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キーワード:
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要旨:
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.
