The stream of stem cell research seems to be turning into a river with cascades, waterfalls, whirlpools and even stem-cell treatment resorts. I comment here on just one small part of the river, which is research on generating induced pluripotent stem cells (iPSCs) that are safe to use in human tissues. I also include a “crazy” idea at the end on how to close the loop in the stem cell supply chain, possibly enabling very very long lives.
Three of the key issues being worked on are 1. making sure that the final iPSC products are free of viral genes or oncogenes or gene translocations, 2. Making sure the iPSCs are free of viral DNA introduced by the process of making them and 3. raising the efficiency of iPSC production. I discussed the first two of these issues before. See June 2009 blog post Update on induced pluripotent stem cells. The original approach to iPSC production was to use a viral vector to insert four genes into the cell to be reverted to iPSC status: Oct4, Sox2, cMyc, and Klf4. The blog post Rebooting cells and longevity mentions the approach and the alternatives that appeared to be visible back in March 2009, but that is a long time ago as far as iPSC stem cell knowledge is concerned.
One of the central problems with the original approach was that the DNA of these genes unpredictably integrates itself into the DNA of the iPSCs and cMyc is known to be a potential oncogene. “When Myc is mutated, or overexpressed, the protein doesn’t bind correctly, and often causes cancer(ref).” If the cells are to be used on humans, traces of those genes are unacceptable. There is a growing perception that “– residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease(ref).” Virus vectors used for gene insertion for gene insertion are also suspect. DNA from virus vectors can integrate into the DNA of the iPSC cells, possibly affecting their transcriptional profiles or sometimes even inducing cell death or tumors. What is desired is iPSC cells that are completely free of “footprints” due to how they were created.
One approach to the issue of foreign DNA in iPSC cells is to eliminate insertion of some of the four genes or eliminate their use altogether. A June 2008 news story tells of four different approaches towards this end being pursued at that time. Here are some more-recent publications, including one that appeared the day-before-yesterday.
The September 2009 paper TgfÎ² Signal Inhibition Cooperates in the Induction of iPSCs and Replaces Sox2 and cMyc offers hope for eliminating use of two of the genes and also addresses the productivity issue. “iPSC derivation is highly inefficient, and the underlying mechanisms are largely unknown. This low efficiency suggests the existence of additional cooperative factors whose identification is critical for understanding reprogramming. –]. Thus, the identification of compounds that enhance rather than solely replace the function of the reprogramming factors will be of great use. Here, we demonstrate that inhibition of TgfbÎ² signaling cooperates in the reprogramming of murine fibroblasts by enabling faster, more efficient induction of iPSCs, whereas activation of TgfÎ² signaling blocks reprogramming. In addition to exhibiting a strong cooperative effect, the TgfÎ² receptor inhibitor bypasses the requirement for exogenous cMyc or Sox2, highlighting its dual role as a cooperative and replacement factor.”
In a recent blog post I mentioned another September 2009 publication relating to an approach to iPSC induction without introducing any genes into cells at all. Induction of Stem Cell Gene Expression in Adult Human Fibroblasts without Transgenes. “Because forced expression of these genes by viral transduction results in transgene integration with unknown and unpredictable potential mutagenic effects, identification of cell culture conditions that can induce endogenous expression of these genes is desirable. – Manipulation of oxygen concentration and FGF2 supplementation can modulate expression of some pluripotency related genes at the transcriptional, translational, and cellular localization level. Changing cell culture condition parameters led to expression of REX1, potentiation of expression of LIN28, translation of OCT4, SOX2, and NANOG, and translocation of these transcription factors to the cell nucleus. We also show that culture conditions affect the in vitro lifespan of dermal fibroblasts, nearly doubling the number of population doublings before the cells reach replicative senescence. Our results suggest that it is possible to induce and manipulate endogenous expression of stem cell genes in somatic cells without genetic manipulation, but this short-term induction may not be sufficient for acquisition of true pluripotency.” This is work-in-progress but the idea of inducing pluripotency purely through manipulating culture conditions is intriguing.
Another approach to getting rid of the cancer genes from iPSCs was described in my recent blog post Toward a genetic cure for Parkinson’s disease which cites the March 2009 report Breakthrough produces Parkinson’s patient-specific stem cells free of harmful reprogramming genes. As I said in that post, The approach used by the researchers is a good example of gene editing. “In the current method, Whitehead researchers used viruses to transfer the four reprogramming genes and a gene coding for the enzyme Cre into skin cells from Parkinson’s disease patients. The reprogramming genes were bracketed by short DNA sequences, called loxP, which are recognized by the enzyme Cre. After the skin cells were reprogrammed to iPS cells, the researchers introduced the Cre enzyme into the cells, which removed the DNA between the two loxP sites, thereby deleting the reprogramming genes from the cells. The result is a collection of iPS cells with genomes virtually identical to those of the Parkinson’s disease patients from whom original skin cells came.”
Yet-another chemical approach for increasing the efficiency of iPSC production is described in the year-old news report Technique for Rapidly Reprogramming Adult Cells Into Stem Cells Published in PLoS Biology.
In September2009, researchers from the University of California, San Diego School of Medicine and the Salk Institute for Biological Studies in La Jolla reported developing “ a safe strategy for reprogramming cells to a pluripotent state without use of viral vectors or genomic insertions.” The cells produced were pluripotent but-not-quite virgin iPSCs. “– these induced pluripotent stem cells (iPSCs) are very similar to human embryonic stem cells, yet maintain a “transcriptional signature.” In essence, these cells retain some memory of the donor cells they once were. “”Working with neural stem cells, we discovered that a single factor can be used to re-program a human cell into a pluripotent state, one with the ability to differentiate into any type of cell in the body” said Muotri (the lead researcher). — “While most of the original genetic memory was erased when the cells were reprogrammed, some were retained,” said Muotri. He added that, in the past, it wasn’t known if this was caused by the use of viral vectors. “By using a footprint-free methodology, we have shown a safe way to generate human iPSCs for clinical purposes and basic research. We’ve also raised an interesting question about what, if any, effect the ‘memory retention’ of these cells might have(ref) .”
Then there is the RepSox approach revealed two days ago. Of course it was Boston-based researchers (from the Harvard Stem Cell Institute) who come up with that name. RepSox is a small-molecule compound the researchers discovered that replaces use of the gene Sox2 (thus the name RepSox) when reprogramming cells to iPSC status. It turns out that RepSox also makes use of the gene c-Myc unnecessary. “– many scientists think the safest approach is to replace the genes altogether with so-called small molecules. In a study published online today in the journal Cell Stem Cell entitled A Small-Molecule Inhibitor of Tgf-Î² Signaling Replaces Sox2 in Reprogramming by Inducing Nanog, researchers from the Harvard Stem Cell Institute report that a single compound they dubbed RepSox can replace two of the four key reprogramming genes. – “We’re halfway home, and remarkably we got halfway home with just one chemical,” senior author Kevin Eggan, a professor in Harvard’s department of stem cell and regenerative biology, said in a statement. — Now the group will turn its attention to finding other small molecules that could replace the remaining genes – Oct4 and Klf4 – as well, “opening a route to purely chemical programming,” they write(ref).”
Whatever the approach that ultimately turns out to be most successful, researchers are tackling the oncogene, the viral DNA and the productivity problems involved with producing iPSCs that are safe to use with human cells. These citations are just a small sample of those that already exist and those that can be expected.
A visionary note
A final note of a personal vision. Suppose, just imagine, that following some of the lines of research described above, a set of small-molecule activators could be identified that selectively causes reversion of a small sub-population of multipotent adult stem cells in-vivo (e.g. hematopoietic stem cells or mesenchymal stem cells) in their niches to return to iPSC status. Those iPSC cells would then likely respond to niche signaling and differentiate to produce fresh niche-specific multipotent adult stem cells, cells free of the age-related epigenetic burden carried by the older multipotent cells. What I am imagining is a supplement that closes the loop in the stem cell supply chain which could have a profound longevity-extending effect. See the blog post The stem cell supply chain – closing the loop for very long lives. It may sound crazy but if anybody wants to take me on I would make a small even-money bet that we will hear about something like this within the next three years.