Non-human primate iPS cells for cell replacement therapies and human cardiovascular disease modeling
von Ignacio Rodriguez Polo
Datum der mündl. Prüfung:2019-10-29
Erschienen:2019-12-03
Betreuer:Prof. Dr. Rüdiger Behr
Gutachter:PD Dr. Rüdiger Behr
Gutachter:Prof. Dr. Gregor Bucher
Gutachter:Prof. Dr. Wolfram-Hubertus Zimmermann
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-7757
Dateien
Name:Ignacio Rodriguez Polo_PhD thesis.pdf
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Zusammenfassung
Englisch
Induced pluripotent stem cells (iPSC) have revolutionized biomedical research due to their versatility. They have potential for regenerative medicine, drug testing, disease modeling and developmental biology. The development of cell-based regenerative therapeutics and the subsequent treatment of human degenerative diseases require testing in predictive animal models like non-human primates (NHP). Therefore NHP-iPSC in conjunction with NHP are highly important to conduct preclinical studies. I have generated and established in the context of this collaborative effort, to our knowledge for the first time, transgene- and feeder-free iPSCs from three NHP species, namely the rhesus macaque (Macaca mulatta), the olive baboon (Papio anubis) and the common marmoset (Callithrix jacchus), as well as human iPSC. For the generation of the different NHP-iPSC lines, we explored different reprogramming approaches and culture conditions. Eventually, we succeeded in establishing a universal reprogramming protocol for primate fibroblasts. Furthermore, we successfully adapted a differentiation protocol for human iPSC into functional cardiomyocytes (iPSC-CM) to NHP-iPSC. The generated NHP-iPSC, besides their potential to test stem cell-based regeneration therapies, can also be used for disease modeling. The generation of genetically modified NHP animal models for human diseases may contribute to drug testing and enhances pathophysiological studies during disease onset and progression in a controlled experimental setup. However, in order to safely introduce predefined mutations into the (embryonic) genome of monkeys, the efficiency and accuracy of a genome editing approach needs to be pre-screened, ideally using iPSC of the respective NHP species. Due to the similarities between iPSC and pluripotent cells from the early embryo, it is possible and suggested to study the efficiency, efficacy and accuracy of the editing approach using iPSC. Furthermore, the capability of the iPSC to differentiate into different somatic cell types may allow the prediction of aspects of the in vivo phenotype of genetically modified NHP. In summary, I have generated and established in the context of these collaborative studies a set of NHP-iPSC-lines that (1) can be used for preclinical testing of cell replacement therapies and (2) forms the basis of an in vitro platform that allows the validation of genome editing approaches, e.g by CRISPR/Cas, for the generation of genetically modified NHP. With this approach, we established an in vitro testing and preselection platform for genetic modifications before their application in vivo in NHP. Besides its scientific value, this platform will also contribute to the 3Rs (reduce, replace, refine) in animal experimentation, which are of particular importance in the context of NHP research. The current dissertation has been written following the pseudo-cumulative structure registered in the Official Bulletin I no. 28 dated 22.06.2018, doctoral degree regulations (RerNat-O) GAUSS. This thesis includes four different chapters / manuscripts (both published and not published), each independent and completely understandable as a single manuscript. As all chapters reflect the evolution of techniques and results during this Ph.D., they are grouped under a general introduction and discussion, giving a full overview and analysis of the thesis.
Keywords: iPSC; Non-human primates; Cardiac regeneration; Genome editing; CRISPR/Cas; Human Disease Modeling; Rhesus macaque, Macaca mulatta; Marmoset monkey, Callithrix jacchus; Olive baboon, Papio anubis; Induced Pluripotent Stem Cells; Directed cardiomyocyte differentiation; Stem Cell Based Therapy
