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      Effective derivation of ventricular cardiomyocytes from hPSCs using ascorbic acid-containing maturation medium

      research-article
      a , b , b , b
      Animal Cells and Systems
      Taylor & Francis
      Cardiomyocyte, ascorbic acid, albumin, Wnt signaling

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          ABSTRACT

          Cardiomyocytes derived from human pluripotent stem cells (hPSCs) can be used in various applications including disease modeling, drug safety screening, and novel cell-based cardiac therapies. Here, we report an optimized selection and maturation method to induce maturation of cardiomyocytes into a specific subtype after differentiation driven by the regulation of Wnt signaling. The medium used to optimize selection and maturation was in a glucose starvation conditions, supplemented with either a nutrition complex or ascorbic acid. Following optimized selection and maturation, more cardiac Troponin T (cTnT)-positive cardiomyocytes were detected using albumin and ascorbic acid than B27. In addition, ascorbic acid enriched maturation of ventricular cardiomyocytes. We compared cardiomyocyte-specific gene expression patterns under different selection and maturation conditions by next-generation sequencing (NGS) analysis. Our optimized conditions will enable simple and efficient maturation and specification of the desired cardiomyocyte subtype, facilitating both biomedical research and clinical applications.

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          Most cited references37

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          Induction of pluripotent stem cells from adult human fibroblasts by defined factors.

          Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts.
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            Chemically Defined and Small Molecule-Based Generation of Human Cardiomyocytes

            Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows the elucidation of cardiomyocyte macromolecular and metabolic requirements whilst providing a minimally complex system for the study of maturation and subtype specification.
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              Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.

              Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.
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                Author and article information

                Journal
                Anim Cells Syst (Seoul)
                Anim Cells Syst (Seoul)
                Animal Cells and Systems
                Taylor & Francis
                1976-8354
                2151-2485
                24 March 2023
                2023
                24 March 2023
                : 27
                : 1
                : 82-92
                Affiliations
                [a ]Dongguk University , Seoul, Republic of Korea
                [b ]Korea Institute of Toxicology , Daejeon, Republic of Korea
                Author notes
                [CONTACT ] Ki-Suk Kim idkks@ 123456kitox.re.kr Korea Institute of Toxicolgoy , 141 Gajeong-ro, Yuseong-gu, Daejeon 34114, Republic of Korea

                Supplemental data for this article can be accessed online at https://doi.org/10.1080/19768354.2023.2189932

                Author information
                https://orcid.org/0000-0002-0931-0924
                Article
                2189932
                10.1080/19768354.2023.2189932
                10044166
                ee1f5fee-6ac9-4e65-a666-83ffa0b1caf7
                © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

                History
                Page count
                Figures: 7, Tables: 2, Equations: 0, References: 38, Pages: 11
                Categories
                Research Article
                Research Article

                cardiomyocyte,ascorbic acid,albumin,wnt signaling
                cardiomyocyte, ascorbic acid, albumin, wnt signaling

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