Current strategies for generating iPS cells rely upon expression of multiple pluripotent stem cell associated transcription factors. We show that a single miRNA cluster, miR302/367, can reprogram fibroblasts more efficiently than the standard OSKM method. With ongoing advances in miRNA biology, these findings may lead to a non-viral, non-transcription factor mediated procedure for generating iPS cells for use not only in basic stem cell biology studies but also for high throughput generation of human iPS clones from large patient populations.
Previous studies have demonstrated the usefulness of iPS cells not only in the study of basic stem cell biology but also in the ability to generate patient specific iPS clones which can then be further differentiated into the cell lineage of choice including hematopoietic, cardiomyocyte, and hepatocyte cell lineages (Moretti et al., 2010a
; Moretti et al., 2010b
; Raya et al., 2010
; Si-Tayeb et al., 2010
). However, at this point the low efficiency of iPS reprogramming is an impediment to adapting the process to high throughput approaches. Such approaches would allow for the generation of iPS clones from large patient populations obtained from genome wide association studies for use in characterizing the identified genomic differences at the cell biological level. Our finding that reprogramming by miR302/367
is up to two orders of magnitude more efficient than the OSKM factors suggests that this method may prove to be amenable for use in large scale iPS cell generation. Several other reports have demonstrated that using techniques including Sendai viral expression as well as direct transfection of synthesized mRNAs for the OSKM factors can improve upon the efficiency of iPS reprogramming (Seki et al., 2010
; Warren et al., 2010
). Based on our data, we obtain efficiencies that are greater than either of these techniques and using human fibroblasts the percent of cells that generate iPS cell clones approaches 10%. Thus, miR302/367
iPS cell reprogramming is more efficient than previously described methods including transfection of synthetic mRNAs for OSKM factors (Warren et al., 2010
The mechanism underlying the increased efficiency of miR302/367
iPS reprogramming is likely to revolve in part around the nature of miRNA biology. First, miRNA expression does not require protein translation and thus leads to a fast response in protein expression based on inhibition of mRNA translation and stability. Second, miRNAs generally target scores or hundreds of mRNAs that coordinate expression of many different proteins which can rapidly impose a dominant phenotypic change in cell identity. This ability to target many different mRNAs simultaneously also increases the complexity underlying the mechanism of miR302/367
collectively targets hundreds of different mRNA targets including those that regulate chromatin remodeling and cell proliferation based on bioinformatic prediction algorithms (Betel et al., 2008
; Grimson et al., 2007
; Krek et al., 2005
). Our data indicate that miR367
expression is essential for iPS cell reprogramming by the miR302/367
cluster. As miR367
has a different seed sequence suggesting a different set of mRNA targets, analysis of the combinatorial regulation of miR302a/b/c/d
targets may provide important information regarding both the pluripotent gene network and also factors whose expression is required to be suppressed for efficient iPS cell reprogramming.
Our studies underscore the role of Hdac2 in iPS cell reprogramming. The specific degradation of Hdac2 protein by VPA is likely the reason that this small molecule has been found to be more efficacious than other Hdac enzymatic inhibitors in enhancing iPS reprogramming (Huangfu et al., 2008a
). Several recent studies have demonstrated the importance of other chromatin remodeling processes in iPS cell reprogramming (Bhutani et al., 2010
; Lagarkova et al., 2010
; Mali et al., 2010
). Hdac2 has also been found to be part of an extended regulatory network for pluripotency in ES cells by interacting with both Oct4 and Myc (Kim et al., 2008
). Since iPS cell reprogramming involves the resetting of the epigenetic state of a differentiated cell to a pluripotent “ground state”, additional studies into the necessity of chromatin remodeling will likely lead to better insight into cell lineage trans-differentiation events. Our finding that human cells, which express much lower levels of Hdac2 protein, do not require VPA for miR302/367
mediated reprogramming suggests that differing levels of Hdac2 may account, at least in part, for the different iPS cell reprogramming efficiencies exhibited by different cell lineages. Moreover, Hdac2 expression may decline during development such that adult cells have little Hdac2 protein resulting in an absence of an affect by VPA. Future studies into whether these correlations exist more broadly in other cell lineages may be beneficial for optimizing reprogramming by other methods including the OSKM factors.
Our studies show that miRNAs can be powerful tools for mediating iPS cell reprogramming without the need for pluripotent factors including the OSKM factors. The current focus on developing miRNAs for therapeutic use could lead to a non-viral mediated method of altering miR302/367 expression, which could in turn allow for a rapid miRNA/small molecule approach for iPS cell reprogramming.