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iPSC Perfection?

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Since the birth of the field of induced pluripotent stem cells (iPSCs), each passing month has brought forth numerous papers reporting new tweaks to further improve the reprogramming process; a new chemical here, a replacement transcription factor there, each time removing one small barrier, making reprogramming more rapid or uncovering a new piece of the machinery that controls it. Now, in what amounts to a quantum leap forward, the groups of Jacob Hanna and Noa Novershtern at the Weizmann Institute of Science, Rehevot, Israel have reported that the removal of a distinct epigenetic repressor element; Mbd3, can boost the reprogramming process to 100% and allows the transition to pluripotency on a remarkably fast, synchronized schedule (Rais, Zviran & Geula et al).

The group initially set out to test if the removal of epigenetic repressor genes could enhance the reprogramming process. Their model was the reversion of murine primed pluripotent epiblast stem cells (EpiSCs) resembling cells of a more advanced, pre-gastrulation stage embryo, to a naive pluripotent state (resembling cells of the inner cell mass) in 2i/LIF growth conditions (Mansour et al) with siRNA screening. Of all genes tested, only Mbd3 knockdown enhanced reversion (up to 80%), a finding which was further validated using EpiSCs generated from ESCs lacking Mbd3.   Mbd3 is a component of the NuRD complex which is known to be switched on in all somatic cells but becomes inactivated after fertilization and throughout pre-implantation development until the pluripotent state is lost, whereafter it is reactivated (Kaji et al). The group were also able to derive ESCs from Mbd3-knockout (KO) embryos under serum-free 2i/LIF conditions indicating that Mbd3 is dispensable for establishing the ground state of pluripotency.

When Mbd3-KO mouse fibroblasts were reprogrammed with Oct4, Sox2, Klf4 and Myc (OSKM) under 2i/LIF conditions, an amazing 95% of cells were Oct4+ by day 10 of reprogramming. Moving into a secondary system, in which Mbd3-KO fibroblasts contain a Doxycycline (dox)-inducible OSKM polycistronic cassette, reprogramming in 2i/LIF plus dox conditions reproducibly yielded 100% iPSC derivation efficiency by day 8, as measured by Oct4 detection.   By comparison, in wild type cells, the efficiency by day 8 was 20%.  Detailed analysis of the timing of reprogramming found that cells were also remarkably synchronised in their transition to pluripotency, with Oct4+ cells arising in unison between days 4.5–5.5. Global gene expression, chromatin analysis and DNA methylation comparisons all demonstrated that these newly-derived iPSCs were indistinguishable from multiple ESC and iPS cell lines. When this knowledge was applied to human reprogramming, MBD3 mutant hiPSCs harbouring doxycycline-inducible OSKM differentiated into fibroblasts and subsequently induced with dox were also observed to reprogram at near 100% efficiency.   In wild type OSKM transgenic secondary fibroblasts, MBD3 siRNA treatment also significantly induced reprogramming. Patient and/or disease-harboring cells can often be refractory to reprogramming, either due to the mutation inherent to the disease or the age/number/state of the somatic cells collected, however MBD3 siRNA permitted the generation of hiPSCs from adult human patient-specific fibroblasts after two rounds of OSKM with LIN28 mRNA transfection (Warren et al).

Finally, the researchers investigated the mechanism of reprogramming and soon established that, surprisingly, the OSKM factors specifically co-immunoprecipitated with Mbd3 after exogenous overexpression in HEK293 cells and also with Mbd3/NuRD components in mouse fibroblasts undergoing reprogramming.   Genome-wide ChIP-seq analysis for Mbd3 found that OSKM induction by dox in wildtype fibroblasts led to a general increase in Mbd3 recruitment, with enrichment for pluripotency associated genes targeted by OSKM. In Mbd3 depleted samples, Mbd3 target gene expression was significantly upregulated following Dox induction while the chromatin landscape of Mbd3 and/or OSKM direct targets was significantly more active and open in Mbd3-depleted samples during reprogramming.  Taken together this suggests that the reprogramming factors themselves mediate the recruitment of a repressive factor (Mbd3) to genes important for the attainment of pluripotency.

In conclusion, this paper represents a milestone in iPSC research. Rapid, synchronous methods of generating iPSCs could allow for the molecular dissection of the intricate mechanisms which lie behind the largely unknown reprogramming process and, furthermore, it could mean the rapid gross amplification of more uniform cells, making iPSCs and their progeny more applicable in a clinical setting. However, the interaction of Mbd3 with the pluripotency factors to repress gene activity must have additional roles which for now we can only surmise; this may be as a dampener or brake to stop the uncontrolled development of the pluripotent state potentially leading to tumourigenesis or a key player in the subsequent differentiation of cells. Perhaps the key may be to find small molecule inhibitors which can be used at precise times to stop the interaction with OSKM factors until pluripotency is obtained, but thereafter allowing the Mbd3 to play its normal role. Indeed, while the headline of "100% efficiency in 8 days" will resound throughout the community, the differentiation and tumourigenetic capabilities of cells generated in this manner require to be tested.

References

Mansour, A. A. et al.
The H3K27 demethylase Utx regulates somatic and germ cell epigenetic reprogramming.
Nature 488,409–413 (2012)

Kaji, K. et al. Mbd3,
a component of the NuRD co-repressor complex, is required for development of pluripotent cells.
Development 134, 1123–1132 (2007)

Warren, L. et al.
Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.
Cell Stem Cell 7, 618–630 (2010)

 

STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.