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Abstract

Developmental Biology

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Published: February 12th, 2021

DOI:

10.3791/62206

1Center for Cardiovascular Research, The Abigail Wexner Research Institute, Nationwide Children’s Hospital, 2The Heart Center, Nationwide Children’s Hospital, 3Department of Physiology and Cell Biology, The Ohio State University College of Medicine, 4Department of Anatomy, The Ohio State University College of Medicine, 5MCDB Graduate Program, The Ohio State University, 6Department of Pediatrics, The Ohio State University College of Medicine

Abstract

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient's blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

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Keywords Cardiomyocyte Generation

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