Chang, Hao-Ming

Gregory, Richard I.

Cell Stem Cell

Harvard University - DASH 

 MicroRNA-induced pluripotent stem cells  (Article begins on next page)The Harvard community has made this article openly available.Please share how this access benefits you. Your story matters.Citation Chang, Hao-Ming, and Richard I. Gregory. 2011. “MicroRNA-induced pluripotent stem cells.” Cell stem cell 7 (1): 31-35.doi:10.1016/j.stem.2010.06.011.http://dx.doi.org/10.1016/j.stem.2010.06.011.Published Version doi:10.1016/j.stem.2010.06.011Accessed February 16, 2015 9:29:43 PM ESTCitable Link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12785973Terms of Use This article was downloaded from Harvard University's DASHrepository, and is made available under the terms and conditionsapplicable to Other Posted Material, as set forth athttp://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAAMicroRNA-induced pluripotent stem cellsHao-Ming Chang and Richard I. Gregory1Stem Cell Program, Children’s Hospital Boston, Department of Biological Chemistry andMolecular Pharmacology, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA02115KeywordsmicroRNA (miRNA); Reprogramming; Induced Pluripotent stem (iPS) cells; Embryonic StemcellsThe ability to reprogram differentiated cells to pluripotency has revolutionized stem cellresearch and provides the foundation for new approaches to model human disease andpossible stem cell-based therapies for degenerative diseases. To fully exploit this potential itwill be necessary to develop simpler and more efficient ways to generate induced pluripotentstem (iPS) cells. Anokye-Danso and colleagues present a novel strategy for generatinghuman and mouse iPS cells that is quicker and 100-fold more efficient than conventionalprotocols1. Remarkably, unlike all previous reports that use transcription factors, theirapproach uses only microRNAs (miRNAs)2. So how do these miRNAs accomplish thisamazing feat? Subramanyam et al., shed some light on this question and identify a set ofmiRNA target genes that when depleted can facilitate iPS cell formation3. Together thesestudies highlight the utility of miRNAs in both iPS generation as well as dissecting themechanisms and pathways underlying cell reprogramming.The groundbreaking findings that differentiated can be reprogrammed to iPS cells byenforced expression of a few defined factors (Oct3/4, Sox2, Klf4, and c-Myc (OSKM))overcomes many of the technical, ethical, and political obstacles in human embryonic stemcell (ESC) research and regenerative medicine4. iPS cells represent an alternative source ofpluripotent cells for possible therapeutic applications. However, the initial reprogrammingefficiencies were low (less than 0.1%) and the original cocktail of reprogramming factorsincludes oncogenes that can lead to tumorigenesis4. Also, retroviral infection of the targetcells causes multiple potentially harmful integrations of the transgenes into the host genome.Since then, efforts to refine and develop alternative approaches include the use of non-integrating systems to deliver the reprogramming genes, and the replacement of individualfactors with small molecules5. However, common to all of these is the use of at least onetranscription factor, usually Oct3/42.Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research,subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms1Corresponding author: Richard I. Gregory, Phone: (617) 919-2273, Fax: (617) 730-0748, rgregory@enders.tch.harvard.edu.Competing financial interests:Authors declare no competing financial interestsNIH Public AccessAuthor ManuscriptCell Stem Cell. Author manuscript; available in PMC 2014 August 05.Published in final edited form as:Cell Stem Cell. 2010 July 2; 7(1): 31–35. doi:10.1016/j.stem.2010.06.011.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptThough the ability of transcription factors to reprogram cells is well established, the role ofmiRNAs in this process is much less well understood. miRNAs represent a large family ofregulatory RNAs that posttranscriptionally repress the expression of large sets of targetgenes and are essential for normal development and ESC biology6. Importantly, ESCsexpress a unique a set of miRNAs with the majority transcribed from two genomic loci, themiR-302 cluster (that contains five miRNAs – miR-302a/b/c/d and miR-367) and themiR-290 cluster in mice (miR-290, miR-291a, miR-292, miR-291b, miR-293, miR-294, andmiR-295) or the miR-371-373 cluster in humans (miR-371, miR-372 and miR-373). ESC-specific miRNAs share a very similar ‘Seed’ sequence and therefore likely regulateoverlapping sets of target genes. These miRNAs are required rapid ESC proliferation andcell-cycle progression6. Previous studies indicated that the miRNA pathway could beexploited for cell reprogramming7,8. Introduction of individual members of the miR-290cluster or miR-302 was found to enhance cell reprogramming by OSK, but not by OSKM8.More recently however, it was demonstrated that miRNAs enhance reprogramming of MEFswith either OSKM or OSK. Furthermore, antagonizing these miRNAs with antisenseoligonucleotides conversely inhibited reprogramming9.An exciting new study demands close attention because instead of using any of the standardOSKM transcription factors the authors simply express a single primary miRNA transcript(the miR-302/367 cluster) to reprogram mouse and human somatic cells1. Importantly theresulting iPS cells exhibit gene expression and functional properties characteristic of fullyreprogrammed pluripotent cells. Remarkably, the reprogramming, mediated bymiR-302/367, is 100-fold more efficient than with OSKM, with ~10% of fibroblasts formingiPS colonies. Furthermore, the temporal kinetics of reprogramming may also be accelerated.Though this approach works for both mouse and human cells, valporic acid (VPA) wasrequired for reprogramming mouse cells and was administered in conjunction with themiRNA-expressing virus. Interestingly, reprogramming of human cells did not require VPAand the miRNAs alone were sufficient for efficient reprogramming. So what is thedifference for the species-specific requirement for VPA? It seems that a key barrier toreprogramming is the histone deacetylase, HDAC2, which is targeted for destruction byVPA and is expressed at considerably higher levels in mouse compared to humanfibroblasts. Accordingly, reprogramming of mouse HDAC2−/− MEFs did not require VPAand miR-302/367 alone could efficiently reprogram these cells.What is the mechanism behind miRNA-mediated cell reprogramming? miRNAs commonlytarget a large group of mRNAs simultaneously, and the combinatorial regulation of morethan a handful of genes is likely required for cell reprogramming. The demonstration thatESC miRNAs enhance reprogramming, strongly suggests that these miRNAs arefunctioning to repress expression of genes that would otherwise work to maintain thedifferentiated cell state and may therefore represent barriers to reprogramming. Using thisrationale, the Blelloch laboratory began to address the mechanism by which ESC miRNAsenhance reprogramming3. Using synthetic miRNA mimics, they find that introduction ofmiR-302b or miR-372 can enhance both OSKM- and OSK-mediated reprogramming ofhuman fibroblasts. Next, using a published data set they chose a group of around thirtypotential miR-302/miR-372 target genes for further analysis7. From this list they focus on asubset of twelve genes that respond to miRNA expression in the context of reprogramming.Chang and Gregory Page 2Cell Stem Cell. Author manuscript; available in PMC 2014 August 05.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptImportantly, they use siRNAs to individually deplete each of these putative miRNA targetsand measure the effect on reprogramming. They show that knockdown of three of thesegenes (RBl2, CDC2L, and RHOC) enhances reprogramming both with OSKM and withOSK. They find that individual depletion of an additional three genes (SMARCC2, MBD2,and MECP2) enhances reprogramming in one of the two conditions (i.e. either OSKM orOSK). Since the gene encoding the TGFß receptor, TGFBR2, was among the selected geneset they use a small molecule inhibitor to confirm the involvement of this signaling pathway.They find that TGFBR2 mRNA is directly repressed by these miRNAs. Mouse Tgfbr2 isalso regulated by similar miRNAs and siRNA-mediated knockdown of Tgfbr2 enhancesreprogramming of MEFs9. The evolutionarily conserved requirement for the TGFß receptorfurther supports data that a mesenchymal-epithelial transition (MET) accompanies humancell reprogramming as it does in mouse. Importantly, since inhibition of any single gene ledto relatively modest enhancement in cell reprogramming compared to the effects of themiRNA itself, as well as the increased efficiency observed by the simultaneous inhibition ofmore than one pathway, support the notion that miRNAs promote reprogramming bytargeting multiple genes in several different downstream pathways (Figure 1).These studies illuminate some of the genes that are targeted by the ESC-specific miRNAs inthe context of reprogramming and demonstrate the remarkable power of miRNAs toefficiently reprogram cells to pluripotency1,3,8,9. Both miR-302 and miR-367 are requiredfor iPS cell formation1. What genes are repressed by miR-367 to promote reprogramming?This missing piece of the puzzle remains to be addressed. However, a highly relatedmiRNA, miR-92b, represses expression of Cdkn1c (p57) and is important for cell cycleprogression of human ESCs10. Therefore it is likely that miR-367 facilitates reprogrammingat least in part by modulating cell proliferation.An obvious next experiment is to try and replace the viral delivery of the miRNA transgene.Since Morrisey’s lab showed that miR-367 is essential for miRNA-mediated reprogrammingit will be interesting to see whether adding this synthetic miRNA to those used in theBlelloch study will enable the generation of iPS cells without the need for viruses or genomeintegration1,3. miRNAs may prove particularly useful in this regard since they are small andeasily synthesized. Moreover, they are relatively easily transfected into cells and do notstimulate an innate immune response in the host cell. Furthermore, once incorporated intothe relevant ribonucleoprotein complexes they are relatively stable with a reported half-lifeof several days in cells. These properties, together with the breakthrough proof-of-conceptexperiments, identify miRNAs as perhaps the ideal mode for reprogramming human cellsand may help unleash the therapeutic potential of iPS cells.References:1. Anokye-Danso F, et al. Highly Efficient miRNA-Mediated Reprogramming of Mouse and HumanSomatic Cells to Pluripotency. Cell Stem Cell. 2011; 8:376–388. [PubMed: 21474102]2. Gonzalez F, Boue S, Izpisua Belmonte JC. Methods for making induced pluripotent stem cells:reprogramming a la carte. Nat Rev Genet. 2011; 12:231–242. [PubMed: 21339765]3. Subramanyam D, et al. Multiple targets of miR-302 and miR-372 promote reprogramming of humanfibroblasts to induced pluripotent stem cells. Nat Biotechnol. 2011Chang and Gregory Page 3Cell Stem Cell. Author manuscript; available in PMC 2014 August 05.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript4. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adultfibroblast cultures by defined factors. Cell. 2006; 126:663–676. [PubMed: 16904174]5. Huangfu D, et al. Induction of pluripotent stem cells by defined factors is greatly improved bysmall-molecule compounds. Nat Biotechnol. 2008; 26:795–797. [PubMed: 18568017]6. Martinez NJ, Gregory RI. MicroRNA gene regulatory pathways in the establishment andmaintenance of ESC identity. Cell Stem Cell. 2010; 7:31–35. [PubMed: 20621047]7. Melton C, Judson RL, Blelloch R. Opposing microRNA families regulate self-renewal in mouseembryonic stem cells. Nature. 2010; 463:621–626. [PubMed: 20054295]8. Judson RL, Babiarz JE, Venere M, Blelloch R. Embryonic stem cell-specific microRNAs promoteinduced pluripotency. Nat Biotechnol. 2009; 27:459–461. [PubMed: 19363475]9. Li Z, Yang CS, Nakashima K, Rana TM. Small RNA-mediated regulation of iPS cell generation.EMBO J. 2011; 30:823–834. [PubMed: 21285944]10. Sengupta S, et al. MicroRNA 92b controls the G1/S checkpoint gene p57 in human embryonicstem cells. Stem Cells. 2009; 27:1524–1528. [PubMed: 19544458]Chang and Gregory Page 4Cell Stem Cell. Author manuscript; available in PMC 2014 August 05.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. How do miRNAs reprogram mouse and human cells to pluripotency?Differentiated cells can be reprogrammed to iPS cells by expressing the miR-302/367 clusterof miRNAs. For mouse cells valproic acid (VPA) is additionally required. Multipledownstream target genes and pathways have been identified for miR-302. miR-367 isessential for reprogramming and Oct4 activation however targets of this miRNA remainunknown.Chang and Gregory Page 5Cell Stem Cell. Author manuscript; available in PMC 2014 August 05.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

MicroRNA-induced pluripotent stem cells

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MicroRNA-induced pluripotent stem cells

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