Genes that prevent iPSC formation

In order to make induce pluripotent stem cells, scientists need to reprogram existing cells to form a cell that is undifferentiated and ready to become whatever we want it to become. Reprogramming is achieved by increasing the concentration of four different proteins within the cells. To increase the intracellular concentration of these proteins, scientists use viruses or other vehicles to import the genes that encode these proteins into the cells and the increased production of these proteins drives cells to become embryonic-like cells that can become anything we want them to be.  Unfortunately, reprogramming, at present, is rather inefficient.

Recently, work from five different labs has shown that two biochemical pathways, the p16INK4A and ARF–p53 pathways, put the brakes on iPSC formation. Five papers in a recent edition of the journal Nature show that the components of this pathway are silenced in iPSCs and strongly expressed in terminally differentiated cells. There are three genes found at the site known as Ink4/Arf (p16Ink4a, p19Arf and p15Ink4b), and these genes are absent in several different types of cancers. For example p16Ink4a is inactive in about 90% of all pancreatic cancers. p15Ink4b is absent from several different blood-based cancers and loss of p19Arf is involved in melanoma formation.  Each one of these gene products acts as a barrier to reprogramming and iPSC formation.

p16INK4A and p19ARF positively regulate the p53 pathway.  p53 inhibits cell proliferation and promotes cellular senescence.  During senescence, the cell essentially takes a nap.  If a you want a cell to grow and participate in healing, the induction of senescence is not a good thing, but if a cell is growing uncontrollably and contributing to a tumor, then forcing a cell into senescence is a good thing.  A protein called p21 (CDKN1A) is also upregulated by p53, and this protein inhibits proliferation and promotes senescence.  Thus p53 is one of the major switches that prevents cell proliferation and promotes senescence.  In 2008, Zhao and coworkers showed that interfering with p53 activity promoted iPSC formation (Zhao, et al., Cell Stem Cell 3, 475-79, 2008).

Hong et al. showed that the absence of, or a reduction in, p53 increases the efficiency of iPS cell generation from mouse and human fibroblasts (Hong, H. et al. Nature 460, 1132–1135 (2009)). Up to 10% of mouse fibroblasts that transiently lacked functional p53 became iPSCs.  Kawamura et al. also enhanced the reprogramming of mouse embryonic fibroblasts (MEFs) by reducing the level of p21 or ARF (Kawamura, T. et al. reprogramming. Nature 460, 1140–1144 (2009)). Since ARF inhibits p53 degradation, ARF knockdown might enhance reprogramming by decreasing p53 stability.  All of these studies, using various approaches, showed that p53 depletion enhances iPS cell generation.

Li et al. and Utikal et al. observed that the INK4/ARF locus is silenced in iPS cells reprogrammed from Mouse Embryonic Fibroblasts (MEFs), as well as in embryonic stem cells, but not in MEFs (Li, H. et al. Nature 460, 1136–1139 (2009) & Utikal, J. et al. Nature 460, 1145–1148 (2009)).  Utikal et al. also observed that older MEFs, which harbor increased levels of p16INK4A, ARF and p21 owing to ageing and the onset of senescence, show a decrease in reprogramming efficiency. Li et al. also linked ageing and expression from the INK4/ARF locus with decreased iPS cell generation. They showed that cells from old mice express genes at this locus at a higher level than cells from young mice and that this is associated with a decreased reprogramming efficiency, which can be rescued by knocking down INK4/ARF.

As a final caveat, Marión et al. found that p53 prevents the reprogramming of MEFs that have various types of DNA damage (Marión, R. M. et al. Nature 460, 1149–1153 (2009)). Although loss of p53 function allows faster and more efficient reprogramming in the presence of DNA damage, it generates iPS cells that contain damaged DNA and chromosomal abnormalities. This emphasizes that, although these studies provide crucial mechanistic insight into how the generation of iPS cells is regulated, it will be important to determine how the p16INK4A and ARF–p53 tumor suppressor pathways can be silenced to allow the efficient production of iPS cells without increasing the possibility of making cell lines that contain mutations that predispose them to malignant tumor formation.

Surely iPSC production represents the future of regenerative medicine.  We need not kill human embryos, and the time required to make iPSCs can be substantially cut with this technology as it is honed and even made safer.

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Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).