Abstract
Besides their putative usage for therapies, stem cells are a promising tool for functional studies of genes involved in human genetic diseases or oncogenesis. For this purpose induced pluripotent stem (iPS) cells can be derived from patients harbouring specific mutations. In contrast to adult stem cells, iPS cells are pluripotent and can efficiently be grown in culture. However, iPS cells are modulated due to the ectopic induction of pluripotency, harbour other somatic mutations accumulated during the life span of the source cells, exhibit only imperfectly cleared epigenetic memory of the source cell, and are often genomically instable. In addition, iPS cells from patients only allow the investigation of mutations, which are not prenatally lethal. Embryonic stem (ES) cells have a high proliferation and differentiation potential, but raise ethical issues. Human embryos, which are not transferred in the course of in vitro fertilization, because of preimplantation genetic diagnosis of a genetic defect, are still rarely donated for the establishment of ES cell lines. In addition, their usage for studies on gene functions for oncogenesis is hampered by the fact the ES cells are already tumorigenic per se. In 2003 amniotic fluid stem (AFS) cells have been discovered, which meanwhile have been demonstrated to harbour the potential to differentiate into cells of all three germ layers. Monoclonal human AFS cell lines derived from amniocenteses have a high proliferative potential, are genomically stable and are not associated with ethical controversies. Worldwide amniocenteses are performed for routine human genetic diagnosis. We here discuss how generation and banking of monoclonal human AFS cell lines with specific chromosomal aberrations or monogenic disease mutations would allow to study the functional consequences of disease causing mutations. In addition, recently a protocol for efficient and highly reproducible siRNA-mediated long-term knockdown of endogenous gene functions in AFS cells was established. Since AFS cells are not tumorigenic, gene modulations not only allow to investigate the role of endogenous genes involved in human genetic diseases but also may help to reveal putative oncogenic gene functions in different biological models, both in vitro and in vivo. This concept is discussed and a "proof of principle", already obtained via modulating genes involved in the mammalian target of rapamycin (mTOR) pathway in AFS cells, is presented.
Highlights
In medical genetics the future development of new prophylactic and therapeutic strategies directly depends on a better understanding of the mechanisms by which genetic variation contributes to disease
These findings prove that human amniotic fluid stem (AFS) cells are pluripotent and able to form embryoid bodies and to differentiate into cell types of all three germ layers
Very recently we already made use of the approach discussed above to functionally investigate the role of components of the mammalian target of rapamycin (mTOR) signalling cascade in human AFS cells. mTOR is the key component of the insulin signalling cascade, which is involved in a wide variety of different processes such as cell growth, proliferation, metabolism, transcription, translation, survival, autophagy, aging, differentiation and oncogenesis
Summary
In medical genetics the future development of new prophylactic and therapeutic strategies directly depends on a better understanding of the mechanisms by which genetic variation contributes to disease It must be a major goal of human genetics to use optimal biological models, that allow the investigation of the consequences of a specific genetic aberration. To be useful in medical genetics human stem cells must fulfil certain criteria They should be available with specific natural occurring genetic aberrations, which are of relevance for certain human pathological phenotypes. They should harbour high proliferative activities and pluripotency, the potential to differentiate in cells of all three germ layers. Destroying a human prenatal genetic diagnosis isolation of amniotic fluid stem (AFS) cells establishment of monoclonal AFS cell lines amniotic fluid cell samples with chromosomal abberations
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