Abstract 16656: How to Routinely Obtain One Billion Human Cardiomyocytes: A Large-Scale Systematic Expansion of Pluripotent Stem Cells and Differentiation Into Cardiomyocytes

Abstract

Background: Tissue engineering a human-sized whole heart requires recellularization of a scaffold with billions of cardiomyocytes, which are post-mitotic cells with limited expansion capability. Induced pluripotent stem cells (iPSCs) provide a new opportunity for cardiac tissue engineering, because iPSCs can be expanded and differentiated into cardiomyocytes. A closed cell culture system is an efficient clinically applicable tool that can be used to expand cells under controlled conditions. We used the Quantum Cell Expansion System (QES) as a human iPSC expansion platform. Methods: Human iPSCs were expanded 30-fold using the QES and karyotyped. The undifferentiated cells were characterized by RT-PCR, immunofluorescence and their ability to undergo spontaneous differentiation. After harvesting, iPSCs were differentiated into the cardiac lineage, and the cardiomyocytes were characterized by RT-PCR, immunofluorescence and flow cytometry. Action potential recordings were obtained through intracellular impalement (MultiClamp 700). Results: The QES provided the necessary environment for exponential iPSC growth, reaching 70.9x10e7 ± 20,75 x10e7 cells in less than 7 days (n=4), with a population doubling of 3.801 ± 0.1956. The cells kept a normal karyotype (46-XX), expressed pluripotency markers (OCT4, NANOG, LIN28, SOX2, REX1, DNMT3B, DPPA4, NODAL, TDGFb, TERT3, and GDF) and had characteristic staining for OCT4, SOX2, NANOG, SSEA4, TRA1-60, and TRA1-81. Spontaneous differentiation into ectoderm (NESTIN, TUBB3, and NEFH), mesoderm (MSX1, BMP4, and T) and endoderm (GATA6, AFP, and SOX17) lineages was detected by RT-PCR. Induction to the cardiac lineage was also characterized [cTNT+ by flow cytometry (with an efficiency of 74.2±6.3%, n=5) and immunohistochemistry, and MYH6, MYH7, MLC2a, MLC2V, KCNA5, KCNQ1, KCNJ3 by RT-PCR]. Electrophysiology analysis demonstrated that the action potential duration with 90% of repolarization (cAPD90) was 506.5 ± 27.99 mV and the beating rate of these cells was 39.08±1.8 bpm (n=31). Conclusion: QES maintains the stemness of iPSCs and is a promising platform to provide the high numbers of cardiomyocytes necessary to recellularize human-sized heart scaffolds.