Unit 1.2 - iPSCs for Disease Modelling, Drug Screening and Cell Therapy

Objectives

Research of the Clinic of Cardiothoracic, Transplantation and Vascular surgery is dedicated to clinically relevant topics of organ transplantation, the development of functionalized implants and regenerative medicine. In the Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO) different aspects of regenerative medicine and organ transplantation are investigated.

Research Focus

Technologies for generation of patient-specific induced pluripotent stem cells (iPSCs) together with exciting new tools for highly efficient targeted genome engineering create new options for basic research and therapeutic application of iPSC derivatives, and are therefore in the focus of this research group.

These technologies include TALE nuclease (TALEN) and CRISPR/Cas-based genome engineering, which is applied to efficiently introduce transgenes or mutations into specific genomic loci, or to correct disease-specific mutations in patient-derived iPSCs. Using these techniques, reporter cell lines can be generated in order to screen for therapeutically relevant compounds, and specific mutations can be tested for their disease phenotype. Also, the ability to efficiently introduce transgenes at specific safe genomic loci represents a straightforward approach for the generation of clinically applicable transgenic pluripotent stem cell lines and will enable the parallel expression of several transgenes with defined expression levels. Additionally, in case of genetic defects of patient-derived iPSCs, cells need to be corrected before they can be re-transplanted into the patient.

Another focus of the group is the investigation of potential risks during therapeutic applications of iPSC derivatives, especially genetic abnormalities and mutations that may be inherited from source cells or arise during reprogramming and expansion. In this context, we are also interested to compare the “biological quality” of iPSCs from old versus young cell sources. We are additionally analyzing the role of human non-LTR retrotransposons (LINE-1 elements) which might induce genetic mutations.

Noteworthy, preclinical large animal studies will be required to assess safety and efficacy of iPSC-based cellular therapies. Among the large animal species frequently used in preclinical efficacy and safety studies, pigs and sheep are of special importance. Consequently, it is aim of the group to establish true pluripotent iPSCs from at least one of these species. Importantly, macaques show the highest similarities to humans at physiological, cellular, and molecular levels and thus iPSCs from Cynomolgus monkeys (cyiPSCs) have been generated and may be applied in highly suitable preclinical large animal models.

Achievements/Planning

In view of future clinical application, we conducted a comprehensive study to investigate in detail the type, frequency and origin of genetic abnormalities in human iPSCs. Importantly we also aimed at answering the question whether iPSCs derived from old individuals harbor more chromosomal abnormalities than iPSCs derived from neonatal cell sources. Karyotype analyses as well as Array CGH were performed in close collaboration with the REBIRTH groups 9.5 and 9.6 (Steinemann, Göhring). For exom sequencing, a new SOLiD 5500 device funded by the BMBF was used and necessary bioinformatics were established in close collaboration with the group of B. Tümmler at MHH.

Disease-specific iPSC lines for modelling cardiomyopathies were established from patients with different mutations. Also several iPSC lines from cystic fibrosis patients with the most common ΔF508 mutation were established. Finally, we successfully generated cyiPSCs from skin fibroblasts, resulting in almost identical growth characteristics as compared to cynomolgus embryonic stem cells. We were also able to differentiate these cells into functional cardiomyocytes and to generate transgenic cyiPSC clones with stable reporter expression to enable cell tracking in recipient animals. Cardiac differentiation of the cyiPSCs is currently optimized together with researchers from Unit 5.7.

Both the Zinc Finger Nuclease (ZFN)- and the TALEN-based approach have already been successfully used in our lab for highly efficient gene engineering (Merkert et al., Stem Cell Reports, 2014) and to correct for genetic defects in patient-derived iPSCs. Moreover, we successfully established a TALEN assembly platform to generate various TALENs for several reporter cell lines for cardiomyocyte and lung-specific differentiation, and further gene correction approaches.

 

 


Future applications of TALEN-based gene editing of patient-derived iPS cells

Collaborations

  • R. Bals, University Medical Center of the Saarland (iPSCs for α1-Antitrypsin Deficiency)
  • R. Behr, German Primate Center, Göttingen
  • T. Cathomen, Universitätsklinikum Freiburg (ZFN)
  • E. Curnow, Washington National Primate Research Center (cyiPSCs)
  • L. Galietta, Ospedale Gaslini, Genoa, Italy (iPSC-based drug screening)
  • G. Göhring, D. Steinemann, Dept. of Mol. Pathol., MHH (Karyotyping, Array CGH)
  • I. Gruh, HTTG, MHH (cardiac differentiation)
  • M. Mall, Heidelberg University Hospital (iPSCs for Cystic Fibrosis)
  • B. Tümmler, Paed. Pneumology, MHH (exom sequencing)
  • G. Schumann, Paul-Ehrlich-Institut, Langen (LINE-1)
  • B. Scholte, Erasmus MC Rotterdam, Netherlands
  • BMWZ – Biomolecular Center of Drug Research, LUH: Collaborations with groups of A. Kirschning and G. Dräger
  • DZL: Close collaborations with RGs of Prof. Dr. M. Ochs, Prof. Dr. G. Hansen, Prof. Dr. B. Tümmler, and Dr. Korossis/ Dr. Wiegmann
  • Gynecology, MHH (young somatic cells)
  • HTTG clinic, MHH (old somatic cells)
  • NIFE - Lower Saxony Centre for Biomedical Engineering, Implant Research and Development: Collaborations with groups of A. Heisterkamp, A. Kirschning, G. Dräger and T. Scheper

Public Relations

Further Research Projects

  • DFG: G. Schumann, Paul-Ehrlich-Institute; U. Martin, MHH; P. Horn, Uniklinikum Essen; V. Benes, EMBL Genomics Core Facility, Heidelberg, Project: LINE-1-mediated retrotransposition in human pluripotent stem cells: Consequences for genomic stability of hES and hiPS cells and its derivatives
  • DFG: I. Gruh, U. Martin, Project: In vitro organisation of cardiovascular cells derived from hiPSCs into vascularised heart tissue
  • DFG: T. Kraft, B. Brenner, R. Zweigerdt, U. Martin, Project: An in vitro model for Familial Hypertrophic Cardiomyopathy based on cardiomyoctes derived from human induced pluripotent stem cells
  • BMBF: J. Gottlieb, G. Hansen, A. Haverich, H. Hedrich, M. Hoeper, E. Hummers-Pradier, S. Janciauskiene, M- Kabesch, U. Martin, U. Maus, M. Ochs, B. Tümmler, F. Wacker, G. Warnecke, T. Welte, MHH; Coordination ELD: Haverich/ Martin, Project: BREATH - Biomedical Research in Endstage And ObsTructive Lung Disease Hannover; Sub-project ELD-3.1: Generation of iPS-derived endothelial cells (EC) for a biohybrid lung and therapies targeting pulmonary hypertension (PH) Sub-project ELD-3.2: Treatment of Pulmonary Diseases based on Pluripotent Stem Cells PH-2.4: Endothelial progenitor cell (EPC)-based revascularisation of the lung
  • IMI/EU: U. Martin, R. Zweigerdt, Project: StemBANCC - Stem cells for Biological Assays of Novel drugs and prediCtive toxicology
  • EU: R. Zweigerdt, A. Haverich, U. Martin (MHH), C. Mummery (LUMC, Netherlands), P. Andrews (USF, UK), G. Pasterkamp (MCU, Netherlands), J. Itskovitz-Eldor (TIT, Israel), D. Strunk, K. Schallmoser (PMU; Austria), K. Kinast, M. Arnold (DASGIP, Germany), P. Mathuis (OVIZIO, Belgium), Project: Tools and Technologies for breakthrough in heart therapies – TECHNOBEAT
  • EU: U. Martin, M. Amaral (FCUL-Faculty of Sciences, University of Lisboa), L. Galietta (Istituto Giannina Gaslini – U.O.C. Genetica Medica), B.J. Scholte (Erasmus MC Rotterdam), J. Rossant (Peter Gilgan Centre for Research and Learning), J.W. Hanrahan (Department of Physiology, McGill University), Project: INSTINCT - Induced pluripotent stem cells for identification of novel drug combinations targeting cystic fibrosis lung and liver disease

Equipment and Service Facilities

[Please find a list of all available equipment here]

Further Information

www.lebao.de

Staff

Martin, Ulrich Prof. Dr. Martin.Ulrich(at)mh-hannover.de
Haase, Alexandra, Dr. Haase.Alexandra(at)mh-hannover.de
Merkert, Sylvia Dr. Merkert.Sylvia(at)mh-hannover.de
Wunderlich, Stephanie Dr. Wunderlich.Stephanie(at)mh-hannover.de
Engels, Lena Engels.Lena(at)mh-hannover.de
Kosanke, Maike Kosanke.Maike(at)mh-hannover.de
Malysheva, Svitlana Malysheva.Svitlana(at)mh-hannover.de
Osetek, Katarzyna Osetek.Katarzyna(at)mh-hannover.de
Schubert, Madline Schubert.Madline(at)mh-hannover.de
Witthuhn, Anett Witthuhn.Anett(at)mh-hannover.de
Beier, Jennifer Beier. Jennifer(at)mh-hannover.de
Kohrn, Tim Kohrn.Tim(at)mh-hannover.de
Opel, Ulrike Opel.Ulrike(at)mh-hannover.de