Reprogramming cells

Abstract

The present invention provides for methods, compositions, and kits for producing an induced pluripotent stem cell from a non-pluripotent mammalian cell using a 3′-phosphoinositide-dependent kinase-1 (PDK1) activator or a compound that promotes glycolytic metabolism as well as other small molecules.

1 - 19 . (canceled) 
     
     
         20 . A method of inducing a non-pluripotent mammalian cell into an induced pluripotent stem cell, the method comprising:
 (a) introducing into the non-pluripotent cell a polynucleotide encoding an Oct4 polypeptide; and   (b) contacting the non-pluripotent cell with
 (i) a small molecule selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and a PDK1 activator (3′-phosphoinositide-dependent kinase-1), and 
 (ii) a HDAC (histone deacetylase) inhibitor, 
   thereby inducing the non-pluripotent mammalian cell into an induced pluripotent stem cell;   wherein contacting the non-pluripotent mammalian cell with the small molecule enhances reprogramming when compared to without the small molecule.   
     
     
         21 . The method of  claim 20 , wherein the small molecule PDK1 activator is:
 (a) an allosteric PDK1 activator; or   (b) (Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (“PS48”), (Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (“PS08”), 2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid, (Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (“12Z”), or (Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (“13Z”).   
     
     
         22 . The method of  claim 20 , wherein the HDAC inhibitor comprises sodium butyrate (NaB) or valproic acid (VPA); and/or wherein the non-pluripotent cell is a human cell. 
     
     
         23 . The method of  claim 20 , further comprising one or more of:
 (a) introducing into the non-pluripotent cell a polynucleotide encoding a Klf-4 polypeptide;   (b) introducing into the non-pluripotent cell a polynucleotide encoding a Sox-2 polypeptide; or   (c) introducing into the non-pluripotent cell a polynucleotide encoding a c-Myc polypeptide.   
     
     
         24 . A mixture comprising:
 (a) isolated mammalian cells;   (b) a small molecule selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator (3′-phosphoinositide-dependent kinase-1), and a small molecule PDK1 activator;   (c) a histone deacetylase (HDAC) inhibitor; and   (d) one or more vectors comprising a polynucleotide encoding one or more exogenous transcription factors selected from the group consisting of an Oct-4 polypeptide, a Klf-4 polypeptide, a c-myc polypeptide, and a Sox-2 polypeptide.   
     
     
         25 . The mixture of  claim 24 , wherein the cells are non-pluripotent cells, and/or human cells. 
     
     
         26 . The mixture of  claim 24 , wherein the small molecule PDK1 activator is:
 (a) an allosteric PDK1 activator; or   (b) (Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (“PS48”), (Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (“PS08”), 2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid, (Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (“12Z”), or (Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (“13Z”).   
     
     
         27 . The mixture of  claim 24 , further comprising a TGFβ receptor/ALK5 inhibitor. 
     
     
         28 . The mixture of  claim 26 , wherein the PDKI activator is PS48. 
     
     
         29 . The mixture of  claim 27 , wherein the TGFβ receptor/ALK5 inhibitor is A-83-01. 
     
     
         30 . A kit comprising a mixture of
 (A) a cell culture medium,   (B) a HDAC inhibitor, and   (C) a small molecule in an amount that enhances reprogramming of a non-pluripotent mammalian cell when used in a method of obtaining an induced mammalian pluripotent stem cell (iPSC) comprising:
 introducing into a non-pluripotent mammalian cell a polynucleotide encoding an Oct4 polypeptide; 
 (ii) contacting the non-pluripotent mammalian cell with
 (a) the small molecule, wherein the small molecule enhancing reprogramming is selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator (3′-phosphoinositide-dependent kinase-1), and 
 (b) the HDAC inhibitor; and 
 (c) culturing the contacted non-pluripotent mammalian cell of 
 
 (b) in the culture medium, thereby reprogramming the non-pluripotent mammalian cell to produce an induced mammalian pluripotent stem cell; wherein contacting the non-pluripotent mammalian cell with the small molecule enhances reprogramming when compared to without the small molecule. 
   
     
     
         31 . The kit of  claim 30 , further comprising one or more of the following:
 (a) a non-pluripotent mammalian cell;   (b) at least one exogenous transcription factor comprising Oct4, and optionally one or both of Sox2 and Klf; wherein the at least one exogenous transcription factor is a polypeptide, or a polynucleotide encoding the polypeptide; and/or   (c) one or more of a TGFβ receptor/ALK5 inhibitor, and a glycogen synthase kinase 3 (GSK3) inhibitor, wherein said method further comprises contacting the non-pluripotent mammalian cell with said one or more of a TGFβ receptor/ALK5 inhibitor, and a glycogen synthase kinase 3 (GSK3) inhibitor.   
     
     
         32 . The kit of  claim 30 , wherein:
 (a) the PDK1 activator is an allosteric PDK1 activator; or   (b) the PDK1 activator is (Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (PS48), (Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (PS08), 2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio) acetic acid, (Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (12Z), or (Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (13Z); or   (c) the HDAC inhibitor is sodium butyrate (NaB) or valproic acid (VPA).   
     
     
         33 . The kit of  claim 31 , wherein
 (a) the TGFβ receptor/ALK5 inhibitor is A-83-01; or   (b) the GSK3 inhibitor is CHIR99021.   
     
     
         34 . The kit of  claim 30 , wherein the non-pluripotent mammalian cell is
 (a) a human cell; or   (b) a somatic cell, a progenitor cell, or a fully differentiated cell.   
     
     
         35 . A method for screening for small molecules that induce reprogramming or dedifferentiation of non-pluripotent mammalian cells into pluripotent stem cells, the method comprising:
 (a) introducing an Oct4 polypeptide into non-pluripotent cells to generate transfected cells;   (b) contacting the transfected cells with a small molecule selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator (3′-phosphoinositide-dependent kinase-1), and a small molecule PDK1 activator;   (c) contacting the transfected cells with a library of different small molecules;   (d) screening the contacted cells for pluripotent stem cell characteristics; and   (e) correlating the development of stem cell characteristics with a particular small molecule from the library, thereby identifying a small molecule that stimulates reprogramming or dedifferentiation of non-pluripotent mammalian cells into pluripotent stem cells.   
     
     
         36 . The method of  claim 35 , wherein the small molecule PDK1 activator is:
 (a) an allosteric PDK1 activator; or   (b) (Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (“PS48”), (Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (“PS08”), 2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid, (Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (“12Z”), or (Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (“13Z”).   
     
     
         37 . The method of  claim 35 , further comprising introducing into the non-pluripotent cell at least one of the following:
 (a) one of a Klf-4 polypeptide, a c-myc polypeptide, and a Sox-2 polypeptide;   (b) one or both of a TGFβ receptor/ALK5 inhibitor and a MEK inhibitor;   (c) a HDAC inhibitor; and/or   (d) one or both of a GSK3 (glycogen synthase kinase 3) inhibitor and a LSD1 inhibitor (lysine-specific demethylase 1).   
     
     
         38 . The method of  claim 37 , wherein
 (a) the TGFβ receptor/ALK5 inhibitor is A-83-01;   (b) the MEK inhibitor is PD0325901;   (c) the LSD1 inhibitor is parnate; and/or   (d) the GSK3 inhibitor is CHIR99021.   
     
     
         39 . The method of  claim 35 , wherein the non-pluripotent cells are:
 (a) human cells;   (b) non-human mammalian cells;   (c) progenitor cells; and/or   (d) neural progenitor cells, skin progenitor cells or hair follicle progenitor cells.