Immortalization of Epithelial Cells and Methods of Use

• US 20150099267A1
• Filed: 09/26/2014
• Published: 04/09/2015
• Est. Priority Date: 11/12/2010
• Status: Active Grant

• Current Assignee: Georgetown University
• Original Assignee: Georgetown University
• Inventors: Schlegel, Richard, Liu, Xuefeng
• Granted Patent: US 9,657,272 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 2500/05 Inorganic components
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides or bases
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0625 Epidermal cells, skin cells; Cells of the oral mucosa
• CPC Class Codes: C12N 5/0631 Mammary cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0683 Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells spermatogonia C12N5/061
• CPC Class Codes: C12N 5/0688 Cells from the lungs or the respiratory tract
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/106 : Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: G01N 33/5091 : for testing the pathological state of an organism

Abstract

The present invention is directed towards methods of culturing non-keratinocyte epithelial cells, with the methods comprising culturing non-keratinocyte epithelial cells in the presence of feeder cells and a calcium-containing medium while inhibiting the activity of Rho kinase (ROCK) in the feeder cell, the non-keratinocyte epithelial cells or both during culturing.

Claims

1-58. -58. (canceled)

59. A method of continuously culturing non-keratinocyte epithelial (NKE) cells, the method comprising

a) culturing the NKE cells in the presence of feeder cells and a calcium-containing medium, and

b) inhibiting the activity of Rho kinase (ROCK) in the feeder cell, the NKE cells or both during culturing.

60. The method of claim 59, wherein the NKE cells are selected from the group consisting of squamous cells, columnar cells, adenomatous cells and transitional epithelial cells.

61. The method of claim 60, wherein the NKE cells are selected from the group consisting of prostate epithelial cells, mammary epithelial cells, hepatocytes, pancreatic islet cells, pulmonary epithelial cells, kidney epithelial cells, bladder epithelial cells, stomach epithelial cells, large intestinal epithelial cells, small intestinal epithelial cells, urethral epithelial cells, retinal epithelial cells, neuroendocrine epithelial cells, testicular epithelial cells, ovarian epithelial cells, thyroid epithelial cells, parathyroid epithelial cells, adrenal epithelial cells, thymus epithelial cells, gall bladder epithelial cells and pituitary epithelial cells.

62. The method of claim 59, wherein the calcium-containing medium comprises serum or a serum replacement.

63. The method of claim 59, wherein the feeder cells are proliferating or non-proliferating fibroblasts.

64. The method of claim 59, wherein inhibiting the activity of ROCK comprises culturing the NKE cells in the presence of a small molecule inhibitor of Rho kinase 1 (ROCK 1), Rho kinase 2 (ROCK 2) or both.

65. The method of claim 64, wherein the small molecule ROCK 1 or ROCK 2 inhibitor is selected from the group consisting of Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

66. The method of claim 59, wherein inhibiting the activity of ROCK comprises genetic manipulation of the expression of Rho kinase 1 (ROCK 1), Rho kinase 2 (ROCK 2) or both.

67. The method of claim 66, wherein inhibiting the activity of ROCK 1 comprises applying to the cells an RNAi molecule directed to the nucleotide sequence of ROCK 1.

68. The method of claim 66, wherein inhibiting the activity of ROCK 1 comprises applying to the cells an RNAi molecule directed to the nucleotide sequence of ROCK 2.

69. The method of claim 59, further comprising

c) passaging the NKE cells after inhibiting ROCK, and

d) placing the passaged NKE cells in an environment in which ROCK is not being inhibited.

70. The method of claim 69, wherein the environment in which ROCK is not being inhibited is a cell culture environment.

71. The method of claim 70, wherein the cell culture environment in which ROCK is not being inhibited is a three-dimensional cell culture environment.

72. The method of claim 70, further comprising inserting a transgene into the passaged NKE cells in which ROCK is not being inhibited.

86. The method of claim 59, further comprising inserting a transgene into the NKE cells when the activity of ROCK is being inhibited.

73. A population of conditionally immortalized non-keratinocyte epithelial (NKE) cells derived from a population of non-immortalized NKE primary cells, wherein the conditionally immortalized NKE cells retain the ability to differentiate into the type of cells from which the non-immortalized NKE primary cells were isolated.

74. The cell population of claim 73, wherein the conditionally immortalized NKE cells are derived from a population of normal NKE primary cells or tumor NKE primary cells.

75. The cell population of claim 73, wherein the conditionally immortalized NKE cells are derived from cells selected from the group consisting of primary squamous cells, primary columnar cells, primary adenomatous cells and primary transitional epithelial cells.

76. The cell population of claim 74, wherein the conditionally immortalized NKE cells are capable of differentiating into cells selected from the group consisting of prostate epithelial cells, mammary epithelial cells, hepatocytes, pancreatic islet cells, pulmonary epithelial cells, kidney epithelial cells, bladder epithelial cells, stomach epithelial cells, large intestinal epithelial cells, small intestinal epithelial cells, urethral epithelial cells, retinal epithelial cells, neuroendocrine epithelial cells, testicular epithelial cells, ovarian epithelial cells, thyroid epithelial cells, parathyroid epithelial cells, adrenal epithelial cells, thymus epithelial cells, gall bladder epithelial cells and pituitary epithelial cells, after the conditionally immortalized NKE cells are placed in a subsequent environment that promotes differentiation thereto.

77. A method of identifying a candidate treatment for a subject in need of treatment of a condition that is marked by the presence of abnormal non-keratinocyte epithelial (NKE) cells, the method comprising

a) obtaining an initial sample of abnormal NKE cells from the subject,

b) culturing the initial sample of abnormal NKE cells in the presence of feeder cells, a calcium-containing medium and at least one Rho kinase (ROCK) inhibitor, to produce an experimental population of abnormal NKE cells in vitro,

c) determining a response profile of at least a portion of the experimental population of abnormal NKE cells in vitro, and

d) identifying a candidate treatment for the subject based on the determined response profile.

78. The method of claim 77, wherein the response profile is at least partially determined by a procedure selected from the group consisting of identifying the sequence of at least one portion of DNA extracted from the experimental population of abnormal NKE cells in vitro, identifying at least one mRNA that is produced in the experimental population of abnormal NKE cells in vitro, identifying at least one mRNA that is not produced in the experimental population of abnormal NKE cells in vitro, identifying one or more proteins that the experimental population of abnormal NKE cells in vitro express, and identifying one or more proteins that the experimental population of abnormal NKE cells in vitro do not express.

79. The method of claim 77, wherein the response profile is at least partially determined by subjecting the experimental population of abnormal NKE cells in vitro to a therapy and determining the therapeutic index of the therapy on the experimental population of abnormal NKE cells in vitro.

80. The method of claim 77, wherein determining the response profile comprises performing a functional cellular assay on the experimental population of abnormal NKE cells in vitro.

81. The method of claim 77, wherein the initial population sample of NKE cells are circulating tumor cells (CTCs) or cells from a tumor metastasis.

82. The method of claim 77, wherein initial sample of abnormal NKE cells are obtained from the subject by a needle biopsy.

83. A method of identifying an abnormal non-keratinocyte epithelial (NKE) cell in a subject, the method comprising

a) culturing an initial sample of candidate abnormal NKE cells isolated from the subject in the presence of feeder cells, a calcium-containing medium and at least one Rho kinase (ROCK) inhibitor, to produce an experimental population of candidate abnormal NKE cells in vitro,

b) determining a tissue origin profile of at least a portion of the experimental population of candidate abnormal NKE cells in vitro, and

c) comparing at least one feature of the experimental population of candidate abnormal NKE cells to the same feature of normal NKE cells that are obtained from the same tissue as that of the determined tissue origin profile of the experimental population of candidate abnormal NKE cells to determine if there is a difference between the experimental population of candidate abnormal NKE cells and the normal NKE cells,

d) wherein a difference indicates in the experimental population of candidate abnormal NKE cells are abnormal compared to normal NKE cells.

84. The method of claim 83, wherein the initial sample of candidate abnormal NKE cells are obtained from the circulation of the subject.

85. The method of claim 83, wherein the tissue origin profile is at least partially determined by a procedure selected from the group consisting of identifying at least one mRNA that is produced in the experimental population of candidate abnormal NKE cells in vitro, identifying at least one mRNA that is not produced in the experimental population of candidate abnormal NKE cells in vitro, identifying one or more proteins that the experimental population of candidate abnormal NKE cells in vitro express and identifying one or more proteins that the experimental population of candidate abnormal NKE cells in vitro do not express.