Generation of clinical‐grade functional cardiomyocytes from human embryonic stem cells in chemically defined conditions

A highly efficient cardiac differentiation from human pluripotent stem cells (hPSCs) is achievable using existing methods, especially with the standard B27 induction system. However, bovine serum albumin (BSA), one of the essential ingredients in B27, may pose significant complications for clinical studies owing to its animal origin and potential risks of virus contamination. Furthermore, the high cost of the B27 induction system also limits the applications of hPSCs‐derived cardiomyocytes. Here, a BSA‐free and chemically defined medium has been developed for differentiating hPSCs to clinical‐grade cardiomyocytes, which generated over 80% cardiac troponin T (cTNT)‐positive cardiomyocytes with high yield. When engrafting the cardiomyocytes into the hearts of myocardial infarction model rats, the rats survived with significantly improved heart functions in Δ ejection fraction and Δ fractional shortening. Importantly, the human embryonic stem cell (hESC) line (Q‐CTS‐hESC‐2) chosen for differentiation was of a clinical‐grade maintained in defined xeno‐free conditions. Compliant with the biological safety requirements, the Q‐CTS‐hESC‐2‐derived cardiomyocytes have passed the sterility and pathogen criteria tests for clinical applications. This study reports, for the first time, the generation of clinical‐grade and functional cardiomyocytes from hPSCs where BSA‐free and chemically defined conditions were maintained throughout the whole process. This provides the possibility of future therapeutic use of clinical‐grade hPSCs‐derived cardiomyocytes in treating heart diseases. Copyright © 2016 John Wiley & Sons, Ltd.

[1]  Liu Wang,et al.  Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions , 2015, Stem Cell Research & Therapy.

[2]  E. Tartour,et al.  Human embryonic stem cell-derived cardiac progenitors for severe heart failure treatment: first clinical case report. , 2015, European heart journal.

[3]  Sean P. Palecek,et al.  Chemically defined, albumin-free human cardiomyocyte generation , 2015, Nature Methods.

[4]  C. Mummery,et al.  Pluripotent stem cell derived cardiovascular progenitors--a developmental perspective. , 2015, Developmental biology.

[5]  Praveen Shukla,et al.  Chemically defined generation of human cardiomyocytes , 2014, Nature Methods.

[6]  Paul W. Burridge,et al.  Human Stem Cells for Modeling Heart Disease and for Drug Discovery , 2014, Science Translational Medicine.

[7]  Paul M. Rindler,et al.  The Oxygen-Rich Postnatal Environment Induces Cardiomyocyte Cell-Cycle Arrest through DNA Damage Response , 2014, Cell.

[8]  Charles E. Murry,et al.  Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate Non-Human Primate Hearts , 2014, Nature.

[9]  Paul M. Rindler,et al.  The Oxygen-Rich Postnatal Environment Induces Cardiomyocyte Cell-Cycle Arrest through DNA Damage Response , 2014, Cell.

[10]  J. Garbern,et al.  Cardiac stem cell therapy and the promise of heart regeneration. , 2013, Cell stem cell.

[11]  Sean P. Palecek,et al.  Insulin Inhibits Cardiac Mesoderm, Not Mesendoderm, Formation During Cardiac Differentiation of Human Pluripotent Stem Cells and Modulation of Canonical Wnt Signaling Can Rescue This Inhibition , 2013, Stem cells.

[12]  Sean P. Palecek,et al.  Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions , 2012, Nature Protocols.

[13]  Norio Nakatsuji,et al.  A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions. , 2012, Cell reports.

[14]  J. I. Izpisúa Belmonte,et al.  Small molecule-mediated TGF-β type II receptor degradation promotes cardiomyogenesis in embryonic stem cells. , 2012, Cell stem cell.

[15]  E. Wolvetang,et al.  Primitive cardiac cells from human embryonic stem cells. , 2012, Stem cells and development.

[16]  Sean P. Palecek,et al.  Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling , 2012, Proceedings of the National Academy of Sciences.

[17]  E. Marbán,et al.  Direct comparison of different stem cell types and subpopulations reveals superior paracrine potency and myocardial repair efficacy with cardiosphere-derived cells. , 2012, Journal of the American College of Cardiology.

[18]  G. Keller,et al.  Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. , 2012, Cell stem cell.

[19]  Liu Wang,et al.  Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells , 2011, Cell Research.

[20]  R. Passier,et al.  NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes , 2011, Nature Methods.

[21]  Xuan Yuan,et al.  A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability , 2011, PloS one.

[22]  Jing Zhang,et al.  Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals , 2011, Cell Research.

[23]  Jennifer M. Bolin,et al.  Chemically defined conditions for human iPS cell derivation and culture , 2011, Nature Methods.

[24]  G. Keller,et al.  Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. , 2011, Cell stem cell.

[25]  M. Noseda,et al.  Cardiopoietic factors: extracellular signals for cardiac lineage commitment. , 2011, Circulation research.

[26]  J. Gold,et al.  Human embryonic stem cell-derived cardiomyocytes engraft but do not alter cardiac remodeling after chronic infarction in rats. , 2010, Journal of molecular and cellular cardiology.

[27]  Wei-Zhong Zhu,et al.  Neuregulin/ErbB Signaling Regulates Cardiac Subtype Specification in Differentiating Human Embryonic Stem Cells , 2010, Circulation research.

[28]  Chad A. Cowan,et al.  A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates. , 2010, The Journal of clinical investigation.

[29]  H. Redl,et al.  Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. , 2010, Cell stem cell.

[30]  Geoffrey L. Francis,et al.  Albumin and mammalian cell culture: implications for biotechnology applications , 2010, Cytotechnology.

[31]  D. Roberts,et al.  Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages , 2009, Nature.

[32]  Elizabeth J. Robertson,et al.  Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo , 2009, Nature Reviews Molecular Cell Biology.

[33]  Eric D. Adler,et al.  Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population , 2008, Nature.

[34]  E. Stanley,et al.  A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies , 2008, Nature Protocols.

[35]  J. I. Izpisúa Belmonte,et al.  Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal , 2008, PloS one.

[36]  P. Doevendans,et al.  Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. , 2007, Stem cell research.

[37]  Lila R Collins,et al.  Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts , 2007, Nature Biotechnology.

[38]  D. Loebel,et al.  Gene function in mouse embryogenesis: get set for gastrulation , 2007, Nature Reviews Genetics.

[39]  P. Burridge,et al.  Improved Human Embryonic Stem Cell Embryoid Body Homogeneity and Cardiomyocyte Differentiation from a Novel V‐96 Plate Aggregation System Highlights Interline Variability , 2007, Stem cells.

[40]  Ying Zhang,et al.  Cellular Cardiomyoplasty: Improvement of Left Ventricular Function Correlates with the Release of Cardioactive Cytokines , 2007, Stem cells.

[41]  M. Goumans,et al.  Endoglin Has a Crucial Role in Blood Cell–Mediated Vascular Repair , 2006, Circulation.

[42]  J. Ingwall,et al.  Evidence supporting paracrine hypothesis for Akt‐modified mesenchymal stem cell‐mediated cardiac protection and functional improvement , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[43]  M. Buckingham,et al.  Building the mammalian heart from two sources of myocardial cells , 2005, Nature Reviews Genetics.

[44]  C. Murry,et al.  Regenerating the heart , 2005, Nature Biotechnology.

[45]  J. Ingwall,et al.  Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells , 2005, Nature Medicine.

[46]  S. Swoap,et al.  Effect of ambient temperature on cardiovascular parameters in rats and mice: a comparative approach. , 2004, American journal of physiology. Regulatory, integrative and comparative physiology.

[47]  A. Terzic,et al.  Stable benefit of embryonic stem cell therapy in myocardial infarction. , 2004, American journal of physiology. Heart and circulatory physiology.

[48]  M. Burnett,et al.  Marrow-Derived Stromal Cells Express Genes Encoding a Broad Spectrum of Arteriogenic Cytokines and Promote In Vitro and In Vivo Arteriogenesis Through Paracrine Mechanisms , 2004, Circulation research.

[49]  Baotong Xie,et al.  The functional domains of human ventricular myosin light chain 1. , 2003, Biophysical chemistry.

[50]  James A Thomson,et al.  Human Embryonic Stem Cells Develop Into Multiple Types of Cardiac Myocytes: Action Potential Characterization , 2003, Circulation research.

[51]  Rene Spijker,et al.  Differentiation of Human Embryonic Stem Cells to Cardiomyocytes: Role of Coculture With Visceral Endoderm-Like Cells , 2003, Circulation.

[52]  Richard T. Lee,et al.  Ascorbic Acid Enhances Differentiation of Embryonic Stem Cells Into Cardiac Myocytes , 2003, Circulation.

[53]  Chunhui Xu,et al.  Characterization and Enrichment of Cardiomyocytes Derived From Human Embryonic Stem Cells , 2002, Circulation research.

[54]  Jürgen Hescheler,et al.  Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types , 1993, Mechanisms of Development.

[55]  Gregory J. Brewer,et al.  Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen , 1989, Brain Research.

[56]  Jan Rodriguez Parkitna,et al.  The FASEB Journal • Research Communication , 2007 .

[57]  D. B. Cooke,et al.  The isolation, enrichment, and comparative electron microscopic characterization of cellular components of the aged rat ventral prostate , 1985, The Prostate.

[58]  M. Rothschild,et al.  Regulation of albumin metabolism. , 1975, Annual review of medicine.