iPSC-derived mice through tetraploid complementation

Progress in iPS cell research continues at a blistering pace. Launched two years ago with the discovery and development of nuclear reprogramming of somatic cells by Yamanaka and colleagues (2007), iPS cell technology has been greeted with great enthusiasm by the scientific community.  There are many reasons for this.  These cells can be used as tools to study developmental biology, they could be ideal for investigating disease mechanisms and even for drug discovery, and of course they have the potential to circumvent the ethical issues surrounding the use of embryonic stems cells to treat human disease since they are derived from adult tissue.  However, one of the lingering debates surrounding iPS cells relates to how “stem-cell like” iPS cells are.  Up to this point, the test for the pluripotency of iPS cells has been their ability to produce teratomas in immunodeficient mice that produce all three germ cell layers, ectoderm, mesoderm, and endoderm, or in mouse studies, the ability to generate mice by injecting mouse iPS cells into blastocysts, and thereby produce chimaeric adult mice in which the iPS cells contribute to all tissue types in the adult.    However, until recently, by far most stringent test and ultimate proof of pluripotency by generating a living organism from iPS cells has been lacking. 

Now, in a close race, three groups have provided compelling, and many would say definitive, proof that iPS cells are indeed fully pluripotent. On July 23^rd 2009, articles from two groups from China (Zhao and colleagues in Nature and Kang and colleagues in Cell Stem Cell) and shortly thereafter on August 2^nd an article from a group in San Diego (Boland and co-workers in Nature) reported the remarkable achievement of successfully generating living mice from iPS cells.  These investigators all used a tetraploid complementation approach which involves using blastocysts that have twice as much chromosomal content as normal cells and therefore can only contribute to extra-embryonic tissue.  iPS cells with a normal karyotype are then injected into the tetraploid blastocyst, and any organism that develops from the blastocyst must be of iPS origin. 

The Kang group used a doxycycline-inducible lentiviral system obtained from the Jaenisch laboratory and transduced mouse embryonic fibroblasts (MEFs) from a ROSA-M2rtTA transgenic mouse strain using Oct4, Sox2, Klf4 and c-Myc . Five iPS cell lines with typical morphology were propagated and characterized for expression of AP, Oct4, Sox2, Nanog, and SSEA-1. These investigators used the production of chimaeric mice as a way to predict success in subsequent tetraploid complementation assays. In the first round of complementation assays (n=200), one of two full-term pups survived for three days. In a second attempt, in which they produced a total of 187 complemented blastocysts, only one out of two pups survived into adulthood, which had an efficiency of ~1% of term pups.

One the other hand, Zhao and colleagues used four viral factors with a pMXs backbone obtained from the Yamanaka group. MEFs expressing Oct4-GFP reporter (B6D2F1 strain) and MEFs with a C57 x 129S2 background were transduced with pMXS-Oct4, Sox, Klf4, and c-Myc. This group generated a total of 37 iPS cell lines.  Three of these cells lines produced a total of 27 live pups from 848 tetraploid complementation experiments.  Apparently some of the first generation mice were not normal and some died.  However, a second generation of mice were successfully bred from the original founder, and a substantial number of mice have been raise which appear to be normal.  Overall, for this group, a success rate of 2.2-3.5% for tetraploid complementation was achieved, which was very similar to that of control embryonic stem cells (ES1 and CL11) of 2-3%.

Finally, the Boland group established a doxycycline-inducible lentiviral system and transduced embryonic fibroblasts from mice that were generated by a cross of the Pcdh21/Cre and Z/EG  mouse line. Valproic acid was added to the culture media to accelerate the reprogramming process. Twenty one iPS cell lines were propagated and characterized for morphology, proliferation, and expression of pluripotency markers. Based on these criteria, six iPSC lines were selected for tetraploid blastocyst complementation assays; two of these lines produced pups that grew into adulthood. The efficiency of iPS cells was comparable to ES cells (0.3-13%) in this set of complementation assays as well.

Thus there are now three independent studies indicating that selected iPS cell lines are indeed truly pluripotent and can produce viable mice.  Furthermore, these mice can live into adulthood and give raise to offspring. As important as these observations are, there are number remaining issues. First, as all three groups note, this process is inefficient as many iPS cell lines were incapable producing live pups through the tetraploid blastocyst approach.  This is probably due to differences in the reprogramming process and the ‘completeness’ of the epigenetic changes during nuclear reprogramming.  It will be important in future studies to compare the expression pattern of the iPS cell lines that are not successful in tetraploid complementation approaches to understand the underlying “molecular signatures” for pluripotency and its implication for developmental biology. Understanding these molecular signatures is also crucial for the generation of iPS cell lines as cellular models of disease.  For example, if we know the underlying molecular expression patterns that entail “true” reprogrammed cells, we may be able to obtain more stable clonal iPS cell lines that will be easier to coax into specific somatic tissues that are pertinent to disease oriented research (e.g., dopaminergic neurons for Parkinson’s disease).

A second concern relates to the fact these iPS cells were created using the Yamanaka factors which are introduced to the skin cells using viral vectors.  What effect this will have over time, such as tumor production, is unknown.  Finally, since these iPS cells have been used create a living organism, it is not unlikely that ethical concerns will be raised similar to those that have been expressed regarding cloning, particularly when it comes to the possibility it might be attempted in humans.  One of the most welcome aspects of working with iPS cells derived from human adult somatic cells is that this approach circumvents the bioethical controversies surrounding the derivation of human embryonic stem cell lines from ‘surplus’ embryos from infertility clinics. It would be sad indeed to see iPS cells swept back into the same political arena that embryonic stem cell research has languished in for the last 8 years, and one can only hope this will not happen.

These concerns aside, it seems very likely that the scientific momentum generated by the discovery of iPS cells will continue to gain ground.  With the new and very powerful evidence that these cells are truly pluripotent described above, iPS cells will likely become even more attractive as tools to study developmental biology and for medical research.  While work with embryonic stem cells should and will continue because the long term outcome of iPS cell research is still unknown, these latest research results put iPS cells technology and research on a more solid footing that ever, and we can expect much more to come.  Stay tuned.