WO2012151309A1

WO2012151309A1 - Methods for regulating induced pluripotent stem cell generation and compositions thereof

Info

Publication number: WO2012151309A1
Application number: PCT/US2012/036169
Authority: WO
Inventors: Tariq M. Rana
Original assignee: Sanford-Burnham Medical Research Institute
Priority date: 2011-05-02
Filing date: 2012-05-02
Publication date: 2012-11-08

Abstract

The present invention is based on identification of key microRNAs (miRs) during the early stage of reprogramming from somatic cells into induced pluripotent stem cells. These key miRs can either induce or repress the reprogramming process. The present invention provides that miR-223 and/or miR-495 can inhibit the reprogramming process, but miR-93 and/or miR-135b can enhance the reprogramming process. The present invention provides methods and compositions for generating an induced pluripotent stem (iPS) cell and treating a subject using iPS generated with methods described.

Description

METHODS FOR REGULATING INDUCED PLURIPOTENT STEM CELL

GENERATION AND COMPOSITIONS THEREOF

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0001] The present invention relates generally to the field of induced pluripotent stem (iPS) cells and more specifically to methods for regulating iPS cell generation, as well as uses for iPS cells generated by such methods.

BACKGROUND INFORMATION

[0002] Since the first report of reprogramming of mouse fibroblasts into mouse embryonic stem (mES)-like induced pluripotent stem (iPS) cells, this phenomenon has been confirmed in many different mouse and human cell types. Currently, the main bottleneck for iPS induction is extreme low efficiency, typically from 0.01% - 0.2%. Tremendous efforts have been made to identify small molecules that enhance the reprogramming process or replace some of the four transgenes commonly used in the protocol. However, the mechanisms underlying reprogramming and how somatic cells can be reprogrammed by a combination of four factors remain largely unknown.

[0003] Recent mechanistic studies show that the p53 pathway is a primary barrier to reprogramming. Systematic analysis of overexpression of the four reprogramming factors and promoters targeted by them demonstrates that transgene targeting is altered in some partially reprogrammed cells, while it is similar in iPSCs and mES cells. Chemical screening has also shown that inhibition of TGFp signaling significantly enhances reprogramming and that some inhibitors of this pathway can replace the Sox2 transgene in inducing Nanog expression. Moreover, a mesenchymal-to-epithelial transition (MET) at an early stage of reprogramming was recently shown to occur. During reprogramming, different markers are either induced or repressed. Typically, MEF-specific markers are down-regulated initially, followed by upregulation of mES markers, such as alkaline phosphatase, Nanog and endogenous Oct4. Moreover, the origin of the iPS Cells apparently influences a certain level of epigenetic "memory" in derived iPSCs, which is gradually lost over continuous passages. However, there is still limited information relevant to how transgenes and other cellular factors function mechanistically to reprogram MEFs to an undifferentiated or ES-like state.

[0004] MicroR As are 18-24 nucleotides single-stranded RNAs associated with a protein complex called the RNA-induced silencing complex (RISC). Small RNAs are usually generated from non-coding regions of gene transcripts and function to suppress gene expression by translational repression and mRNA destabilization. Recent work indicates that mES-specific microRNAs can enhance iPS induction and, specifically, that the hES-specific miR-302 can antagonize the senescence response to four-factor expression in human fibroblasts. In addition, our recent findings suggest that the microRNA biogenesis machinery is required for efficient reprogramming. Furthermore, blocking expression of certain microRNAs, such as miR-93 and 106b, can dramatically decrease reprogramming efficiency, while blocking members of the let-7 family of microRNAs apparently enhances

reprogramming. Despite these findings, it is not yet clear how other microRNAs function in the process or whether they cooperate to regulate reprogramming.

SUMMARY OF THE INVENTION

[0005] The present invention is based on the seminal discovery that microRNAs are involved in modulating iPSC induction. Interference of the microRNA biogenesis machinery results in changes of reprogramming efficiency. Key microRNAs are identified and key regulators used by reprogramming cells are also identified that may be advantageously targeted to significantly increase reprogramming efficiency as well as direct differentiation of iPS cells.

[0006] Accordingly, in one embodiment, the present invention provides method of generating an induced pluripotent stem (iPS) cell. The method includes contacting a somatic cell with a nuclear reprogramming factor; and contacting the cell with a microRNA that alters RNA levels or activity within the cell, thereby generating an iPS cell. In one aspect, the microRNA or RNA is modified. In another aspect, the microRNA is in a vector. In another aspect, the microRNA is miR-93, miR-135b, or a combination thereof. In another aspect, the microRNA has a polynucleotide sequence comprising SEQ ID NO: 1.

[0007] In one aspect, the microRNA regulates expression or activity of Wisp 1, Tgfbr2, Igfbp5, or a combination thereof. In another aspect, the nuclear reprogramming factor is encoded by a gene contained in a vector. In another aspect, the nuclear reprogramming factor is a SOX family gene, a KLF family gene, a MYC family gene, SALL4, OCT4, NANOG, LIN28, or a combination thereof. In another aspect, the nuclear reprogramming factor is one or more of OCT4, SOX2, KLF4, C-MYC. In another aspect, induction efficiency is at least doubled as compared without the microRNA.

[0008] In one aspect, the somatic cell is contacted with the reprogramming factor prior to, simultaneously with or following contacting with the microRNA. In another aspect, the somatic cell is a mammalian cell. In an additional aspect, the somatic cell is a human cell or a mouse cell.

[0009] In another embodiment, the present invention provides an induced plunpotent stem (iPS) cell produced using the method described herein. In another embodiment, the present invention provides an enriched population of induced pluripotent stem (iPS) cells produced by the method described herein. In another embodiment, the present invention provides a differentiated cell derived by inducing differentiation of the pluripotent stem cell produced by the method described herein. In one aspect, the somatic cell is derived by inducing differentiation by contacting the iPSC with an RNA molecule or antisense oligonucleotide. In one aspect, the RNA molecule is selected from the group consisting of microRNA, dsRNA, siRNA, stRNA, or shRNA.

[0010] In another embodiment, the present invention provides a method of treating a subject. The method includes (a) generating an induced pluripotent stem (iPS) cell from a somatic cell of the subject by the method described herein; (b) inducing differentiation of the iPS cell of step (a); and (c) introducing the cell of (b) into the subject, thereby treating the condition. In another embodiment, the present invention provides the use of microRNA for increasing efficiency of generating of iPS cells. In one aspect, the microRNA is selected from the group consisting of miR-93, miR-135b, miR-223, miR-495, or a combination thereof. In abother aspect, the microRNA is selected from the group consisting of miR-93, miR-135b, or a combination thereof.

[0011] In another embodiment, the present invention provides a method of generating an induced pluripotent stem (iPS) cell. The method includes contacting a somatic cell with a nuclear reprogramming factor; and contacting the cell of with an inhibitor of microRNA, thereby generating an iPS cell. In one aspect, the microRNA is miR-223, miR-495, or a combination thereof. In another aspect, the nuclear reprogramming factor is a SOX family gene, a KLF family gene, a MYC family gene, SALL4, OCT4, NANOG, LIN28, or a combination thereof. In another aspect, the somatic cell includes a fibroblast. In another aspect, the inhibitor is a small molecule, a peptide or a nucleic acid molecule. In another aspect, the nucleic acid molecule is an siRNA. In another embodiment, the present invention provides an induced pluripotent stem (iPS) cell produced using the method described herein.

[0012] In another embodiment, the present invention provides an agent for altering mRNA level in a cell during reprogramming. In one aspect, the agent is a polynucleotide, polypeptide, or small molecule. In an additional aspect, the polynucleotide is an antisense oligonucleotide, chemically modified oligonucleotides, locked nucleic acid (LNA), or DNA. In another aspect, the polynucleotide is RNA. In an additional aspect, the RNA is selected from the group consisting of microRNA, dsRNA, siRNA, stRNA, or shRNA. In another aspect, the somatic cell is a mouse embryonic fibroblast (MEF). In various embodiments, the RNA is non-coding RNA (ncRNA), including microRNA.

[0013] In another embodiment, the present invention provides a method of generating an induced pluripotent stem (iPS) cell. The method includes contacting a somatic cell with a nuclear reprogramming factor; and contacting the cell with an agonist of microRNA, thereby generating an iPS cell. In one aspect, the microRNA is miR-93, miR-135b, or a combination thereof. In another aspect, the agonist is a peptide, small molecule or a nucleic acid. In another embodiment, the present invention provides an induced pluripotent stem (iPS) cell produced using the method described herein.

[0014] In another embodiment, the present invention provides a method of generating an induced pluripotent stem (iPS) cell. The method includes contacting a cell with a microRNA or miRNA mimic that enhances reprogramming of an induced pluripotent stem (iPS) cell in combination with an agent that enhances reprogramming of an induced pluripotent stem (iPS) cell. In one aspect, the method further includes administering the cell a pluripotency transcription factor.

[0015] In one aspect, the agent is a small molecule, a peptide, a nucleic acid, a

pluripotency transcription factor or a combination thereof. In another aspect, the nucleic acid is an siRNA. In another aspect, the agent is an miRNA inhibitor. In another aspect, the miRNA inhibitor is an inhibitor of miRNA selected from the group consisting of miR-223, miR-543, miR-542-5p, miR-665, miR-142-5p, miR-450b-5p, miR-184, miR-370, miR-431, miR-376a, miR-495, and a combination thereof. In another aspect, the miRNA inhibitor is a peptide, small molecule, or nucleic acid molecule. In an additional aspect, the nucleic acid molecule is a siRNA. In another aspect, the agent is a NSAID or kinase inhibitor. In another aspect, the agent is selected from the group consisting of nabumetone, 4-hydroxytamoxifen (OHTM), corynanthine, moclobemide, nickel sulfate hexahydrate (NiS0₄), lectin, and a combination thereof. In another aspect, the miRNA or miRNA mimic is selected from the group consisting of miR-135b, miR-302b, miR-124, miR-547, miR-701, miR-302d, miR-92a, miR-20a, miR-93, miR-491, miR-367, or a combination thereof.

[0016] In one aspect, the small molecule is selected from the group consisting of nabumetone, 4-hydroxytamoxifen (OHTM), corynanthine, moclobemide, nickel sulfate hexahydrate (NiS0₄), lectin, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin, inhibitor of TGF-β, Acitretin, Retinoic acid p-hydroxyanilide, Diacerein, Phorbol 12-myristate 13 -acetate, Progesterone, Tolazamide, 15-deoxy-A¹²' ^-prostaglandin J_2> (-)-Norepinephrine, β-estradiol, and a combination thereof. In another as ect, the small molecule i selected from the group

consisting of £-616452 E-616451 Ef-275

[0017] In another embodiment, the present invention provides a method of generating an induced pluripotent stem (iPS) cell. The method includes contacting a cell with an inhibitor of miRNA selected from the group consisting of miR-223, miR-543, miR-542-5p, miR-665, miR-142-5p, miR-450b-5p, miR-184, miR-370, miR-431, miR-376a, miR-495, or a combination thereof. In one aspect, the method further includes administering the cell a pluripotency transcription factor. In another aspect, the inhibitor of miRNA is a peptide, small molecule, or nucleic acid molecule. In an additional aspect, the nucleic acid molecule is a siRNA.

[0018] In another embodiment, the present invention provides a method of generating an induced pluripotent stem (iPS) cell. The mthod includes contacting a cell with a miRNA or miRNA mimic selected from the group consisting of miR-135b, miR-302b, miR-124, miR- 547, miR-701, miR-302d, miR-92a, miR-20a, miR-93, miR-491, and miR-367 in

combination with a miRNA inhibitor selected from miR-223, miR-543, miR-542-5p, miR- 665, miR-142-5p, miR-450b-5p, miR-184, miR-370, miR-431, miR-376a, and miR-495. In one aspect, the method further includes contacting the cell with a small molecule that enhances reprogramming of an induced pluripotent stem (iPS) cell. In another aspect, the method further includes administering the cell a pluripotency transcription factor. In another aspect, the inhibitor of miRNA is a peptide, small molecule, or nucleic acid molecule. In an additional aspect, the nucleic acid molecule is a siRNA.

[0019] In another embodiment, the present invention provides a method of generating an induced pluripotent stem (iPS) cell. The method includes contacting a cell with an agent regulates expression of activity of Wispl, Tgfbr2, Igfbp5, or a combination thereof. In one aspect, the method further includes administering the cell a pluripotency transcription factor.. In another aspect, the agent is a peptide, small molecule, or nucleic acid molecule. In an additional aspect, the nucleic acid molecule is a siRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows that potential iPS cells are enriched in the thyl - population during early reprogramming stages. Figure 1 A shows exemplary analysis of iPSC potential. MEFs are infected with 4F (the four factors OSKM) virus and incubated 5 days before sorting. Day 5 MEFs are sorted based on surface antigen thyl expression. Both thyl - and thyl+ cells are harvested for microRNA expression profile analysis. Figure IB shows representative gating for day 5 4F-infected MEF sorting. PE-conjugated thyl antibody was used to detect dryland thyl+ populations. Figure 1C shows that iPS cells are enriched in thyl - population from 4F-infected MEFs at day 5. Equal numbers of cells (10,000 cells) sorted from 4F infected MEFs are replated into feeder plates and cultured until day 14 for GFP+ colony counting. Figure ID shows that AP staining confirms that iPS cells generated in (c) are enriched in the thyl- population. Cells can be harvested for AP staining at day 14 post infection.

[0021] Figure 2 shows identification of both induced and repressed microRNAs during iPSC induction. Figure 2A shows that induced or repressed microRNAs are identified in thyl - cells. Both thyl - and thyl+ cells are harvested for microR A expression profiling. Data from the thyl - population is compared with the original MEFs, and microRNAs showing a 2-fold change and p < 0.05 are identified using a volcano map. Hits are labeled as red dots. Figure 2B shows an exemplary set of significantly induced microRNAs.

microRNAs induced at least 2-fold are shown based on fold induction. Figure 2C shows an exemplary set of significantly repressed microRNAs. MicroRNAs repressed by at least 2- fold are shown.

[0022] Figure 3 shows that miR-135b enhances reprogramming of MEFs to iPSCs. Figure 3 A shows that miR-135b enhances Oct4-GFP+ colony formation. The indicated microRNA mimics are transfected twice into MEFs on day 0 and day 5 post 4F transduction at a final concentration of 50 nM. GFP+ colonies are counted at day 11-12. Data represents an experiment with triplicate wells. Let-7a is used as a control. Figure 3B shows that miR-135b increases the overall percentage of Oct4-GFP+ cells. Cells from different treatments are harvested at day 14 post-infection with 4F and paraformaldehyde-fixed prior to FACS analysis to determine the percentage of GFP+ cells. Data represents an experiment with triplicate wells. Figure 3C shows that blocking of miR-135b can compromise

reprogramming. MicroRNA inhibitors are transfected into MEFs using the same schedule as miR mimics. GFP+ colonies are counted at day 11-12 post infection. MiR-135b iPSCs can reach a fully reprogrammed state. MiR- 135b transfected iPS cells can be fixed with paraformaldehyde and stained for alkaline phosphatase, Nanog and SSEA1 expression.

Endogenous Oct4 expression can be monitored by GFP expression.

[0023] Figure 4 shows genome- wide identification of potential genes regulated by miR- 135b. Figure 4 A shows Volcano maps from miR- 135b transfected MEFs. MEFs are transfected with siControl and miR- 135b for two days and analyzed by mRNA expression array. Hits are gated for at least 2-fold expression change and p < 0.05. Figure 4B shows that miR-135b-repressed genes are enriched with genes suppressed in ES/iPS cells. MiR- 135b regulated genes are separated into two groups (induced or repressed) and then compared with existing iPS/ES/MEF expression profiles. "Correlated genes" indicates that genes which are changed upon miR-135b transfection show similar changes from MEFs to iPS/mES cells. "Uncorrelated genes" indicates a group of genes which are changed upon miR-135b transfection but had a different (reversed) expression pattern change from MEFs to iPS/mES cells. Figure 4C shows fold change of correlated miR-135b-repressed genes. Signals from miR-135b transfected samples for various genes are normalized to those of siControl transfected samples. Almost all genes show a 2 to 3-fold change.

[0024] Figure 5 shows that miR-135b represses expression of Tgfbr2, Wispl and Igfbp5. Figure 5 A shows raw data of several miR-135b repressed genes. Genes with high relative expression level in MEFs are chosen as potential candidates for further validation. Figure 5B shows that TGFBR2 protein expression is efficiently repressed by miR-135b. Total cell lysates of miR-135b transfected MEFs are harvested for western blotting. Let7a and miR-93 transfected samples serve as negative and positive controls, respectively. Figure 5C shows that repressed genes are confirmed by RT-qPCR of miR-135b-transfected MEFs. MEFs are transfected with miR-135b and harvested at 48 hours. RT-qPCR is used to quantify the relative expression of target genes. GAPDH serves as the normalization standard. Figure 5D shows that IGFBP5 is repressed by miR-135b. A miR-93-transfected sample is included as negative control.

[0025] Figure 6 shows that Tgfbr2, Wispl or Igfbp5 knockdown can enhance

reprogramming. Figure 6A shows that potential target genes are efficiently knocked down by siRNAs. Smartpool siRNAs are used to transfect MEFs at a final concentration of 50 nM. Total RNAs are harvested at day 2 for RT-qPCR to evaluate knockdown efficiency of each siRNA. Figure 6B shows that knockdown of Tgfbr2 or Igfbp5 enhances Oct4-GFP+ colony formation, while knockdown of Eif4ebpl and Cxcll4 does not have such effects. MEFs are transfected with siRNAs at day 0 and day 5 together with 4F infection. GFP+ colonies are counted at day 11-12 post-infection. Error bars represent three independent experiments with triplicate wells. The p value is calculated using Student's t-test. Figure 6C shows that knockdown of Wispl shows stage-specific effects. Knockdown of Wispl at day 0 post 4F transduction dramatically decreases the reprogramming efficiency by -70% percent while the same transfection at day 5 enhances reprogramming by -3 fold. [0026] Figure 7 shows a model for microRNA functions during somatic cell reprogramming process. A set of microRNAs are either induced or suppressed by four pluripotency transcription factors (OSKM) at early stage of reprogramming. Repression of MEF-specific microRNAs increases reprogramming efficiency. Induction of ES-specific microRNAs such as miR-93 and 135b can also enhance reprogramming by directly targeting and downregulating expression of barrier genes such as Tgfbr2, Igfbp5 and Wispl.

[0027] Figure 8 shows that miR-135b can enhance iPS induction in suboptimal conditions. MEFs are infected with low-titer 4F virus and transfected with siControl, Let7a, miR-93 and miR-135b. Let7a is used as a negative control under such condition. GFP+ colonies are counted at day 11-12 post-infection. Possibly due to low transgene expression, no GFP+ colonies are identified from cells transfected with siControl. Let7a-transfected cells only have ~0.5 colony per well. However, the presence of miR-93 or 135b promotes iPSC induction.

[0028] Figure 9 shows that miR-135b can enhance the overall percentage of Oct4-GFP+ cells during reprogramming. MEFs are transfected with indicated microRNA mimics 3 hours before infection with 4 transcription factors, and cells are trypsinized at day 14 for FACS analysis. Single cells are collected by filtering through a cell strainer. Wild-type MEFs serve as negative controls.

[0029] Figure 10 shows that miR-135b iPSCs show expression profiles similar to mES cells. Total RNAs from miR-135b iPSCs are used for mRNA expression profile analysis and compared with original MEFs and with mES cells. All the three tested miR-135b-iPSC clones (clone 1, 3 and Nl) show similar expression pattern as mES cells, which are quite different from expression profile of original starting MEFs.

[0030] Figure 11 shows that Tgfbr2, Wispl and Igfbp5 are directly regulated by miR- 135b. Results from dual luciferase assay support direct regulation by miR-135b. The full length Tgfbr2 3'UTR, a Wispl fragment, and the Igfbp5 3'UTR are cloned into pGL3 luciferase reporter and transfected into Hela cells together with pRL-TK. Relative luciferase activity is calculated by the GL/RL signal and normalized to siControl-transfected cells, p values are calculated using Student's t-test from at least two independent experiments with duplicate wells. List of predicted miR-135b target sites identified by both miRanda software and Targetscan in Tgfbr2, Wispl and Igfbp5 3'UTRs is also shown in Table 2.

[0031] Figure 12 shows exemplary miR-135b target site analysis. Genes whose expression is significantly repressed upon miR-135b transfection are analyzed with miRanda and TargetScan (see Rehmsmeier, M. et ah, 2004, Fast and effective prediction of microRNA/target duplexes. RNA 10, 1507-1517) to identify potential miR-135b target sites in their 3'UTR regions. Sites with good seed match and significant predicted energy are listed.

[0032] Figure 13 A displays the structures of six small molecules used in iPS cell reprogramming. Figure 13B is a plot of GFP+ colony number showing effects on reprogramming efficiency. Error bars represent standard deviations of three independent experiments. * p value < 0.05; ** p value < 0.005. Figure 13C is a plot of GFP+ colony number at day 12 ~ 14. Error bars represent standard deviations of six independent experiments. * p value < 0.05; ** p value < 0.005; *** p value < 0.0005. siNT serves as control.

[0033] Figure 14A shows eleven barrier candiadates and three inhibitors, B6, B8, and B10. Figures 14B and 14C show that compounds Bl-Bl 1 are tested for their effects on reprogramming efficiency.

[0034] Figure 15 shows that other members of the TGFP signaling pathway are predicted to be regulated by miR-135b. Other members of TGFP signaling pathway are analyzed by miRanda or TargetScan to identify potential direct miR-135b target sites in their 3'UTR regions. Potential direct targets by miR-135b and miR-93 family microRNAs are marked as shown in the figure.

[0035] Figure 16 shows the inhibition of MEF-enriched microRNAs, miR-21 and miR- 29a, enhances reprogramming efficiency. (A) miR-29a, miR-21, and let7a are highly expressed in MEFs. Total RNAs were isolated from Oct4-EGFP MEFs and mouse ES cells and resolved by gel electrophoresis. Specific radioactive-labeled probes against the indicated miRNAs were used to detect expression. U6 snRNA served as a loading control. (B) miRNA inhibition enhances reprogramming efficiency. Oct4-EGFP MEFs were transduced with OSKM. GFP-positive colonies were identified and counted by fluorescence microscopy at day 14 after transduction. GFP+ colony number was normalized to the number of anti-miR nontargeting control treatment and is reported as fold-change. Error bars, SD of three independent experiments. *P-value<0.05; **P-value <0.005.

[0036] Figure 17 shows that c-Myc is the primary repressor of MEF-enriched miRNAs during reprogramming. (A) Northern analysis of selected miRNAs at day 5 after

reprogramming. Oct4-EGFP MEFs were transduced with a single factor or various combinations of reprogramming factors, as indicated. IF indicates one factor; 2F, two factors; 3F, three factors. OSKM indicates Oct3/4, Sox2, Klf4, and c-Myc. U6 is used as a loading control RNA. Total RNA from embryonic stem (ES) cells serve as negative control to MEF and transduced cells. Various probes were used to detect specific miRNAs as indicated on the right side. miR-291 blotting is a positive control for ES RNA. (B)

Quantitative representation of miRNA expression in the presence of various reprogramming factors. Signal intensity was normalized to intensity of U6 snRNA. The expression ratio is calculated as the percentage of expression of each miRNA relative to expression in MEFs, which was arbitrarily set to 100%. Various miRNAs were quantified (from panel A) and indicated on the right side. (C) Real-time RT-PCR analysis of selected miRNAs in Oct4- EGFP MEFs at various time points following OSK- or OSKM-reprogramming. RNA was isolated at the indicated day (D) after transduction for real time RT-PCR analysis. Signals were normalized to U6 and are shown as a percentage of miRNAs expressed in MEFs, which was arbitrarily set to 100. Error bars, SD of two independent experiments.

[0037] Figure 18 shows that Mouse iPS cells derived with mlR-21 and miR-29a inhibitors are pluripotent. (A) Staining with ES cell markers of OSKM/anti miR-29a or miR-21 iPS cells. GFP+ colonies derived following OSKM and various miR inhibitor treatments were picked for further analysis. Representative colonies expressing the embryonic stem cell markers Nanog and SSEA1 are shown. Endogenous Oct3/4 was also activated, as indicated by the EGFP expression. Strong alkaline phosphatase (AP) activity is shown as one of the ES markers. Anti-miR NT (nontargeting) serves as miR inhibitor control. (B) In vitro differentiation of OSKM/anti miR- 29a or miR-21 iPS cells. Embryoid bodies were formed in vitro and cultured for 2 wk. Cells were fixed and stained with anti-a fetoprotein (for mesoderm) and anti-b-tubulin III (for ectoderm). Nuclei are shown as counter stain by Hoescht staining. (C) Teratoma formation analysis of OSKM/anti miR-29a or miR-21 iPS cells. We injected l.X 3 10⁶ iPSCs subcutaneously into athymic nude female mice. Tumor masses were collected at 3 wk after injection and fixed for histopathologic analysis. Various tissues derived from three germ layers were identified, including gut-like epithelium and pancreatic islet-like structure (endoderm); adipose tissue, cartilage, and muscle (mesoderm); and neural tissue and epidermis (ectoderm). (D) Chimera analysis of OSKM/anti miR-29a and OSK/anti miR-21 iPS cells. Eight to 14 iPS cells were injected into E3.5 mouse blastocysts. iPS cell contribution to each chimera was estimated by assessing black coat color and is shown as a percentage.

[0038] Figure 19 shows inhibition of miR-21 or miR-29a enhances iPS cell

reprograrnming by decreasing p53 protein levels and up-regulating p85a and CDC42 pathways. (A) Western analysis of expression of p53, CDC42, and p85a following inhibition of various miRNAs. We transfected 1 X 10⁵ Oct4-EGFP MEFs with the indicated miRNA inhibitors. Cells were harvested and analyzed 5 d later. (B) Quantitative representation of protein expression in the presence of indicated miR inhibitors. Signal intensity was normalized to GAPDH intensity and is shown as a percentage relative to expression in control (NT) cells, which was set arbitrarily to 100. Error bars, SD of at least three independent experiments. *P-value <0.05. (C) Immunoblot analysis of p53, CDC42, and p85a expression following inhibition of various miRNAs and OSKM transduction. We transfected 1 X 10⁵ Oct4-EGFP MEFs with the indicated miRNA inhibitors. Cells were harvested 5 d later and analyzed by immunoblot. Signal intensity was normalized as described in B. Error bars, SD of at least three independent experiments. *P-value <0.05. (D) Depleting miR-29a or p53 enhances reprograrnming efficiency. We transfected 4 3 104 Oct4- EGFP MEFs with the indicated siRNAs and miRNA inhibitors, as well as OSKM reprograrnming factors. GFP -positive cells were counted at day 12 after transduction. Error bars, SD of at least three independent experiments. *P-value <0.05.

[0039] Figure 20 shows that depleting miR-21 and miR-29a promotes reprograrnming efficiency by downregulating the ERKl/2 pathway. (A) Western analysis of phosphorylated and total ERKl/2 following inhibition of various miRNAs in MEFs. We transfected 1 3 105 Oct4-EGFP MEFs with the indicated miRNA inhibitors, harvested 5 d later, and

immunoblotted. Signal intensity normalized to actin and shown as percentage relative to expression of anti-miR NT control. Error bars, SD of three independent experiments. *P- value <0.05; **P-value <0.005. (B) Western blot analysis of Spryl expression ratio shows that depleting miR-21 and miR-29a increases Spryl protein levels. MEFs were transfected with various miRNA inhibitors as indicated. Cells were harvested at day 5 after transfection for Western blot analysis. Signal intensity normalized to actin and shown as described in A. Error bars, SD of three independent experiments. *P-value <0.05; **P-value <0.005. (C) Fold-change in reprogramming efficiency following ERK1/2 or GSK3P knock-down. We transfected 4 X 10⁴ Oct4-EGFP MEFs with the indicated siRNAs, as well as OSKM. GFP- positive cells were counted 2 wk later. Transfection with siNT serves as control for the reprogramming efficiency. Error bars, SD of three independent experiments. *P-value <0.05; **P-value <0.005. (D) Western analysis of phosphorylated and total GSK3 β following inhibition of various miRNAs in MEFs. We transfected 1 X 10⁵ Oct4-EGFP MEFs with the indicated miRNA inhibitors, harvested 5 d later, and analyzed by immunoblot.

Signal intensity normalized as described in A. Error bars, SD of three independent experiments. (E) Schematic representation showing that c-Myc enhances reprogramming by down-regulating the MEF-enriched miRNAs, miR-21 and miR- 29a. The p53 and ERK1/2 pathways function as barriers to reprogramming, and miR-21 and miR-29a indirectly activate those pathways through down-regulating CDC42, p85a, and Spryl. The cross-talk between miR-21/p53 and miR-29a/ERKl/2 pathways is also shown. c-Myc represses expression of these miRNAs and in turn compromises induction of ERKl/2 and p53. The dotted lines indicate p53 and ERKl/2 effects on iPS generation.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention is based on identification of key microRNAs (miRs) during the early stage of reprogramming from somatic cells into induced pluripotent stem cells. These key miRs can either induce or repress the reprogramming process. The present invention provides that miR-223 and/or miR-495 can inhibit the reprogramming process, but miR-93 and/or miR-135b can enhance the reprogramming process. The present invention provides methods and compositions for generating an induced pluripotent stem (iPS) cell and treating a subject using iPS generated with methods described.

[0041] The present invention is based on the discovery of key regulatory mechanisms involved in iPSC induction. A key aspect being the discovery of a link between cellular microRNAs to the induction of iPSCs. [0042] Somatic cells can be reprogrammed to reach an ES-like state by overexpression of defined factors. Currently, the reprogramming process suffers from extremely low efficiency, requiring further understanding of underlying mechanisms in order to develop new reprogramming methods and understand the transitions to a pluripotent state.

MicroRNAs (miRs) are small non-coding RNAs that primarily regulate target gene expression post-transcriptionally. The present invention provides systematic miRs analysis identifying key miRs either induced or repressed during the early stage of reprogramming. The present invention also provides that MEF-specific miRs such as miR-223 and 495 inhibit the reprogramming process, while miR-135b, like miR-93, enhances it. MiR-135b is the most highly induced microRNA at the early stage and enhanced both Oct4-GFP+ colony formation and the overall percentage of GFP+ cells. Genome-wide mRNA microarray and bioinformatics analyses identifies a set of genes potentially targeted by miR-135b, and they are enriched with MEF-specific genes. Among them, Wispl, Tgfbr2 and Igfbp5 are further confirmed to be likely direct targets of miR-135b, and siRNA-mediated knockdown of these genes enhanced reprogramming. Moreover, Wispl shows a dual role, differentially enhancing or inhibiting reprogramming in a stage-dependent manner. The present invention provides that microRNAs play critical roles in the early stages of reprogramming process and analyzing miR targets can identify several new barriers genes.

[0043] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0044] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.

[0046] As discussed herein, the discovery that microRNAs are involved in reprogramming process and iPSC induction efficiency leads to the ability of one to greatly enhance iPSC induction efficiency by manipulating the level of these microRNAs in the cells. Accordingly, the present invention provides a method of generating an iPS cell having improved induction efficiency as compared to know methods. The method includes contacting a somatic cell with a nuclear reprogramming factor, and an agent that alters microRNA levels or activity within the cell, with the proviso that the agent is not a nuclear reprogramming factor, thereby generating an iPS cell.

[0047] Given the regulatory involvement of RNA in generation of iPSC, it is

contemplated that induction may occur using agents that regulate RNA levels other than nuclear reprogramming factors. Accordingly, the present invention provides a method of generating an induced pluripotent stem (iPS) cell by contacting a somatic cell with an agent that alters RNA levels or activity within the cell, wherein the agent induces pluripotency in the somatic cell, with the proviso that the agent is not a nuclear reprogramming factor, thereby generating an iPS cell. In various embodiments, the RNA is non-coding RNA (ncRNA), such microRNA.

[0048] In various embodiments, one or more nuclear reprogramming factors can be used to induce reprogramming of a differentiated cell without using eggs, embryos, or ES cells. Efficiency of the induction process is enhanced by utilizing an agent that alters microRNA levels or activity within the cell during the induction process. The method may be used to conveniently and highly reproducibly establish an induced pluripotent stem cell having pluripotency and growth ability similar to those of ES cells. For example, the nuclear reprogramming factor may be introduced into a cell by transducing the cell with a

recombinant vector comprising a gene encoding the nuclear reprogramming factor along with a recombinant vector comprising a polynucleotide encoding an RNA molecule, such as a microRNA. Accordingly, the cell can express the nuclear reprogramming factor expressed as a product of a gene contained in the recombinant vector, as well as expressing the microRNA expressed as a product of a polynucleotide contained in the recombinant vector thereby inducing reprogramming of a differentiated cell at an increased efficiency rate as compare to use of the nuclear reprogramming factor alone.

[0049] As used herein, pluripotent cells include cells that have the potential to divide in vitro for an extended period of time (greater than one year) and have the unique ability to differentiate into cells derived from all three embryonic germ layers, including the endoderm, mesoderm and ectoderm.

[0050] Somatic cells for use with the present invention may be primary cells or immortalized cells. Such cells may be primary cells (non-immortalized cells), such as those freshly isolated from an animal, or may be derived from a cell line (immortalized cells). In an exemplary aspect, the somatic cells are mammalian cells, such as, for example, human cells or mouse cells. They may be obtained by well-known methods, from different organs, such as, but not limited to skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, or generally from any organ or tissue containing living somatic cells. Mammalian somatic cells useful in the present invention include, by way of example, adult stem cells, Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, other known muscle cells, and generally any live somatic cells. In particular embodiments, fibroblasts are used. The term somatic cell, as used herein, is also intended to include adult stem cells. An adult stem cell is a cell that is capable of giving rise to all cell types of a particular tissue. Exemplary adult stem cells include hematopoietic stem cells, neural stem cells, and mesenchymal stem cells.

[0051] As used herein, reprogramming is intended to refer to a process that alters or reverses the differentiation status of a somatic cell that is either partially or terminally differentiated. Reprogramming of a somatic cell may be a partial or complete reversion of the differentiation status of the somatic cell. In an exemplary aspect, reprogramming is complete wherein a somatic cell is reprogrammed into an induced pluripotent stem cell.

However, reprogramming may be partial, such as reversion into any less differentiated state. For example, reverting a terminally differentiated cell into a cell of a less differentiated state, such as a multipotent cell. [0052] In various aspects of the present invention, nuclear reprogramming factors or pluripotency transcription factors are genes that induce pluripotency and utilized to reprogram differentiated or semi-differentiated cells to a phenotype that is more primitive than that of the initial cell, such as the phenotype of a pluripotent stem cell. Such genes are utilized with agents that alter microRNA levels or activities in the cell and/or inhibit p21 expression or activity to increase induction efficiency. Such genes and agents are capable of generating a pluripotent stem cell from a somatic cell upon expression of one or more such genes having been integrated into the genome of the somatic cell. As used herein, a gene that induces pluripotency is intended to refer to a gene that is associated with pluripotency and capable of generating a less differentiated cell, such as a pluripotent stem cell from a somatic cell upon integration and expression of the gene. The expression of a pluripotency gene is typically restricted to pluripotent stem cells, and is crucial for the functional identity of pluripotent stem cells.

[0053] An agent useful in any of the methods of the invention can be any type of molecule, for example, a polynucleotide, a peptide, a peptidomimetic, peptoids such as vinylogous peptoids, chemical compounds, such as organic molecules or small organic molecules, or the like. Accordingly, in one aspect, an agent for use in the method of the present invention is a polynucleotide, such as an antisense oligonucleotide or RNA molecule. In various aspects, the agent may be a polynucleotide, such as an antisense oligonucleotide or RNA molecule, such as microRNA, dsRNA,, siRNA, stRNA, and shRNA.

[0054] MicroRNAs (miRNA) are single-stranded RNA molecules, which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but mlRNAs are not translated into protein; instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are either fully or partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression. MicroRNAs can be encoded by independent genes, but also be processed (via the enzyme Dicer) from a variety of different RNA species, including introns, 3' UTRs of mRNAs, long noncoding RNAs, snoRNAs and transposons. As used herein, microRNAs also include "mimic" microRNAs which are intended to mean a microRNA exogenously introduced into a cell that have the same or substantially the same function as their endogenous counterpart. Thus, while one of skill in the art would understand that an agent may be an exogenously introduced RNA, an agent also includes a compound or the like that increase or decrease expression of microRNA in the cell.

[0055] The terms "small interfering RNA" and "siRNA" also are used herein to refer to short interfering RNA or silencing RNA, which are a class of short double-stranded RNA molecules that play a variety of biological roles. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi- related pathways (e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome).

[0056] Polynucleotides of the present invention, such as antisense oligonucleotides and RNA molecules may be of any suitable length. For example, one of skill in the art would understand what lengths are suitable for antisense oligonucleotides or RNA molecule to be used to regulate gene expression. Such molecules are typically from about 5 to 100, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, or 10 to 20 nucleotides in length. For example the molecule may be about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45 or 50 nucleotides in length. Such polynucleotides may include from at least about 15 to more than about 120 nucleotides, including at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides or greater than 120 nucleotides.

[0057] The term "polynucleotide" or "nucleotide sequence" or "nucleic acid molecule" is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. As such, the terms include RNA and DNA, which can be a gene or a portion thereof, a cDNA, a synthetic

polydeoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. Furthermore, the terms as used herein include naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic polynucleotides, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR). It should be recognized that the different terms are used only for convenience of discussion so as to distinguish, for example, different components of a composition.

[0058] In general, the nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'- deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose. Depending on the use, however, a polynucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs. The covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond. However, depending on the purpose for which the polynucleotide is to be used, the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides.

[0059] A polynucleotide or oligonucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template.

[0060] In various embodiments antisense oligonucleotides or RNA molecules include oligonucleotides containing modifications. A variety of modification are known in the art and contemplated for use in the present invention. For example oligonucleotides containing modified backbones or non-natural internucleoside linkages are contemplated. As used herein, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified

oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0061] In various aspects modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates,

phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,

selenophosphates and borano-phosphates having normal 3 -5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Certain oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0062] In various aspects modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0063] In various aspects, oligonucleotide mimetics, both the sugar and the

internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. In various aspects, oligonucleotides may include phosphorothioate backbones and oligonucleosides with heteroatom backbones. Modified oligonucleotides may also contain one or more substituted sugar moieties. In some embodiments oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N- alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C_\ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are 0[(CH₂)_nO]_mCH₃, 0(CH₂)_nOCH₃, 0(CH₂)_nNH₂, 0(CH₂)_nCH₃, 0(CH₂)_nONH₂ and 0(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: d to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, S0₂CH₃, ON0₂, N0₂, N3, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Another modification includes 2'-methoxyethoxy(2'OCH CH₂OCH₃, also known as 2'-0-(2-methoxyethyl) or 2'- MOE).

[0064] In related aspects, the present invention includes use of Locked Nucleic Acids (LNAs) to generate antisense nucleic acids having enhanced affinity and specificity for the target polynucleotide. LNAs are nucleic acid in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (-CH₂-)_n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.

[0065] Other modifications include 2'-methoxy(2'-0-CH₃), 2'-aminopropoxy(2'- OCH₂CH₂CH₂NH₂), 2'-allyl (2'-CH-CH-CH₂), 2'-0-allyl (2'-0-CH₂-CH-CH₂), 2'-fluoro (2*- F), 2'-amino, 2'-thio, 2'-Omethyl, 2'-methoxymethyl, 2'-propyl, and the like. The 2'- modification may be in the arabino (up) position or ribo (down) position. A preferred 2'- arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'- 5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0066] Oligonucleotides may also include nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraeil and cytosine, 5- propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 8-halo, 8-arnino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (lH-pyrimido[5,4- b][l,4]benzoxazi-n-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4- b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9- (2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3- d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases are known in the art. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds described herein. These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.

[0067] Another modification of the antisense oligonucleotides described herein involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The antisense oligonucleotides can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids,

phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,

fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific

hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., dihexadecyl-rac- glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylaminocarbonyloxycholesterol moiety.

[0068] Several genes have been found to be associated with pluripotency and suitable for use with the present invention as reprogramming factors. Such genes are known in the art and include, by way of example, SOX family genes (SOXl, SOX2, SOX3, SOX15, SOX18), KLF family genes (KLF1, KLF2, KLF4, KLF5), MYC family genes (C-MYC, L-MYC, N- MYC), SALL4, OCT4, NANOG, LIN28, STELLA, NOBOX or a STAT family gene. STAT family members may include for example STAT1, STAT2, STAT3, STAT4, STAT5

(STAT5A and STAT5B), and STAT6. While in some instances, use of only one gene to induce pluripotency may be possible, in general, expression of more than one gene is required to induce pluripotency. For example, two, three, four or more genes may be simultaneously integrated into the somatic cell genome as a polycistronic construct to allow simultaneous expression of such genes. In an exemplary aspect, four genes are utilized to induce pluripotency including OCT4, SOX2, KLF4 and C-MYC. Additional genes known as reprogramming factors suitable for use with the present invention are disclosed in U.S. Patent Application No. 10/997,146 and U.S. Patent Application No. 12/289,873, incorporated herein by reference.

[0069] All of these genes commonly exist in mammals, including human, and thus homologues from any mammals may be used in the present invention, such as genes derived from mammals including, but not limited to mouse, rat, bovine, ovine, horse, and ape.

Further, in addition to wild-type gene products, mutant gene products including substitution, insertion, and/or deletion of several (e.g., 1 to 10, 1 to 6, 1 to 4, 1 to 3, and 1 or 2) amino acids and having similar function to that of the wild-type gene products can also be used. Furthermore, the combinations of factors are not limited to the use of wild-type genes or gene products. For example, Myc chimeras or other Myc variants can be used instead of wild-type Myc.

[0070] The present invention is not limited to any particular combination of nuclear reprogramming factors. As discussed herein a nuclear reprogramming factor may comprise one or more gene products. The nuclear reprogramming factor may also comprise a combination of gene products as discussed herein. Each nuclear reprogramming factor may be used alone or in combination with other nuclear reprogramming factors as disclosed herein. Further, nuclear reprogramming factors of the present invention can be identified by screening methods, for example, as discussed in U.S. Patent Application No. 10/997,146, incorporated herein by reference. Additionally, the nuclear reprogramming factor of the present invention may contain one or more factors relating to differentiation, development, proliferation or the like and factors having other physiological activities, as well as other gene products which can function as a nuclear reprogramming factor.

[0071] The nuclear reprogramming factor may comprise a protein or peptide. The protein may be produced from a gene as discussed herein, or alternatively, in the form of a fusion gene product of the protein with another protein, peptide or the like. The protein or peptide may be a fluorescent protein and/or a fusion protein. For example, a fusion protein with green fluorescence protein (GFP) or a fusion gene product with a peptide such as a histidine tag can also be used. Further, by preparing and using a fusion protein with the TAT peptide derived from the virus HIV, intracellular uptake of the nuclear reprogramming factor through cell membranes can be promoted, thereby enabling induction of reprogramming only by adding the fusion protein to a medium thus avoiding complicated operations such as gene transduction. Since preparation methods of such fusion gene products are well known to those skilled in the art, skilled artisans can easily design and prepare an appropriate fusion gene product depending on the purpose.

[0072] The nucleic acid construct of the present invention, such as recombinant vectors may be introduced into a cell using a variety of well known techniques, such as non- viral based transfection of the cell. In an exemplary aspect the construct is incorporated into a vector and introduced into the cell to allow expression of the construct. Introduction into the cell may be performed by any viral or non- viral based transfection known in the art, such as, but not limited to electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include proprietary transfection reagents such as

Lipofectamine™, Dojindo Hilymax™, Fugene™, jefPEI™, Effectene™ and DreamFect™

[0073] In various aspects, reprogramming induction efficiency may be increased by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or ever 500 percent as compared with convention methods. For example, induction efficiency may be as high as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 50 percent (e.g., percent of induced cells as compared with total number of starting somatic cells).

[0074] In various aspects, the somatic cell is contacted with the reprogramming factor about 1, 2, 3, 4, 5, 7, 8, 9, 10, 1 1, 12, 13, 14 or more days before the cell is contacted with any other agent or inhibitor. In an exemplary aspect, the somatic cell is contacted with the reprogramming factor about 1 , 2, 3, 4 or 5 days before the cell is contacted with any other agent or inhibitor.

[0075] Further analysis may be performed to assess the pluripotency characteristics of a reprogrammed cell. The cells may be analyzed for different growth characteristics and embryonic stem cell like morphology. For example, cells may be differentiated in vitro by adding certain growth factors known to drive differentiation into specific cell types.

Reprogrammed cells capable of forming only a few cell types of the body are multipotent, while reprogrammed cells capable of forming any cell type of the body are pluripotent.

[0076] Expression profiling of reprogrammed somatic cells to assess their pluripotency characteristics may also be conducted. Expression of individual genes associated with pluripotency may also be examined. Additionally, expression of embryonic stem cell surface markers may be analyzed. Detection and analysis of a variety of genes known in the art to be associated with pluripotent stem cells may include analysis of genes such as, but not limited to OCT4, NANOG, SALL4, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, or a combination thereof. iPS cells may express any number of pluripotent cell markers, including: alkaline phosphatase (AP); ABCG2; stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; β-tubulin III; a-smooth muscle actin (a-SMA); fibroblast growth factor 4 (FGF4), Cripto, Daxl; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Natl); ES cell associated transcript 1 (ECAT1); ESG 1 /DPP A5/EC AT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthll7; Sall4; undifferentiated embryonic cell transcription factor (Utfl); Rexl; p53; G3PDH; telomerase, including TERT; silent X chromosome genes;

Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbxl5); Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1;

developmental pluripotency-associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tell); DPPA3/Stella; DPPA4; as well as other general markers for Pluripotency, for example any genes used during induction to reprogram the cell. IPS cells can also be characterized by the down-regulation of markers characteristic of the differentiated cell from which the iPS cell is induced.

[0077] The invention further provides iPS cells produced using the methods described herein, as well as populations of such cells. The reprogrammed cells of the present invention, capable of differentiation into a variety of cell types, have a variety of applications and therapeutic uses. The basic properties of stem cells, the capability to infinitely self-renew and the ability to differentiate into every cell type in the body make them ideal for therapeutic uses. [0078] Accordingly, in one aspect the present invention further provides a method of treatment or prevention of a disorder and/or condition in a subject using induced pluripotent stem cells generated using the methods described herein. The method includes obtaining a somatic cell from a subject and reprogramming the somatic cell into an induced pluripotent stem (iPS) cell using the methods described herein. The cell is then cultured under suitable conditions to differentiate the cell into a desired cell type suitable for treating the condition. The differentiated cell may then be introducing into the subject to treat or prevent the condition.

[0079] In one aspect, the iPS cells produced using the methods described herein, as well as populations of such cells may be differentiated in vitro by treating or contacting the cells with agents that alter microRNA levels or activities in the cells. Since microRNAs have been identified as key regulators in iPSC induction, it is expected that manipulation of individual microRNAs or populations of microRNAs may be used in directing differentiation of such iPSCs. Such treatment may be used in combination with growth factors or other agents and stimuli commonly known in the art to drive differentiation into specific cell types.

[0080] One advantage of the present invention is that it provides an essentially limitless supply of isogenic or synegenic human cells suitable for transplantation. The iPS cells are tailored specifically to the patient, avoiding immune rejection. Therefore, it will obviate the significant problem associated with current transplantation methods, such as, rejection of the transplanted tissue which may occur because of host versus graft or graft versus host rejection. Several kinds of iPS cells or fully differentiated somatic cells prepared from iPS cells from somatic cells derived from healthy humans can be stored in an iPS cell bank as a library of cells, and one kind or more kinds of the iPS cells in the library can be used for preparation of somatic cells, tissues, or organs that are free of rejection by a patient to be subjected to stem cell therapy.

[0081] The iPS cells of the present invention may be differentiated into a number of different cell types to treat a variety of disorders by methods known in the art. For example, iPS cells may be induced to differentiate into hematopoetic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, neuronal cells, and the like. The differentiated cells may then be transplanted back into the patient's body to prevent or treat a condition. Thus, the methods of the present invention may be used to treat a subject having a myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, wound healing, immunodeficiency, aplastic anemia, anemia, Huntington's disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases, multiple sclerosis, spinal cord injuries, genetic disorders, and similar diseases, where an increase or replacement of a particular cell type/ tissue or cellular de-differentiation is desirable.

[0082] In various embodiments, the method increases the number of cells of the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the method increases the biological activity of the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the method increases blood vessel formation in the tissue or organ by at least about 5%, 10%, 25%, 50%, 15% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the cell is administered directly to a subject at a site where an increase in cell number is desired.

[0083] The present invention further provides a method for evaluating a physiological function or toxicity of an agent, compound, a medicament, a poison or the like by using various cells obtained by the methods described herein.

[0084] Oct4-GFP mouse embryonic fibroblasts (MEFs) are derived from mice carrying an IRES-EGFP fusion cassette downstream of the stop codon of pou5fl (Jackson lab,

Stock#008214) at D13.5. These MEFs are cultured in DMEM (Invitrogen, 11995-065) with 10% FBS (Invitrogen) plus glutamine and NEAA. For iPSC induction, only MEFs with passage of 0 to 4 are used.

[0085] The present invention provides a systematic analysis of microRNA expression profiles during the early reprogramming stage and identify a set of microRNAs that are either induced or repressed at that stage. The present invention provides that blocking some of the highly induced microRNAs, such as miR-135b, inhibits reprogramming, while blocking MEF-specific microRNAs, such as miR-495 and miR-223, enhances the process. Among the four factor-induced microRNAs, miR-135b is the most highly induced, and transfection of a miR mimic enhanced both Oct4-GFP+ colony formation and the overall percentage of GFP+ cells. Analysis of a genome-wide mRNA array identifies genes potentially regulated by miR- 135b, and those repressed by miR- 135b are enriched among MEF-specific genes. Among repressed miR- 135b targets, Tgfbr2, the Wnt effector Wispl, and Igfbp5 are confirmed to be likely direct targets and reprogramming barriers. Notably, Wispl knockdown at day 0 of transduction significantly inhibited reprogramming, while the same treatment at day 5 has the opposite effect, indicating that Wispl, and possibly Wnt signaling overall, play dual roles in the process. The present invention provides that miR- 135b is a key regulator of the reprogramming process and identify two previously unreported barrier genes, Wispl and Igfbp5, as its direct targets.

[0086] The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE 1

Cell Culture, Vectors, and Virus Transduction

[0087] Oct4-GFP MEFs are derived from mouse embryos harboring an IRES-EGFP fusion cassette downstream of the stop codon of pou5fl (Jackson lab, Stock#008214) at E13.5. MEFs are cultured in DMEM (Invitrogen, 11995-065) with 10% FBS (Invitrogen) plus glutamine and NEAA. Only MEFs at passage of 0 to 4 are used for iPS induction.

pMXs-Oct4, Sox2, Klf4 and cMyc are purchased from Addgene. To generate retrovirus, PLAT-E cells are seeded in 10 cm plates. Nine μg of each factor is transfected the next day using Lipofectamine (Invitrogen, 18324-012) and PLUS (Invitrogen, 11514-015). Viruses are harvested and combined two days later. For iPS induction, MEFs are seeded in 12-well plates and transduced with "four factor" virus the next day with 4 μg/ml Polybrene. One day later, the medium is changed to fresh MEF medium, and 3 days later it is changed to mES culture medium supplemented with LIF (Millipore, ESG1107). GFP+ colonies are picked at day 14 post-transduction, and expanded clones are cultured in DMEM with 15% FBS

(Hyclone) plus LIF, thioglycerol, glutamine and NEAA. Irradiated CF1 MEFs serve as feeder layers to culture mES and derived iPS clones. [0088] MicroRNAs, siRNAs and MEF transfection: microRNA mimics and inhibitory siRNAs are purchased from Dharmacon. To transfect MEFs, microRNA mimics are diluted in Opti-MEM (Invitrogen, 11058-021) to the desired final concentration. Two μΐ/well of Lipofectamine 2000 (Invitrogen, 11668-019) is added to the mix and incubated 20 minutes at room temperature. For 12-well plate transfections, 80 μΐ of the miR mixture is added to each well with 320 μΐ of Opti-MEM. Three hours later, 0.8 ml of the virus mixture (for iPS) or fresh medium is added to each well, and the medium is changed to fresh MEF medium the next day.

[0089] Western blotting: Total cell lysates are prepared using MPER buffer (PIERCE, 78503) on ice for 20 minutes and cleared by centrifuging at 13,000 rpm for 10 minutes. Equal amounts of lysate are loaded onto 10% SDS-PAGE gels. Proteins are transferred to PVDF membranes (Bio-Rad, 1620177) using the semi-dry system (Bio-Rad) and then blocked with 5 % milk in TBST for at least 1 hour at room temperature or overnight at 4 °C. Antibodies used are: anti-niNanog (R&D, AF2729), anti-h/mSSEAl (R&D, MAB2156), anti-TGFBR2 (Cell signaling, #3713), anti-IGFBP5 (R&D systems, AF578), anti-Actin (Thermo, MS1295P0), anti-AFP (Abeam, ab7751), anti-Beta III tubulin (R&D systems, MAB1368), and anti-alpha actinin (Sigma, A7811).

[0090] mRNA and microRNA RT and quantitative PCR: Total RNAs are extracted using Trizol (Invitrogen), and then one μg total RNA is used for RT using Superscript II

(Invitrogen). Quantitative PCR is performed using Roche LightCycler480 II and the Sybr green mixture from Abgene (Ab-4166). For microRNA quantitative analysis, total RNA is also extracted using the method described above. 1.5-3.0 μg of total RNA is then used for microRNA reverse transcription using QuantiMir kit following the manufacturer's protocol (SBI, RA420A-1). RT products then are used for quantitative PCR using the mature microRNA sequence as a forward primer and the universal primer provided with the kit.

[0091] Immunostaining: Cells are washed twice with PBS and fixed with 4 %

paraformaldehyde at room temperature for 20 minutes. Fixed cells are permeabilized with 0.1% Triton X-100 for 5 minutes. Cells are then blocked in 5 % BSA in PBS containing 0.1% Triton X-100 for 1 hour at room temperature. Primary antibody is diluted in 2.5% BSA PBS containing 0.1% Triton X-100 at 1:100 to 1:400, according to the manufacturer's suggestion. Cells are stained with primary antibody for 1 hour and then washed three times with PBS. Secondary antibody is diluted 1 :400 and cells are stained for 45 minutes at room temperature.

[0092] EB formation and differentiation assay: iPS cells are trypsinized to a single cell suspension, and the hanging drop method is used to generate embryoid bodies. For each drop, 4000 iPS cells in 20 μΐ EB differentiation medium are used. EBs are cultured in hanging drops for 3 days before being reseeded onto gelatin-coated plates. After reseeding, cells are further cultured until day 14, when apparent beating areas can be identified.

[0093] Teratoma formation: To generate teratomas, iPS cells are trypsinized and resuspended at a concentration of 1 x 10⁷ cells/ml. Athymic nude mice are anesthetized with avertin, and then 150 μΐ of iPS cells are injected into each mouse. Tumors are checked every week for 3-4 weeks. Tumors are then harvested and fixed in z-fix solution for 24 hours at room temperature before paraffin embedding and H&E staining. To further evaluate pluripotency of derived iPSC clones, iPS cells are injected into C57BL/6J-Tyr(C-2J)/J (albino) blastocysts. Generally, each blastocyst receives 12-18 iPS cells. ICR recipient females are used for embryo transfer.

EXAMPLE 2

A Set of microRNAs Is Induced or Repressed During the Early Reprogramming Stage

[0094] Potential iPS cells are enriched in the thyl - population during early reprogramming stages: The present invention provides that at different reprogramming stages, potential iPS cells express distinct sets of microR As that regulate how these cells reach a fully reprogrammed stage and represent unique marker signatures. The present invention provides that stem cell markers are upregulated sequentially at different reprogramming stages. For example, the cell surface antigen thyl, which is highly expressed in Oct4-GFP MEFs, is repressed first when reprogramming is initiated, followed by upregulation of the niES markers alkaline phosphatase and SSEA1 and then of self-renewal genes Nanog and endogenous Oct4. To demonstrate the role of microRNAs in reprogramming, on the present example provides data in early reprogramming stages (before post-transduction day 5), as that period is reportedly critical for the process. Oct4-GFP MEFs are infected with 4F virus and then sorted by flow cytometry five days later (Figure 1 A). PE-conjugated thyl antibody is used to isolate thyl+ and thyl - populations. To isolate pure populations, some intermediate cells are uncollected (Figure IB). Equal numbers (10,000 cells) of thyl+ and thyl- cells are reseeded in 12-well plates on CF1-MEF feeders and evaluated their potential for iPS induction based on reporter activity and marker expression. Potential iPS Cells are mainly enriched in the thyl - population, as determined by both GFP+ colony counting and AP staining (Figures 1C & ID). No GFP+ colonies are detected and only a few AP+ colonies in the thyl + population in any given condition. The present invention provides that the fate of 4F-infected MEFs is determined before day 5 post infection and that iPS cells are enriched in thyl -population.

[0095] For whole microRNA expression profiling, total RNAs are collected from sorted thyl -population at day 5 post 4 factor transduction. To identify microRNAs whose expression is significantly altered relative to that seen in starting MEFs, a gate of at least a 2- fold change is set and p < 0.05 is used to filter the data (Figure 2A). Indeed, a set of microRNAs are identified in the thyl - population that are significantly induced after 5 days of infection (Figure 2B). Among them, miR-135b is the most highly induced and shows significant expression levels (Table 1). Thus miR-135b is chosen for analysis of its direct targets and its role in the reprogramming process. The present invention also provides that other microRNAs, such as miR-93 which belongs to miR-25~106b cluster, miR-92a which belongs to miR-17~92 cluster, and miR-302b which belongs miR-302 cluster, are also highly induced at early stage of reprogramming.

[0096] Table 1 shows original microRNA expression profile data of the present example. List of microRNAs significantly (2-fold, p < 0.05) alters at reprogramming day 5 in Thyl- cells.

Table 1. microRNA expression profile data.

Group MEF D0 Thyl&D5 Thyl-D5

Systematic/ Common/ ChromoGenbank Norm Norm Norm Description some

ILMN_3166979 23609.834 11127.709 5009.208 mmu-miR-223 X

ILMN_3167887 2198.5 1020.5833 467 mmu-miR-543 12

ILMN_3167409 186.45834 177.125 45.958336 mmu-miR-542-5p X

ILMN 3167846 2968.25 3031.4583 756.5416 mmu-miR-665 12

ILMN 3168509 9271.25 5771.125 2394.125 mmu-miR- 142-5p 11

ILMN_3167397 1008.0833 618.4167 291.58334 mmu-miR-450b-5p X ILMN_3167698 332 295.5 104.75 mmu-miR-184 9

ILMN_3167215 1054.2916 709.2291 341.58334 mmu-miR-370 12

ILMN_3167383 3672.5 3614.625 1195.1666 mmu-miR-431 12

ILMN_3167419 10785 11485.334 4213.4585 mmu-miR-376a 12

ILMN_3167052 9908.291 7695.708 4398.333 mmu-miR-495 12

ILMN_3167553 1670.4167 1320.9583 3398.25 mmu-miR-491 4

ILMNJ168517 10821.084 18965.5 22198.043 mmu-miR-93 5

ILMN_3166986 5989.583 6450.375 12607.75 mmu-miR-92a 14,X

ILMN_3167510 17315.918 33186.332 36422.082 mmu-miR-20a 14

ILMN_3167938 94.625 197.79167 236.04166 mmu-miR-302d 3

ILMN 3167918 5277.917 7928.625 14486.333 rnmu-miR-701 5

ILMN_3168117 166.47916 1955.6667 743.75 mmu-miR-547 X

ILMN_3168303 696.3333 6182.8335 5252 mmu-miR-124 2,14,3

ILMNJ 167279 222.91666 757.625 3749.1665 mmu-miR-302b 3

ILMN_3167874 446.2083 8373.084 13559.25 mmu-miR-135b 1

[0097] The data also reveals microRNAs that are significantly repressed (Figure 2C), suggesting that they may serve as reprogramming barriers. To further evaluate potential barrier function, miR-223 and miR-495 are chosen as examples, as they are highly expressed in MEFs.

EXAMPLE 3

MiR-135b Enhances Reprogramming, But miR-223 and 495 Inhibits Reprogramming

[0098] To determine whether miR- 135b enhances reprogramming, a miR- 135b microRNA mimic is transfected into Oct4-GFP MEFs infected with 4F virus and GFP+ colonies are counted at day 11-12 post-transduction. Transfection of the miR- 135b mimic results in a ~2-fold increase in the number of Oct4-GFP+ colonies, as does transfection with miR-93 which is previously characterized as an enhancer for reprogramming (Figure 3 A). In similar experiments, cells are transfected with miR-223 or 495 mimics, which compromise reprogramming, although the effect is minor (Figure 3 A). This observation can be potentially due to the saturation effect of endogenous miRs. In cases in which the 4F virus is not sufficiently competent to induce GFP+ colonies in control samples, transfection of miR-93 or miR- 135b results in a significant number of GFP+ colonies (Figure 8), further supporting a role in lowering programming barriers.

[0099] The percentage of GFP+ cells in miR-transfected reprogrammed cells is analyzed. Although both miR-93 and miR- 135b increase GFP+ colony formation, only miR- 135b increases the percentage of GFP+ cells by ~2 fold (Figures 3B and 9). In the same assay, miR-223 transfection significantly decreased the GFP+ population (Figure 3B); supporting the idea that miR-223 serves as a reprogramming barrier. MicroRNA inhibitors are also used for analysis. Blocking miR- 135b compromises reprogramming efficiency, while inhibiting miR-223 results in a significant increase in the number of Oct4-GFP+ colonies (Figure 3C). The present invention provides that, as the microRNA most highly induced by the 4F factors, miR- 135b enhances reprogramming, while miR-223, which the data show to be the most highly repressed microRNA, serves as a barrier.

[0100] Since GFP positivity can result from inappropriate reactivation of the Oct4 locus, the present invention provides that 135b-transfected iPSCs can reach a fully reprogrammed state. Analysis of miR- 135b transfected iPSCs indicates that these iPSCs turned on all endogenous markers, including alkaline phosphatase, SSEA1, Nanog and endogenous Oct4 (Figure 3D). Lineage markers are expressed in differentiated EBs from miR-135b induced iPSCs. Embryoid bodies are formed using the hanging drop method for two days and replated onto gelatin-coated plates until day 12-14. Cells are then fixed and stained for AFP (endoderm), Tubulin III (ectoderm), and a-Actin (mesoderm) staining. DAPI is used for nuclear staining. Teratoma formation confirms the pluripotency of miR- 135b iPSCs. lxl 0⁶ iPSCs are injected into athymic nude mice and tumors are harvested for H&E staining 3-4 weeks later. These miR- 135b iPSCs have the full capacity to differentiate into three germ layers as indicated by marker analysis and to form heterogeneous teratomas when injected into athymic nude mice. These cells also contributed to chimeric mice. Genome-wide mRNA profiling confirms that miR- 135b iPSCs resemble mES cells and differ significantly from MEFs in terms of gene expression (Figure 10). The present example provides that miR- 135b transfection does not adversely affect iPSC pluripotency. EXAMPLE 4

Identification of mi R- 135b Regulated Genes

[0101] Initially, microRNAs are thought to simply repress translation. However, recent findings suggest that niRNA destabilization mediated by microRNAs comprises a major underlying cause of repression. Thus, a genome-wide mRNA expression array is used to detect potential miR-135b targets. MiR-135b or control siRNA is transfected into Oct4-GFP MEFs, and total RNAs are harvested 48 hours later for array analysis. Raw data is filtered by at least two-fold changes (both increased and decreased) and p < 0.05 (Figure 4A).

[0102] Candidate genes are then compared with published mES/iPS/MEF expression profiles and divided into two groups: genes induced (group 1) or repressed (group 2) after miR-135b transfection, the latter being more likely to contain direct targets. The present invention provides that genes repressed by miR-135b transfection are enriched with genes normally silenced as MEFs are reprogrammed to iPS/mES cells (correlated) (Figure 4B). This enrichment is not observed in genes induced by miR-135b transfection (group 1), in which approximately half are normally suppressed during reprogramming (uncorrelated) and the other half is increased (correlated). The present invention provides that miR-135b targets a subset of barrier genes, which are normally repressed during reprogramming.

[0103] To confirm mRNA microarray analysis, total RNAs from an independent experiment are harvested, and RT-qPCR is used to assess representative mRNA levels.

Indeed, very similar mRNA decrease is detected upon miR-135b transfection, in agreement with the mRNA array data (Figures 5A & 5C). In the case of Tgfbr2 and Igfbp5, -70% decrease for their mRNA levels is detected upon miR-135b transfection and thus protein levels are analyzed. Indeed, western analysis indicates a dramatic decrease in Tgfbr2 and Igfbp5 protein expression following miR-135b transfection (Figures 5B & 5D).

[0104] To identify direct targets of miR-135b, the "correlated" genes in group 1 (Figures 4B & 4C) are analyzed using both miRanda (Enright, A.J. et ah, 2003, MicroRNA targets in Drosophila. Genome Biol 5, Rl) and Targetscan (Lewis, B.P. et ah, 2005, Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15-20). Potential target sites are identified based on seed region matches and overall predicted binding energy. Of 27 genes repressed by miR- 135b by at least twofold, 14 exhibit at least one predicted miR- 135b target site (Table 2 and Figure 13).

[0105] Table 2 shows a list of predicted miR- 135b target sites identified by both miRanda software and Targetscan in the 3'UTRs of Tgfbr2, Wispl, and Igfbp5. The portion of miR- 135b used for interacting with the target sites is shown as

5'-UAUGGCUUUUCAUUCCUAUGUGA-3' (SEQ ID NO: 1).

[0106] The 3'UTR of several potential targets are cloned into the pGL3 luciferase reporter vector and co-transfected the reporter plus the pRL-TK plasmid into HeLa cells. Among candidates chosen for analysis, miR- 135b decreases luciferase activity of both Tgfbr2 and Wispl reporters by -80%. A 30% decrease is detected in similar assays of an Igfbp5 reporter (Figure 11). The present invention provides that several genes harbor predicted miR- 135b target sites, and that Tgfbr2, Wispl and Igfbp5 are direct miR- 135b targets.

Table 3. Exemplary genes affected by miR- 135b mimic transfection.

siControl miR-135b 135b/ctrl

1 Mmpl3 2439.7168 538.7083 0.22080772

2 Ssr2 9958.9 2575.1333 0.25857608

3 Gpcl 17654.266 5494.533 0.31122976 MmplO 898.45 280.84583 0.31258927

Ecel 3008.7 1005.7916 0.33429441

1190002H23Rik 885.3833 321.30835 0.36290311

Rgsl6 7778.225 2841.0166 0.36525256

Entpd4 4623.3667 1703.7334 0.36850493

Sema7a 980.56665 373.225 0.38062176

Inhba 12432.358 4787.7 0.38509991

Adra2a 1711 668.8833 0.39093121

Creldl 2141.55 837.1625 0.39091429

Eif4ebpl 14258.191 5669.408 0.39762464

Sprr2k 6371.658 2581.3 0.40512218

CxclH 5082.5 2109.0833 0.41496966

Tgfbr2 6950.125 2935.2915 0.4223365

Wispl 11202.6 4737.1167 0.42285868

Cell 2 1894.2 812.8458 0.42912354

2310001A20Rik 1022.275 441.84998 0.43222223

Ogfr 2861.7666 1272.846 0.44477631

Aqp5 1304.9917 584.11664 0.4476018

Nptx2 925.5 421.3333 0.45524938

Dusp4 1473.9667 691.94995 0.46944748

Greml 1314.4417 616.475 0.4690014

1200015F23Rik 898.875 432.25833 0.48088814

Nras 7671.4497 3686.5417 0.48055346

Trib3 2113.9917 1017.7708 0.48144503

Ifit3 2212.2583 1080.0833 0.48822658

St3gal3 3312.25 1609.75 0.48599894

Mtbfd2 1443.7 716.5416 0.49632306

Igfbp5 10339.175 5151.6 0.49826026

Nbll 4588.85 2290.6416 0.49917552

Ndufa4 25525.383 12770.292 0.50029776

Ly6a 20063.092 40192.016 2.00328125

BC031353 960.2375 1930.3 2.01023184

Btbdl 1216.2666 2449.933 2.01430591

Kiflb 1098.5917 2218.4043 2.01931646

Adamtsl2 1180.025 2405.308 2.03835342

Tmem43 2145.7917 4415.1 2.05756225

4933439C20Rik 1743.2167 3600.1167 2.06521467 41 Ncald 178.2 368.55832 2.06822851

42 Nnmt 2737.9917 5777.225 2.11002283

43 Notch3 243.07083 513.575 2.11286151

44 Cxadr 208.13751 440.57083 2.11672961

45 Ptprv 540.675 1150.1792 2.12730235

46 Mxd4 588.1625 1261.9333 2.14555212

47 Gabarap 7238.6416 15543.541 2.14730081

48 Tle6 489.025 1058.2334 2.16396585

49 Fas 751.06665 1629.8501 2.17004723

50 Ephxl 6257.4834 14002.008 2.23764205

51 Ddit41 1087.9624 2482.025 2.28135182

52 Nipal 718.1333 1642.9209 2.28776593

53 Ctxn 457.96667 1069.1458 2.33454937

54 Tcea3 1136.9917 2677.8252 2.35518447

55 117 292.79166 692.87085 2.3664296

56 Asahl 10981.791 26243.625 2.38973998

57 Hmoxl 889.9375 2178.9 2.44837418

58 1700007K13Rik 630.12085 2569.5 4.07778921

59 Sncg 691.1 2831.7793 4.09749573

[0107] Table 3 shows mR A expression profile upon miR-135b transfection, where significantly altered mRNAs upon miR-135b mimic transfection are listed.

EXAMPLE 5

Knockdown of miR-135b Targets Increases Reprogramming Efficiency

[0108] Potential miR-135b targets are examined as reprogramming barriers. Tgfbr2 is previously reported to be a reprogramming barrier and a potential target of miR-93 and its family microRNAs. In addition to Tgfbr2, Wispl and Igfbp5, several other genes such as Eif4ebpl and Cxcll4, are also included in the analysis. There genes are potentially indirectly regulated by miR-135b. RT-qPCR confirms that each candidate mRNA is efficiently knocked down by at least 60% by siRNAs (Figure 6A).

[0109] The present invention provides that knocking down candidate targets can increase reprogramming efficiency. Respective siRNAs are transfected into Oct4-MEFs at both day 0 and day 5 and GFP+ colonies are counted at days 11-12. Among candidates tested, a significant increase in the number of GFP+ colonies can be detected after transfection of siR A targeting Igfbp5 (Figure 6B). Surprisingly, when si Wispl is transfected at both day 0 and day 5, a dramatic decrease in reprogramming is observed, although when si Wispl is introduced at day 5 only, a 3-fold increase in the number of GFP+ colonies is detected (Figure 6C). Thus, Wispl may play a dual role during reprogramming, potentially enhancing it at early stages and inhibiting it later. In addition, bioinformatics analysis identifies several other members of TGFP signaling pathway that can be targeted by miR-135b, such as Rock kinases, LIMK2 and p38 (Figure 12).

[0110] The present invention provides that during reprogramming, MEF-specific microRNAs are repressed, while mES-specific ones are induced. The induced microR As target different barrier genes and help establish mES regulatory networks, changing the fate of fibroblasts to that of iPS Cells (see Figure 7 for a model for microR A functions during somatic cell reprogramming process).

EXAMPLE 6

MicroRNA Modulate IPS Cell Reprogramming

[0111] Since the discovery of direct reprogramming of MEFs to iPS Cells, efforts have been made to understand how the four reprogramming transcription factors shut down endogenous MEF genes and gradually re-establish mES-like regulatory networks. Due to the extremely low efficiency of the process, understanding how reprogramming factors overcome key barriers is critical for development of novel technologies or compounds to improve efficiency. The present invention provides that several microRNAs function in the reprogramming process and constitute key mechanisms regulated by OSKM factors to overcome endogenous regulatory networks in MEFs.

[0112] By analyzing potential-iPSCs enriched thy 1 ^" cell population during early reprogramming stages, a set of microRNAs is identified that are either induced or repressed during the process. Manipulating levels of some of those microRNAs with miR mimics or inhibitors dramatically altered iPSC induction efficiency. Typically, blocking of MEF- specific microRNAs increases reprogramming efficiency as introducing mES-specific miR mimics. Among the microRNAs analyzed, miR- 135b is the most highly induced. The present invention provides that miR-135b enhances both Oct4-GFP+ colony formation and the overall percentage of GFP+ cells. Moreover, genome-wide mRNA expression profiling identifies candidate genes whose expression is significantly repressed by miR-135b transfection, and those genes are enriched in MEF-specific genes. Such genes are generally expressed at very low levels in mES/iPSCs. Among them, Tgfbr2, Wispl and Igfbp5 are provided to be direct targets of miR-135b and can function as reprogramming barriers.

[0113] The TGFP pathway has been recently reported to inhibit iPS induction, and small molecules inhibiting TGFP can enhance reprogramming efficiency. The reprogramming factors can suppress expression of both Tgfbrl and Tgfbr2 and induce a mesenchymal to epithelial transition (MET) during the process. Initiation of TGFP signaling usually requires formation of a heterodimer of TGFP receptor I and receptor II after ligand binding. In MEFs, both receptors are highly expressed compared to mES cells or fully reprogrammed iPSCs, supporting the idea TGFP signaling must be silenced to achieve fully reprogrammed cells and suggesting that incomplete silencing of the pathway may underlie formation of partial iPSCs. Indeed, genome-wide mRNA expression analysis has shown that in partial iPSCs, expression of TGF receptor II is as high as that seen in MEFs.

[0114] The present invention provides that similar to miR-93 family microRNAs, miR- 135b is highly induced during the reprogramming process and can function to inhibit TGFP signaling, based on inhibition of Tgfbr2 expression. Moreover, bioinformatics analysis identifies several other members of TGFP signaling pathway that can be targeted by miR- 135b, such as Rock kinases, LIMK2 and p38 (Figure 12).

[0115] The Wnt signaling pathway also functions in the reprogramming process. Wnt signaling can enhance iPS induction, since addition of Wnt3a to mES culture medium enhances reprogramming. The present invention provides that a member of the Wnt signaling pathway, Wispl, unexpectedly has a dual role during the reprogramming process, a finding never before reported. Wispl is one of the highest expressed genes in MEFs, and its expression is dramatically decreased in established as well as partially reprogrammed iPSCs. Wispl reportedly functions as a pro-survival factor in cardiomyocytes and promotes fibroblast proliferation. It can also activate some cell survival pathways, such as PI3K/Akt, and interacts with p53 pathways. This observation can explain why reprogramming efficiency dramatically decreased when Wispl is knocked down at day 0, since it likely regulates MEF proliferation, which is critical for reprogramming. The present invention provides that Wispl later becomes a barrier after transduction of the four reprogramming factors and that knocking it down by siRNAs at day 5 significantly increases reprogramming efficiency. Wispl is regulated by miR-135b, which is significantly induced in 4F-infected MEFs at day 5, and that regulation likely requires the Wispl 3'UTR region.

[0116] The insulin-like growth factors are important regulators of cell growth, as they can bind insulin/insulin-like growth factor- 1 (IGF-1) with high affinity, and thus block IGF-1 signaling. Igfbp5 is the most conserved member of the six IGF-1 binding proteins, and it has been shown that IGFBP5 overexpression induces cell senescence in a p53-dependent way. Igfbp5 is highly expressed in fibroblasts, and its expression is further increased upon senescence. The present invention provides that the p53 pathway is a major barrier to reprogramming and miR-93 and its family members inhibit p53 downstream effectors, such as p21, during reprogramming.

[0117] Interestingly, miR-93 expression in MEFs promotes IGFBP5 induction (Figures 5C & 5D), resulting in a -40% increase in mRNA levels and even higher protein levels. This increase can be due to p21 suppression by miR-93. The present invention provides that miR- 135b can act cooperatively with miR-93 to target multiple effectors of the p53 pathway and therefore repress intrinsic cellular barriers to activity of the four reprogramming factors, shifting the balance between MEFs and iPSCs and significantly increasing reprogramming efficiency (Figure 6D).

[0118] MiR-135b is reportedly expressed in many cancer cells, such as human colon, breast and prostate cancer. Its expression has been shown to be regulated by core self- renewal regulators of embryonic stem cells. However, there is limited information about its targets and how it functions in oncogenesis or stem cell fate determination. The present invention provides that miR-135b can directly target Tgfbr2, Wispl and IGFBP5 and decreases their mRNA and protein levels. The present invention provides the role of miR- 135b in tumor cells and stem cell differentiation. EXAMPLE 7

NSAID for IPS Cell Reprogramming

[0119] The NSAID Nabutone enhances iPS cell generation: a genomics database drug discovery strategy was developed to identify small molecules that enhance reprogramming.

[0120] To shorten the list without extensive shot-gun screening, candidate molecules that potentially either antagonized MEF-specific genes or upregulated MES-specific/

reprogramming genes are focused on. To do so, computational screening by utilizing

NextBio (nextbio.com) data-mining tools to collect information from public data sources is conducted as in Kupershmidt et al. (PLoS One 5 (2010)). Using highly enriched genes in either MES or MEF as queries, 17 molecules (Table 4) are acquired that either negatively regulated MEF genes or positively affected MES genes from the NextBio meta-analysis.

Table 4. Molecules that either negatively regulate MEF genes or positively affect MES genes.

ID Molecules CAS # Predicted targets

1 Nickel sulfate hexahydrate (NiS0₄) 10101-97-0 WISP1, PRRX1,

LYZS

2 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin 1746-01-6 TGF- 3

3 Nabumetone 42924-53-8 COX2

4 4-hydroxytamoxifen (OHTM) 68047-06-3 Sox2

5 Moclobemide 71320-77-9 Nanog

6 Lectin DPPA5

7 Corynanthine hydrochloride 66634-44-4 TDGF1

8 TGF-β Oct3/4

9 Acitretin 55079-83-9 Oct3/4

10 Retinoic acid p-hydroxyanilide 65646-68-6 Oct3/4

11 Diacerein 13739-02-1 Nanog

12 Phorbol 12-myristate 13 -acetate 16561-29-8 Nanog

13 Progesterone 57-83-0 Nanog

14 Tolazamide 1156-19-0 Nanog

15 15-deoxy-A¹²' ^-prostaglandin J₂ 89886-60-2 Klf4

16 (-)-Norepinephrine 51-41-2 c-Myc

17 β-estradiol 50-28-2 c-Myc [0121] All 17 molecules are tested by examining alkaline phosphatase (AP) + colony formation during reprogramming while these molecules were applied. Molecules not showing adverse effect on AP+ colony formation are picked for further study. To that end, 6 molecules were picked— Nabumetone, 4-hydroxytamoxifen (OHTM), Corynanthine, Moclobemide, NiS04, and lectin-— for further analysis (Figure 13 A). To evaluate their effect on induction of mature GFP+ iPS cells, OSKM-transduced Oct4-EGFP MEFs are treated four days after transduction with each of these factors separately. Among the six, the NS AID prostaglandin-endoperoxide synthase (PTGS) and the cyclooxygenase (COX) inhibitor Nabumetone greatly increased the number of reprogrammed colonies by at least 2.8-fold (Figure 13B) compared with DMSO controls, while Lectin shows minor but consistent improvement on iPSC formation.

[0122] Figure 13B shows that Nabumetone significantly boosts OSKM-induced reprogramming while lectin shows minor but consistent increase as well. Oct4-EGFP MEFs are transduced with OSKM and four days later treated with individual small molecules for at least 10 days. GFP+ colonies are identified. Error bars represent standard deviations of three independent experiments. * p value < 0.05; ** p value < 0.005.

[0123] Figure 13C shows that Nabumetone improves reprogramming through blocking COX2. Oct4-EGFP MEFs are transduced with OSKM. Four days later, cells are treated with Nabumetone or DMSO. The next day, cells are transfected with various siRNAs as indicated. GFP+ colonies are identified at day 12 ~ 14. Error bars represent standard deviations of six independent experiments. * p value < 0.05; ** p value < 0.005; *** p value < 0.0005. siNT serves as control.

EXAMPLE 8

Kinase Inhibitors for IPS Cell Reprogramming

[0124] Eleven barrier candidates for iPS cell reprogramming are shown in Figure 14A, where kinase inhibitors B4, B8, and B10 consistently and significantly enhanced

reprogramming (Figures 14B and 14C). Figure 14C shows results using a lower

concentration (~1 μΜ) during secondary screening stidues. Inhibitor B4 is identified as able to enhance reprogramming and/or replace the transcription factor Sox2 in four pluripotency factor cocktail (Ichida et al, 2009, Cell Stem Cell 5:491-503). Surprisingly, inhibitor B6 dramatically decreses reprogramming efficiency at 2 μΜ but robustly enhances reprogramming at 1 μΜ. Dose/reponse analysis confirms that B6, B8, and BIO act as potent enhancers at 0.5 μΜ. Since these inhibitors can inhibit multiple kinases at a given concentration, their specificity in targeting barrier kinases is validated. MEFs are transfected with siR Ss to individual targets of the inhibitors and quantified reprogramming efficiency. Indeed, Mapkl 1 (p38beta), ItpkA, Stk6 and SyK, which are targets of inhibitors B6, B8, and BIO, act as barrier proteins: knockdown of any one of these genes during reprogramming results in significant increases in iPS induction. It is noteworthy that knockdown of some B6 targets, such as Bmx, IgflP and Lck, comprise iPS induction,k which may explain why B6 both inhibits and enhances reprogramming, depending on concentration.

EXAMPLE 9 microRNAs modulate iPS cell generation

[0125] MATERIALS AND METHODS

[0126] MEF derivation

[0127] Oct4-EGFP MEFs were derived from the mouse strain B6; 129S4- PouSfl^^Jae/J (Jackson Laboratory; stock no. 008214) using the protocol provided on the WiCell Research Institute website ht p://www■wicell.or . Oct4-EGFP MEFs were maintained in MEF complete medium (DMEM with 10% FBS, nonessential amino acids, L- glutamine, but without sodium pyruvate).

[0128] Reprogramming using retrovirus

[0129] Reprogramming was conducted as described (Takahashi and Yamanaka 2006). In brief, 4 X 10⁴ Oct4-EGFP MEFs were transduced with pMX retroviruses to overexpress Oct4, Sox2, Klf4, and c-Myc (Addgene). Two days later, transduced Oct4-EGFP MEFs were fed with ES medium (DMEM with 15% ES screened FBS, nonessential amino acids, L- glutamine, monothioglycerol, and 1000 U/mL LIF), and the media were changed every other day. Reprogrammed pluripotent stem cells (defined as EGFP+ iPSC colonies) were scored by fluorescence microscopy ~2 wk after transduction, unless otherwise stated. To derive iPSCs, EGFP+ colonies were manually picked under a stereo microscope (Leica). [0130] miRNA inhibitor or siRNA transfection

[0131] Inhibitors of let-7a, miR-21 , and miR-29a miRNAs were purchased from

Dharmacon. We transfected 4 X 10⁴ Oct4-EGFP MEFs with lipofectamine and inhibitors according to manufacturer's instruction (Invitrogen). Three to 5 hr later, the medium was discarded and replaced with MEF complete medium; for reprogramming, retrovirus encoding reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) was added and the medium was changed to complete medium the next day. Inhibitors or siRNAs were introduced again at day 5 after transfection/transduction, unless otherwise stated.

[0132] For Northern analysis, 1 X 10⁵ Oct4-EGFP MEFs were transfected and harvested 5 d later. Total RNA was isolated by TRIZOL (Invitrogen) and ~9 mg of total RNA was resolved on a 14% denaturing polyacrylamide gel (National Diagnostics). RNAs were transferred onto Hybond-XL membranes (GE healthcare), and miRNAs were detected by isotopically labeled specific DNA probes. Signal intensity was visualized by phospho-imager and analyzed using Multi Gauge V3.0 (FUJIFILM). miRNA signal intensity was normalized to that of U6 snRNA. Experiments were performed in triplicate.

[0133] For Western analysis, 1 X 10⁵ Oct4-EGFP MEFs were transfected and harvested 5 d later. Total proteins were prepared in M-PER buffer (Pierce), and equal amounts of total protein were separated on 10% SDS-PAGE gels. Proteins were transferred to PVDF membranes, and bands were detected using the following antibodies: GAPDH (Santa Cruz; catalog no. sc-20357), p53 (Santa Cruz; catalog no. sc-55476), PI3 kinase p85 (Cell

Signaling; catalog no. 4257), Cdc42 (Santa Cruz; catalog no. sc-8401), p-ERKl/2 (Cell Signaling; catalog no. 9101), ERK1/2 (Cell Signaling; catalog no. 9102), p-GSK3p (Cell Signaling; catalog no. 9323), GSK3 (Cell Signaling; catalog no. 9315), and β-actin (Thermo Scientific; catalog no. MS- 1295). Signal intensity was quantified by Multi Gauge V3.0 (FUJIFILM) and normalized to GAPDH or β-actin. Experiments were repeated three to five times.

[0134] In vitro differentiation and teratoma formation assay

[0135] For in vitro differentiation, iPSCs were dissociated by trypsin/EDTA and resuspended in EB medium (DMEM with 15% FBS, nonessential amino acid, L-glutamine) to a final concentration of 5 X 10⁴ cells/mL. To induce EB formation, 1000 iPS cells in 20 μΐ, were cultured in hanging drops on inverted Petri dish lids. Three to 5 d later, EBs were collected and transferred onto 0.1% gelatincoated six-well plates at about 10 EBs per well. Two weeks after formation of EBs, beating cardiomyocytes (mesoderm) were identified by microscopy, and cells derived from endoderm and ectoderm were identified by a-fetoprotein (R&D; catalog no. MAB1368) and neuron-specific βΙΙΙ-tubulin (abeam; catalog no. ab7751) antibodies, respectively.

[0136] For teratoma assays, 1.5 X 10⁶ iPSCs were trypsinized and resuspended in 150 μΐ, and then injected subcutaneously into the dorsal hind limbs of athymic nude mice anesthetized with avertin. Three weeks later, mice were killed to collect teratomas. Tumor masses were fixed, dissected, and analyzed in the Cell Imaging-Histology core facility at the Sanford-Burnham Institute.

[0137] Chimera analysis

[0138] iPSC media was changed 2 hr before harvest. Trypsinized iPSCs were cultured on 0.1% gelatin-coated plates for 30 min to remove feeder cells. iPSCs were injected into E3.5 C57 L/6-cBrd/cBrd blastocysts and then transferred into pseudopregnant recipient females. After birth, the contribution of iPSCs was evaluated by pup coat color: black is from iPSCs.

[0139] Immunofluorescence and alkaline phosphatase staining

[0140] iPSCs were seeded and cultured on 0.1% gelatin-coated six- well plates. Four days later, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences; catalog no. 15710-S). For immunofluorescence staining, fixed cells were permeablized with 0.1% Trixton X-100 in PBS and blocked in 5% BSA/PBS. Antibodies against SSEA-1 (R&D; catalog no. MAB2155) and Nanog (R&D; catalog no. AF2729) served as ES markers.

Nuclei were visualized by Hoechst 33342 staining (Invitrogen). For alkaline phosphatase (AP) staining, fixed cells were treated with AP substrate following the manufacturer's instruction (Vector Laboratories; catalog no. SK-5100).

[0141] RESULTS

[0142] Inhibition of miR-21 or miR-29a enhances reprogramming efficiency [0143] To determine whether inhibiting MEF-specific miRNAs lowers reprogramming barriers, we first analyzed MEF-enriched miRNAs and compared their levels with those seen in mouse ES cells. As shown in Figure 16 A, let-7a, miR-21, and miR-29a were highly expressed in MEFs compared with ES cells. In contrast, miR-291 was highly abundant in ES but absent in MEFs (Figure 16A). Next, we introduced miRNA inhibitors against let-7a, miR-21, and miR-29a into Oct4-EGFP MEFs (MEFs harboring Oct4-EGFP reporter) together with retroviruses expressing Oct 3/4, Sox2, Klf4, and c-Myc (OSKM). At day 14 post-transduction, cells treated with miR-21 inhibitors showed an about 2.4 fold increase in reprogramming efficiency compared with a nontargeting (NT) control (Figure 16B).

Similarly, reprogramming efficiency increased significantly by about threefold following inhibition of miR-29a (Figure 16B). Under similar antagomir treatments as used for miR-29a or miR-21 inhibition, we observed a minor effect on OSKM reprogramming following let-7a inhibition (Figure 16B). To further test whether miRNA inhibition enhances reprogramming with three factors in the absence of c-Myc, cells were transduced with the miRNA inhibitor together with Oct3/4, Sox2, and Klf4 (abbreviated as OSK), which reprograms cells at much lower efficiency than OSKM (Nakagawa et al. 2008). The number of OSK-reprogrammed iPS cell colonies increased in the presence of the miR-21 inhibitor relative to treatment with OSK alone. These results demonstrate that the depletion of the MEF-enriched miRNAs miR- 21 and miR-29 enhances 4F-reprogramming significantly and that blocking miR-21 moderately increases the efficiency of OSK reprogramming.

[0144] c-Myc represses expression of miRNAs let-7a, miR-16, miR-21, miR-29a, and miR-143 during reprogramming

[0145] Recent work indicates that the OSKM factors alter cell identity through both epigenetic and transcriptional mechanisms (Sridharan et al. 2009). Therefore, we

hypothesized that the OSKM reprogramming factors could down-regulate MEFenriched miRNAs. To evaluate the potential effect of each reprogramming factor on miRNA expression, MEFs were transduced with various combinations of the OSKM factors and subjected to Northern blot analysis (Figure 17A). Interestingly, Sox2 alone induced expression level of miR-21, miR-29a, and let-7a by more than twofold, compared with the MEF control (Figure 17B, left). Klf4 also induced miR-29a and let-7a by about 1.5-fold and 1.8-fold, respectively (Figure 17B, left). With Oct3/4 overexpression only, miRNAs did not change expression level (Figure 17B, left). In contrast to Oct4, Sox2, and Klf4, the single factor c-Myc down regulated expression of miR-21 and miR-29a, the most abundant miRNAs in MEFs, by -70% of the MEF control (Figures 17A, B, left).

[0146] Furthermore, among various combinations of two factors (2F) shown in Figure 17B (middle), inclusion of c-Myc enhanced decreases in all three miRNAs, including miR- 21, miR-29a, and let-7a, by about 25%-80% (Figure 17B, middle). Similar to the single- factor effect on miRNA expression, Sox2 and Oct3/4 together increased miR-21 and miR-29a by 1.5 -fold and 2.3 -fold of the MEF control, respectively, while OK and SK had no obvious effects on miRNA expression (Figure 17B, middle). Moreover, among various three-factor (3F) combinations, the expression of miRNA-21 decreased by about 70% and 78% in SKM and OKM cells, respectively, relative to the expression seen in MEFs (Figure 17B, right). Similarly, miR-29a expression decreased by about 48%-70% in 3F combinations containing c-Myc (Figure 17B, right). Inclusion of c-Myc in 3F combinations also slightly decreased let-7a levels (Figure 17B, right). OSK without c-Myc had little effect on miRNA expression (Figure 17B, right). Therefore, these results strongly suggest that c-Myc plays an important role in regulating miRNA expression during the reprogramming.

[0147] To further confirm that c-Myc is the primary factor antagonizing miRNA expression, cells were transduced with OSK with or without c-Myc, and miRNA expression was examined by real-time quantitative reverse transcription polymerase chain reaction (RTqPCR) at various time points posttransduction. In contrast to OSK, OSKM transduction greatly decreased expression of let-7a, miR-16, miR-21, miR-29a, and miR-143 during reprogramming (Figure 17C), indicating that c-Myc plays a predominant role in regulating expression of MEF-enriched miRNAs, including the most abundant ones, let-7a, miR-21, and miR-29a. These data also suggest that c-Myc boosts reprogramming, in part, through miRNA down-regulation.

[0148] c-Myc regulates miRNA expression at transcriptional level during

reprogramming

[0149] c-Myc has been shown to influence miRNA expression in multiple human and mouse cancer models (Chang et al. 2008, 2009), and Lin28b is one of the key intermediate modulators to post-transcriptionally regulate let-7 biogenesis (Chang et al. 2009). Therefore, we examined whether Lin28b-mediated regulation of miRNA expression plays a role during reprogramming. To address this question, we collected reprogrammed cells at various time intervals from days 3-15 after transduction of reprogramming factors. RT-qPCR analysis showed that n RNA expression of Lin28a and Lin28b were undetectable in MEF and during the reprogramming process, while ES cells exhibited a high level of Lin28a and Lin28b expression. Control marker gene expression analysis established the successful progression of reprogramming process where the levels of Thy 1 and Fibrillin-2 were downregulated and Nanog and Fboxl5 were up-regulated. These findings strongly suggest that c-Myc regulation of miR As is Lin28-independent.

[0150] Next, to determine whether the c-Myc effect on MEF-enriched miRNAs is post- transcriptional, we examined miRNA biogenesis by Northern blotting and quantified the amounts of pre-miRNAs and mature miRNAs. Our analysis showed that the ratio between pre-miRNA and mature miRNA in the presence of OSKM was not changed, suggesting that the miRNA maturation process of miR-21 and miR-29a was not compromised by c-Myc during reprogramming. Altogether, these data show that the c-Myc-mediated

downregulation of miR-21 and miR-29a is Lin28a/b-independent and occurs at the transcriptional level.

[0151] iPS cells derived via miRNA depletion attain pluripotency

[0152] To investigate whether blocking miR-21 or miR-29a compromises iPS cell pluripotency, we derived iPS cells treated with miR antagomirs and evaluated them for pluripotency (Li et al. 2011). Since OSKM-derived iPS cells were already well characterized in numerous studies, we decided to thoroughly examine our OSKM/anti miR-29a and OSKM/anti miR21 iPS cells, as well as OSK/anti miR- 21 clones. First, iPS cells were manually picked ~2 wk after reprogramming and were expanded to examine morphology and expression of ES-specific markers. Cells exhibited an ES-like morphology and a highly expressed Oct4-EGFP (Figure 18 A), indicating establishment of endogenous ES cell signaling. In addition, anti-miR-derived iPS cells expressed ES cell-specific markers, including Nanog and SSEA1, and exhibited alkaline phosphatase activity (Figure 18A). To test whether those iPS cells showed pluripotent potential comparable to that of ES cells, those iPS cells were induced to form embryoid bodies (EBs) (Figure 18B) or were injected into nude mice (Figure 18C) and allowed to differentiate into various tissues. After 2 wk of in vitro differentiation, typical cell types derived from all three germ layers were observed (Figure 18B). Teratoma tumors, formed 3 wk after injection, were subjected to

histopathologic analysis. Various tissues originating from all three germ layers (Figure 18C) were generated, confirming that anti-miR-derived iPS cells obtained pluripotency. To use the most stringent test of pluripotency, iPS cells were injected into embryonic day (E) 3.5 blastocysts to create chimeric mice. Mouse derived from anti miR-29a iPS cells showed a significant -15% black coat color attributable to iPS cells (Figure 18D). Since OSK in combination with miR 21 inhibitors resulted in high reprogramming efficiency, we also determined the pluripotency of OSK/anti miR-21 iPS cells by chimera analysis. Mouse generated from OSK/anti miR-21 iPS cells showed -25% black coat color (Figure 18D). These data show that depleting miR-21 and miR-29a had no adverse effect on pluripotency of derived iPS cells.

[0153] Inhibiting miR-29a down-regulates p53 through p85a and CDC42 pathways

[0154] To understand mechanisms underlying miR-29a's effect on reprogramming, we first examined expression of p85a and CDC42, which are reportedly direct targets of miR-29 in HeLa cells (Park et al. 2009). To do so, we transfected miR A inhibitors into MEFs and analyzed p85a and CDC42 protein expression by Western blot at day 5 after transfection. p85a and CDC42 protein levels increased slightly following the miR-29a block, whereas a let-7a inhibitor had little effect (Figures 19A,B). The transformation related protein 53 (Trp53 or p53) is also reportedly a direct target of p85a and CDC42 (Park et al. 2009).

Therefore, we asked whether p53 is indirectly regulated by miR-29a in MEFs as well. To test that, MEFs were transfected with miRNA inhibitors and harvested 5 d for immunoblotting to evaluate expression of p53. p53 protein levels decreased by -30% (Figures 19A,B) following miR-29a inhibition but were not altered by the NT control or by let-7a inhibition.

Significantly, depleting miR-21 also released p85a and CDC42 protein repression, and consequently, the levels of p85a and CDC42 increased, which resulted in down-regulation of p53 expression by -25% (Figures 19A,B).

[0155] To further confirm that p53 levels decrease with inhibition of miR-21 or miR-29a during reprogramming, we examined p53 expression at reprogramming day 5 by Western blot analysis. To initiate reprogramming we introduced miRNA inhibitors together with OSKM. Consistent with observations in MEFs alone, p53 protein levels decreased by -25% or -40% following miR-21 or miR-29a depletion, respectively, during reprogramming, compared with OSKM controls (Figure 19C). In summary, our data showed that blocking miR-29a reduced p53 protein levels by about 30%- 0% through p85a and CDC42 pathways during reprogramming. In addition, depletion of miR-21 had a similar effect on both p85a and CDC42 and lowered p53 protein levels by about 25% to about 30%.

[0156] Inhibition of miR-29a enhances reprogramming efficiency through p53 down- regulation

[0157] It was recently reported that p53 deficiency can greatly increase reprogramming efficiency (Banito et al. 2009; Hong et al. 2009; Judson et al. 2009; Kawamura et al. 2009; Marion et al. 2009; Utikal et al. 2009). Since depleting miR-29a significantly decreased p53 levels and increased reprogramming efficiency by about threefold (Figure 16), we asked whether the effect of miR-29a knockdown is mediated primarily by p53 down-regulation. To that end, we transfected p53 siRNA and/or the miR- 29a inhibitor into Oct4-EGFP MEFs together with OSKM to initiate reprogramming. Down-regulation (-80%) of p53 by small interfering RNA (siRNA) had a similar positive effect on reprogramming efficiency as did miR-29a inhibition (Figure 19D). We did not observe an increase in reprogramming efficiency when miR inhibitors were added in the presence of p53 siRNA (Figure 19D). These results suggest that inhibition of miR- 29a acts, in part (see below), through down- regulation of p53 to increase reprogramming efficiency.

[0158] Inhibition of miR-21 and miR-29a decreases phosphorylation of ERK1/2, but not GSK3b, to enhance reprogramming

[0159] miR21 reportedly activates MAPK/ERK through inhibition of the sprouty homolog 1 (Spryl) in cardiac fibroblasts (Thum et al. 2008). Blocking MAPK/ ERK activity promotes reprogramming of neural stem cells (Silva et al. 2008) and secures the ground state of ESC self-renewal (Nichols et al. 2009; Ying et al. 2008). Therefore, we asked whether miR-21 regulates the MAPK/ERK pathway during reprogramming by evaluating ERK 1/2

phosphorylation in MEFs following the introduction of miRNA inhibitors. To test that, MEFs were transfected with miRNA inhibitors and then harvested for Western blot analysis to determine the phosphorylated ERKl/2 level. Western blot analysis showed that blocking miR-21 significantly decreased by -45% the ERK 1/2 phosphorylation relative to the NT controls, while let-7a inhibitors had no effect (Figure 20A). Interestingly, depleting MEFs of miR-29a also significantly reduced ERK1/2 phosphorylation by 60% relative to the NT control (Figure 20A). Next we determined whether miR-21 and miR-29a affected ERK1/2 phosphorylation by altering Spryl levels. We depleted miR-21 or miR-29a in MEFs by transfecting various miRNA inhibitors, and quantified Spryl expression levels by

immunoblotting. Our results showed that inhibiting miR-21 and miR-29a enhanced Spryl expression levels (Figure 20B). Therefore, our data demonstrate that depleting miR-21 and miR-29a down-regulates phosphorylation of ERK1/2 by modulating Spryl protein levels.

[0160] To address whether ER 1/2 downregulation enhances reprogramming efficiency, we introduced siRNAs targeting ERKl or ERK2 into Oct4-EGFP MEFs in the course of 4F- reprogramming. Depletion of either ERKl or ERK2 significantly enhanced the generation of mature iPS cells (Figure 20C). Our data showed that miR-21 acts as an inducer of ERKl 12 activation in MEFs, since blocking miR-21 reduced ERKl/2 phosphorylation. Depleting miR-29a also significantly diminished ERKl/2 phosphorylation. These results strongly suggest that miR-21 and miR-29a regulate ERKl/2 activity to modulate reprogramming efficiency (Figures 20A-C).

[0161] The GSK3P pathway also represses ES self-renewal and reprogramming of neural stem cells (Ying et al. 2008). Depleting GSK3P with siRNA greatly increased mature iPS cell generation (Figure 20C). Therefore, we asked whether miRNA depletion regulated GSK3P activation. Immunoblotting showed that blocking miRNAs in Oct4-EGFP MEFs had no significant effect on GSK3P activation (Figure 20D). We then asked whether miRNA depletion alters apoptosis or cell proliferation during reprogramming by using flow cytometry to assess cell viability and replication rate. Blocking miRNA-21, miRNA-29a, or let-7 during reprogramming with OSKM did not alter apoptosis or proliferation rates. Overall, our results demonstrate that miR-29a and miR-21 modulate p53 and ERKl/2 pathways to regulate iPS cell reprogramming efficiency.

[0162] DISCUSSION

[0163] To develop alternatives for transgenes currently used for induced reprogramming, it is crucial to understand how signaling pathways are regulated by these factors. This is the first report to show that c-Myc represses MEF-enriched miRNAs, such as miR-21, let-7a, and miR-29a, during reprogramming (Figure 16). Depleting miR-29a with inhibitors decreased p53 protein levels most likely by releasing p85a and CDC42 repression (Figure 19). In addition, depleting miR- 21 decreased ERK1/2 phosphorylation (Figure 20). Interestingly, we found that miR-21 inhibition reduced p53 protein levels and that inhibiting miR-29a also reduced ERK1/2 phosphorylation level. Both p53 and ERKl/2 signaling antagonizes reprogramming (Banito et al. 2009; Hong et al. 2009; Judson et al. 2009; Kawamura et al. 2009; Marion et al. 2009; Silva et al. 2008; Utikal et al. 2009). Blocking miR-21 and miR- 29a or knockdown of p53 and ERKl/2 can enhance reprogramming efficiency (Figures 19, 20). Thus, we propose that c-Myc facilitates reprogramming in part by suppressing the MEF- enriched miRNAs, miR-21 and miR-29a, that act as reprogramming barriers through induction of p53 protein levels and ERKl/2 activation (Figure 20E).

[0164] Forced expression of ES-specific miRNAs of the miR-290 family can replace c- Myc to promote reprogramming (Judson et al. 2009). c-Myc also binds the promoter region of the miR- 290 cluster (Chen et al. 2008; Judson et al. 2009). However, early expression of the c-Myc transgene is effective to initiate reprogramming but dispensable at the transition stage or later in mature iPS cells (Sridharan et al. 2009), where miR-290 clusters start to express. Therefore, it is unlikely that c-Myc promotes early stages of reprogramming through activating the miR-290 family.

[0165] We also found that expression level of MEF-enriched miRNAs, including miR- 29a, miR-21, miR- 143, and let-7a, decreases when c-Myc is introduced for reprogramming. c-Myc has a profound transcriptional effect (Wanzel et al. 2003) on miRNAs in promoting tumorigenesis (Chang et al. 2008, 2009) or sustaining the plunpotency ground state (Lin et al. 2009; Smith et al. 2010). Therefore, c-Myc repression of miRNA expression is the likely mechanism underlying reprogramming. miR-21 acts as positive mediator to enhance fibrogenic activity through the TGFpl (Liu et al. 2010) and ERKl/2 (Thum et al. 2008) pathways, both of which have been shown to influence reprogramming and the ES cell ground state (Ichida et al. 2009; Nichols et al. 2009; Ying et al. 2008). Notably, among validated miR-29a targets, the protein level of p53 is indirectly induced by miR-29a (Park et al. 2009). In addition, recent studies show that the Ink4-Arf/p53/p21 pathway compromises reprogramming and that p53 deficiency greatly enhances reprogramming efficiency (Banito et al. 2009; Hong et al. 2009; Judson et al. 2009; Kawamura et al. 2009; Marion et al. 2009; Utikal et al. 2009). Thus these signaling pathways are likely the primary barriers to the reprogramming process.

[0166] Depleting the c-Myc-targeted miRNAs, miR-21 and miR-29a, enhanced reprogramming efficiency about 2.4- fold to about threefold (Figure 16), suggesting that MEF-enriched miRNAs also function as reprogramming barriers. Let-7 inhibition has been recently reported to enhance reprogramming (Melton et al. 2010); however, in several attempts we observed a minor effect in reprogramming when let-7 was inhibited by antagomirs (Figure 16). Moreover, our data showed that the induction of p53 during reprogramming was compromised by miR-29a inhibition, enhancing reprogramming efficiency. Similarly, reprogramming can be greatly promoted by depleting either miR-21 or ERKl/2. c-Myc is a major contributor to the early stage of reprogramming and is not required to sustain the process at transition and late stages (Sridharan et al. 2009), indicating that c- Myc-regulated miRNAs may be employed to initiate high efficiency reprogramming. c-Myc reportedly directly binds to and represses the miR-29a promoter (Chang et al. 2008).

However, further studies are needed to understand how c-Myc regulates miR-21 expression. Our data show that c-Myc can be only partially replaced by depleting miR-21 and suggest that c-Myc has other functions in reprogramming. Thus the regulation of multiple pathways or wide repression of MEF-enriched miRNAs may be required to replace c-Myc function during reprogramming. In summary, here we show that c-Myc reduces the threshold for reprogramming by decreasing p53 levels and antagonizing ERKl/2 activation through miR- 21 and miR-29a down-regulation. Additionally, factors downstream from c-Myc may serve as targets for manipulation by siRNA, miRNA, or small molecules, to improve

reprogramming. These approaches could be extended to replace all four reprogramming factors.

[0167] Based on the information provided, one skilled in the art would be able to design and synthesize similar compounds (including peptides, nucleic acids, and small molecules) for tragetting the same barrier candidates for iPS cell reprogramming.

[0168] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A method of generating an induced pluripotent stem (iPS) cell comprising: a) contacting a somatic cell with a nuclear reprogramming factor; and b) contacting the cell of (a) with a microR A that alters RNA levels or activity within the cell, thereby generating an iPS cell.

2. The method of claim 1, wherein the microRNA or RNA is modified.

3. The method of claim 1 , wherein the microRNA is in a vector.

4. The method of claim 1, wherein the microRNA is miR-93, miR-135b, or a combination thereof.

5. The method of claim 1 , wherein the microRNA has a polynucleotide sequence comprising SEQ ID NO: 1.

6. The method of claim 1, wherein the microRNA regulates expression or activity of Wispl, Tgfbr2, Igfbp5, or a combination thereof.

7. The method of claim 1, wherein the nuclear reprogramming factor is encoded by a gene contained in a vector.

8. The method of claim 1, wherein the nuclear reprogramming factor is a SOX family gene, a KLF family gene, a MYC family gene, SALL4, OCT4, NANOG, LIN28, or a combination thereof.

9. The method of claim 1 , wherein the nuclear reprogramming factor is one or more of OCT4, SOX2, KLF4, C-MYC.

10. The method of claim 1 , wherein the somatic cell is contacted with the reprogramming factor prior to, simultaneously with or following contacting with the microRNA.

11. The method of claim 1 , wherein the somatic cell is a mammalian cell.

12. An induced pluripotent stem (iPS) cell produced using the method of claim 1.

13. An enriched population of induced pluripotent stem (iPS) cells produced by the method of claim 1.

14. A differentiated cell derived by inducing differentiation of the pluripotent stem cell produced by the method of claim 1.

15. A method of treating a subj ect comprising: a) generating an induced pluripotent stem (iPS) cell from a somatic cell of the subject by the method of claim 1 ; b) inducing differentiation of the iPS cell of step (a); and c) introducing the cell of (b) into the subject, thereby treating the condition.

16. The use of microRNA for increasing efficiency of generating of iPS cells.

17. The use of claim 16, wherein the microRNA is selected from the group consisting of miR-93, miR-135b, or a combination thereof.

18. A method of generating an induced pluripotent stem (iPS) cell comprising: a) contacting a somatic cell with a nuclear reprogramming factor; and b) contacting the cell of (a) with an inhibitor of microRNA, thereby generating an iPS cell.

19. The method of claim 18, wherein the microRNA is miR-223, miR-495, or a combination thereof.

20. The method of claim 18, wherein the nuclear reprogramming factor is a SOX family gene, a KLF family gene, a MYC family gene, SALL4, OCT4, NANOG, LIN28, or a combination thereof.

21. The method of claim 18, wherein the somatic cell comprises a fibroblast.

22. The method of claim 18, wherein the inhibitor is a small molecule, a peptide or a nucleic acid molecule.

23. The method of claim 22, wherein the nucleic acid molecule is an siRNA.

24. A method of generating an induced pluripotent stem (iPS) cell comprising: a) contacting a somatic cell with a nuclear reprogramming factor; and b) contacting the cell of (a) with an agonist of microRNA, thereby generating an iPS cell.

25. The method of claim 24, wherein the microRNA is miR-93, miR-135b, or a combination thereof.

26. The method of claim 24, wherein the agonist is a peptide, small molecule or a nucleic acid.

27. An induced pluripotent stem (iPS) cell produced using the method of claim 18 or 24.

28. A method of generating an induced pluripotent stem (iPS) cell comprising: contacting a cell with a microRNA or miRNA mimic that enhances reprogramming of an induced pluripotent stem (iPS) cell in combination with an agent that enhances

reprogramming of an induced pluripotent stem (iPS) cell.

29. The method of claim 28, wherein the agent is a small molecule, a peptide, a nucleic acid, a pluripotency transcription factor or a combination thereof.

30. The method of claim 29, wherein the nucleic acid is an siRNA.

31. The method of claim 29, wherein the agent is an miRNA inhibitor.

32. The method of claim 31 , wherein the miRNA inhibitor is an inhibitor of miRNA selected from the group consisting of miR-223, miR-543, miR-542-5p, miR-665, miR-142- 5p, miR-450b-5p, miR-184, miR-370, miR-431, miR-376a, miR-495, and a combination thereof.

33. The method of claim 31 , wherein the miRNA inhibitor is a peptide, small molecule, or nucleic acid molecule.

34. The method of claim 33, wherein the nucleic acid molecule is a siRNA.

35. The method of claim 28, wherein the agent is an NSAID or kinase inhibitor.

36. The method of claim 29, wherein the small molecule is selected from the group consisting of nabumetone, 4-hydroxytamoxifen (OHTM), corynanthine, moclobemide, nickel sulfate hexahydrate (N1SO₄), lectin, and a combination thereof.

37. The method of claim 29, wherein the small molecule is selected from the group consisting of nabumetone, 4-hydroxytamoxifen (OHTM), corynanthine, moclobemide, nickel sulfate hexahydrate (NiS0₄), lectin, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin, inhibitor of TGF- β, Acitretin, Retinoic acid p-hydroxyanilide, Diacerein, Phorbol 12-myristate 13-acetate, Progesterone, Tolazamide, 15-deoxy-A¹²' ¹⁴-prostaglandin J_2, (-)-Norepinephrine, β-estradiol, and a combination thereof.

38. The method of claim 29, wherein the small molecule is selected from the group consisting of:

, an

39. The method of claim 28, wherein the miRNA or miRNA mimic is selected from the group consisting of miR-135b, miR-302b, miR-124, miR-547, miR-701, miR-302d, miR-92a, miR-20a, miR-93, miR-491 , miR-367, or a combination thereof.

40. A method of generating an induced pluripotent stem (iPS) cell comprising: contacting a cell with an inhibitor of miRNA selected from the group consisting of miR-223, miR-543, miR-542-5p, miR-665, miR-142-5p, miR-450b-5p, miR-184, miR-370, miR-431, miR-376a, miR-495, or a combination thereof.

41. The method of claim 40, further comprising administering the cell a pluripotency transcription factor.

42. The method of claim 40, wherein the inhibitor of miRNA is a peptide, small molecule, or nucleic acid molecule.

43. The method of claim 42, wherein the nucleic acid molecule is a siR A.

44. A method of generating an induced pluripotent stem (iPS) cell comprising: contacting a cell with a miRNA or miRNA mimic selected from the group consisting of miR-135b, miR-302b, miR-124, miR-547, miR-701, miR-302d, miR-92a, miR-20a, miR- 93, miR-491, and miR-367 in combination with a miRNA inhibitor wherein the miRNA is selected from miR-223, miR-543, miR- 542-5p, miR-665, miR-142-5p, miR-450b-5p, miR-184, miR-370, miR-431, miR-376a, and miR-495.

45. The method of claim 44, further comprising contacting the cell with a small molecule that enhances reprogramming of an induced pluripotent stem (iPS) cell.

46. The method of claim 44, further comprising administering to the cell a pluripotency transcription factor.

47. The method of claim 44, wherein the inhibitor of miRNA is a peptide, small molecule, or nucleic acid molecule.

48. The method of claim 47, wherein the nucleic acid molecule is a siRNA.

49. A method of generating an induced pluripotent stem (iPS) cell comprising: contacting a cell with an agent that regulates expression of activity of Wisp 1, Tgfbr2, Igfbp5, or a combination thereof.

50. The method of claim 49, wherein the agent is a peptide, small molecule, or nucleic acid molecule.

51. The method of claim 50, wherein the nucleic acid molecule is an siR A.