?iPS cells for the study of dyskeratosis congenita
By SarahLepage, Section Biology Posted on Fri Apr 30th, 2010 at 04:15:18 PM PST
Dyskeratosis congenita (DC) is a very rare congenital disorder that is characterized by symptoms that resemble premature aging.  DC is a disorder of defective telomere maintenance, related to mutations in the vertebrate telomerase RNA component (TERC).A recent paper by Agarwal et al. successfully generated induced pluripotent stem (iPS) cells from DC patients, renewing their telomerase function.  I propose an experiment to study at what point the DC iPS cells lose their renewed telomerase function by inducing these DC iPS cells to differentiate and monitoring their TERC expression over time as compared to normal iPS cells.

Dyskeratosis congenita (DC) is a very rare congenital disorder that is characterized by symptoms that resemble premature aging: cutaneous pigmentation, premature graying, thrombocytopenia, anemia, and predisposition to cancer. DC is a disorder of defective telomere maintenance, related to mutations in the vertebrate telomerase RNA component (TERC). The early embryo begins with an abundance of telomerase, and as it continues through development, the amount of telomerase usually declines temporally, and by the time that a baby is born, the genes for telomerase are pretty much turned off. In DC, the early abundance of TERC is not present, leading to early shortening of the telomeres. A recent paper by Agarwal et al. successfully generated induced pluripotent stem (iPS) cells from DC patients. iPS cells are a type of pluripotent cell that greatly resemble embryonic stem cells, but are derived from differentiated cells (usually skin fibroblasts). They are often used nowadays in the study of genetic diseases. Fibroblasts are reprogrammed by the induced expression of four transcription factors (Oct-3/4, c-Myc, Klf4, Sox2), which returns the differentiated cell back to its pluripotent state. The purpose of generating iPS cells from DC patients was to see if the defect in TERC affected the reprogramming; surprisingly, these cells could be reprogrammed at a similar rate to normal cells. Also surprising, telomerase function was restored, as well as telomere length. Proposal: As a follow-up to this paper, I propose an experiment to study at what point the DC iPS cells lose their renewed telomerase function by inducing these DC iPS cells to differentiate and monitoring their TERC expression over time as compared to normal iPS cells. To do this, I would perform qRT-PCR on TERC RNA as well as a western blot (should there be an appropriate antibody for TERC) to determine transcript and protein abundance before, during, and after induced differentiation. I would probably just allow the cells to form embryoid bodies first, then move on to testing TERC expression in cells induced to become skin cells, blood cells, etc. Telomere length will be compared as well via Southern blot, qPCR, and/or qFISH. If we can determine which stage during cell differentiation the renewed telomerase function declines compared to normal, we may be able to tease out the molecular mechanism of its downregulation. This may result in future therapies to help treat this accelerated aging disease. Agarwal et al. (2010) Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients. Nature, 464:292-296.

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