Mass Production of Pluripotent Stem Cells and Derivatives

Objectives

We aim at

  • Establishing human pluripotent stem cell (hPSC) culture (hiPSCs and hESCs) and directed lineage-specific differentiation in clinical scale and GMP grade.
  • Establishing efficient cardiomyogenic differentiation and providing cardiomyocytes for tissue engineering and heart repair in animal models and envisioned therapies.
  • Advancing process development and upscaling in controlled stirred tank bioreactor.
  • Achieving advanced in vitro modeling of congenital cardiomyopathies.
  • Supporting process development for mass production of other lineages including endothelial cells, macrophages and megakaryocytes in collation with REBIRTH PIs.

Research Focus

Our research resulted in recent milestone papers on the defined single cell inoculated cultivation of hESCs and hiPSCs as “matrix-free cells only aggregates” in stirred suspension culture (i.e. independent of microcarriers or other extracellular matrices). This technique, for the first time, enables straightforward upscaling of hPSC culture in fully controlled stirred tank bioreactors providing the required “raw material” for the mass production of any required hPSC progenies. We work on the generation of pure hPSC cardiomyocytes (CMs) in chemically defined media at GMP compliant conditions followed by process transition to a cleanroom environment. We are producing cells “on-demand” in frame of numerous collaborative projects within REBIRTH and beyond, including ongoing network projects funded by DFG (e.g. KFO311), BMBF (iCARE, iMAC, BioPace) and EU H2020 (TECHNOBEAT) focusing on cardiac tissue engineering, in vitro modelling of cardiomyopathies and process development for hematopoietic lineage production.

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)

  • hPSC proteomics; F. Büttner, REBIRTH-Unit Stem Cell Glycomics and Proteomics, MHH
  • In vitro modeling of Cardiomyopathies; T. Kraft, U Martin, REBIRTH-Unit Large Animal Models, MHH, D. Hilfiker-Kleiner , K. Wollert, and T. Thum.
  • Cardiac Tissue Engineering & Animal Models; I. Gruh, A. Haverich, REBIRTH-Unit Myocardial Tissue Engineering, MHH
  • Advanced imaging, printing and manipulation of hPSC- and cardiac- aggregates in 3D-culture; H. Meyer, REBIRTH-Unit Laser Manipulation and Cellular Engineering, Laser Zentrum Hannover , A. Heisterkamp and S. Kalies, B. Chichkov
  • Chemical compounds and recombinant factors for culture media development; T. Scheper, REBIRTH-Unit Production and Purification of Recombinant Polymers, A. Kirschning, G. Dräger, REBIRTH-Unit Functional Polymers and Regenerative Agents, Leibniz Universität Hannover
  • Hematopoietic differentiation: N. Lachmann, T. Moritz, C. Figueiredo
  • Genetic engineering: D. Wirth, A. Schambach
  • International TECHNOBEAT partners: C. Mummery (Leiden University), Peter Andrews (University Sheffield) D. Strunk (Paracelsus Medizinische Privanuniversität, Salzburg), J. Sluijter (University Medical Center Utrecht).

Grants (selection)

  • StemBANCC: Large-scale, 5 year academic-industry partnership. Aims: hiPSC lines for in vitro modeling of chronic diseases and drug testing.
  • TECHNOBEAT: Large EU H2020 network grant, 4 year academic-industry collaboration: Aims: Develop novel Tools and Technologies for Breakthrough in hiPS cell based Heart Therapies
  • iCARE: BMBF network grant. Aims: Induced pluripotent stem cells for Clinically Applicable heart Repair
  • iMAC: BMBF network grant. Aims: Genetically corrected iPSC-derived macrophages (i-MAC) for innovative gene therapeutic strategies.
  • KFO311: DFG funded network. Aims: Establish novel clinical treatments by research on Advanced cardiac and pulmonary failure: mechanical unloading and repair

Extra

Established industrial collaborations on process development & reactor technologies and clinical translation:

  • DASGIP / Eppendorf (Jülich / Hamburg, Germany)
  • Ovizio Imaging Systems (Brussels, Belgium)
  • Miltenyi Biotec (Bergisch Gladbach, Germany)
  • KadimaStem (Rehovot, Israel)

Publications

2013 - ongoing

2018

Christoffersson J, Meier F, Kempf H, Schwanke K, Coffee M, Beilmann M, Zweigerdt R, Mandenius C-F. A Cardiac Cell Outgrowth Assay for Evaluating Drug Compounds Using a Cardiac Spheroid-on-a-Chip Device. Bioengineering. 2018;5(2).

Olmer R, Engels L, Usman A, Menke S, Malik MNH, Pessler F, Göhring G, Bornhorst D, Bolten S, Abdelilah-Seyfried S, Scheper T, Kempf H, Zweigerdt R, Martin U. Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture. Stem Cell Reports. 2018;10(5):1657-72.

Koch L, Deiwick A, Franke A, Schwanke K, Haverich A, Zweigerdt R, Chichkov B. Laser bioprinting of human induced pluripotent stem cells—the effect of printing and biomaterials on cell survival, pluripotency, and differentiation. Biofabrication. 2018;10(3):035005.

Lipps C, Klein F, Wahlicht T, Seiffert V, Butueva M, Zauers J, Truschel T, Luckner M, Köster M, MacLeod R, Pezoldt J, Hühn J, Yuan Q, Müller PP, Kempf H, Zweigerdt R, Dittrich-Breiholz O, Pufe T, Beckmann R, Drescher W, Riancho J, Sañudo C, Korff T, Opalka B, Rebmann V, Göthert JR, Alves PM, Ott M, Schucht R, Hauser H, Wirth D, May T. Expansion of functional personalized cells with specific transgene combinations. Nature Communications. 2018;9:994.

Iorga B, Schwanke K, Weber N, Wendland M, Greten S, Piep B, dos Remedios CG, Martin U, Zweigerdt R, Kraft T, Brenner B. Differences in Contractile Function of Myofibrils within Human Embryonic Stem Cell-Derived Cardiomyocytes vs. Adult Ventricular Myofibrils Are Related to Distinct Sarcomeric Protein Isoforms. Frontiers in Physiology. 2017;8:1111.

Weist R, Flörkemeier T, Roger Y, Franke A, Schwanke K, Zweigerdt R, Martin U, Willbold E, Hoffmann A. Differential Expression of Cholinergic System Components in Human Induced Pluripotent Stem Cells, Bone Marrow-Derived Multipotent Stromal Cells, and Induced Pluripotent Stem Cell-Derived Multipotent Stromal Cells. Stem Cells and Development. 2017;27(3):166-83.

Kempf H, Zweigerdt R. Scalable Cardiac Differentiation of Pluripotent Stem Cells Using Specific Growth Factors and Small Molecules. In: Martin U, Zweigerdt R, Gruh I, editors. Engineering and Application of Pluripotent Stem Cells. Cham: Springer International Publishing; 2018. p. 39-69.

2017

Sgodda M, Dai Z, Zweigerdt R, Sharma AD, Ott M, Cantz T. A Scalable Approach for the Generation of Human Pluripotent Stem Cell-Derived Hepatic Organoids with Sensitive Hepatotoxicity Features. Stem Cells and Development. 2017;26(20):1490-504.

Massai D, Bolesani E, Diaz DR, Kropp C, Kempf H, Halloin C, Martin U, Braniste T, Isu G, Harms V, Morbiducci U, Dräger G, Zweigerdt R. Sensitivity of human pluripotent stem cells to insulin precipitation induced by peristaltic pump-based medium circulation: considerations on process development. Scientific Reports. 2017;7:3950.

Rojas SV, Kensah G, Rotaermel A, Baraki H, Kutschka I, Zweigerdt R, Martin U, Haverich A, Gruh I, Martens A. Transplantation of purified iPSC-derived cardiomyocytes in myocardial infarction. PLoS ONE. 2017;12(5):e0173222.

Konze SA, Werneburg S, Oberbeck A, Olmer R, Kempf H, Jara-Avaca M, Pich A, Zweigerdt R, Buettner FFR. Proteomic Analysis of Human Pluripotent Stem Cell Cardiomyogenesis Revealed Altered Expression of Metabolic Enzymes and PDLIM5 Isoforms. Journal of Proteome Research. 2017;16(3):1133-49.

Gentemann L, Kalies S, Coffee M, Meyer H, Ripken T, Heisterkamp A, Zweigerdt R, Heinemann D. Modulation of cardiomyocyte activity using pulsed laser irradiated gold nanoparticles. Biomedical Optics Express. 2017;8(1):177-92.

Jara-Avaca M, Kempf H, Rückert M, Robles-Diaz D, Franke A, de la Roche J, Fischer M, Malan D, Sasse P, Solodenko W, Dräger G, Kirschning A, Martin U, Zweigerdt R. EBIO Does Not Induce Cardiomyogenesis in Human Pluripotent Stem Cells but Modulates Cardiac Subtype Enrichment by Lineage-Selective Survival. Stem Cell Reports. 2017;8(2):305-17.

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.

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