and ascorbic acid resulted in enhanced cardiac differentiation outcome. The later resulted in a high yield (up to 92%) of cardiac troponin-T (cTnT) + CMs contracting spontaneously as organized sheets in 15 independent experiments. They were validated structurally and functionally using immunofluorescent staining for sarcomeric α-actinin, cTnT, MLC2v and Connexin 43. Reverse-transcriptase PCR revealed cardiac transcription factors and cardiac-specific genes expression. CMs were electrically connected to one another. Recorded action potential (AP) showed waves of relatively mature ventricular-like phenotype. SIGNIFICANCE: We demonstrated that hiPSC lines respond differently to a standard cardiac differentiation protocol and that a well-orchestrated interplay between Wnt, BMP4, FGF/MEK and Ascorbic acid MEK/ERK1/2 signaling pathways is beneficial in enhancing the differentiation outcome.
Human induced pluripotent stem cells (hiPSCs) act as a promising therapeutic alternative for cardiovascular diseases. They yield a large number of functional cardiomyocytes (CMs) from autologous cell sources without ethical or immunological problems. However, significant limitations still remain in terms of line-to-line variability in CM yield and reproducibility. AIM: To efficiently enhance NP0040 hiPSCs differentiation into CMs. MAIN METHODS: Following a standard cardiac differentiation protocol using small molecules targeting the canonical Wnt signaling, growth factors (BMP4 and FGF2) and ascorbic acid were added further in order to increase the cardiac differentiation efficiency. All cultures were conducted in serum-free, feeder-free monolayer system followed by lactate purification. KEY FINDINGS: Using NP0040 hiPSCs, the CM yield resulting from modulation of the Wnt signaling pathway alone was inefficient compared to previous studies while the addition of BMP4, FGF2 and ascorbic acid resulted in enhanced cardiac differentiation outcome. The later resulted in a high yield (up to 92%) of cardiac troponin-T (cTnT) + CMs contracting spontaneously as organized sheets in 15 independent experiments. They were validated structurally and functionally using immunofluorescent staining for sarcomeric α-actinin, cTnT, MLC2v and Connexin 43. Reverse-transcriptase PCR revealed cardiac transcription factors and cardiac-specific genes expression. CMs were electrically connected to one another. Recorded action potential (AP) showed waves of relatively mature ventricular-like phenotype. SIGNIFICANCE: We demonstrated that hiPSC lines respond differently to a standard cardiac differentiation protocol and that a well-orchestrated interplay between Wnt, BMP4, FGF/MEK and Ascorbic acidMEK/ERK1/2 signaling pathways is beneficial in enhancing the differentiation outcome.
Authors: Andreas Brodehl; Hans Ebbinghaus; Marcus-André Deutsch; Jan Gummert; Anna Gärtner; Sandra Ratnavadivel; Hendrik Milting Journal: Int J Mol Sci Date: 2019-09-06 Impact factor: 5.923