4 results on '"Induced pluripotent stem cell"'
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2. Optimierung von Kieferperiostzellen für das Knochen-Tissue Engineering: Herstellung von iPS-Zellen und Analyse von Progenitor-Oberflächenmarkern
- Author
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Umrath, Felix and Stevanović, Stefan (Prof. Dr.)
- Subjects
induced pluripotent stem cell ,Kieferperiost ,jaw periosteum ,human platelet lysate ,Mesenchymale Stammzelle ,osteogenic differentiation ,MSC-ähnliche Zelle ,iMSCs ,Induzierte pluripotente Stammzelle , Differenzierung , Knochen , Knochenbildung , Tissue Engineering , Periost ,humanes Thrombozytenlysat ,surface marker ,Osteogene Differenzierung ,Oberflächenmarker ,mesenchymal stem cell ,iMSC - Abstract
Die Knochengewebezüchtung im Labor (engl.: Bone Tissue Engineering, BTE) könnte in Zukunft autologe Knochentransplantate in der Mund-, Kiefer- und Gesichtschirurgie (MKGC) ersetzen. Für die Herstellung klinisch anwendbarer BTE- Produkte sind die Qualität und die Verfügbarkeit der dabei eingesetzten Stammzellen entscheidende Faktoren. Die Verfügbarkeit von Stammzellen ist einerseits durch die Größe der Gewebebiopsie, andererseits durch die Abnahme der Vitalität und des osteogenen Potenzials im Laufe der in vitro Kultivierung limitiert. Da für die Herstellung von BTE-Konstrukten zur Regeneration größerer Knochendefekte, z.B. nach Tumorresektionen, große Mengen osteogener Vorläuferzellen benötigt werden, sollten in der vorliegenden Arbeit induzierte pluripotente Stammzellen (iPSCs) aus Kieferperiostzellen (JPCs) generiert, und als Zellquelle für das BTE etabliert werden. iPSCs besitzen die Fähigkeit zur Selbsterneuerung und sind daher in großen Mengen verfügbar. Im Hinblick auf zukünftige klinische Anwendungen der Zellen, wurde zur Generierung von iPSCs eine besonders sichere xenogen-freie RNA-basierte Reprogrammierungstechnik entwickelt. Durch die Differenzierung von iPSCs zu MSC-ähnlichen Zellen (iMSCs) ist es möglich osteogene Vorläuferzellen herzustellen, welche analog zu normalen MSCs (mesenchymale Stamm-/Stromazellen) eingesetzt werden können. In der vorliegenden Arbeit wurden daher aus JPCs generierte iPSCs zu iMSCs differenziert, und ihre Eignung für Anwendungen im BTE anhand der osteogenen Differenzierung in vitro getestet. Neben der Verfügbarkeit von Stammzellen ist auch die Qualität des zur Verfügung stehenden Zellmaterials ein entscheidender Faktor. Die Gewinnung von Stammzellen aus Gewebebiopsien führt jedoch meistens zur Isolation von heterogenen Zellpopulationen aus verschiedenen Zelltypen, wodurch die Qualität sowie das Stammzellpotenzial der für das BTE benötigten Zellkulturen beeinträchtigt werden kann. Zur Qualitätskontrolle und zur Isolation osteogener Vorläuferzellen aus solchen Mischpopulationen werden Oberflächenmarker benötigt, mit denen sich osteogene Vorläuferzellen in heterogenen Zellpopulationen identifizieren und ggf. mithilfe von Fluorescence-activated Cell Sorting (FACS) isolieren lassen. In der vorliegenden Arbeit wurden die Oberflächenmarker MSCA-1 (Mesenchymal Stromal Cell Antigen-1) und CD146 (Melanoma Cell Adhesion Molecule (MCAM)) auf ihre Eignung für die Separation osteogener Vorläuferzellen aus der Periostzelllinie TAg58 getestet. Dabei zeigte sich, dass obwohl beide Marker in der Literatur als osteogene Marker beschrieben waren, sich nur MSCA-1 zur Identifikation und Sortierung osteogener Vorläuferzellen in der verwendeten Zelllinie als geeignet erwies. Zur Herstellung von BTE-Produkten unter GMP-gerechten Bedingungen sollte bei der Zellkultur auf xenogene Bestandteile verzichtet werden. Daher wurde im Zuge dieser Arbeit die Kultivierung von JPCs von FBS- (fötales Kälberserum) auf hPL- (humanes Plättchenlysat) supplementiertes Medium umgestellt. Dabei zeigte sich, dass sich hPL wesentlich besser für die Kultivierung von JPCs eignete und Proliferation sowie Differenzierungspotenzial deutlich verbessert wurden. Zudem konnte für die osteogene Induktion von Periostzellen auf die Zugabe von Dexamethason unter hPL-Supplementierung verzichtet werden.
- Published
- 2020
3. Human stem cell-based three-dimensional microtissues for advanced cardiac cell therapies
- Author
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Benedikt Weber, Alexis Bosman, Marisa Jaconi, Volkmar Falk, Gino Gemayel, Maximilian Y. Emmert, Chad Brokopp, Nadine Wickboldt, Petra Wolint, Simon P. Hoerstrup, Alessandro Boni, University of Zurich, and Hoerstrup, Simon P
- Subjects
Cellular differentiation ,Induced Pluripotent Stem Cells ,Biophysics ,Cell- and Tissue-Based Therapy ,Clinical uses of mesenchymal stem cells ,Bioengineering ,610 Medicine & health ,2503 Ceramics and Composites ,ddc:616.07 ,Biology ,Biomaterials ,2211 Mechanics of Materials ,Induced Pluripotent Stem Cells/cytology ,Tissue Engineering/methods ,Humans ,Induced pluripotent stem cell ,Mesenchymal Stromal Cells/cytology ,Cells, Cultured ,Embryonic Stem Cells ,Stem cell transplantation for articular cartilage repair ,Cell Proliferation ,Embryonic Stem Cells/cytology ,Tissue Engineering ,1502 Bioengineering ,Myocardium ,Myocardium/cytology ,Mesenchymal stem cell ,2502 Biomaterials ,Cell Differentiation ,Mesenchymal Stem Cells ,Embryonic stem cell ,Cell biology ,10020 Clinic for Cardiac Surgery ,Endothelial stem cell ,10022 Division of Surgical Research ,Mechanics of Materials ,Ceramics and Composites ,Stem cell ,Biomedical engineering ,1304 Biophysics - Abstract
Cardiac stem cell therapy has been proposed as a therapy option to treat the diseased myocardium. However, the low retention rate of transplanted single-cell suspensions remains a major issue of current therapy strategies. Therefore, the concept of scaffold-free cellular self-assembly into three-dimensional microtissues (3D-MTs) prior to transplantation may be beneficial to enhance retention and survival. We compared clinically relevant, human stem cell sources for their ability to generate 3D-MTs with particular regards to formation characteristics, proliferation-activity, viability and extracellular-matrix production. Single-cell suspensions of human bone marrow- and adipose tissue-derived mesenchymal stem cells (hBMMSCs and hATMSCs), Isl1(+) cardiac progenitors derived from human embryonic stem cells (hESC-Isl1(+) cells), and undifferentiated human induced pluripotent cells (hiPSCs) were characterized before to generate 3D-MTs using a hanging-drop culture. Besides the principal feasibility of cell-specific 3D-MT formation, a detailed head-to-head comparison between cell sources was performed using histology, immunocyto- and histo-chemistry as well as flow cytometry. Round-oval shaped and uniform 3D-MTs could be successfully generated from all cell types starting with a loose formation within the first 24 h that fully stabilized after 3 days and resulting in a mean 3D-MT diameter of 194.56 ± 18.01 μm (hBMMSCs), 194.56 ± 16.30 μm (hATMSCs), 159.73 ± 19.20 μm (hESC-Isl1(+) cells) and 120.95 ± 7.97 μm (hiPSCs). While all 3D-MTs showed a homogenous cell distribution, hiPSC-derived 3D-MTs displayed a compact cell formation primarily located at the outer margin. hESC-Isl1(+) and hiPSC-derived 3D-MTs maintained their proliferation-activity which was rather limited in the MSC-based 3D-MTs. All four 3D-MT types revealed a comparable viability in excess of 70% and showed a cell-specific expression profile being comparable to their single-cell counterparts. Extracellular matrix (ECM) production during 3D-MT formation was observed for all cell-specific 3D-MTs, with hiPSC-derived 3D-MTs being the fastest one. Interestingly, ECM distribution was homogenous for hATMSC- and hiPSC-based 3D-MTs, while it appeared to be primarily concentrated within in the center of hESC-Isl1(+) and hBMMSC-based 3D-MTs. The results of this head-to-head comparative study indicated that 3D-MTs can be successfully generated from hESC-derived Isl1(+) cells, hiPSCs and MSC lines upon hanging drop culture. Cell-specific 3D-MTs displayed sufficient viability and instant ECM formation. The concept of 3D-MT in vitro generation prior to cell transplantation may represent a promising delivery format for future strategies to enhance cellular engraftment and survival.
- Published
- 2013
4. DNA Repair in Embryonic Stem Cells
- Author
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Christine Blattner and Volker Middel
- Subjects
Life sciences ,biology ,DNA repair ,Cellular differentiation ,ddc:570 ,Embryoid body ,Stem cell ,Biology ,Induced pluripotent stem cell ,Embryonic stem cell ,Stem cell transplantation for articular cartilage repair ,Cell biology ,Adult stem cell - Abstract
DNA is the largest and most important molecule of a cell. Due to its chemical nature, DNA is particularly prone to numerous lesions. These lesions comprise different sugar and base modifications, deletion of bases as well as single (SSBs) and double DNA strand breaks (DSBs). All alterations in the DNA that a cell experiences sum up to more than 10.000 lesions per day (Lindahl, 1993). DNA damage can be brought about by several endogenous and exogenous factors including reactive oxygen species (ROS), ultraviolet light (UV), ionizing radiation (IR) or by DNA damaging chemicals. The different lesions are removed by several repair mechanisms that help the cell to preserve structure and sequence of the DNA. These repair pathways include mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and homologous recombination (HR). If DNA damage is too severe, cells can also initiate a cell death program that removes cells with damaged DNA from the population (reviewed in: Roos & Kaina, 2006). Stem cells comprise a group of self-renewing cells including embryonic and adult stem cells. Embryonic stem cells (ESCs) have the ability of indefinite self-renewal and rapid proliferation. They are pluripotent and can differentiate into cells of all three germ layers (ectoderm, mesoderm and endoderm) as well as of the germ cell lineage. Adult or tissue stem cells, in contrast, are multipotent and differentiate into only one or several specific cell lines (reviewed in: Barilleaux et al, 2006; Thomson & Marshall, 1998; Weissman et al, 2001). By proliferation and differentiation, adult stem cells replenish tissue cells that are lost during normal wear and tear or after injury and thus are key for tissue and organ regeneration as well as for the preservation of homeostasis in a living organism (Reya et al, 2001). ESCs can be isolated from different species, but the most investigated ones are human (hESCs) and murine (mESCs) ESCs. Both, hESCs and mESCs are derived from the inner cell mass of a blastocyst, an early stage of embryonic development (Martin, 1981; Thomson et al, 1998). Human ESCs are of particular interest because of their high potential for medical applications like tissue and organ regeneration (reviewed in: Donovan & Gearhart, 2001; Thomson & Odorico, 2000; Wobus, 2001). However, since hESCs are derived from human embryos, they also raise ethical, social and juristic problems (McLaren, 2000). A potential alternative to the use of hESCs is the employment of induced pluripotent stem (iPS) cells. By transfection of a combination of stem cell markers, iPS cells can be made from basically every differentiated cell (Kim et al, 2009; Okita et al, 2007; Takahashi et al, 2007; Takahashi & Yamanaka, 2006; Wernig et al, 2007; Yu et al, 2007). Thus, differentiated cells can be taken from a patient, modified and induced for pluripotency in vitro, and transplanted back into
- Published
- 2011
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