Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPSC state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPSCs display similar characteristics to Oct4/Sox2/Klf4/Myc-iPSCs, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can cooperate in a powerful way to reprogram somatic cells to pluripotency.

Of the miRNAs expressed at high levels in ESCs and iPSCs, the miR302/367 cluster has been shown to be a direct target of Oct4 and Sox2 (), two of the critical factors required for iPSC reprogramming. Levels of miR302/367 correlate with Oct4 transcripts in ESCs and early embryonic development, indicating an important role in ESC homeostasis and maintenance of pluripotency (). Despite their ability to enhance iPSC reprogramming in the presence of several of the OSKM factors (), the ability of these miRNAs to directly reprogram somatic cells to an iPSC phenotype is unclear. We show that expression of the miR302/367 cluster can directly reprogram mouse and human somatic cells to a pluripotent stem cell state in the absence of any of the previously described pluripotent stem cell transcription factors. Reprogramming by miR302/367 is up to two orders of magnitude more efficient than that with the OSKM factors. We also show that valproic acid (VPA) is required for reprogramming mouse fibroblasts by specifically degrading Hdac2 protein, a finding that is supported by the efficient reprogramming of Hdac2−/− fibroblasts in the absence of VPA. Thus, the expression of miR302/367 along with Hdac2 suppression allows for highly efficient iPSC reprogramming without the expression of the known reprogramming factors.

The current standard strategy for iPSC generation relies upon ectopic expression of Oct4, Sox2, Klf4, and Myc (OSKM) (). Although there are several alternatives to some of these factors, including the use of other transcription factors, signaling factors, and pharmacological molecules, at least one pluripotent stem cell transcription factor—usually Oct4—is required for efficient iPSC reprogramming (). Recently, several microRNAs (miRNAs) have been shown to enhance iPSC reprogramming when expressed along with combinations of the OSKM factors (). These miRNAs belong to families of miRNAs that are expressed preferentially in embryonic stem cells and are thought to help maintain the ESC phenotype (;). How these miRNAs enhance iPSC reprogramming is unclear but may have to do with their ability to regulate the cell cycle ().

The transformation of differentiated cells to induced pluripotent stem cells (iPSCs) has revolutionized stem cell biology by providing a more tractable source of pluripotent cells for regenerative therapy. Although powerful, there are currently several limitations to iPSC generation, including the rather low efficiency of the process (0.2%–1.0%) and the necessity of forced expression of at least one pluripotent stem cell transcription factor, including Oct4, Nanog, Sox2, Klf4, and/or Myc. These limitations hamper the use of iPSC technology in high throughput formats such as generation of human iPSC clones from large patient populations.

To test whether suppression of Hdac2 is specifically required for efficient reprogramming by miR302/367, we generated Hdac2−/− MEFs from Hdac2flox/flox mice using adenoviral-mediated cre excision of Hdac2 and determined whether loss of Hdac2 altered the efficiency of miR302/367 reprogramming of MEFs in the absence of VPA ( Figure S5 ). We found that in Hdac2−/− MEFs transduced with the miR302/367 virus, Oct4-GFP-positive clones were observed as early as 6 days post-viral infection ( Figure 7 C). Eight days after viral transduction, Hdac2−/− MEFs had formed significant numbers of iPSC clones in the absence of VPA, whereas wild-type MEFs in the absence of VPA did not generate any viable clones ( Figure 7 D). VPA addition to Hdac2−/− MEFs did not change the number of iPSC clones obtained ( Figure 7 D). The number of iPSC clones generated and the percentage of clones that were Oct4-GFP positive with miR302/367-transduced wild-type MEFs plus VPA and miR302/367-transduced Hdac2−/− MEFs lacking VPA were similar ( Figure 7 D and 7E). Loss of Hdac2 expression or VPA addition did not affect proliferation rates in MEFs ( Figure S6 ). Q-PCR to assess expression of pluripotency-related genes also shows increased reprogramming by miR302/367 in Hdac2−/− MEFs compared to wild-type MEFs without VPA ( Figure 7 F). Thus, low levels of Hdac2 or suppression of Hdac2 is required for efficient pluripotent stem cell reprogramming by miR302/367.

Recent evidence has pointed to an important role for chromatin remodeling factors in regulating the ESC pluripotent state (). Previous data have shown that VPA, a known Hdac inhibitor, enhances OSKM reprogramming, suggesting an important role for Hdac-mediated chromatin remodeling in iPSC reprogramming (). We initially found that, in the absence of VPA, miR302/367 was unable to efficiently reprogram MEFs to iPSCs and of the few clones that did develop, none survived clonal replating ( Figures 7 D and 7F and data not shown). Interestingly, VPA was not necessary for reprogramming of human foreskin or dermal fibroblasts ( Figure 5 ). VPA has been reported to specifically degrade Hdac2 protein (). Therefore, we assessed whether expression of class I Hdacs was altered by miR302/367 or VPA treatment by performing western blots for Hdac1, -2, and -3 expression during miR302/367-mediated reprogramming. While Hdac1 and Hdac3 expression levels were unchanged in all conditions, VPA caused degradation of Hdac2 protein in MEFs ( Figure 7 A). Expression of miR302/367 did not affect the levels of Hdac1, -2, or -3 in the presence or absence of VPA in MEFs ( Figure 7 A). In contrast, human foreskin fibroblasts expressed much lower levels of Hdac2 protein, and the protein levels of Hdac2 were not affected by VPA in these cells ( Figure 7 B). These data suggest that low levels of Hdac2 may significantly enhance or even be required for miR302/367 reprogramming and that human fibroblasts express much lower levels of Hdac2 than MEFs.

(F) Q-PCR for pluripotent stem cell marker genes shows enhanced expression of pluripotency markers at day 8 of reprogramming by miR302/367 in wild-type (Hdac2+/+) and Hdac2−/− MEFs versus WT MEFs without VPA treatment. Data are the average of three assays ± SEM.

(D) Number of clones generated with Hdac2−/− MEFs in the absence of VPA is similar to Hdac2+/+ MEFs with VPA at 8 days postviral transduction. Hdac2+/+ MEFs without VPA treatment did not generate any viable clones and VPA addition to Hdac2−/− MEFs did not increase the number of clones generated.

(C) Hdac2−/− MEFs, in the absence of VPA, start to reprogram between 6 and 7 days postviral transduction, which is similar to wild-type MEFs treated with VPA.

(A) VPA specifically degrades Hdac2, but not Hdac1 or Hdac3 proteins. Expression of miR302/367 alone did not have any effect on Hdac1, -2, or -3 protein levels.

The miR302/367 cluster contains five different miRNAs, miR302a/b/c/d and miR367. All are expressed from a common promoter located in intron 8 of the Larp7 gene (). miR302a/b/c/d all share a common seed sequence suggesting that they target a similar set of mRNAs and thus may act redundantly ( Figure 1 A). However, miR367 has a different seed sequence and thus may target a different set of mRNAs ( Figure 1 A). Therefore, we tested whether miR367 expression is required for miR302/367 iPSC reprogramming. Using a lentivirus lacking the miR367 sequence, we infected Oct4-GFP MEFs alongside the miR302/367 lentivirus and assessed pluripotent reprogramming by colony counts, Q-PCR, and FACS analysis. The miR302a/b/c/d virus lacking miR367 is expressed at high levels in MEFs ( Figure 6 A ). However, miR302a/b/c/d did not generate any iPSC colonies when expressed in MEFs at day 10 of reprogramming ( Figure 6 B). Continued culture for up to 3 weeks did not result in formation of any iPSC colonies from miR302a/b/c/d-transduced MEFs (data not shown). Moreover, expression of miR367 alone did not reprogram fibroblasts (data not shown). Q-PCR of primary induction plates 8 days after viral transduction shows that several important pluripotent genes were expressed at lower levels in miR302a/b/c/d-transduced MEFs versus miR302/367-transduced MEFs ( Figure 6 C). Importantly, Oct4 expression is not observed at detectable levels in response to miR302a/b/c/d expression ( Figure 6 C, arrow). Using FACS analysis and Oct4-GFP MEFs, we show that there is no induction of Oct4 gene expression when expressing miR302a/b/c/d without miR367 while miR302/367 expression induces robust Oct4-GFP expression by day 8 ( Figure 6 D). These data show that without miR367 expression, miR302a/b/c/d expression was unable to reprogram mouse MEFs and that this correlated with a lack of induction of Oct4 gene expression. Thus, the coordinated action of the miR302a/b/c/d family along with miR367 is required for iPSC reprogramming.

(C) Pluripotent gene expression from primary induction plates 8 days after viral induction of miR302a/b/c/d or miR302/367 viruses. Note lack of Oct4 gene expression in miR302a/b/c/d-expressing cells (red arrow). Data are the average of three assays ± SEM.

(B) Number of colonies generated after 10 days of miR302a/b/c/d or miR302/367 expression. Data are the average of four assays ± SEM.

We next assessed whether there was an increase in human reprogramming efficiency similar to what we observed in MEFs. Starting with the same number of human foreskin fibroblasts and OSKM and miR302/367 viral titers, the number of colonies with ES-like morphology formed at 18 and 26 days after starting viral transduction is two orders of magnitude greater for miR302/367 than when using OSKM expression ( Figure 5 M). Based on the cell counts, approximately 10% of human fibroblasts used for viral transduction produce iPSC clones ( Figure 5 M). Q-PCR from primary induction plates also reveals a dramatic increase in pluripotent gene expression in miR302/367-expressing versus OSKM-expressing human foreskin fibroblasts ( Figure 5 N). These data indicate that miR302/367 can reprogram human as well as mouse fibroblasts to an iPSC state with greatly increased efficiency.

To assess whether miR302/367 can reprogram human fibroblasts, we transduced human foreskin and dermal fibroblasts with the miR302/367 lentivirus. Within 12–14 days, we observed clones with the classic human ESC morphology ( Figure 5 A ). Immunostaining of these clones showed they expressed OCT4, SSEA4, TRA-1-60, and TRA-1-81 ( Figures 5 B–5E). Q-PCR using three different miR302/367 hiPSC cell clones shows that they all express pluripotent markers at levels equivalent to the hESC line HUES13 ( Figure 5 F). We reprogrammed the human foreskin fibroblast cell line BJ and performed DNA fingerprinting to show that clones from miR302/367 reprogramming are derived from the original parental BJ line ( Figure S4 ). Moreover, these human clones did not contain any integrants of the OSKM viruses, and the miR302/367 virus was silenced in later passages ( Figures S1 and S2 ). Interestingly, VPA was not required for reprogramming human fibroblasts and its addition did not affect the efficiency of reprogramming (see below and data not shown). Teratomas were generated from seven different miR302/367 hiPSC clones and all exhibited formation of mesoderm, endoderm, and ectoderm ( Figures 5 G–5L). A summary of human clones tested for pluripotency is found in Table S1

(N) Q-PCR of pluripotent gene expression in miR302/367-reprogrammed human foreskin fibroblasts at 18 and 26 days postviral transduction. Data are the average of three assays ± SEM.

(M) Efficiency of miR302/367 reprogramming in human foreskin fibroblasts by colony counts of clones with human ES-like morphology at 18 and 26 days postviral transduction. Data are the average of three assays ± SEM.

(G–I) Hematoxylin and eosin staining of teratomas derived from miR302/367 human iPSC clones showing endoderm (gut)-, mesoderm (muscle)-, and ectoderm (neural epithelium)-like structures. These data represent the results from seven human miR302/367 iPSC clones.

To test whether miR302/367 iPSCs could contribute to the germline of mice, we injected three different mouse miR302/367 iPSC clones derived from Oct4-GFP MEFs. Mouse gonads were collected at E13.5 and E15.5 and visualized both by whole-mount fluorescence and then fixed and sectioned for immunostaining for GFP expression. All three clones contributed efficiently to germ cells in the gonads of chimeric mice ( Figures 4 D–4J). Moreover, miR302/367 iPSC clones generated from C57BL/6 MEFs can generate high-percentage postnatal chimeras, although germline transmission has not yet been examined ( Figure 4 K). Thus, miR302/367 iPSC clones are pluripotent, are competent to generate all three germ layers, and contribute efficiently to the germline of mice. A summary of mouse clones tested for pluripotency is found in Table S1

To more fully characterize the pluripotent characteristics of miR302/367 iPSCs, we generated teratomas in immune-deficient mice with multiple miR302/367 iPSC clones. miR302/367 iPSC-derived teratomas formed readily and exhibited tissues representing all three germ layers, as noted by structures resembling muscle fibers, keratinized epidermal cells, and luminal structures lined with gut-like epithelium ( Figure 4 A ). Supporting these morphological findings, neural epithelial-like structures were positive for βIII-tubulin expression, muscle-like structures were positive for myosin heavy-chain expression, and gut-like epithelium was positive for E-cadherin expression ( Figure 4 B). A more stringent assay for pluripotency is determining whether miR302/367 iPSCs can generate tissues within the developing embryo using chimeric embryo analysis. Therefore, we generated miR302/367 iPSC clones from MEFs made from the Rosa26lacZ mouse line which expresses β-galactosidase ubiquitously (). Injection of these miR302/367 iPSC clones generated high-percentage chimeras in more than 50% of the injected embryos ( Figure 4 C and data not shown). Most of these chimeras exhibited 80%–95% contribution from miR302/367 iPSCs to all tissues examined ( Figure 4 C and Figure S3 ).

(D–J) Both whole-mount fluorescence (D) and immunostaining for Oct4-GFP protein expression (E–J) show high-level contribution of miR302/367 iPSC clones to the germline within the gonads of recipient mice. The data are representative of three clones (C6, C7, C10), which were injected into blastocysts and all three contributed to the germline.

(C) miR302/367 iPSC clones can generate all tissues within the developing mouse embryo as shown by lacZ histochemical staining of high-percentage chimeric embryos derived from Rosa26-miR302/367 iPSC clones at both E9.5 and E13.5.

(A) Hematoxylin and eosin staining of teratomas derived from mouse miR302/367 iPSC clones showing skin epidermal-like structures, muscle, and gut-like epithelium. These data are representative of five different miR302/367 iPSC clones, all of which were injected and produced teratomas.

To better quantify this increase in iPSC reprogramming efficiency, we performed quantitative real-time PCR (Q-PCR) for pluripotent marker genes during the first 8 days of the reprogramming process on primary induction plates. The experiment used the same number of starting MEFs and viral titer for infection. These data indicate that while cells transduced with the OSKM factors expressed only very low levels of pluripotent marker genes during this time period, miR302/367-transduced cells expressed all of the genes examined at robust levels by day 8 ( Figure 3 D). The numbers of clones were such that after 8–10 days, the plates containing the miR302/367 iPSC clones became overcrowded, resulting in decreased cell viability unless they were isolated and expanded. We also assessed the efficiency of reprogramming by miR302/367 using fluorescence-activated cell sorting (FACS) for expression of GFP from the Oct4 locus in Oct4-GFP MEFs (). OSKM-reprogrammed MEFs do show Oct4-GFP expression at both 6 and 8 days of the reprogramming process, with up to 17% of cells expressing GFP by day 8, which is in the same range as previously reported ( Figure 3 E;). However, miR302/367 is able to activate Oct4-GFP expression in up to 80% of MEFs after 8 days of reprogramming ( Figure 3 E). These data support the conclusion that miR302/367 is able to reprogram fibroblasts to a pluripotent state up to two orders of magnitude more efficiently than OSKM factors.

The rapid appearance of miR302/367-reprogrammed iPSCs suggested that expression of these miRNAs improved the temporal kinetics of reprogramming. To test this hypothesis, we expressed in parallel miR302/367 and the OSKM genes using an identical number of starting MEFs and viral titer. VPA was included in both OSKM as well as miR302/367-reprogramming experiments. Previous studies have demonstrated that using the OSKM factors, an average colony-forming reprogramming efficiency of 0.2%–0.8% is observed (). Using miR302/367, we consistently observed Oct4-GFP-positive clones 7 days after starting viral transduction, which is sooner than cells transduced in parallel with the OSKM factors ( Figure 3 A ). By counting the number of clones with ES-like morphology at 8 and 10 days after starting viral transduction, we show that expression of miR302/367 produces two orders of magnitude more iPSC clones than when the OSKM factors are used ( Figure 3 B). At day 10, 79.8% of miR302/367 iPSC clones exhibited robust expression of Oct4-GFP, which is greater than clones expressing the OSKM factors, of which only approximately 50% express Oct4-GFP ( Figure 3 C).

(C) Percentage of Oct4-GFP-positive clones 10 days after viral transduction with OSKM or miR302/367. Data are the average of three assays ± SEM.

(B) Counts of clones with ES-like morphology from transduction of 1.75 × 10 4 Oct4-GFP MEFs with equivalent amounts of either OSKM or miR302/367 virus at 8 and 10 days after viral transduction. Data are the average of three assays ± SEM.

(A) miR302/367 iPSC clones are readily observed 6 to 7 days after starting viral transduction and express high levels of Oct4-GFP while OSKM-induced clones are not observed until 8–10 days, are very rare, and do not express significant levels of GFP from the Oct4 locus.

miR302/367 Plus VPA is Two Orders of Magnitude More Efficient Than OSKM Factors in iPSC Reprogramming of Mouse Fibroblasts

Figure 3 miR302/367 Plus VPA is Two Orders of Magnitude More Efficient Than OSKM Factors in iPSC Reprogramming of Mouse Fibroblasts

We further characterized the miR302/367-generated iPSC clones by microarray analysis for their similarity at the global gene expression level to the mouse ESC line R1. We used clones at passage 15 for these analyses. These data show a very high degree of correlation with global gene expression in the R1 ESC line ( Figures 2 A and 2B ). These clones lacked integration of any of the OSKM factors that we use as controls, but did contain viral integration of the miR302/367 lentivirus into the genome (see Figure S1 available online). miR302/367 iPSC clones that have been passaged serially maintain their ESC-like morphology and Q-PCR shows that they exhibit identical expression of pluripotent genes as mouse ESCs ( Figure 2 C and data not shown). Moreover, the miR302/367 lentivirus is silenced at later passages ( Figure S2 ). These results suggest that expression of miR302/367 in addition to VPA was able to reprogram mouse MEFs to an iPSC state without expression of other previously described pluripotent factors.

(A) Microarray experiments were used to show the similarity between miR302/367 iPSC clones C6, C7, and C10 at passage 15 and the mouse ESC line R1.

Previous studies have shown that the miR302/367 cluster comprises five miRNAs, four of which—miR302a/b/c/d—have identical seed sequences ( Figure 1 A ). The miR302/367 cluster is located in intron 8 of the Larp7 gene on chromosome 3 and is transcribed as a single polycistronic primary transcript (). The sequences of the miR302/367 miRNAs are highly conserved across species (). To determine whether expression of miR302/367 could reprogram somatic cells, we generated a lentiviral vector, which expressed the 690 bp region encoding the mouse miR302/367 sequences, and used it to transfect mouse embryonic fibroblasts (MEFs) derived from the Oct4-GFP mouse line ( Figure 1 B). We included the Hdac inhibitor VPA in these experiments, as this has been shown to enhance iPSC reprogramming (). Surprisingly, we observed clones derived from miR302/367-transduced MEFs within 6 to 8 days after the start of a viral infection that had already assumed an ESC-like morphology (Figures 1 C and 3 A). Most of these clones were Oct4-GFP positive and alkaline-phosphatase positive ( Figures 1 C and 1D). These clones also expressed Nanog, Sox2, and SSEA1 ( Figure 1 E). In comparison, parallel expression of OSKM-expressing viruses in addition to VPA did not result in any visible clones until at least 8–10 days after starting viral transduction ( Figure 3 and data not shown). Use of a polycistronic virus did not alter the timing or overall number of colonies generated by OSKM expression (data not shown and). Moreover, in the absence of VPA, miR302/367 was unable to reprogram MEFs efficiently (see below and data not shown).

(E) Immunostaining for Nanog, Oct4, Sox2, and SSEA1 in both mouse ES and primary induction samples of miR302/367 iPSCs at day 10, showing expression of pluripotent genes.

(A) The sequences of the miR302/367 cluster showing the similarity between members of the miR302a/b/c/d subfamily. miR367 has a different seed sequence than miR302a/b/c/d.

Discussion

Current strategies for generating iPSCs 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 nonviral, nontranscription-factor mediated procedure for generating iPSCs for use not only in basic stem cell biology studies, but also for high throughput generation of human iPSC clones from large patient populations.

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Orkin S.H. An extended transcriptional network for pluripotency of embryonic stem cells. Our studies underscore the role of Hdac2 in iPSC 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 iPSC reprogramming (). Several recent studies have demonstrated the importance of other chromatin remodeling processes in iPSC reprogramming (). Hdac2 has also been found to be part of an extended regulatory network for pluripotency in ESCs by interacting with both Oct4 and Myc (). Since iPSC 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 transdifferentiation 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 iPSC reprogramming efficiencies exhibited by different cell lineages. Moreover, Hdac2 expression may decline during development such that adult cells have little Hdac2 protein, resulting in the 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 iPSC reprogramming without the need for pluripotent factors including the OSKM factors. The current focus on developing miRNAs for therapeutic use could lead to a nonviral mediated method of altering miR302/367 expression, which could in turn allow for a rapid miRNA/small molecule approach for iPSC reprogramming.