Methods for promoting cell reprogramming

Abstract

The present invention is based on the seminal discovery that several kinases play important roles in barrier pathways in somatic cell reprogramming. The present invention provides that modulating expression or activity of these kinases can significantly promote or enhance cell reprogramming efficiency. Key kinases are identified and key regulation networks involving such kinases are also identified that may be advantageously targeted to significantly increase reprogramming efficiency as well as direct differentiation of induced pluripotent stem (iPS) cells.

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 an agent which modulates expression or activity of at least one kinase within the cell, thereby generating an IPS cell.   
     
     
         2 . The method of  claim 1 , wherein the agent stimulates or inhibits expression or activity of at least one kinase. 
     
     
         3 . The method of  claim 1 , wherein the agent is a small molecule, a peptide, a nucleic acid, a pluripotency transcription factor or a combination thereof. 
     
     
         4 . The method of  claim 3 , wherein the nucleic acid is an siRNA, shRNA, miRNA, Locked Nucleic Acid (LNA), antisense oligonucleotide, a chemically modified oligonucleotide, or a combination thereof. 
     
     
         5 . The method of  claim 1 , wherein the kinase phosphorylates cofilin. 
     
     
         6 . The method of  claim 1 , wherein the kinase is selected from the group of kinases listed in Table 1, Table 3, Table 4, Table 6, Table 7 or Table 8. 
     
     
         7 . The method of  claim 1 , wherein the kinase is selected from the group consisting of AURKA, P38, IP3K, BUB1B, IRAK3, BMPR2, IRAK2, LIMK2, BMPR1A, TESK1, PRKCA, MAPK1, SRPK1, RAGE, AATK, EPHA5, CDC2L6, DDR1, JAK1, EPHA1, SNF ILK, PIM2, FRAP1, DAPK2, TRIB3, DAPK3, CAMKV, STK25, MAP2K1, PAK7, STK24, CSNK2B, KHK, 6330514A18RIK, NPR2, BMP2K, EIF2AK2, MOS, NEK2, NEK6, PLK1, PLK2, RNASEL, SCYL1, TBK1, TLK2, UHMK1, RPS6KB1, AK3, DGKε, PIK3C2G, GALK2, NME1, GTF2F1, PAX8, PKIG, PIK3R5, GIT2, PIK3AP1, CNKSR3, PKIB, PER2, FASTKD5, and a combination thereof. 
     
     
         8 . The method of  claim 1 , wherein the kinase is selected from the group consisting of DGKε, PLK2, TESK1, BMP2K, BMPR2, MAPK1, and a combination thereof. 
     
     
         9 . The method of  claim 1 , wherein the kinase is selected from the group consisting of DGKε, PLK2, TESK1, and a combination thereof. 
     
     
         10 . The method of  claim 1 , wherein the agent is selected from the group of compounds listed in Table 5, Table 7, or Table 8. 
     
     
         11 . The method of  claim 1 , wherein the agent is selected from the group consisting of KN-62, Alsterpaullone, Arcyriaflavin A, IP3K inhibitor, ML-7, PP3, Syk inhibitor III, Aurora kinase inhibitor III, Sphingosine kinase inhibitor, TGF-13 RI inhibitor III, p38 MPA kinase inhibitor IV, and a combination thereof. 
     
     
         12 . The method of  claim 1 , wherein the agent is selected from the group consisting of AGL 2043, Cdk2/9 inhibitor, Cdk/Crk inhibitor, Fascaplysin, Rho kinase inhibitor IV, K-252a Nocardiopsis, UCN-01, PI 3-K inhibitor VIII, Reversine, and a combination thereof. 
     
     
         13 . The method of  claim 3 , wherein the small molecule is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         14 . The method of  claim 1 , further comprising contacting the cell of (a) with a second agent that enhances reprogramming of an induced pluripotent stem (iPS) cell. 
     
     
         15 . The method of  claim 14 , wherein the second agent is a small molecule, a peptide, a nucleic acid, a pluripotency transcription factor or a combination thereof. 
     
     
         16 . The method of  claim 14 , wherein the second agent is an microRNA, miRNA mimic, miRNA inhibitor, Locked Nucleic Acid (LNA), antisense oligonucleotide, a chemically modified oligonucleotide, or a combination thereof. 
     
     
         17 . The method of  claim 14 , wherein the second agent is an nonsteroidal anti-inflammatory drug (NSAID). 
     
     
         18 . The method of  claim 14 , wherein the second agent is selected from the group consisting of nabumetone, 4-hydroxytamoxifen (OHTM), corynanthine, moclobemide, nickel sulfate hexahydrate (NiSO 4 ), lectin, and a combination thereof. 
     
     
         19 . The method of  claim 14 , wherein the second agent is selected from the group consisting of nabumetone, 4-hydroxytamoxifen (OHTM), corynanthine, moclobemide, nickel sulfate hexahydrate (NiSO 4 ), 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-Δ 12,14 -prostaglandin J 2 (−)-Norepinephrine, β-estradiol, and a combination thereof. 
     
     
         20 . 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. 
     
     
         21 . The method of  claim 1 , wherein the nuclear reprogramming factor is one or more of OCT4, SOX2, KLF4, C-MYC. 
     
     
         22 . The method of  claim 1 , wherein the somatic cell is contacted with the reprogramming factor prior to, simultaneously with or following contacting the agent. 
     
     
         23 . The method of  claim 1 , wherein the somatic cell is a mammalian cell. 
     
     
         24 . The method of  claim 1 , wherein the somatic cell is a fibroblast. 
     
     
         25 . An enriched population of induced pluripotent stem (iPS) cells produced by the method of  claim 1 . 
     
     
         26 . A differentiated cell derived by inducing differentiation of the pluripotent stem cell produced by the method of  claim 1 . 
     
     
         27 . A method of treating a subject 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.   
     
     
         28 . The use of an agent inhibiting expression or activity of at least one kinase within the cell for increasing efficiency of generating of iPS cells. 
     
     
         29 . The use 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 use of  claim 29 , wherein the nucleic acid is an siRNA, shRNA, miRNA, Locked Nucleic Acid (LNA), antisense oligonucleotide, a chemically modified oligonucleotide, or a combination thereof. 
     
     
         31 . The use of  claim 28 , wherein the kinase is involved in a barrier pathway for somatic cell reprogramming. 
     
     
         32 . The use of  claim 28 , wherein the kinase is selected from the group of kinases listed in Table 1, Table 3, Table 4, Table 6, Table 7 or Table 8. 
     
     
         33 . The use of  claim 28 , wherein the kinase is selected from the group consisting of AURKA, P38, IP3K, BUB1B, IRAK3, BMPR2, IRAK2, LIMK2, BMPR1A, TESK1, PRKCA, MAPK1, SRPK1, RAGE, AATK, EPHA5, CDC2L6, DDR1, JAK1, EPHA1, SNF1LK, PIM2, FRAP1, DAPK2, TRIB3, DAPK3, CAMKV, STK25, MAP2K1, PAK7, STK24, CSNK2B, KHK, 6330514A18RIK, NPR2, BMP2K, EIF2AK2, MOS, NEK2, NEK6, PLK1, PLK2, RNASEL, SCYL1, TBK1, TLK2, UHMK1, RPS6KB1, AK3, DGKε, PIK3C2G, GALK2, NME1, GTF2F1, PAX8, PKIG, PIK3R5, GIT2, PIK3AP1, CNKSR3, PKIB, PER2, FASTKD5, and a combination thereof. 
     
     
         34 . The use of  claim 28 , wherein the kinase is selected from the group consisting of DGKε, PLK2, TESK1, BMP2K, BMPR2, MAPK1, and a combination thereof. 
     
     
         35 . The use of  claim 28 , wherein the kinase is selected from the group consisting of DGKs, PLK2, TESK1, and a combination thereof. 
     
     
         36 . The use of  claim 28 , wherein the agent is selected from the group of compounds listed in Table 5, Table 7, or Table 8. 
     
     
         37 . The use of  claim 28 , wherein the agent is selected from the group consisting of KN-62, Alsterpaullone, Arcyriaflavin A, IP3K inhibitor, ML-7, PP3, Syk inhibitor III, Aurora kinase inhibitor III, Sphingosine kinase inhibitor, TGF-β RI inhibitor III, p38 MPA kinase inhibitor IV, and a combination thereof. 
     
     
         38 . The use of  claim 28 , wherein the agent is selected from the group consisting of AGL 2043, Cdk2/9 inhibitor, Cdk/Crk inhibitor, Fascaplysin, Rho kinase inhibitor IV, K-252a Nocardiopsis, UCN-01, PI 3-K inhibitor VIII, Reversine, and a combination thereof. 
     
     
         39 . The use of  claim 29 , wherein the small molecule is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         40 . A method of identifying barrier pathways in somatic cell reprogramming in a subject:
 a) generating a lentiviral shRNA library, wherein the lentiviral shRNA library targets selected genes of the subject;   b) preparing shRNA lentiviruses using cell culture; and   c) contacting the shRNA lentiviruses with a cell of a reporter cell line for somatic cell reprogramming.   
     
     
         41 . The method of  claim 40 , wherein the lentiviral shRNA library targets kinase genes of an entire kinome of the subject. 
     
     
         42 . The method of  claim 40 , wherein the subject is a mammalian. 
     
     
         43 . The method of  claim 40 , wherein the subject is a mouse. 
     
     
         44 . The method of  claim 40 , wherein the reporter cell line for somatic cell reprogramming is a somatic cell line, wherein the cells comprise a nucleic acid sequence encoding a promoter of an endogenous nuclear reprogramming factor operably linked to a reporter gene. 
     
     
         45 . The method of  claim 44 , the reporter gene encodes luciferase (LUC), β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), β-galactosidase (β-gal), and xanthine guanine phophoribosyltransferase (XGPRT), an affinity or epitope tag, or a fluorescent protein. 
     
     
         46 . The method of  claim 45 , wherein the reporter gene encodes a fluorescent protein. 
     
     
         47 . The method of  claim 46 , wherein the fluorescent protein is green fluorescent protein (GFP) or enhanced green fluorescent protein (eGFP). 
     
     
         48 . The method of  claim 44 , wherein the promoter is an Oct4, Klf-4, c-Myc, Sox2 or Nanog promoter. 
     
     
         49 . The method of  claim 40 , wherein the reporter cell line for somatic cell reprogramming comprises Oct4-GFP mouse embryonic fibroblast (MEF). 
     
     
         50 . The method of  claim 40 , wherein the cell culture comprises 293FT cells. 
     
     
         51 . A method of generating an induced pluripotent stem (iPS) cell comprising:
 contacting a cell with an agonist of microRNA in combination with an agent which modulates expression or activity of at least one kinase within the cell, thereby generating an iPS cell.   
     
     
         52 . The method of  claim 51 , wherein the agonist of microRNA comprises a microRNA or miRNA mimic that enhances reprogramming of an induced pluripotent stem (iPS) cell. 
     
     
         53 . The method of  claim 51 , wherein the agonist is a small molecule, a peptide, a nucleic acid, a pluripotency transcription factor or a combination thereof. 
     
     
         54 . A method of generating an induced pluripotent stem (iPS) cell comprising:
 contacting a cell with an microRNA inhibitor in combination with an agent which modulates expression or activity of at least one kinase within the cell, thereby generating an iPS cell.   
     
     
         55 . The method of  claim 54 , wherein the microRNA inhibitor is a small molecule, a peptide, a nucleic acid, a pluripotency transcription factor or a combination thereof. 
     
     
         56 . The method of  claim 55 , wherein the nucleic acid is an siRNA, shRNA, Locked Nucleic Acid (LNA), antisense oligonucleotide, a chemically modified oligonucleotide, or a combination thereof. 
     
     
         57 . The method of  claim 4 ,  16 ,  56  or the use of  claim 30 , wherein the chemically modified oligonucleotide comprises a 2′-deoxyribonucleotide, 2′-O-methyl ribonucleotide, 2′-fluoro ribonucleotide, 2′-amino ribonucleotide, 2′-O-amino ribonucleotide, 2′-C-allyl ribonucleotide, 2′-O-allyl ribonucleotide, 2′-methoxyethyl ribonucleotide, 5′-C-methyl ribonucleotide, or a combination thereof. 
     
     
         58 . A method of screening for a modulator of somatic cell reprogramming, comprising:
 a) contacting a cell of a reporter cell line for somatic cell reprogramming with one or more nuclear reprogramming factors;   b) contacting the cell of (a) with a test agent;   c) detecting a reporter of the cell which is indicative of pluripotency; and   d) comparing the level of the reporter with that of a corresponding cell not contacted with the test agent, wherein a test agent that increases or decreases the level of the reporter as compared to the level of the reporter in the corresponding cell is identified as a modulator of somatic cell reprogramming.   
     
     
         59 . The method of  claim 58 , wherein the test agent is a small molecule, a peptide, a nucleic acid, a pluripotency transcription factor or a combination thereof. 
     
     
         60 . The method of  claim 59 , wherein the nucleic acid is an siRNA, shRNA, miRNA, Locked Nucleic Acid (LNA), antisense oligonucleotide, a chemically modified oligonucleotide, or a combination thereof. 
     
     
         61 . The method of  claim 59 , wherein the test agent is a small molecule. 
     
     
         62 . The method of  claim 58 , wherein the reporter cell line for somatic cell reprogramming is mammalian. 
     
     
         63 . The method of  claim 58 , wherein the reporter cell line for somatic cell reprogramming is a somatic cell line, wherein the cells comprise a nucleic acid sequence encoding a promoter of an endogenous nuclear reprogramming factor operably linked to a reporter gene. 
     
     
         64 . The method of  claim 63 , the reporter gene encodes luciferase (LUC), β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), β-galactosidase (β-gal), and xanthine guanine phophoribosyltransferase (XGPRT), an affinity or epitope tag, or a fluorescent protein. 
     
     
         65 . The method of  claim 64 , wherein the reporter gene encodes a fluorescent protein. 
     
     
         66 . The method of  claim 65 , wherein the fluorescent protein is green fluorescent protein (GFP) or enhanced green fluorescent protein (eGFP). 
     
     
         67 . The method of  claim 63 , wherein the promoter is an Oct4, Klf-4, c-Myc, Sox2 or Nanog promoter. 
     
     
         68 . The method of  claim 58 , wherein the reporter cell line for somatic cell reprogramming is Oct4-GFP mouse embryonic fibroblast (MEF). 
     
     
         69 . The method of  claim 58 , wherein the test agent activates or inhibits the expression or activity of a kinase. 
     
     
         70 . The method of  claim 58 , wherein the kinase is selected from the group of kinases listed in Table 1, Table 3, Table 4, Table 6, Table 7 or Table 8. 
     
     
         71 . The method of  claim 58 , wherein the nuclear reprogramming factor is one or more of Oct4, Klf4, c-Myc, Sox2 and Nanog. 
     
     
         72 . The method of  claim 58 , wherein the method is conducted in a multi-well format. 
     
     
         73 . The method of  claim 58 , wherein the reporter cell line is cultured continuously for 1, 2, 3, or 4 days before contact with the test agent. 
     
     
         74 . The method of  claim 58 , wherein the test agent inhibits expression or activity of a kinase. 
     
     
         75 . The method of  claim 58 , wherein the reporter is detected about 10 days after the cell line is contacted with the one or more nuclear reprogramming factors.