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.

What is claimed is: 
     
         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 breast 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 the components listed in Table II. 
     
     
         5 . A method of isolating mammalian primary cells, comprising:
 (a) providing tissue containing primary cells from a mammal;   (b) separating or enriching primary cells from other cells in the tissue;   (c) plating primary cells produced in (b) on a tissue culture container with mixed (+-) charge surface, in culture medium for about a week with frequent (e.g. daily) medium change; and   (d) harvesting primary cells by using high concentration of trypsin at about 0.15%, 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.   
     
     
         6 - 15 . (canceled) 
     
     
         16 . A method of producing tumorigenic cells from corresponding normal primary cells, the method comprising:
 (a) isolating normal primary cells using the method of  claim 5 , thereby producing . isolated normal primary cells;   (b) introducing into isolated normal primary cells produced in (a) exogenous DNA which, when expressed in the isolated cells, transforms the cells into tumorigenic cells which form tumors in immunocompromised mice into which they are introduced.   
     
     
         17 . The method of  claim 16 , wherein the exogenous DNA comprises:
 (a) DNA that encodes human telomerase catalytic subunit;   (b) DNA that encodes a first oncogene or inhibitor of a first tumor suppressor (TS) gene;   and   (c) DNA that encodes a second oncogene or inhibitor of a second TS gene, wherein the first oncogene the first TS gene and the second oncogene second TS gene function in two distinct biochemical pathways in human somatic cells.   
     
     
         18 . The method of  claim 17 , wherein the DNA of (b) is cDNA that encodes the H-ras oncogene product and the DNA of (c) is cDNA that encodes the SV40 large T oncogene product. 
     
     
         19 .- 28 . (canceled)