A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability

Background The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34+ cord blood using non-viral, non-integrating methods. Methodology/Principal Findings We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs. Conclusion/Significance This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.

[1]  Katja Schenke-Layland,et al.  Identification of the critical extracellular matrix proteins that promote human embryonic stem cell assembly. , 2009, Stem cells and development.

[2]  Ronald A. Li,et al.  Distinct cardiogenic preferences of two human embryonic stem cell (hESC) lines are imprinted in their proteomes in the pluripotent state. , 2008, Biochemical and biophysical research communications.

[3]  George Q. Daley,et al.  Reprogramming of human somatic cells to pluripotency with defined factors , 2008, Nature.

[4]  T. Kamp,et al.  Human embryonic stem cell-derived cardiomyocytes can be maintained in defined medium without serum. , 2006, Stem cells and development.

[5]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[6]  D. Frank Culture of Animal Cells: A Manual of Basic Technique , 1984, The Yale Journal of Biology and Medicine.

[7]  K. Krause,et al.  Fetal bovine serum enables cardiac differentiation of human embryonic stem cells. , 2007, Differentiation; research in biological diversity.

[8]  W. Lowry,et al.  Roadblocks en route to the clinical application of induced pluripotent stem cells , 2010, Journal of Cell Science.

[9]  H. Hermersdörfer R. I. Freshney: Culture of Animal Cells. A Manual of Basic Technique. Third Edition. 486 Seiten, zahlr. Abb. und Tab. Wiley‐Liss, a John Wiley and Sons, Inc., Publication. New York, Chichester, Brisbane, Toronto. Singapore 1994. Preis: 69.95 US $ , 1995 .

[10]  Alon Spira,et al.  High-Resolution Electrophysiological Assessment of Human Embryonic Stem Cell-Derived Cardiomyocytes: A Novel In Vitro Model for the Study of Conduction , 2002, Circulation research.

[11]  Alexander Meissner,et al.  Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. , 2010, Cell stem cell.

[12]  K. Hochedlinger,et al.  Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells , 2010, Nature Biotechnology.

[13]  H. Ichikawa,et al.  Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body development. , 2009, American journal of physiology. Heart and circulatory physiology.

[14]  Mike J. Mason,et al.  Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. , 2009, Cell stem cell.

[15]  Chad A. Cowan,et al.  Marked differences in differentiation propensity among human embryonic stem cell lines , 2008, Nature Biotechnology.

[16]  M. Simon,et al.  The role of oxygen availability in embryonic development and stem cell function , 2008, Nature Reviews Molecular Cell Biology.

[17]  S. Yamanaka,et al.  Cell line-dependent differentiation of induced pluripotent stem cells into cardiomyocytes in mice. , 2010, Cardiovascular research.

[18]  T. Self,et al.  Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7. , 2006, The International journal of developmental biology.

[19]  Lin Chen,et al.  Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells. , 2008, Blood.

[20]  Robert Passier,et al.  Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes. , 2010, Stem cell research.

[21]  Sean P. Palecek,et al.  Functional Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells , 2009, Circulation research.

[22]  Robert Passier,et al.  Increased Cardiomyocyte Differentiation from Human Embryonic Stem Cells in Serum‐Free Cultures , 2005, Stem cells.

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

[24]  M. Wiles,et al.  Embryonic stem cell development in a chemically defined medium. , 1999, Experimental cell research.

[25]  Martin J. Aryee,et al.  Epigenetic memory in induced pluripotent stem cells , 2010, Nature.

[26]  R. Stewart,et al.  Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences , 2009, Science.

[27]  C. Allegrucci,et al.  Differences between human embryonic stem cell lines. , 2007, Human reproduction update.

[28]  Ann Peters,et al.  Erythropoietic differentiation of a human embryonic stem cell line harbouring the sickle cell anaemia mutation. , 2010, Reproductive biomedicine online.

[29]  Eugenia Kumacheva,et al.  Generation of human embryonic stem cell‐derived mesoderm and cardiac cells using size‐specified aggregates in an oxygen‐controlled bioreactor , 2009, Biotechnology and bioengineering.

[30]  G. Proetzel,et al.  The use of a chemically defined media for the analyses of early development in ES cells and mouse embryos. , 2002, Methods in molecular biology.

[31]  Lior Gepstein,et al.  Modelling the long QT syndrome with induced pluripotent stem cells , 2011, Nature.

[32]  Leslie Tung,et al.  In vitro electrophysiological mapping of stem cells. , 2010, Methods in molecular biology.

[33]  L Gepstein,et al.  Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. , 2001, The Journal of clinical investigation.

[34]  Olga K Afanasiev,et al.  Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells , 2010, PloS one.

[35]  A. Trounson,et al.  Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro , 2000, Nature Biotechnology.

[36]  R. Lahesmaa,et al.  Gene Expression Signatures of Seven Individual Human Embryonic Stem Cell Lines , 2005, Stem cells.

[37]  Yunyu Zhang,et al.  Wnt3a‐Induced Mesoderm Formation and Cardiomyogenesis in Human Embryonic Stem Cells , 2009, Stem cells.

[38]  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.

[39]  C. Mummery,et al.  Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells. , 2008, Differentiation; research in biological diversity.

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

[41]  P. Zandstra,et al.  Reproducible, Ultra High-Throughput Formation of Multicellular Organization from Single Cell Suspension-Derived Human Embryonic Stem Cell Aggregates , 2008, PloS one.

[42]  Elias T. Zambidis,et al.  Generation of Nonviral Integration-Free Induced Pluripotent Stem Cells from Plucked Human Hair Follicles , 2011 .

[43]  E. Stanley,et al.  Differentiating Embryonic Stem Cells Pass through ‘Temporal Windows’ That Mark Responsiveness to Exogenous and Paracrine Mesendoderm Inducing Signals , 2010, PloS one.

[44]  G. Churchill,et al.  Characterization of human embryonic stem cell lines by the International Stem Cell Initiative , 2007, Nature Biotechnology.

[45]  R. Passier,et al.  Insulin Redirects Differentiation from Cardiogenic Mesoderm and Endoderm to Neuroectoderm in Differentiating Human Embryonic Stem Cells , 2008, Stem cells.

[46]  L. Fink,et al.  Generation and characterization of functional cardiomyocytes using induced pluripotent stem cells derived from human fibroblasts , 2009, Cell biology international.

[47]  Fred H. Gage,et al.  Transcriptional Signature and Memory Retention of Human-Induced Pluripotent Stem Cells , 2009, PloS one.

[48]  Shau-Ping Lin,et al.  Hypoxic culture maintains self-renewal and enhances embryoid body formation of human embryonic stem cells. , 2010, Tissue engineering. Part A.

[49]  Chunhui Xu,et al.  Feeder-free growth of undifferentiated human embryonic stem cells , 2001, Nature Biotechnology.

[50]  Kenneth R Chien,et al.  Regeneration next: toward heart stem cell therapeutics. , 2009, Cell stem cell.

[51]  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.

[52]  Karl-Ludwig Laugwitz,et al.  Patient-specific induced pluripotent stem-cell models for long-QT syndrome. , 2010, New England Journal of Medicine.

[53]  Jarno M. A. Tanskanen,et al.  Substantial variation in the cardiac differentiation of human embryonic stem cell lines derived and propagated under the same conditions—a comparison of multiple cell lines , 2009, Annals of medicine.

[54]  M. Barron,et al.  Requirement for BMP and FGF signaling during cardiogenic induction in non‐precardiac mesoderm is specific, transient, and cooperative , 2000, Developmental dynamics : an official publication of the American Association of Anatomists.

[55]  S. Cole,et al.  Sequences Human Induced Pluripotent Stem Cells Free of Vector and Transgene , 2012 .

[56]  J. Itskovitz‐Eldor,et al.  Molecular analysis of cardiomyocytes derived from human embryonic stem cells , 2005, Development, growth & differentiation.

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