Hormone responsive tissue culture system and uses thereof

Abstract

The invention provides tissue culture system for primary cells (e.g. normal mammalian primary epithelial progenitors). This system includes: a) a serum-free, chemically defined cell culture media; and, b) methods for isolation and in vitro long-term propagation of primary cells (e.g. primary epithelial cells). Primary cells so isolated and cultured can be kept undifferentiated and proliferate for many weeks (>15 weeks) or population doubling (>35 PD) without senescence, or any detectable genetic alterations. Upon changing media/culture conditions, these cells can be induced to differentiate. The invention also provides methods to transform normal primary cells so cultured into “cancer stem cells.” The genetically defined cancer stem cell tumor model mimics the behavior of the disease closely, e.g., the cells are invasive, hormone responsive and metastatic when injected into mice. The tumor cells express genes that are specific to cancer stem cells identified in patient samples.

1 . A culture medium comprising:
 (a) one or more lipid synthesis precursors;   (b) one or more protein synthesis precursors;   (c) one or more carbohydrate synthesis and energy metabolism precursors;   (d) one or more monovalent and/or divalent cations, ions, trace metals and enzyme cofactors/vitamins;   (e) one or more agents that induce increased intracellular 3′-5′ cyclic adenosine monophosphate (cAMP) levels; and   (f) insulin,   wherein the medium supports undifferentiated growth and/or proliferation of primary epithelial progenitor cells for at least about 4 weeks or at least about 15 population doubling (PD) in vitro, without a significant decrease in differentiation potential.   
     
     
         2 . The medium of  claim 1 , also comprising one or more of:
 (a) one or more antioxidants;   (b) one or more nucleotide salvage pathway synthesis precursors;   (c) one or more buffers;   (d) one or more carrier proteins;   (e) one or more detergents, and   (f) one or more non-insulin hormones and growth factors,   
     
     
         3 . The medium of  claim 1 , wherein
 (a) the one or more antioxidants comprise at least one antioxidant selected from the group consisting of: glutathione (reduced), dithiothreitol (DTT), vitamin E, vitamin K3, vitamin D2 or calciferol, niacin, niacinamide, and ascorbic acid;   (b) the one or more nucleotide salvage pathway synthesis precursors is at least one member selected from the group consisting of: hypoxanthine, xanthine, adenine, guanine, and thymidine;   (c) wherein the one or more lipid synthesis precursors is at least one member selected from the group consisting of: cholesterol, linoleic acid, lipoic acid, and o-phosphoryl ethanolamine; and   (d) the one or more hormones is at least one selected from the group consisting of: progesterone, testosterone, hydrocortisone, and estrogen.   
     
     
         4 . The medium of  claim 1 , comprising at least 10 of the components listed as optional in Table II or at least 10 of the components listed in the medium of Example XIX. 
     
     
         5 . The medium of  claim 1 , comprising the at least one component listed in Table XI (Medium 199) at between 0.3 and 3 times the listed concentration. 
     
     
         6 . The medium of  claim 4 , comprising all or at least 90% of the components listed in Table XI (Medium 199) at between 0.3 and 3 times the listed concentrations. 
     
     
         7 . The medium of  claim 6 , further comprising at least one component listed in Table XII (F12 Medium) at between 0.3 and 3 times the listed concentration. 
     
     
         8 . The medium of  claim 7 , comprising at least 90% of the components listed in Table XII (F12), optionally at concentrations varying from between 0.3 and 3 times the listed concentrations. 
     
     
         9 . The medium of  claim 3 , wherein the medium comprises 30%-70% by volume of F12 medium. 
     
     
         10 . The medium of  claim 3 , wherein the medium comprises about 50% (by volume) of F12 medium and about 50% (by volume) of Medium 199. 
     
     
         11 . A method of isolating mammalian primary cells, comprising:
 (a) providing tissue containing primary cells from a mammal;   (b) plating primary cells obtained from the tissue on a tissue culture container with mixed (+/−) charge surface, in culture medium for between 3 days to 4 weeks with medium change at reasonable frequency, wherein said medium change optionally occurs at least once every 12 hours to 3 days; and   (c) harvesting primary cells and transferring the harvested the primary cells to a new tissue culture container with mixed (+/−) charge surface in the medium of  claim 1 , thereby isolating the primary cells from the mammal.   
     
     
         12 . A primary cell isolated by the method of  claim 11 . 
     
     
         13 . The primary cell of  claim 12 , which is a primary glandular epithelial cell. 
     
     
         14 . A tumorigenic cell derived from the primary cell of  claim 12 , wherein the tumorigenic cell expresses:
 (a) a telomerase catalytic subunit;   (b) a first polypeptide that functions in the same signaling pathway(s) as does the SV40 large T antigen; and   (c) a second polypeptide that functions in the same signaling pathway as does the mutant H-ras oncogene product.   
     
     
         15 - 16 . (canceled) 
     
     
         17 . A method for long-term culturing and maintenance of mammalian hormone-responsive primary cells, the method comprising:
 (a) isolating primary cells from a mammal using the method of  claim 11 ;   (b) culturing and subculturing primary cells isolated in (a) in culture medium comprising
 i. one or more lipid synthesis precursors; 
 ii. one or more protein synthesis precursors; 
 iii. one or more carbohydrate synthesis and energy metabolism precursors; 
 iv. one or more monovalent and/or divalent cations, ions, trace metals and enzyme cofactors/vitamins; 
 v. one or more agents that induce increased intracellular 3′-5′ cyclic adenosine monophosphate (cAMP) levels; and 
 vi. insulin, 
   wherein the medium supports undifferentiated growth and/or proliferation of primary epithelial progenitor cells for at least about 4 weeks or at least about 15 population doubling (PD) in vitro, without a significant decrease in differentiation potential.   
     
     
         18 - 27 . (canceled) 
     
     
         28 . An in vitro method of identifying an agent which reduces proliferation of tumorigenic cells, comprising:
 (a) contacting tumorigenic cells of  claim 14  with a candidate agent to be assessed for its ability to reduce proliferation of the tumorigenic cells, under conditions appropriate for the candidate agent to enter cells;   (b) determining the extent to which proliferation of the tumorigenic cells occurs in the presence of the candidate agent to be assessed; and   (c) comparing the extent determined with the extent to which proliferation of the tumorigenic cells occurs under the same conditions, but in the absence of the candidate agent to be assessed,   wherein if proliferation occurs to a lesser extent in the presence of the candidate agent to be assessed than in its absence, the candidate agent to be assessed is an agent which reduces proliferation of tumorigenic human somatic cells.   
     
     
         29 - 41 . (canceled) 
     
     
         42 . Cell culture medium comprising:
 (a) one or more lipid synthesis precursors;   (b) one or more protein synthesis precursors;   (c) one or more carbohydrate synthesis and energy metabolism precursors;   (d) one or more monovalent and/or divalent cations, ions, trace metals and enzyme cofactors/vitamins; and   (e) insulin,   wherein the medium supports undifferentiated growth and/or proliferation of primary epithelial progenitor cells transformed by telomerase catalytic subunit for at least about 4 weeks or at least about 15 population doubling (PD) in vitro, without a significant decrease in differentiation potential.   
     
     
         43 . Cell culture medium comprising
 (a) None or trace amount of calf/fetal bovine serum;   (b) Epidermal Growth Factor at a final concentration of about 10 ng/mL;   (c) Hydrocortisone at a final concentration of about 0.5 μg/mL;   (d) Cholera toxin at a final concentration of about 100 ng/mL;   (e) Insulin at a final concentration of about 20 μg/mL,   wherein the medium supports undifferentiated growth and/or proliferation of human primary breast cells for at least about 4 weeks or at least about 15 population doubling (PD) in vitro, without a significant decrease in differentiation potential.   
     
     
         44 . Cell culture medium comprising
 (a) none or trace amount of calf/fetal bovine serum;   (b) epidermal growth factor at a final concentration of about 10 ng/mL;   (c) hydrocortisone at a final concentration of about 0.5 μg/mL;   (d) cholera toxin at a final concentration of about 25 ng/mL;   (e) insulin at a final concentration of about 20 μg/mL;   wherein the medium supports undifferentiated growth and/or proliferation of human ovarian & fallopian tube epithelial cells for at least about 4 weeks or at least about 15 population doubling (PD) in vitro, without a significant decrease in differentiation potential.   
     
     
         45 . Cell culture medium comprising
 (a) calf/fetal bovine serum at a final concentration of about 0.1%-0.2% by volume;   (b) epidermal growth factor at a final concentration of about 10 ng/mL;   (c) hydrocortisone at a final concentration of about 0.5 μg/mL;   (d) cholera toxin at a final concentration of about 25 ng/mL;   (e) insulin at a final concentration of about 20 μg/mL;   wherein the medium supports undifferentiated growth and/or proliferation of human mesothelial cells for at least about 4 weeks or at least about 15 population doubling (PD) in vitro, without a significant decrease in differentiation potential.   
     
     
         46 - 51 . (canceled)