Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds Danwei Huangfu1, Rene �� Maehr1, Wenjun Guo2, Astrid Eijkelenboom1,3, Melinda Snitow1, Alice E Chen1 & Douglas A Melton1 Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors, but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular, valproic acid (VPA), an HDAC inhibitor, improves reprogramming efficiency by more than 100-fold, using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc. Stem cells specific to an individual may be created by reprogramming somatic cells to a pluripotent state. Recently, pioneering work showed that forced expression of just four transcription factors, Oct4, Klf4, Sox2 and c-Myc, reprograms mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells that closely resemble embryonic stem (ES) cells1���4. Reprogramming human somatic cells has now been achieved through similar means5���9, suggesting that the mechanism of reprogramming is conserved between humans and mice. However, reprogramming by viral infection is a slow and inefficient process. In addition, genetic transformation with exogenous genes, in particular oncogenes such as c-Myc and Klf4, and the use of viral delivery systems handicap this method with regard to human therapeutic applications. Previous studies have shown that histone deacetylase (HDAC) inhibitors and DNA demethylation have a modest effect (two- to fivefold) on the efficiency of reprogramming mediated by somatic cell nuclear transfer (SCNT)10���12. We speculated that reprogramming by defined factors may share common mechanisms with SCNT. Using an Oct4-GFP transgenic reporter13, we tested whether small molecules involved in chromatin modification have any effect on reprogramming (Supplementary Fig. 1 online). Retroviral expression of four tran- scription factors, Oct4, Sox2, Klf4 and c-Myc, in MEFs hemizygous for the Oct4-GFP transgene (Oct4-GFP/+) induced 0.03% �� 0.02% 18% 104 104 103 103 102 102 PE-A 101 0.04% Not treated GFP FITC-A FITC-A 101 100 100 104 104 103 103 102 102 PE-A 101 0.53% 101 100 100 14% 10% 6% 2% *** *** *** ** * Percent of GFP-positive cells Percent of GFP-positive cells Control fluorescence 1% No treatment DMSO��� 5 -azaC 5 ��� -azaC+dex 5���-azaC treated 5 ��� -azaC ** *** VPA FITC-A 104 104 103 103 102 102 PE-A 101 11.4% 101 100 100 VPA treated 3.0% 2.5% 2.0% 1.5% 1.0% 0.5% 0.0% No treatme nt Not treated VPA treated dex VPASAHA TSA a b c d Figure 1 Chemicals that promote reprogramming efficiency. (a) The percentages of GFP+ cells induced in four factor (Oct4, Sox2, Klf4 and c-Myc)���infected Oct4-GFP/+ MEFs treated with chemicals. Chemical treatments were: 5��-azaC (2 mM), dexamethasone (dex, 1 mM), 5��-azaC and dexamethasone, VPA (2 mM), SAHA (5 mM) and TSA (20 nM). The controls were infected MEFs without chemical treatment or treated with DMSO (the solvent for dexamethasone, SAHA and TSA). The y axis is truncated to accommodate the high percentage from the VPA treatment. For all figures in this study, s.d. are indicated by error bars, and P values by two-tailed student t-test o0.05, 0.01 and 0.001 are indicated by one, two and three asterisks, respectively. (b) Representative FACS plots from four factor���infected MEFs treated with 5��-azaC and VPA compared to the control-infected MEFs without treatment. Signal from the PE channel was used as a control for autofluorescence. (c) MEFs infected with genes for the three factors (Oct4, Sox2, Klf4, but not c-Myc) were treated with 5��-azaC or VPA for 1 week and the percentage of Oct4-GFP+ cells induced was measured by FACS analysis at 10 d post-infection, and compared to three factor���infected MEFs without chemical treatment. (d) Representative pictures at 16 d post-infection in three factor���infected MEFs with VPA treatment compared to the control-infected MEFs without VPA treatment. Received 19 February accepted 5 June published online 22 June 2008 doi:10.1038/nbt1418 1Department of Stem Cell and Regenerative Biology, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA. 2Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA. 3Biomedical Sciences, Utrecht University, The Netherlands. Correspondence should be addressed to D.A.M. (dmelton@harvard.edu). NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 7 JULY 2008 795 B R I E F C O M M U N I C AT I O N S �� 200 8 Nature Publishing Group http://www.nature.com/naturebiotechnology

(mean �� s.d.) of the cells to become GFP+, typically starting at 7 d post-infection, and the percentage of GFP+ cells remained at similar levels between 7 and 13 d post-infection (Supplementary Fig. 2 online). Treating four factor���infected MEFs with 2 mM 5��-azacytidine (5��-azaC), a DNA methyltransferase inhibitor, increased the percentage of GFP+ cells by approximately tenfold to 0.50% �� 0.06% (mean �� s.d.) (Fig. 1a,b). 5��-azaC promoted reprogramming efficiency in a dose-dependent manner, with an effective concentration (EC)50 of B2.4 mM (Supplementary Fig. 3a online). Dexamethasone (1 mM), a synthetic glucocorticoid, improved the effect of 5��-azaC by 2.6-fold when used in combination, although dexamethasone alone had no significant effect. Three known HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA) and VPA also greatly improved reprogramming efficiency. VPA was the most potent of the three. Treating four factor���infected MEFs with 2 mM VPA for 1 week induced B11.8% �� 2.2% GFP+ cells, which amounts to 4100-fold improvement over the control (Fig. 1a,b). This reprogramming efficiency approached the estimated 13���41% viral co-transduction rate, arguing that most if not all cells infected with all four factors can be reprogrammed. VPA promoted reprogramming efficiency in a dose-dependent manner, with an EC50 of B1.9 mM (Supplementary Fig. 3b). The effect of VPA is much stronger than that of 5��-azaC and other HDAC inhibitors tested. This could be due to toxicity of other chemicals at higher dosages. Alternatively, VPA may have additional activities, beyond inhibition of HDACs. Chemical treatment induced GFP+ iPS colonies in greater numbers, consistent with the fluorescence-activated cell sorting (FACS) data. Eight days post-infection, an average of 10 and 241 colonies were observed in 5��-azaC��� and VPA-treated MEF cultures (out of 270,000 cells seeded), respectively. No GFP+ colonies were observed 8 d post- infection without chemical treatment, though some did emerge after 10 d post-infection in untreated cells. The dramatic difference in colony numbers was maintained as more GFP+ iPS colonies emerged in both the chemical-treated and nontreated MEF cultures during the following days more than 8- and 40-fold increases in colony number were observed with 5��-azaC and VPA treatment at 2 weeks post- infection, respectively. In addition to improving the efficiency of reprogramming four factor���infected MEFs, chemical treatment allowed efficient induction of iPS cells without the oncogene c-Myc, which may be tumorigenic in cells derived from iPS cells2. Although reprogramming is possible with three factors (Oct4, Sox2 and Klf4) without c-Myc, the efficiency is extremely low and the appearance of iPS colonies significantly delayed compared to reprogramming with four factors. A previous study found that o1 iPS colony was formed from 100,000 human dermal fibroblasts infected (o0.001%)7, an efficiency that can make it difficult to derive individual-specific iPS cells from a small starting population of cells. Similar low efficiency was also reported for induction of iPS cells from mouse fibroblasts without c-Myc14. We tested whether treating the cells with 5��-azaC or VPA could improve the efficiency of iPS colony formation without the need for c-Myc. Oct4-GFP/+ MEFs were first infected with Oct4, Sox2 and Klf4, and then treated with 5��-azaC or VPA for 1 week starting 1 d post- infection. FACS analysis 10 d post-infection showed that treatment with 5��-azaC increased reprogramming efficiency threefold, a small improvement (Fig. 1c). Treatment with VPA improved reprogram- ming efficiency by 50-fold (Fig. 1c). Notably, this reprogramming efficiency is superior to that achieved when MEFs were infected with Bright field Oct4-GFP AP staining 104 104 103 103 102 104 103 102 102 104 103 102 iPS m82 iPS m82 ES (AV3) MEF Oct4 Nanog Sox2 Oct4 Nanog Sox2 Acinar gland Tubular gland Cartilage Muscle Skin Neural epithelium Noninjected control Chimera Embryos from a chimera mouse nt g lb a b d e g f c Figure 2 c-Myc-free iPS cells induced by VPA treatment resemble ES cells in gene expression and pluripotency. (a) iPS colonies exhibited typical ES cell morphology and expressed Oct4-GFP homogeneously. (b) iPS colonies exhibited high alkaline phosphatase activity. (c) Scatter plots comparing global gene expression patterns between iPS cells and ES cells and between iPS cells and MEFs. Red lines indicate the linear equivalent and twofold changes in gene expression levels between the samples. (d) Hematoxylin and eosin staining of teratoma sections showed differentiation of iPS cells to various tissues. (e) lacZ staining of a mid-gestation chimeric embryo from donor iPS cells carrying the Rosa26-lacZ allele compared to the noninjected control. (f) Sections of chimeric embryos showed contribution of donor iPS cells to tissues derived from all three germ layers, including the neural tube (nt, ectoderm derivative), gut endoderm (g) and limb bud (lb, mesoderm derivative). (g) Shown here is a lacZ-positive e8.5 embryo with a littermate control on the left from a mating between a wild-type female and a chimera from blastocyst injection of iPS cells. Both embryos have yolk sacs attached and are oriented with the anterior to the left. Scale bars, 100 mm in a,b,d,f 1 mm in e,g. 796 VOLUME 26 NUMBER 7 JULY 2008 NATURE BIOTECHNOLOGY B R I E F C O M M U N I C AT I O N S �� 200 8 Nature Publishing Group http://www.nature.com/naturebiotechnology