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Viral and non-viral generation and characterization of induced pluripotent stem cells from human amniotic fluid cells

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons50648

Drews,  Katharina
Molecular Embryology and Aging (James Adjaye), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons50409

Lehrach,  Hans
Molecular Embryology and Aging (James Adjaye), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

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DrewsK_THESIS.pdf
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Citation

Drews, K. (2012). Viral and non-viral generation and characterization of induced pluripotent stem cells from human amniotic fluid cells. PhD Thesis.


Cite as: http://hdl.handle.net/11858/00-001M-0000-000E-F55D-8
Abstract
The ability to induce pluripotency in somatic cells offers unprecedented opportunities in basic and applied research. The implementation of induced pluripotent stem cells (iPSCs) into clinical settings, however, is hampered by genetic modifications associated with retro- or lentivirus-mediated reprogramming. The quest for efficient alternative reprogramming approaches has been closely connected with the identification of cell sources, which readily acquire the pluripotent stem cell (PSC) state. Human amniotic fluid cells (AFCs) represent routinely available cells with stem cell-like features, which could presumably facilitate efficient reprogramming even by non-integrating techniques. The goal of this project was to generate and comparatively characterize iPSCs derived from human AFCs by viral and non-viral techniques with respect to human embryonic stem cells (ESCs), the golden standard of PSCs, and iPSCs generated from cells of other tissues of origin. Retrovirus-mediated overexpression of the reprogramming factors in primary human AFCs resulted in fast and efficient generation of iPSCs (AFiPSCs), which resembled human ESCs with regards to morphology, proliferation and marker expression. Their ability to differentiate into derivatives of the three embryonic germ layers was demonstrated in vitro and in vivo and upon BMP2 and BMP4-treatment expression of trophoblast markers, including CDX2, KRT7 and HAND1, was confirmed. Detailed microarray-based transcriptome analysis of ESCs, AFiPSCs, fibroblast-derived iPSCs (FiPSCs) and the respective parental cell lines revealed the activation of a transcriptional regulatory network common to all PSCs but also highlighted, for example, residual gene expression signatures in iPSCs from different tissues of origin. These findings were summarized in a concept coined the LARGE Principle of Cellular Reprogramming. Genetic manipulation of AFiPSCs was not accomplished. Attempts to reprogram human AFCs by non-viral, non-integrating methods included nucleofection of episomal plasmids and lipofection of mRNAs encoding the reprogramming factors. Despite multiple trials fully reprogrammed iPSCs could not be established. In depth analysis of the cellular response to the transfected mRNAs uncovered an extensive induction of interferon-regulated immune-related genes to be the key roadblock in mRNA-mediated reprogramming. Subsequent efforts to identify chemicals which could suppress this innate immune reaction did not yield potent candidates. The data presented herein, however, provide the basis for further investigations into this effect. In summary, this work highlights the value of human AFCs for the derivation of iPSCs and emphasizes the obstacles that need to be overcome before AFiPSCs can potentially be employed into clinical settings.