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.

iPSCs with just one factor

Several different labs have managed to streamline the production of induced pluripotent stem cells (iPSCs). Originally, scientists inserted four different genes into cells to push them into the pluripotent state. However, by using a variety of new techniques and soaking cells in various chemicals, several labs have managed to lower the number of genes required to generate iPSCs.

Now Hans Schöler and his colleagues at the Max Planck Institute for Molecular Biomedicine in Münster, Germany, have shown that neural cells, which already express high levels of three of the four standard pluripotency factors (SOX2, KLF4 and C-MYC), can be converted into iPSCs by transfection with only OCT4 (Kim, J. B. et al. Oct4-induced pluripotency in adult neural stem cells. Cell 136, 411–419 (2009)). This worked in mouse cells and the resulting iPSCs all passed every test of pluripotency.

Thus the production of iPSCs is getter easier and easier.

iPSCs from other sources

Nature News has a fascinating article on induction of pluripotent stem cells by means of genetic reprogramming.  Typically, skin cells have been used for reprogramming experiments.  Cells called fibroblasts, which are prevalent in skin and help heal the skin when injured, have been the cell of choice for induced pluripotent stem cell (iPSC) production.

Recently, fat cells and pigmented skin cells appear to produce iPSCs much more efficiently and quickly.  Reprogramming procedures with skin fibroblasts are rather inefficient and slow.  Reprogramming human skin cells takes about a month for 1 in 10,000 fibroblasts to form iPSCs. Other cell types can do the job besides fibroblasts.  For example blood, hair, bone marrow, and neural stem cells can be converted into iPSCs, but their conversion rate is not any better than that of skin fibroblasts.  However, foreskin fibroblasts from a baby are better at making iPSCs (see Aasen, T. et al. Nat. Biotechnol. 26, 1276-1284), but this is hardly a good source of cells for adults.

Is there are better way?  Apparently there is.  Joseph Wu and Michael Langaker at Stanford University School of Medicine in California have converted fat tissue into iPSCs, and it took them only two days to acquire enough material for reprogramming.  Compare that to about a month of biopsies to get enough fibroblasts.  Additionally, cellular reprogramming of fat cells took only two more weeks and was 20-times more efficient than fibroblast reprogramming.  By using fat cells, they were able to reduce the time required for the procedure by six to eight weeks (Sun, N. et al. Proc. Natl. Acad. Sci. USA, doi:10.1073/pnas.0908450106).  Likewise, Konrad Hochedlinger and his co-workers at the Massachusetts General Hospital in Boston reprogrammed melanocytes, the skin cells that produce pigmented skin.  Melanocytes undergo reprogramming after just 10 days and with five-fold greater success rates compared with fibroblasts (Utikal, J., Maherali, N., Kulalert, W. & Hochedlinger, K. J. Cell Sci., doi:10.1242/jcs.054783).

These findings suggest that making iPSCs is easier than previously thought.  The therapeutic uses of iPSCs just became even more attractive.

About that Hold

Geron has revealed the reason for the FDA hold on its Spinal Cord Injury Investigational New Drug application. In an August 27th press release, Geron scientists revealed that the implanted GRNOPC1 cells caused cysts in a small proportion of the animals injected with them. These cysts were not cancerous. The report calls them “non-proliferative,” which simply means that they are not growing. Additionally. the cysts are very small – microscopic in size. Finally, the cysts were confined to the region of the injury and did not adversely affect the laboratory animals.

Why the hub-hub? A recent animal study reported a greater frequency of cysts. Once again, they are non-proliferative (non-growing), restricted in location to the site of injury and do not affect the animals.

What’s going on? Cyst formation is common in spinal cord injury. Once the spinal cord is injured, inflammation ensues and this involves the invasion of the spinal cord by immune cells that mop up the dead cells and debris from the injury. Unfortunately, immune cells are sloppy eaters and they do a great deal of damage to the spinal cord. The damage they cause also tends to summon more immune cells, which come to the scene of the injury and damage the spinal cord even more. the whole thing is a positive feedback mess.

To put an end to it the spinal cord makes a plug called a glial scar. The glial scar comes from the stem cell population in the spinal cord. These stem cells form support cells called “glial cells” and these cells plug the hole in the spine and shut out the immune response, thus saving the spinal cord. The formation of this glial scar, however has a severe downside for spinal cord regeneration: the glial scar is loaded with chemicals that repel growing neurons. Therefore, those neurons that were severed by the injury could not regrow their extensions if they wanted to. The glial scar acts like a bunch of burly security guards that prevent the neuronal extensions from getting to the other side.

These cysts are probably the result of the GRNOPC1 cells forming tiny glial scars to help the injured spinal cord heal. Now they do not seem to affect the laboratory animals, but they are inhibitors of neuronal healing. Therefore, while they may not affect the laboratory animals, they may represent a fix that sentences the spinal cord to never being fixed by anything else again.