Large Animal Models

Objectives

A long term goal is to produce well characterized large animal models for the above purpose, i.e. transgenic pigs from which humanized cardiomyocytes and hepatocytes can be harvested for cell therapeutic use. 

The project can be approached in a stepwise manner. In addition, it requires significant consideration of the ethical aspects. The following steps are envisaged: 

  • Discussion of the ethical dimension of the use of human iPS cells in this thematic context. 
  • Production of viable offspring from injection of human induced pluripotent stem cells (iPS cells) into porcine preimplantation embryos. 
  • Production of human iPSCs with a knockout for master genes of the neuronal lineage. 
  • Production of transgenic pigs with an inducible knockdown of mesodermal or cardiovascular master genes. 
  • Production of viable interspecies chimeras and ultimately the production of porcine offspring with hiPSC derived heart or liver tissue. 

Research Focus

With the advent of improved maps of the genome of the large domestic species and the availability of molecular tools for precise genetic modification, the creation of large transgenic animals models for specific research purposes has become feasible. This enables to overcome limitations that are inherent to transgenic mouse models. The most prominent example is the recently reported homozygous knockout for the CFTR gene in the domestic pig. Pigs with a homozygous knockout for CFTR develop the cystic fibrosis symptoms seen in human patients, which were not seen in transgenic mice. In this project we will use up-to-date molecular tools and technology to produce transgenic pigs with a humanized cardiomyocyte and hepatocyte population. The main goal is to produce human cardiomyocytes and hepatocytes, respectively, in the pig for use in cell therapies in human patients. In addition, we plan for a large animal model for familial hypertrophic cardiomyopathy (FHC). 

Publications

2013 - ongoing

2018

Montag J, Petersen B, Flögel AK, Becker E, Lucas-Hahn A, Cost G, Mühlfeld C, Kraft T, Niemann H, Brenner B. Successful knock-in of Hypertrophic Cardiomypathy mutation R723G into the MYH7 gene mimics HCM pathology in pigs. Scientific Reports. 2018 (accepted)

2017

Petkov SG, Glage S, Niemann H. Mouse iPSC generated with porcine reprogramming factors as a model for studying the effects of non-silenced heterologous transgenes on pluripotency. Journal of Stem Cells & Regenerative Medicine. 2017;13(1):20-8.

Mall EM, Herrmann D, Niemann H. Murine pluripotent stem cells with a homozygous knockout of Foxg1 show reduced differentiation towards cortical progenitors in vitro. Stem Cell Research. 2017;25:50-60.

Chatterjee A, Saha D, Niemann H, Gryshkov O, Glasmacher B, Hofmann N. Effects of cryopreservation on the epigenetic profile of cells. Cryobiology. 2017;74:1-7.

2016

Petersen B, Frenzel A, Lucas-Hahn A, Herrmann D, Hassel P, Klein S, Ziegler M, Hadeler K-G, Niemann H. Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes. Xenotransplantation. 2016;23(5):338-46.

Ramm R, Niemann H, Petersen B, Haverich A, Hilfiker A. Decellularized GGTA1-KO pig heart valves do not bind preformed human xenoantibodies. Basic Research in Cardiology. 2016;111(4):39.

Petkov S, Glage S, Nowak-Imialek M, Niemann H. Long-Term Culture of Porcine Induced Pluripotent Stem-Like Cells Under Feeder-Free Conditions in the Presence of Histone Deacetylase Inhibitors. Stem Cells Dev. 2016;25(5):386-94.

Garrels W, Mukherjee A, Holler S, Cleve N, Talluri TR, Barg-Kues B, Diederich M, Kohler P, Petersen B, Lucas-Hahn A, Niemann H, Izsvak Z, Ivics Z, Kues WA. Identification and re-addressing of a transcriptionally permissive locus in the porcine genome. Transgenic Res. 2016;25(1):63-70.

Fischer K, Kraner-Scheiber S, Petersen B, Rieblinger B, Buermann A, Flisikowska T, Flisikowski K, Christan S, Edlinger M, Baars W, Kurome M, Zakhartchenko V, Kessler B, Plotzki E, Szczerbal I, Switonski M, Denner J, Wolf E, Schwinzer R, Niemann H, Kind A, Schnieke A. Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing. Scientific reports. 2016;6:29081.

2015

Petkov S, Glage S, Nowak-Imialek M, Niemann H. Long-Term Culture of Porcine Induced Pluripotent Stem-Like Cells Under Feeder-Free Conditions in the Presence of Histone Deacetylase Inhibitors. Stem Cells and Development. 2015;25(5):386-94.

Hinkel R, Lange P, Petersen B, Gottlieb E, Ng JK, Finger S, Horstkotte J, Lee S, Thormann M, Knorr M, El-Aouni C, Boekstegers P, Reichart B, Wenzel P, Niemann H, Kupatt C. Heme Oxygenase-1 Gene Therapy Provides Cardioprotection Via Control of Post-Ischemic Inflammation: An Experimental Study in a Pre-Clinical Pig Model. Journal of the American College of Cardiology. 2015;66(2):154-65.

Petersen B, Niemann H. The future of transgenic farm animal production with emphasis on pigs. CAB Reviews 9, Nr. 44, 1-16. 2015.

Petersen B, Niemann H. Advances in Genetic Modification of Farm Animals Using Zinc-Finger Nucleases (Zfn). Chromosome Res. 2015;23(1):7-15. Epub 2015/01/19.

Denner J, Petersen B, Niemann H. Tolerance and Immune Response to the Porcine Endogenous Retrovirus in German Landrace Pigs Immunised with Viral Proteins. Virus Res. 2015;208:39-43. Epub 2015/06/06.

Ahrens HE, Petersen B, Ramackers W, Petkov S, Herrmann D, Hauschild-Quintern J, Lucas-Hahn A, Hassel P, Ziegler M, Baars W, Bergmann S, Schwinzer R, Winkler M, Niemann H. Kidneys from Α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected from Rejection During Ex Vivo Perfusion with Human Blood. Transplantation Direct. 2015;1(6):1-8.

Hinkel R, Lange P, Petersen B, Gottlieb E, Ng JK, Finger S, Horstkotte J, Lee S, Thormann M, Knorr M, El-Aouni C, Boekstegers P, Reichart B, Wenzel P, Niemann H, Kupatt C. Heme Oxygenase-1 Gene Therapy Provides Cardioprotection Via Control of Post-Ischemic Inflammation: An Experimental Study in a Pre-Clinical Pig Model. J Am Coll Cardiol. 2015;66(2):154-65. Epub 2015/07/15.

Ahrens HE, Petersen B, Herrmann D, Lucas-Hahn A, Hassel P, Ziegler M, Kues WA, Baulain U, Baars W, Schwinzer R, Denner J, Rataj D, Werwitzke S, Tiede A, Bongoni AK, Garimella PS, Despont A, Rieben R, Niemann H. Sirna Mediated Knockdown of Tissue Factor Expression in Pigs for Xenotransplantation. Am J Transplant. 2015;15(5):1407-14.

Talluri TR, Kumar D, Glage S, Garrels W, Ivics Z, Debowski K, Behr R, Niemann H, Kues WA. Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming. Cell Reprogram. 2015;17(2):131-40.

Reichart B, Niemann H, Chavakis T, Denner J, Jaeckel E, Ludwig B, Marckmann G, Schnieke A, Schwinzer R, Seissler J, Tonjes RR, Klymiuk N, Wolf E, Bornstein SR. Xenotransplantation of Porcine Islet Cells as a Potential Option for the Treatment of Type 1 Diabetes in the Future. Horm Metab Res. 2015;47(1):31-5. Epub 2014/12/17.

Petersen B, Niemann H. Molecular Scissors and Their Application in Genetically Modified Farm Animals. Transgenic Res. 2015;24(3):381-96. Epub 2015/01/22.

Heinzmann J, Mattern F, Aldag P, Bernal-Ulloa SM, Schneider T, Haaf T, Niemann H. Extended in Vitro Maturation Affects Gene Expression and DNA Methylation in Bovine Oocytes. Mol Hum Reprod. 2015;21(10):770-82.

2014

Keller M, Petersen B, Niemann H, Denner J. Lack of antibody response in pigs immunised with the transmembrane envelope protein of porcine endogenous retroviruses (PERV). J Gen. 2014. Virol 95, 1827-1831.

Garrels W, Holler S, Taylor U, Herrmann D, Niemann H, Ivics Z, Kues WA. Assessment of Fetal Cell Chimerism in Transgenic Pig Lines Generated by Sleeping Beauty Transposition. PLoS One. 2014;9(5):e96673.

Amrute-Nayak M, Lambeck KA, Radocaj A, Huhnt HE, Scholz T, Hahn N, Tsiavaliaris G, Walter WJ, Brenner B. Atp Turnover by Individual Myosin Molecules Hints at Two Conformers of the Myosin Active Site. Proc Natl Acad Sci U S A. 2014;111(7):2536-41. Epub 2014/02/20.

Hinkel R, Trenkwalder T, Petersen B, Husada W, Gesenhues F, Lee S, Hannappel E, Bock-Marquette I, Theisen D, Leitner L, Boekstegers P, Cierniewski C, Muller OJ, le Noble F, Adams RH, Weinl C, Nordheim A, Reichart B, Weber C, Olson E, Posern G, Deindl E, Niemann H, Kupatt C. Mrtf-a Controls Vessel Growth and Maturation by Increasing the Expression of Ccn1 and Ccn2. Nat Commun. 2014;5:3970.

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.

Urrego R, Rodriguez-Osorio N, Niemann H. Epigenetic Disorders and Altered Gene Expression after Use of Assisted Reproductive Technologies in Domestic Cattle. Epigenetics. 2014;9(6):803-15.

Petkov S, Hyttel P, Niemann H. The Small Molecule Inhibitors Pd0325091 and Chir99021 Reduce Expression of Pluripotency-Related Genes in Putative Porcine Induced Pluripotent Stem Cells. Cell Reprogram. 2014;16(4):235-40.

Brenner B, Seebohm B, Tripathi S, Montag J, Kraft T. Familial Hypertrophic Cardiomyopathy: Functional Variance among Individual Cardiomyocytes as a Trigger of Fhc-Phenotype Development. Front Physiol. 2014;5:392. Epub 2014/10/28.

Witjas-Paalberends ER, Ferrara C, Scellini B, Piroddi N, Montag J, Tesi C, Stienen GJ, Michels M, Ho CY, Kraft T, Poggesi C, van der Velden J. Faster Cross-Bridge Detachment and Increased Tension Cost in Human Hypertrophic Cardiomyopathy with the R403q Myh7 Mutation. J Physiol. 2014;592(Pt 15):3257-72. Epub 2014/06/15.

Merkert S, Wunderlich S, Bednarski C, Beier J, Haase A, Dreyer AK, Schwanke K, Meyer J, Gohring G, Cathomen T, Martin U. Efficient Designer Nuclease-Based Homologous Recombination Enables Direct Pcr Screening for Footprintless Targeted Human Pluripotent Stem Cells. Stem Cell Reports. 2014;2(1):107-18.

2013

Petkov S, Hyttel P, Niemann H. The Choice of Expression Vector Promoter Is an Important Factor in the Reprogramming of Porcine Fibroblasts into Induced Pluripotent Cells. Cell Reprogram. 2013;15(1):1-8.

Kues WA, Herrmann D, Barg-Kues B, Haridoss S, Nowak-Imialek M, Buchholz T, Streeck M, Grebe A, Grabundzija I, Merkert S, Martin U, Hall VJ, Rasmussen MA, Ivics Z, Hyttel P, Niemann H. Derivation and Characterization of Sleeping Beauty Transposon-Mediated Porcine Induced Pluripotent Stem Cells. Stem Cells Dev. 2013;22(1):124-35.

Herrmann D, Dahl JA, Lucas-Hahn A, Collas P, Niemann H. Histone Modifications and Mrna Expression in the Inner Cell Mass and Trophectoderm of Bovine Blastocysts. Epigenetics. 2013;8(3):281-9.

Hauschild-Quintern J, Petersen B, Cost GJ, Niemann H. Gene Knockout and Knockin by Zinc-Finger Nucleases: Current Status and Perspectives. Cell Mol Life Sci. 2013;70(16):2969-83.

Kraft T, Witjas-Paalberends ER, Boontje NM, Tripathi S, Brandis A, Montag J, Hodgkinson JL, Francino A, Navarro-Lopez F, Brenner B, Stienen GJ, van der Velden J. Familial Hypertrophic Cardiomyopathy: Functional Effects of Myosin Mutation R723g in Cardiomyocytes. J Mol Cell Cardiol. 2013;57:13-22. Epub 2013/01/16.

Pfaff N, Lachmann N, Ackermann M, Kohlscheen S, Brendel C, Maetzig T, Niemann H, Antoniou MN, Grez M, Schambach A, Cantz T, Moritz T. A Ubiquitous Chromatin Opening Element Prevents Transgene Silencing in Pluripotent Stem Cells and Their Differentiated Progeny. Stem Cells. 2013;31(3):488-99.

2006 - 2012

2012

Garrels W, Kues WB, Herrmann D, Holler S, Baulain U, Niemann H. Ectopic Expression of Human Telomerase Rna Component Results in Increased Telomerase Activity and Elongated Telomeres in Bovine Blastocysts. Biol Reprod. 2012;87(4):95.

Pfaff N, Lachmann N, Kohlscheen S, Sgodda M, Arauzo-Bravo MJ, Greber B, Kues W, Glage S, Baum C, Niemann H, Schambach A, Cantz T, Moritz T. Efficient Hematopoietic Redifferentiation of Induced Pluripotent Stem Cells Derived from Primitive Murine Bone Marrow Cells. Stem Cells Dev. 2012;21(5):689-701.

Semaan M, Kaulitz D, Petersen B, Niemann H, Denner J. Long-Term Effects of Perv-Specific Rna Interference in Transgenic Pigs. Xenotransplantation. 2012;19(2):112-21.

Brenner B, Hahn N, Hanke E, Matinmehr F, Scholz T, Steffen W, Kraft T. Mechanical and Kinetic Properties of Beta-Cardiac/Slow Skeletal Muscle Myosin. J Muscle Res Cell Motil. 2012;33(6):403-17. Epub 2012/08/01.

Nowak-Imialek M, Niemann H. Pluripotent Cells in Farm Animals: State of the Art and Future Perspectives. Reprod Fertil Dev. 2012;25(1):103-28.

Niemann H, Kind A, Schnieke A. Production of Biopharmaceuticals in Transgenic Animals. Pharmaceutical Biotechnology: Wiley-VCH Verlag GmbH & Co. KGaA; 2012. p. 71-111.

Wunderlich S, Haase A, Merkert S, Beier J, Schwanke K, Schambach A, Glage S, Gohring G, Curnow EC, Martin U. Induction of Pluripotent Stem Cells from a Cynomolgus Monkey Using a Polycistronic Simian Immunodeficiency Virus-Based Vector, Differentiation toward Functional Cardiomyocytes, and Generation of Stably Expressing Reporter Lines. Cell Reprogram. 2012;14(6):471-84. Epub 2012/12/01.

2011

Deshmukh RS, Ostrup O, Ostrup E, Vejlsted M, Niemann H, Lucas-Hahn A, Petersen B, Li J, Callesen H, Hyttel P. DNA Methylation in Porcine Preimplantation Embryos Developed in Vivo and Produced by in Vitro Fertilization, Parthenogenetic Activation and Somatic Cell Nuclear Transfer. Epigenetics. 2011;6(2):177-87.

Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath JW, Lucas-Hahn A, Zhang L, Meng X, Gregory PD, Schwinzer R, Cost GJ, Niemann H. Efficient Generation of a Biallelic Knockout in Pigs Using Zinc-Finger Nucleases. Proc Natl Acad Sci U S A. 2011;108(29):12013-7.

El Hajj N, Trapphoff T, Linke M, May A, Hansmann T, Kuhtz J, Reifenberg K, Heinzmann J, Niemann H, Daser A, Eichenlaub-Ritter U, Zechner U, Haaf T. Limiting Dilution Bisulfite (Pyro)Sequencing Reveals Parent-Specific Methylation Patterns in Single Early Mouse Embryos and Bovine Oocytes. Epigenetics. 2011;6(10):1176-88.

Nowak-Imialek M, Kues WA, Petersen B, Lucas-Hahn A, Herrmann D, Haridoss S, Oropeza M, Lemme E, Scholer HR, Carnwath JW, Niemann H. Oct4-Enhanced Green Fluorescent Protein Transgenic Pigs: A New Large Animal Model for Reprogramming Studies. Stem Cells Dev. 2011;20(9):1563-75.

Nowak-Imialek M, Kues W, Carnwath JW, Niemann H. Pluripotent Stem Cells and Reprogrammed Cells in Farm Animals. Microsc Microanal. 2011;17(4):474-97.

Kuetemeyer K, Lucas-Hahn A, Petersen B, Niemann H, Heisterkamp A. Femtosecond Laser-Induced Fusion of Nonadherent Cells and Two-Cell Porcine Embryos. J Biomed Opt. 2011;16(8):088001.

Garrels W, Mates L, Holler S, Dalda A, Taylor U, Petersen B, Niemann H, Izsvak Z, Ivics Z, Kues WA. Germline Transgenic Pigs by Sleeping Beauty Transposition in Porcine Zygotes and Targeted Integration in the Pig Genome. PLoS One. 2011;6(8):e23573.

Iqbal K, Kues WA, Baulain U, Garrels W, Herrmann D, Niemann H. Species-Specific Telomere Length Differences between Blastocyst Cell Compartments and Ectopic Telomere Extension in Early Bovine Embryos by Human Telomerase Reverse Transcriptase. Biol Reprod. 2011;84(4):723-33.

Petersen B, Ramackers W, Lucas-Hahn A, Lemme E, Hassel P, Queisser AL, Herrmann D, Barg-Kues B, Carnwath JW, Klose J, Tiede A, Friedrich L, Baars W, Schwinzer R, Winkler M, Niemann H. Transgenic Expression of Human Heme Oxygenase-1 in Pigs Confers Resistance against Xenograft Rejection During Ex Vivo Perfusion of Porcine Kidneys. Xenotransplantation. 2011;18(6):355-68.

2010

Kuetemeyer K, Lucas-Hahn A, Petersen B, Lemme E, Hassel P, Niemann H, Heisterkamp A. Combined Multiphoton Imaging and Automated Functional Enucleation of Porcine Oocytes Using Femtosecond Laser Pulses. J Biomed Opt. 2010;15(4):046006.

Niemann H, Carnwath JW, Herrmann D, Wieczorek G, Lemme E, Lucas-Hahn A, Olek S. DNA Methylation Patterns Reflect Epigenetic Reprogramming in Bovine Embryos. Cell Reprogram. 2010;12(1):33-42.

Nowak-Imialek M, Kues WA, Rudolph C, Schlegelberger B, Taylor U, Carnwath JW, Niemann H. Preferential Loss of Porcine Chromosomes in Reprogrammed Interspecies Cell Hybrids. Cell Reprogram. 2010;12(1):55-65.

2008

Niemann H, Tian XC, King WA, Lee RS. Epigenetic Reprogramming in Embryonic and Foetal Development Upon Somatic Cell Nuclear Transfer Cloning. Reproduction. 2008;135(2):151-63. Epub 2008/02/02.

Kues WA, Sudheer S, Herrmann D, Carnwath JW, Havlicek V, Besenfelder U, Lehrach H, Adjaye J, Niemann H. Genome-Wide Expression Profiling Reveals Distinct Clusters of Transcriptional Regulation During Bovine Preimplantation Development in Vivo. Proc Natl Acad Sci U S A. 2008;105(50):19768-73.

2004

Schaetzlein S, Lucas-Hahn A, Lemme E, Kues WA, Dorsch M, Manns MP, Niemann H, Rudolph KL. Telomere Length Is Reset During Early Mammalian Embryogenesis. Proc Natl Acad Sci U S A. 2004;101(21):8034-8. Epub 2004/05/19.

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