Compositions and methods of generating reprogrammed adipocyte cells and methods of use therefore

Abstract

The invention provides therapeutic compositions comprising reprogrammed adipocyte cells for use as disease models, therapeutic compositions comprising reprogrammed adipocyte cells for the treatment of conditions characterized by a reduction in cell number or tissue mass, and methods of generating such cells.

1 - 46 . (canceled) 
     
     
         47 . A method for generating a reprogrammed adipocyte, the method comprising
 exogenously expressing in a pluripotent stem cell one or more adipogenic transcription factor polypeptides; and   contacting the cell with one or more of insulin, rosiglitazone, dexamethasone and isobutylmethylxanthine, thereby generating a reprogrammed adipocyte.   
     
     
         48 . The method of  claim 47 , wherein the adipogenic transcription factor polypeptide is selected from the group consisting of PPARγ2, CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBPβ, C/EBPδ, C/EBPα and CDEC. 
     
     
         49 . The method of  claim 48 , wherein the pluripotent stem cell is selected from the group consisting of induced pluripotent stem cell, human embryonic stem cell, mesenchymal stem cell, adipocyte-derived mesenchymal stem cell, bone marrow derived stem cell and other mesenchymal stem cell. 
     
     
         50 . The method of  claim 47 , wherein the induced pluripotent stem cell is derived from a somatic cell. 
     
     
         51 . The method of  claim 50 , wherein the somatic cell is selected from the group consisting of adipocyte, keratinocyte, epidermal cell, fibroblast, hematopoietic cell, peripheral blood mononuclear cell and their progenitor cells. 
     
     
         52 . The method of  claim 47 , wherein pluripotency is induced by expression of one or more of OCT4, SOX2 and either cMYC and KLF4 or NANOG and LIN28 in a somatic cell. 
     
     
         53 . The method of  claim 47 , wherein the pluripotent stem cell is contacted in vitro. 
     
     
         54 . The method of  claim 47 , wherein the method further comprises identifying an adipocyte phenotype by detecting an increase in an adipocyte marker, an adipocyte morphology, or adipocyte function that is not detectably expressed or expressed only nominally in a corresponding control cell. 
     
     
         55 . The method of  claim 47 , wherein the reprogrammed adipocyte expresses one or more adipocyte markers selected from the group consisting of CIDEC, FABP4, PPARγ2, adiponectin, leptin, and perilipin. 
     
     
         56 . The method of  claim 47 , wherein the reprogrammed adipocyte comprises lipid droplets. 
     
     
         57 . The method of  claim 47 , wherein the reprogrammed adipocyte responds to insulin, has lipolytic activity, displays de novo synthesis of fatty acids and/or incorporates free fatty acids. 
     
     
         58 . A reprogrammed adipocyte generated according to the method of  claim 47 . 
     
     
         59 . The reprogrammed adipocyte of  claim 58 , wherein the cell comprises a genetic alteration associated with a disease selected from the group consisting of Type 2 diabetes mellitus, insulin resistance, obesity, lipodystrophy, metabolic disorders, cardiac disease, early-onset myocardial infarction and laminopathies. 
     
     
         60 . The reprogrammed adipocyte of  claim 58 , wherein the cell comprises a single nucleotide polymorphism listed in Table 1, Table 2, or Table 3. 
     
     
         61 . The reprogrammed adipocyte of  claim 58 , wherein the cell is derived from an FPLD2 patient, a patient with type 2 diabetes, or a patient with early onset myocardial infarction. 
     
     
         62 . The reprogrammed adipocyte of  claim 58 , wherein the cell from the FPLD2 subject has a LMNA mutation at R482W, H506D, R399H, R582H, T655fxX49, or L387V L421P. 
     
     
         63 . A reprogrammed adipocyte that exogenously expresses an adipogenic transcription factor polypeptide selected from the group consisting of PPARγ, CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBPβ, C/EBPδ C/EBPα, and CDEC, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function. 
     
     
         64 . A reprogrammed adipocyte that exogenously expresses a PPARγ2 or C/EBPα polypeptide, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function. 
     
     
         65 . A reprogrammed adipocyte that exogenously expresses a polypeptide selected from the group consisting of C/EBPα, C/EBPβ and C/EBPδ, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function. 
     
     
         66 . A method for identifying a therapeutic agent, the method comprising contacting the reprogrammed adipocyte of  claim 65  with a candidate agent and identifying an alteration in a disease marker. 
     
     
         67 . A method of ameliorating cell or tissue loss in a subject in need thereof, the method comprising delivering to the subject a cell generated according to the method of  claim 47 . 
     
     
         68 . The method of  claim 67 , wherein the cell or tissue loss is associated with trauma, cell death, or a congenital defect. 
     
     
         69 . A collection of at least two expression vectors, wherein each vector comprises a distinct nucleic acid sequence encoding a polypeptide selected from the group consisting of PPARγ2, C/EBPα, C/EBPβ, C/EBPδ, SREBP1c, CREB1 and KROX20. 
     
     
         70 . A host cell comprising one or more of the expression vectors of  claim 69 . 
     
     
         71 . A pharmaceutical composition comprising a reprogrammed adipocyte according to  claim 58  in a pharmaceutically acceptable excipient.