COMPOSITIONS AND METHODS OF GENERATING REPROGRAMMED ADIPOCYTE CELLS AND METHODS OF USE THEREFORE

• US 20120219530A1
• Filed: 09/26/2011
• Published: 08/30/2012
• Est. Priority Date: 04/15/1999
• Status: Abandoned Application

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Lum, David H., Ahfeldt, Tim D., Cowan, Chad
• Application Number:
  US13/245,716
• Publication Number:
  US 20120219530A1
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: B81B 2203/0353 Holes
• CPC Class Codes: B81B 2203/0384 sloped profile
• CPC Class Codes: B81C 1/00103 Structures having a predefined profile, e.g. sloped or rounded grooves

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.

Claims

1-46. -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 PPARγ

2, 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, PPARγ

2, 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.

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.

71. A pharmaceutical composition comprising a reprogrammed adipocyte according to claim 58 in a pharmaceutically acceptable excipient.

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 PPARγ

2 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.

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 PPARγ

2, 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.