Mass Production of Pluripotent Stem Cells and Derivatives

Objectives

We aim to

  • Establish human pluripotent stem cell (hPSC) culture (including hiPSCs and hESCs) in clinical scale and grade.
  • Establish efficient cardiomyogenic differentiation of hPSCs and provide. cardiomyocytes for tissue engineering and heart repair in pre-clinical animal model.
  • Perform process development for hPSC production and differentiation in controlled bioreactor.
  • Achieve in vitro modeling of congenital cardiomyopathie.
  • Achieve process up-scaling to 1 Liter scale in stirred bioreactors.

Research Focus

Our research resulted in recent milestone papers on the defined, single cell inoculated cultivation of human pluripotent stem cells (hPSC, including hESCs and hiPSCs) as “cells only” aggregates in suspension culture, independent of micro carriers or other extracellular matrices. This technique, for the first time, enables straightforward up-scaling of hPSC culture in, fully controllable stirred-tank bioreactors providing the required “raw material” for the mass production of any differentiated human PSC-derived progenies. Independent research allowed the generation of pure human hPSC-derived cardiomyocytes (CMs) in chemically defined, clinically compliant culture media in suspension culture. We are currently working on combining the hPSC suspension culture process with our CMs differentiation protocol in controlled bioreactors to develop clinically relevant conditions for CMs production. Cells will be provided “on-demand” in frame of REBIRTH-2 collaborative projects, particularly for cardiac tissue engineering and in vitro modelling of cardiomyopathies.

Achievements

  • Robust hPSC expansion and maintenance of pluripotency when cultured in suspension as “cell only” aggregates in 100 ml scale stirred bioreactors in defined media
  • Efficient cardiomyogenic differentiation of hPSCs in stirred bioreactors achieving 80-90% cardiomyocyte purity in chemically defined media

Collaborations (selection)

Grants (selection)

  • StemBANCC: Large-scale, 5 year academic-industry partnership. Aims: hiPSC lines for in vitro modeling of chronic diseases and drug testing.

Extra

Established industrial collaborations on process development & reactor technology with:

  • STEMCELL Technologies (Vancouver, Canada)
  • DASGIP / Eppendorf (Jühlich / Hamburg, Germany)

Publications

2013 - ongoing

2016

Weber N, Schwanke K, Greten S, Wendland M, Iorga B, Fischer M, Geers-Knorr C, Hegermann J, Wrede C, Fiedler J, Kempf H, Franke A, Piep B, Pfanne A, Thum T, Martin U, Brenner B, Zweigerdt R, Kraft T. Stiff matrix induces switch to pure beta-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes. Basic research in cardiology. 2016;111(6):68.

Viereck J, Kumarswamy R, Foinquinos A, Xiao K, Avramopoulos P, Kunz M, Dittrich M, Maetzig T, Zimmer K, Remke J, Just A, Fendrich J, Scherf K, Bolesani E, Schambach A, Weidemann F, Zweigerdt R, de Windt LJ, Engelhardt S, Dandekar T, Batkai S, Thum T. Long noncoding RNA Chast promotes cardiac remodeling. Science translational medicine. 2016;8(326):326ra22.

Kropp C, Kempf H, Halloin C, Robles-Diaz D, Franke A, Scheper T, Kinast K, Knorpp T, Joos TO, Haverich A, Martin U, Zweigerdt R, Olmer R. Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors. Stem cells translational medicine. 2016;5(10):1289-301.

Kempf H, Olmer R, Haase A, Franke A, Bolesani E, Schwanke K, Robles-Diaz D, Coffee M, Gohring G, Drager G, Potz O, Joos T, Martinez-Hackert E, Haverich A, Buettner FF, Martin U, Zweigerdt R. Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells. Nature communications. 2016;7:13602.

Kempf H, Andree B, Zweigerdt R. Large-scale production of human pluripotent stem cell derived cardiomyocytes. Adv Drug Deliv Rev. 2016;96:18-30.

Andree B, Zweigerdt R. Directing Cardiomyogenic Differentiation and Transdifferentiation By Ectopic Gene Expression - Direct Transition Or Reprogramming Detour? Curr Gene Ther. 2016;16(1):14-20.

2015

Kempf H, Kropp C, Olmer R, Martin U, Zweigerdt R. Cardiac Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture. Nat Protoc. 2015;10(9):1345-61.

Bergstrom G, Christoffersson J, Schwanke K, Zweigerdt R, Mandenius CF. Stem Cell Derived in Vivo-Like Human Cardiac Bodies in a Microfluidic Device for Toxicity Testing by Beating Frequency Imaging. Lab Chip. 2015;15(15):3242-9.

Rojas SV, Martens A, Zweigerdt R, Baraki H, Rathert C, Schecker N, Rojas-Hernandez S, Schwanke K, Martin U, Haverich A, Kutschka I. Transplantation Effectiveness of Induced Pluripotent Stem Cells Is Improved by a Fibrinogen Biomatrix in an Experimental Model of Ischemic Heart Failure. Tissue Eng Part A. 2015;21(13-14):1991-2000. Epub 2015/04/14.

2014

Antonopoulos GC, Pscheniza D, Lorbeer1 R-A, Heidrich M, Schwanke K, Zweigerdt, Ripken T, Meyer H. Correction of image artifacts caused by refractive cylindrical surfaces in scanning optical tomography. Biomedizinische Technik 09/2014; 59(s1). DOI: 10.1515/bmt-2014-4219

Zweigerdt R, Gruh I, Martin U. Your Heart on a Chip: Ipsc-Based Modeling of Barth-Syndrome-Associated Cardiomyopathy. Cell Stem Cell. 2014;15(1):9-11.

Schwanke K, Merkert S, Kempf H, Hartung S, Jara-Avaca M, Templin C, Gohring G, Haverich A, Martin U, Zweigerdt R. Fast and Efficient Multitransgenic Modification of Human Pluripotent Stem Cells. Hum Gene Ther Methods. 2014;25(2):136-53.

Martens A, Rojas SV, Baraki H, Rathert C, Schecker N, Zweigerdt R, Schwanke K, Rojas-Hernandez S, Martin U, Saito S, Schmitto JD, Haverich A, Kutschka I. Substantial Early Loss of Induced Pluripotent Stem Cells Following Transplantation in Myocardial Infarction. Artif Organs. 2014;38(11):978-84.

Martens A, Rojas SV, Baraki H, Rathert C, Schecker N, Hernandez SR, Schwanke K, Zweigerdt R, Martin U, Saito S, Haverich A, Kutschka I. Macroscopic Fluorescence Imaging: A Novel Technique to Monitor Retention and Distribution of Injected Microspheres in an Experimental Model of Ischemic Heart Failure. PLoS One. 2014;9(8):e101775.

Konze SA, van Diepen L, Schroder A, Olmer R, Moller H, Pich A, Weissmann R, Kuss AW, Zweigerdt R, Buettner FF. Cleavage of E-Cadherin and Beta-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells. Mol Cell Proteomics. 2014;13(4):990-1007.

Kempf H, Olmer R, Kropp C, Ruckert M, Jara-Avaca M, Robles-Diaz D, Franke A, Elliott DA, Wojciechowski D, Fischer M, Roa Lara A, Kensah G, Gruh I, Haverich A, Martin U, Zweigerdt R. Controlling Expansion and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture. Stem Cell Reports. 2014;3(6):1132-46.

Andree B, Bela K, Horvath T, Lux M, Ramm R, Venturini L, Ciubotaru A, Zweigerdt R, Haverich A, Hilfiker A. Successful Re-Endothelialization of a Perfusable Biological Vascularized Matrix (Biovam) for the Generation of 3d Artificial Cardiac Tissue. Basic Res Cardiol. 2014;109(6):441.

Ackermann M, Lachmann N, Hartung S, Eggenschwiler R, Pfaff N, Happle C, Mucci A, Gohring G, Niemann H, Hansen G, Schambach A, Cantz T, Zweigerdt R, Moritz T. Promoter and Lineage Independent Anti-Silencing Activity of the A2 Ubiquitous Chromatin Opening Element for Optimized Human Pluripotent Stem Cell-Based Gene Therapy. Biomaterials. 2014;35(5):1531-42.

2013

Kensah G, Roa Lara A, Dahlmann J, Zweigerdt R, Schwanke K, Hegermann J, Skvorc D, Gawol A, Azizian A, Wagner S, Maier LS, Krause A, Drager G, Ochs M, Haverich A, Gruh I, Martin U. Murine and Human Pluripotent Stem Cell-Derived Cardiac Bodies Form Contractile Myocardial Tissue in Vitro. Eur Heart J. 2013;34(15):1134-46.

Hartung S, Schwanke K, Haase A, David R, Franz WM, Martin U, Zweigerdt R. Directing Cardiomyogenic Differentiation of Human Pluripotent Stem Cells by Plasmid-Based Transient Overexpression of Cardiac Transcription Factors. Stem Cells Dev. 2013;22(7):1112-25.

Dahlmann J, Kensah G, Kempf H, Skvorc D, Gawol A, Elliott DA, Drager G, Zweigerdt R, Martin U, Gruh I. The Use of Agarose Microwells for Scalable Embryoid Body Formation and Cardiac Differentiation of Human and Murine Pluripotent Stem Cells. Biomaterials. 2013;34(10):2463-71.

2006 - 2012

2012

Templin C, Zweigerdt R, Schwanke K, Olmer R, Ghadri JR, Emmert MY, Muller E, Kuest SM, Cohrs S, Schibli R, Kronen P, Hilbe M, Reinisch A, Strunk D, Haverich A, Hoerstrup S, Luscher TF, Kaufmann PA, Landmesser U, Martin U. Transplantation and Tracking of Human-Induced Pluripotent Stem Cells in a Pig Model of Myocardial Infarction: Assessment of Cell Survival, Engraftment, and Distribution by Hybrid Single Photon Emission Computed Tomography/Computed Tomography of Sodium Iodide Symporter Transgene Expression. Circulation. 2012;126(4):430-9.

Olmer R, Lange A, Selzer S, Kasper C, Haverich A, Martin U, Zweigerdt R. Suspension Culture of Human Pluripotent Stem Cells in Controlled, Stirred Bioreactors. Tissue Eng Part C Methods. 2012;18(10):772-84.

Chen R, John J, Lavrentieva A, Müller S, Tomala M, Zhao Y, Zweigerdt R, Beutel S, Hitzmann B, Kasper C, Martin U, Rinas U, Stahl F, Scheper T. Cytokine Production Using Membrane Adsorbers: Human Basic Fibroblast Growth Factor Produced by Escherichia Coli. Engineering in Life Sciences. 2012;12(1):29-38.

2011

Zweigerdt R, Olmer R, Singh H, Haverich A, Martin U. Scalable Expansion of Human Pluripotent Stem Cells in Suspension Culture. Nat Protoc. 2011;6(5):689-700.

Palecek J, Zweigerdt R, Olmer R, Martin U, Kirschning A, Drager G. A Practical Synthesis of Rho-Kinase Inhibitor Y-27632 and Fluoro Derivatives and Their Evaluation in Human Pluripotent Stem Cells. Org Biomol Chem. 2011;9(15):5503-10.

Mauritz C, Martens A, Rojas SV, Schnick T, Rathert C, Schecker N, Menke S, Glage S, Zweigerdt R, Haverich A, Martin U, Kutschka I. Induced Pluripotent Stem Cell (Ipsc)-Derived Flk-1 Progenitor Cells Engraft, Differentiate, and Improve Heart Function in a Mouse Model of Acute Myocardial Infarction. Eur Heart J. 2011;32(21):2634-41.

Kempf H, Lecina M, Ting S, Zweigerdt R, Oh S. Distinct Regulation of Mitogen-Activated Protein Kinase Activities Is Coupled with Enhanced Cardiac Differentiation of Human Embryonic Stem Cells. Stem Cell Res. 2011;7(3):198-209.