WO2015192035A1 - Compositions and methods for immortalization of epithelial cells - Google Patents

Abstract

The present invention is directed towards methods of culturing epithelial cells, with the methods comprising culturing epithelial cells in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, while inhibiting the activity of Rho kinase (ROCK) in the epithelial cells during culturing.

Description

COMPOSITIONS AND METHODS FOR IMMORTALIZATION OF

EPITHELIAL CELLS

Background of the Invention

Field of the Invention

[0001] The present invention is directed towards methods of culturing epithelial cells, with the methods comprising culturing epithelial cells in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, while inhibiting the activity of ho kinase (ROCK) in the epithelial cells during culturing. The present invention is also directed towards methods of using these

immortalized epithelial cells.

Background of the Invention

[0002] Vital organs like the lung, the kidney, liver, pancreas and the skin are characterized by, among other things, the presence of organ-specific differentiated epithelial cells. The differentiated epithelial cells are of course related to the specific function of each such organ. The specific functions may be as varied as, for example, gas exchange in the lung, filtration in the kidney, detoxification and conjugation in the liver, insulin production in the pancreatic islet cells or protection against a hazardous environment by the skin. Disease or degeneration of such an organ is often life threatening because degenerated or lost organ structure is often poorly replaced and because the specialized cells of one organ cannot take over the function of another organ.

[0003] Differentiated cells such as kidney epithelial cells, insulin-producing cells in the Islets of Langerhans of the pancreas, and glandular and/or hair follicle cells of the dermis are difficult to recover, if possible at all, and even more difficult to maintain once taken out of their context in the body. Indeed, differentiated epithelial cells have a very limited lifespan in culture. Generally speaking, epithelial cells, other than keratinocytes, harvested from animals can be grown in culture perhaps through only one or two passages.

[0004] To study epithelial cells in vitro, some type of genetic manipulation such as inserting viral or cellular oncogenes, is required to allow the cells to survive more than a few passages. These genetic manipulations, however, change the cells' genetic background as well as physiology such that cells may not resemble or function like normal epithelial cells. Moreover, these genetically-modified cells would not be candidates for implantation into an intact animal.

[0005] What is needed in the art are methods of culturing epithelial cells harvested from subjects for extended periods of time, without having to genetically alter the cells. The present invention solves the problems associated with culturing epithelial cells for extended periods of time without the need for genetic manipulation.

Summary of the Invention

[0006] The present invention is directed towards methods of culturing epithelial cells, with the methods comprising culturing epithelial cells in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, in the presence of a collagen while inhibiting the activity of Rho kinase (ROCK) in the epithelial cells during culturing.

[0007] The present invention is also directed towards methods of stimulating growth of epithelial cells, with the methods comprising culturing epithelial cells in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, while inhibiting the activity of Rho kinase (ROCK) in the epithelial cells during culturing.

Brief Description of the Drawings

[0008] Fig. 1 shows relative proliferation of primary human ectocervical cells (HECs) cultured with conditioned medium (CM) or lysed medium. IR-: cells not irradiated; IR+: cells irradiated; J2: J2 murine fibroblasts; 3T3: NIH 3T3 murine fibroblasts .

[0009] Fig. 2 shows relative proliferation of cultured HECs. 80S supt: soluble fraction of fractionated feeder cells. 80S pellet: particulate fraction of fractionated feeder cells.

[0010] Fig. 3 shows representative photographs of cultured HECs with CM and Efn A5 constructs. F: F-medium; Y: Y27632; His-Efn A5: His-tagged Efn-A5; Efn A5-Fc: recombinant Efn A5 (chimera with Fc domain of mouse IgG).

[0011] Fig. 4 shows proliferation of cultured HECs with CM or different amounts of Fc-Efn A5 (Fc- A5). pA: protein A.

[0012] Fig. 5 shows relative protein levels of screened factors after irradiation compared to protein levels before irradiation.

[0013] Fig. 6 is a schematic graph showing the activation cascade by inflammatory cytokines.

[0014] Fig. 7 is a schematic graph showing the activation cascade by IL-6.

[0015] Fig. 8 shows the western blot results of Janus kinase 1 (JAK1), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) activation. [0016] Fig. 9 shows relative proliferation of cultured HECs when cultured with different factors.

[0017] Fig. 10 shows relative proliferation of cultured HECs with CM made from irradiated human foreskin fibroblasts (HFF).

Detailed Description of the Invention

[0018] The present invention is directed towards methods of culturing epithelial cells, with the methods comprising culturing epithelial cells in a calcium-containing medium with lnterleukin-6 (IL6) (uniprot accession number P05231) or ephrin A5 (EfnA5) (uniprot accession number P52803), or both, while inhibiting the activity of ho kinase (ROCK) in the epithelial cells during culturing. The uniprot database records are incorporated by reference in their entirety.

[0019] As used herein, the term "epithelium" or "epithelial cell" refers to a cell or cells that line hollow organs, as well as those that make up glands and the outer surface of the body. In general, there can be considered four types of epithelial cells: squamous epithelial cells, columnar epithelial cells, adenomatous epithelial cells and transitional epithelial cells. Epithelial cells can be arranged in single or multiple layers, depending on the organ and location. Keratinocytes are the cells that compose the squamous epithelium that is found at anatomic sites such as the skin, esophagus and cervix. Keratinocytes terminally differentiate into flat, highly keratinized, non-viable cells that help protect against the environment and infection by forming a protective barrier. The present invention is directed to keratinocytes epithelial cells ("KE cells") as well as non-keratinocyte epithelial cells ("NKE cells").

[0020] NKE cells form the glandular epithelium of the body such as found in the breast, prostate, liver, and gastrointestinal tract. NKE cells differentiate into functional, viable cells that can either function in absorption and/or secretion and these cells do not form highly keratinized structures characteristic of squamous epithelial cells. The phrase "non-keratinocyte epithelial cell" is well- understood in the art and one of ordinary skill in the art would readily understand the common, ordinary meaning of the term. The NKE cells used in the methods of the present invention can be of any type or tissue of origin.

[0021] Examples of NKE cells that are encompassed by the term as used herein include but are not limited to prostate cells, mammary cells, hepatocytes, pancreatic islet cells including beta cells, pulmonary epithelial cells, kidney cells, bladder cells, stomach epithelial cells, large and small intestinal epithelial cells, urethral epithelial cells, testicular epithelial cells, ovarian epithelial cells, cervical epithelial cells, thyroid cells, parathyroid cells, adrenal cells, thymus cells, gall bladder cells, pituitary cells.

[0022] The epithelial cells can be from any animal, including but not limited to any mammal, such as mouse, rat, canine, feline, bovine, equine, porcine, non-human and human primates. Mammalian cells particularly suitable for cultivation in the present media include epithelial cells of human origin, which may be primary cells derived from a tissues such as but not limited to skin, mammary glands, prostate glands, liver, pancreas, kidney, bronchi and trachea. In addition, transformed cells or established cell lines, e.g., HeLa cervical epithelial cell lines can also be used. The cells used in the present invention may be normal, healthy cells that are not diseased or not genetically altered, or the cells may be diseased or genetically altered. Accordingly, "diseased epithelial cells" are a subset of epithelial cells herein. "Diseased cells" means that the cells are from abnormal tissue, such as from a neoplasia, a hyperplasia or malignant tumor or benign tumor including, but not limited to, diseased cells isolated from the circulation, i.e., circulating tumor cells (CTC's), of an animal. Other mammalian cells such as but not limited to CHO cells, COS cells, VE O cells, BHK cells (including BHK- 21 cells) and derivatives or subclones thereof are also suitable for the methods of the present invention. In one embodiment, the cells are primary or secondary human epithelial (KE or NKE) cells from a sample of normal or abnormal tissue. In another embodiment, the epithelial (KE or NKE) cells are not primary cells, such as cells from an established cell line, transformed cells, thawed cells from a previously frozen collection and the like. Animal cells for culturing by the present invention may be obtained commercially, for example from ATCC (Rockville, Md.), Cell Systems, Inc. (Kirkland, Wash.), Clonetics Corporation (San Diego, Calif.), BioWhittaker (Walkersville, Md.) or Cascade Biologicals (Portland, Oreg.).

[0023] As used herein, primary cells are cells that have been taken directly from living tissue, such as a biopsy or isolated from circulation, and have not been passaged or only passaged one time. Thus, primary cells have been freshly isolated, often through tissue digestion and plated. Provided the cells have been passaged one time or less, primary cells may or may not be frozen and then thawed at a later time. In addition, the tissue from which the primary cells are isolated may or may not have been frozen of preserved in some other manner immediately prior to processing.

[0024] The epithelial (KE or NKE) cells for use in the present invention are not undifferentiated, embryonic stem cells. Thus, the phrases keratinocyte epithelial cells (or keratinocytes) and non- keratinocyte epithelial cells as used herein automatically excludes undifferentiated embryonic stem cells. As used herein and in the art, embryonic stem cells are undifferentiated cells that have the capacity to regenerate or self-renew indefinitely. The KE or NKE cells used in the methods herein may or may not be adult stem cells. As used herein, adult stem cells are isolated from tissues of an animal and are less differentiated than completely differentiated cells, but are more differentiated than embryonic stem cells. In one embodiment, the KE or NKE cells cultured according to the methods of the present invention are adult stem cells. In another embodiment of the present invention the KE or NKE cells cultured according to the methods of the present invention are not adult stem cells. The KE or NKE cells used in the present invention would not normally have the capacity for indefinite self-renewal. Moreover, the KE or NKE cells are not completely

undifferentiated cells upon initial isolation and plating in that the cells will possess cell surface markers not typically associated with undifferentiated stem cells, or conversely the KE or NKE cells do not possess cell surface markers typically associated with undifferentiated stem cells.

[0025] When isolating primary cells, tissue should ideally be handled using standard sterile techniques and a laminar flow safety cabinet. In one embodiment, a single needle biopsy is sufficient to isolate enough primary cells to begin the cell culture methods of the present invention. In the case of a tissue biopsy, tissue can be cut into small pieces using sterile instruments. In another embodiment, a single cell isolated from the circulation of a subject is sufficient material to begin the cell culture methods of the present invention. The small pieces can then be washed several times with sterile saline solution or other buffer, such as PBS, that may or may not be supplemented with antibiotics or other ingredients. After washing, the pieces are often, but need not be, treated with an enzymatic solution such as, but not limited to collagenase, dispase or trypsin, to promote dissociation of cells from the tissue matrix.

[0026] Dispase is often used to dissociate epithelium from the underlying tissue. This intact epithelium may then be treated with trypsin or collagenase. These digestion steps often results in a slurry containing dissociated cells and tissue matrix. The slurry can then be centrifuged with sufficient force to separate the cells from the remainder of the slurry. The cell pellet can then be removed and washed with buffer and/or saline and/or cell culture medium. The centrifuging and washing can be repeated any number of times. After the final washing, the cells can then be washed with any suitable cell culture medium. Of course, the digestion and washing steps need not be performed if the cells are sufficiently separated from the underlying tissue upon isolation, such as the case in a needle biopsy or if isolated from the circulation. For example, cells such as tumor cells may be isolated from the circulation of the organism using currently available techniques for isolating cells that express cell markers that are specific for a specific type of tumor cell. See Lu. J., et al., Int'l. J. Cancer, 126(3):669-683 (2010) and Yu, M., et al., J. Cell Biol., 192(3): 373-382 (2011), which are incorporated by reference. Cells may or may not be counted using an electronic cell counter, such as a Coulter Counter, or they can be counted manually using a hemocytometer. Of course, the cells need not be counted at all.

[0027] For the purposes of the present invention cells are no longer considered to be primary cells after the cells have been passaged more than once. In addition, cells passaged once or more and immediately frozen after passaging are also considered not to be primary cells when thawed. In select embodiments of the present invention, the KE or NKE cells are initially primary cells and, through the use of the methods of the present invention, become non-primary cells after passaging.

[0028] By "cell culture" or "culture" is meant the maintenance of cells in an artificial, in vitro environment. The term "cell culture" also encompasses cultivating individual cells and tissues.

[0029] The cells being cultured according to the present invention, whether primary or not, can be cultured and plated according to the experimental conditions as needed by the technician. The examples herein demonstrate at least one functional set of culture conditions that can be used in conjunction with the methods described herein. If not known, plating and culture conditions for a given animal cell type can be determined by one of ordinary skill in the art using only routine experimentation. Cells may or may not be plated onto the surface of culture vessels using attachment factors. If attachment factors are used, the culture vessels can be precoated with a natural, recombinant or synthetic attachment factor or factors or peptide fragments thereof, such as but not limited to collagen, fibronectin and natural or synthetic fragments thereof.

[0030] The cell seeding densities for each experimental condition can be manipulated for the specific culture conditions needed. For routine culture in plastic culture vessels, an initial seeding density of from about lxlO⁴ to about 1-lOxlO⁵ cells per cm² is fairly typical, e.g., 1 x 10^s cells are often cultured in a 75cm² culture flask. Cell density can be altered as needed at any passage.

[0031] Mammalian cells are typically cultivated in a cell incubator at about 37°C at normal atmospheric pressure. The incubator atmosphere is normally humidified and often contain about from about 3-10% carbon dioxide in air. Temperature, pressure and C0₂ concentration can be altered as necessary, provided the cells are still viable. Culture medium pH can be in the range of about 7.1 to about 7.6, in particular from about 7.1 to about 7.4, and even more particular from about 7.1 to about 7.3.

[0032] Cell culture medium is normally replaced every 1-2 days or more or less frequently as required by the specific cell type. As the KE or NKE cells approach confluence in the culture vessel, they are normally passaged. As used herein a cell passage is used as it is in the art and means splitting or dividing the cells and transferring a portion of the cells into a new culture vessel or culture environment. Most likely, the KE or NKE cells used in the methods of the present invention will be adherent to the cell culture surface and will need to be detached. Methods of detaching adherent cells from the surface of culture vessels are well-known and commonly employed and can include the use of enzymes such as trypsin.

[0033] A single passage refers to when a technician splits or manually divides the cells one time and transfers a smaller number of cells into a new vessel or environment. When passaging, the cells can be split into any ratio that allows the cells to attach and grow. Thus, at a single passage the cells can be split in a 1:2 ratio, 1:3, 1:4, 1:5 etc. Passaging cells, therefore, is not equivalent to population doubling. As used herein a population doubling is when the cells divide in culture one time such that the number of cells in culture is doubled. Cells need to be counted to determine if a population of cells has doubled, tripled or multiplied by some other factor. In other words, passaging the cells and splitting them in a 1:3 ratio for further culturing in vitro is not to be taken as the equivalent that the cell population has tripled.

[0034] In one embodiment of the present invention, the KE or NKE cells are continuously cultured in vitro. As used herein, "continuous culturing" is the notion that the cells continually divide and reach or approach confluence in the cell culture vessel such that the cells require passaging and fresh medium to maintain their health. Thus, the concept of "continuously culturing" is similar to the concept that the KE or NKE cells would be immortalized. In one embodiment, when cultured using the present methods and conditions of the present invention, normal KE or NKE cells can continue to grow and divide for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250 or 300 passages or more.

[0035] The present invention is also directed towards methods of stimulating growth of KE or NKE cells, in particular normal KE or NKE cells, in vitro with the methods comprising culturing epithelial (KE or NKE) cells in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, while inhibiting the activity of ho kinase (ROCK) in the epithelial cells during culturing.

Culturing the KE or NKE cells in such conditions will stimulate the KE or NKE cells to grow, whereas otherwise the cells may not grow. In one specific embodiment, the cells grow in tight clusters, i.e., the cells become tightly adherent. In one embodiment, the cultured KE or NKE cells form junctions involving e-cadherin, non muscle myosin, and pl20 catenin. These types of junctions can be assayed according to Li, D. et al., J. Cell Biol., 191(3):631-644 (2010), which is incorporated by reference. [0036] As used herein and throughout the specification, "cell growth" refers to cell division, such that one "mother cell" divides into two "daughter cells." As used herein, "cell growth" does not refer to an increase in the actual size of the cells. Stimulation of cell growth can be assayed by plotting cell populations over time. A cell population with a steeper growth curve can said to be growing faster than a cell population with a curve not as steep. Growth curves can be compared for various treatments between the same cell types, or growth curves can be compared for different cell types with the same conditions.

[0037] The late passage KE or NKE cells, in particular late passage normal KE or NKE cells, of the present invention may or may not be characterized by their telomere length. As normally happens, the length of the telomeres generally shortens as cells divide. A cell will normally stop dividing when the average length of telomeres is reduced to a critical length, e.g., 4kb. In the present invention, the average telomere length of late passage cells may or may not be reduced to a length of as little as 2kb and continue to grow. The average telomere length is readily determined using routine methods and techniques in the art. Thus in one embodiment, the present invention provides KE or NKE cells, in particular normal KE or NKE cells, capable of dividing in the culture conditions of the present invention, wherein the average telomere length of the KE or NKE cells is shorter than the average telomere length of KE or NKE cells that would normally not divide when placed under different or heretofore routine culture conditions. For example, the average telomere length of senescent human prostate epithelial cells (HPECs) is about 4kb, thus when the average telomere length in HPECs is reduced to about 4kb, the cells will normally not divide when placed in culture conditions currently considered in the art to be acceptable or even optimal for culturing prostate cells. Using the culture conditions of the present invention, however, the average telomere length of the HPECs can be reduced to a length as little as 2kb, or even lower, and still divide and grow. Thus, the methods of the present invention are capa ble of generating conditionally immortalized KE or NKE cells, in particular normal conditionally immortalized KE or NKE cells, whereby the cells have an average telomere length that is less than the average telomere length of KE or NKE cells that are normally capable of dividing and whereby the conditionally immortalized KE or NKE cells are capable of still dividing in spite of their reduced telomere length. To be clear, KE or NKE cells, in particular normal KE or NKE cells will normally stop dividing when the average telomere length is reduced to a certain length even when placed in culture conditions currently considered in the art to be acceptable or even optimal for culturing prostate cells. The average telomere length can vary from cell type to cell type. [0038] Such currently acceptable or optimal conditions for culturing epithelial cells generally include culturing cells in well-defined, or synthetic, serum-free medium. For examples, culturing prostate cells normally involves culturing in prostate cell-specific medium, without added serum. In addition, many other types of KE or NKE cells are often cultured in the absence of feeder cells. Thus the methods of the present invention provide the unexpected results of being able to culture and passage KE or NKE cells, in particular normal KE or NKE cells, long after one would have been able to do so using currently acceptable or currently optimal conditions.

[0039] As used herein, the term "conditionally immortalized" indicates that the KE or NKE cells have a reduced average telomere length over the average telomere length of normal senescent KE or NKE yet are still capable of unlimited growth, provided the conditionally immortalized KE or NKE cells, including conditionally immortalized normal KE or NKE cells, are maintained in the culture conditions of the present invention. When determining if a cell is conditionally immortalized, it may be necessary to compare the average telomere length of the conditionally immortalized cells with the average telomere length of non-conditionally immortalized KE or NKE cells that would normally be senescent in vitro. The phrase "normally senescent" is used to mean a population of cells that, but for the conditions outlined herein, would be incapable of further divisions in vitro and thus would not need to be passaged any further. Therefore, the invention provides methods of conditionally immortalizing KE or NKE cells, in particular normal KE or NKE cells, comprising culturing the KE or NKE cells, in particular normal KE or NKE cells, in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, while inhibiting the activity of ho kinase (ROCK) in the epithelial cells during culturing.

[0040] The KE or NKE cells can grow, become in need of continuous culturing and/or become conditionally immortalized in vitro without apparent change to the karyotype of the cells after any number of passages. Accordingly, the methods of the present invention comprise continuously culturing KE or NKE cells, in particular normal KE or NKE cells, whereby the cells' karyotype at any passage is not altered or is not substantially altered when compared to the karyotype of the same types of primary cells or early passage cells. An alteration of a cell's karyotype includes but is not limited to duplication or deletion of chromosomes or portions thereof and/or translocation of a portion of one chromosome to another. Identifying a karyotype and alterations thereof are common techniques in the art. Accordingly, one embodiment of the present invention is directed to late passage KE or NKE cells, in particular late passage normal KE or NKE cells wherein the late passage KE or NKE cells have (a) an unaltered karyotype when compared to the karyotype of primary KE or NKE cells of the same origin or (b) an unaltered karyotype when compared to the karyotype of initially thawed KE or NKE cells of the same origin. As used herein, a late passage KE or NKE cell is defined as a KE or NKE cell that has gone through at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250 or 300 passages or more.

[0041] The present invention is also directed to conditionally immortalized KE or NKE cells, in particular conditionally immortalized normal KE or NKE cells. In select embodiments, the conditionally immortalized KE or NKE cells, in particular the conditionally immortalized normal KE or NKE cells have (a) an unaltered karyotype when compared to the karyotype of primary KE or NKE cells of the same origin or (b) an unaltered karyotype when compared to the karyotype of initially thawed KE or NKE cells of the same origin.

[0042] The methods of the present invention do not require the use of feeder cells. Accordingly, in select embodiments, the methods described herein are performed when the epithelial (KE or NKE) cells are not co-cultured with feeder cells. In other select embodiments, the methods described herein are performed when the epithelial (KE or NKE) cells are not cultured in media conditioned by feeder cells ("conditioned medium").

[0043] In other more specific embodiments, the epithelial (KE or NKE) cells are initially cultured in the presence of feeder cells or in conditioned medium. For example, the KE or NKE cells are harvested as primary cells and treated as described herein and as is routine in the art and placed in co-culture with feeder cells conditioned medium. At the first passage, the cells can then transferred into the culture conditions described herein, without feeder cells and/or without conditioned medium. Of course, the cells can be transferred from feeder culture conditions to feeder-free culture conditions after two, three, four, five, six, seven, eight, nine or ten passages. As used herein, the term "feeder culture conditions" is used to mean culturing the KE or NKE cells in the presence of feeder cells, i.e., co-culturing the cells with feeder cells, culturing the KE or NKE cells in conditioned medium or culturing the cells with particulate and/or soluble fractions of feeder cells. As used herein, the term "feeder-free" culture conditions is used to mean culturing the KE or NKE cells in the absence of feeder cells, i.e., not co-culturing the cells with feeder cells, culturing the KE or NKE cells without the use of conditioned medium or culturing without particulate and/or soluble fractions of feeder cells.

[0044] In another embodiment, cells are harvested as primary cells and are cultured in the presence of one or more fractions of fractionated feeder cells. As used herein, the term "fractionated feeder cells" is used to mean feeder cells that have been disrupted, e.g., lysed. A "fraction" of fractionated feeder cells is prepared by disrupting the feeder cells, e.g., lysing, and further processing the contents of the disrupted cells to create a "particulate fraction" and/or a "soluble fraction" of the cell contents. Methods of lysing cells are well-known and include but are not limited to such processes as irradiating the cells, sonicating the cells, or combining irradiation with sonication. Some feeder cells will spontaneously disintegrate into cell culture medium after irradiation; other cells, however, may require additional treatment, e.g., sonication, to complete the disruption (disintegration) of the cells into the cell culture medium.

[0045] In general, the "particulate fraction" of cellular contents includes components that are greater than or equal to the size of a typical eukaryotic 80S ribosome. The term "80S ribosome" is readily understood in the art and refers to the size of the ribosome in terms of its sedimentation coefficient in Svedberg units. In general the "soluble fraction" of cellular contents includes components that are smaller than the size of a typical eukaryotic 80S ribosome. Fractionating cell contents based on size, particularly based on size in relation to eukaryotic ribosomes, is routine in the art.

[0046] The term "feeder cells" is used herein as it is in the art. Namely, feeder cells are cells that are cultured with the KE or NKE cells of the present invention. In one embodiment, the feeder cells are non-proliferating feeder cells. In one embodiment of the present invention, the feeder cells can be treated to inhibit proliferation of the feeders, while still keeping them alive and metabolically active. For example, feeder cells can be irradiated with gamma irradiation and/or treated with mitomycin C, which will arrest cell division but maintain the cells in a metabolically active state. Methods of treating cells to arrest cell division but maintain a metabolically active state are well- known in the art.

[0047] Feeder cells can be from any mammal and the animal source of the feeder cells need not be the same animal source as the KE or NKE cells being cultured. For example feeder cells may be, but are not limited to mouse, rat, canine, feline, bovine, equine, porcine, non-human and human primate feeder cells. The types of feeder cells used are typically spleenocytes, macrophages thymocytes and/or fibroblasts. In one embodiment, the spleenocytes, macrophages thymocytes and/or fibroblasts have been treated such that they are non-proliferating. One example of a feeder cell that may be used in the methods of the present invention is a population of J2 cells. The J2 cells are a subclone of mouse fibroblasts derived from the established Swiss 3T3 cell line. In one embodiment, the J2 cells are gamma irradiated. In another embodiment, the J2 cells are treated with mitomycin C. In another embodiment, the feeder cells are human feeder cells. [0048] In other embodiments, the KE or NKE cells are cultured with fractions of fractionated feeder cells prior to transferring the cells into the feeder-free conditions described herein. In specific embodiments, the KE or NKE cells are cultured with particulate fractions of fractionated feeder cells prior to transferring the cells into the feeder-free conditions described herein. In specific embodiments, the KE or NKE cells are cultured with soluble fractions of fractionated feeder cells prior to transferring the cells into feeder-free conditions described herein. In specific embodiments, the KE or NKE cells are cultured with particulate and soluble fractions of fractionated feeder cells prior to transferring the cells into feeder-free conditions described herein. In additional specific embodiments, the KE or NKE cells that are cultured with particulate and/or soluble fractions of fractionated feeder cells prior to transferring the cells into feeder-free conditions are primary KE or NKE cells. In other words, the cells are harvested and initially plated in conditions include the particulate and/or soluble fraction of the feeder cells. At the first passage, for example, the cells are then transferred to the feeder-free conditions described herein. In additional specific embodiments, the KE or NKE cells that are cultured with particulate and/or soluble fractions of fractionated feeder cells prior to transferring the cells into feeder-free conditions are not primary KE or NKE cells, e.g., the cells have been passage two, three, four, five, six, seven, eight, nine, ten or more times and are re-plated in the presence of particulate and/or soluble fractions of fractionated feeder cells, but are eventually transferred to the feeder-free conditions described herein.

[0049] In other embodiments, the KE or NKE cells are cultured with fractions of fractionated human feeder cells prior to transferring the cells into the feeder-free conditions described herein. In specific embodiments, the fractionated human feeder cells are prepared by irradiating the cells and subsequently sonicating the cells to disrupt the cells. The contents of the disrupted human feeder cells are then processed to produce the cell fractions. In specific embodiments, the KE or NKE cells are cultured with particulate fractions of fractionated human feeder cells prior to transferring the cells into the feeder-free conditions described herein. In specific embodiments, the KE or NKE cells are cultured with soluble fractions of fractionated human feeder cells prior to transferring the cells into feeder-free conditions described herein. In specific embodiments, the KE or NKE cells are cultured with particulate and soluble fractions of fractionated human feeder cells prior to transferring the cells into feeder-free conditions described herein. In additional specific

embodiments, the KE or NKE cells that are cultured with particulate and/or soluble fractions of fractionated human feeder cells prior to transferring the cells into feeder-free conditions are primary KE or NKE cells. In other words, the cells are harvested and initially plated in conditions include the particulate and/or soluble fraction of the human feeder cells. At the first passage, for example, the cells are then transferred to the feeder-free conditions described herein. In additional specific embodiments, the KE or NKE cells that are cultured with particulate and/or soluble fractions of fractionated human feeder cells prior to transferring the cells into feeder-free conditions are not primary KE or NKE cells, e.g., the cells have been passage two, three, four, five, six, seven, eight, nine, ten or more times and are re-plated in the presence of particulate and/or soluble fractions of fractionated human feeder cells, but are eventually transferred to the feeder-free conditions described herein.

[0050] In other embodiments, the species from which the feeder cells or fractionated feeder cells derive are the same species of KE or NKE cells. For example, canine feeder cells can be generated and processed to produce fractions of fractionated canine feeder cells, and these fractions can then be applied to canine KE or NKE cells. Method of preparing the fractionated feeder cells may be dependent on the cell types used. For example, J2 cells spontaneously undergo apoptosis and release their cell contents in response to radiation, i.e., sonication is not required, where as 3T3 cells generally require sonication for cell disruption.

[0051] In another embodiment, medium conditioned with feeder cells, "conditioned medium," is used in place of culturing with feeder cells. Preparing conditioned medium is routine in the art. Generally, preparation of conditioned medium involves culturing cells in a medium, e.g., F-medium as defined herein, for a few days and collecting this medium. The conditioned medium is often, but need not be, combined with fresh medium in a diluted fashion. Discovering the optimal dilution ratios of conditioned medium to "fresh medium" is routine, but the ratios can be from about 1:99 to about 99:1 of "conditioned medium" to "fresh medium." As used herein, "conditioned medium" is any medium where all or a percentage of the medium has been previously used in culture.

[0052] The cell culture media of the present invention can be any aqueous-based medium and can include any "classic" media such as, but not limited to DMEM (Dulbecco's Modified Essential Medium), Ham's F12 medium, Ham's F-10 medium, RPMI 1640, Eagle's Basal Medium (EBM), Eagle's Minimum Essential Medium (MEM), HEPES, Medium 199 and the like. The culture medium can also be combinations of any of the classical medium, such as but not limited to, a combination of DM EM and F12 Media.

[0053] Additional ingredients may be added to the culture medium used in the methods of the present invention. Such additional ingredients include but are not limited to, amino acids, vitamins, inorganic salts, adenine, ethanolamine, D-glucose, heparin, N-[2-hydroxyethyl]piperazine-N'-[2- ethanesulfonic acid] (HEPES), hydrocortisone, insulin, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, triiodothyronine (T3), thymidine and transferrin. Alternatively, insulin and transferrin may be replaced by ferric citrate or ferrous sulfate chelates. Each of these additional ingredients is commercially available.

[0054] Amino acid ingredients which may be included in the media of the present invention include but are not limited to, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

[0055] Vitamin that may be added include but are not limited to biotin, choline chloride, D-Ca⁺²- pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine and vitamin B12.

[0056] Inorganic salt ingredients which may be added include but are not limited to calcium salt (e.g., CaCI₂), CuS0₄, FeS0₄, KCI, a magnesium salt, e.g., MgCI₂, a manganese salt, e.g., MnCI₂, sodium acetate, NaCI, NaHC0₃, Na₂HP0₄, Na₂S0₄ and ions of the trace elements selenium, silicon, molybdenum, vanadium, nickel, tin and zinc. These trace elements may be provided in a variety of forms, preferably in the form of salts such as Na₂Se0₃, Na₂ Si0₃, (NH₄)6Mo₇ 0₂₄, NH₄ V0₃, NiS0₄, SnCI and ZnSO.

[0057] Additional ingredients include but are not limited to heparin, epidermal growth factor (EGF), at least one agent increasing intracellular cyclic adenosine monophosphate (cAMP) levels, and at least one fibroblast growth factor (FGF). Heparin, EGF, the cAMP-increasing agent(s) and FGF(s) may be added to the basal medium or they may be admixed in a solution of, for example, Dulbecco's Phosphate Buffered Saline (DPBS) and stored frozen until being added to basal medium to formulate the medium to be used in the methods of the present invention.

[0058] Heparin may be obtained commercially. Heparin is added to the present media primarily to stabilize the activity of the growth factor components, for example FGF. If heparin is used, it may be added to the basal medium at a concentration of a bout 1-500 U.S. P. units/liter. EGF is available commercially. If EGF is used, it may be added to the basal medium at a concentration of about 0.00001-10 mg/L.

[0059] Still other ingredients may be added to the medium. In select embodiments, one or more of the components selected from the group consisting of granulocyte macrophage colony-stimulating factor (GM-CSF) (uniprot accession number P04141), granulocyte colony stimulating factor (G-CSF) (uniprot accession number P09919), hepatocyte growth factor (HGF) (uniprot accession number P14210), neuregulin 1 (N G1) (uniprot accession number Q6ICV5), neuregulin 2 (NRG2) (uniprot accession number Q3M I86), neuregulin 3 (NRG3) (uniprot accession number B9EGV5), neuregulin 4 (N G4) (uniprot accession number Q0P6N6), epiregulin (ERG) (uniprot accession number 014944), betacellulin (BC) (uniprot accession number Q86UF5), lnterleukin-11 (IL11) (uniprot accession number P20809), a collagen and heparin-binding EGF-like growth factor (HB-EGF) (uniprot accession number Q14487). The uniprot database records are incorporated by reference herein in their entirety.

[0060] The origin and identity of these components are well known. In some embodiments, the origin of the components of the culture conditions matches the origin of the cells being culture. For example, if human KE or NKE cells are being cultured according to the methods of the invention disclosed herein, one or more, including all, of the additional components may also be of human origin.

[0061] The invention therefore provides methods of using the peptide/protein components listed herein and variants thereof. Variants of any of the peptide/protein components include peptides/proteins comprising an amino acid sequence at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of the human sequences referenced in the Uniprot accession numbers listed herein. In one specific embodiment, methods comprise the use of a peptide with an amino acid sequence at least 95% identical to the human sequences referenced in the Uniprot accession numbers listed herein. In further embodiments, methods using the peptides of the present invention comprise an amino acid sequence 100% identical to the human sequences referenced in the Uniprot accession numbers listed herein.

[0062] A polypeptide having an amino acid sequence at least, for example, about 95% "identical" to a reference an amino acid sequence, e.g., SEQ ID NO:2, is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to about five modifications per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a peptide having an amino acid sequence at least about 95% identical to a reference amino acid sequence, up to about 5% of the amino acid residues of the reference sequence may be deleted or substituted with another amino acid or a number of amino acids up to about 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the N- terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. [0063] As used herein, "identity" is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using pu blished techniques. (See, e.g., Computational Molecular Biology, Lesk, A. M ., ed., Oxford University Press, New York (1988); Biocomputing: Informatics And Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); von Heinje, G., Sequence Analysis In Molecular Biology, Academic Press (1987); a nd Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York (1991)). While there are several methods to measu re identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math 48: 1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994) a nd Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988). Computer programs may also contain methods and algorithms that calculate identity and similarity. Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP, ExPASy, BLASTN, FASTA (Atschul, S. F., et a l., J Molec Biol 215 :403 (1990)) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels, G. and Garian, R., Current Protocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000), which is incorporated by reference.

[0064] In one em bodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is BLASTP. In another embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990), incorporated by reference). In a FASTDB sequence alignment, the query and reference sequences are a mino sequences. The resu lt of sequence alignment is in percent identity. In one em bodiment, parameters that may be used in a FASTDB alignment of amino acid sequences to calculate percent identity include, but are not limited to: Matrix=PAM, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the su bject amino sequence, whichever is shorter.

[0065] If the reference sequence is shorter or longer than the query sequence because of N- terminus or C-terminus additions or deletions, but not because of internal additions or deletions, a manual correction can be made, because the FASTDB program does not account for N-terminus and C-terminus truncations or additions of the reference sequence when calculating percent identity. For query sequences truncated at the N- or C- termini, relative to the reference sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are island C- terminus to the reference sequence that are not matched/aligned, as a percent of the total bases of the query sequence. The results of the FASTDB sequence alignment determine

matching/alignment. The alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score can be used for the purposes of determining how alignments "correspond" to each other, as well as percentage identity. Residues of the reference sequence that extend past the N- or C-termini of the query sequence may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N- or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.

[0066] For example, a 90 amino acid residue query sequence is aligned with a 100 residue reference sequence to determine percent identity. The deletion occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the reference sequence (number of residues at the N- and C-termini not matched/total number of residues in the reference sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched (100% alignment) the final percent identity would be 90% (100% alignment - 10% unmatched overhang). In another example, a 90 residue query sequence is compared with a 100 reference sequence, except that the deletions are internal deletions. In this case the percent identity calculated by FASTDB is not manually corrected, since there are no residues at the N- or C-termini of the subject sequence that are not matched/aligned with the query. In still another example, a 110 amino acid query sequence is aligned with a 100 residue reference sequence to determine percent identity. The addition in the query occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment may not show a match/alignment of the first 10 residues at the N-terminus. If the remaining 100 amino acid residues of the query sequence have 95% identity to the entire length of the reference sequence, the N-terminal addition of the query would be ignored and the percent identity of the query to the reference sequence would be 95%. [0067] As used herein, the terms "correspond(s) to" and "corresponding to," as they relate to sequence alignment, are intended to mean enumerated positions within the reference protein, e.g., wild-type IL6, and those positions in a mutant or related IL6 that align with the positions on the reference protein. Thus, when the amino acid sequence of a subject peptide is aligned with the amino acid sequence of a reference IL6, the amino acids in the subject sequence that "correspond to" certain enumerated positions of the reference sequence are those that align with these positions of the reference sequence, but are not necessarily in these exact numerical positions of the reference sequence. Methods for aligning sequences for determining corresponding amino acids between sequences are described herein. Accordingly, the invention provides for the use of novel peptides whose sequences correspond to the sequence of the human sequences referenced in the Uniprot accession numbers listed herein.

[0068] The invention further embraces other species, preferably mammalian, homologs with amino acid sequences that correspond to the the human sequences referenced in the Uniprot accession numbers listed herein. Species homologs, sometimes referred to as "orthologs," in general, share at least 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the human version of the proteins. Such corresponding sequences account for a variety of species, such as canine, feline, mouse, rat, rabbit, monkey, etc. of, e.g. IL6.

[0069] Variants resulting from insertion of the polynucleotide encoding the human sequences referenced in the Uniprot accession numbers listed herein into an expression vector system are also contemplated. For example, variants (usually insertions) may arise from when the amino terminus and/or the carboxy terminus of the human sequences referenced in the Uniprot accession numbers listed hereinis/are fused to another polypeptide.

[0070] In another aspect, the invention provides deletion variants wherein one or more amino acid residues in the human sequences referenced in the Uniprot accession numbers listed herein protein are removed. Deletions can be effected at one or both termini of the peptide, or with removal of one or more non-terminal amino acid residues of the peptide. Deletion variants, therefore, include all fragments of the human sequences referenced in the Uniprot accession numbers listed herein.

[0071] Within the confines of the disclosed percent identity, the invention also relates to substitution variants of the disclosed polypeptides of the invention. Substitution variants include those polypeptides wherein one or more amino acid residues of the human sequences referenced in the Uniprot accession numbers listed herein are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in the tables below. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in below.

Table I - Conservative Substitutions

SIDE CHAIN CHARACTERISTIC AMINO ACID

Aliphatic

Non-polar G A P

1 L V

Polar - uncharged C S T M

N Q

Polar - charged D E

K R

Aromatic H F w Y

Other N Q D E

[0072] Alternatively, conservative amino acids can be grouped as described in Lehninger,

[Biochemsitry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71 77] as set out below.

Table II - Conservative Substitutions

SIDE CHAIN CHARACTERISTIC AMINO ACID

Non-polar (hydrophobic)

A. Aliphatic: V

B. Aromatic: F W

C. Sulfur-containing: M

D. Borderline: G Uncharged-polar

A. Hydroxyl: B. Amides: N Q

C. Sylfhydryl: C

D. Borderline: G

Positively Charged (Basic): K H Negatively Charged (Acidic) D E

[0073] And still other alternative, exemplary conservative substitutions are set out below. Table III - Conservative Substitutions

Original Residue Exemplary Substitution

Ala (A) Val, Leu, lie

Arg (R) Lys, Gin, Asn

Asn (N) Gin, His, Lys, Arg

Asp (D) Glu

Cys (C) Ser

Gin (Q) Asn

Glu (E) Asp

His (H) Asn, Gin, Lys, Arg

He (1) Leu, Val, Met, Ala, Phe

Leu (L) lie, Val, Met, Ala, Phe

Lys (K) Arg, Gin, Asn

Met (M) Leu, Phe, lie

Phe (F) Leu, Val, lie, Ala

Pro (P) Gly

Ser (S) Thr

Thr (T) Ser

Trp (W) Tyr

Tyr (Y) Trp, Phe, Thr, Ser

Val (V) lie, Leu, Met, Phe, Ala

[0074] It should be understood that the definition of peptides or polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic and inorganic moieties. Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues or organs. Similarly, the invention further embraces IL4il peptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol,

polyoxyethylene glycol or polypropylene glycol.

[0075] A variety of agents that increase intracellular cAMP levels may be used in formulating the media of the present invention. Included are agents which induce a direct increase in intracellular cAMP levels (e.g., dibutyryl cAMP), agents which cause an increase in intracellular cAMP levels by an interaction with a cellular G-protein (e.g., cholera toxin and forskolin), agents which cause an increase in intracellular cAMP levels by acting as agonists of β-adrenergic receptors (e.g., isoproterenol) and agents which cause an increase in intracellular cAMP levels by inhibiting the activities of cAMP phosphodiesterases (e.g., isobutylmethylxanthine (IBMX) and theophylline). These cAMP-increasing agents are available commercially.

[0076] The culture medium used in the methods of the present invention comprises a calcium source. In one embodiment, the calcium source is serum or a serum replacement. In another embodiment, the calcium source is a calcium-containing salt that is added to the medium. If serum is used as a calcium source, the serum can be in a concentration (v/v) of from a bout 1% to about 35%. In select embodiments, the serum is at a concentration of from about 1% to about 20%, or from about 1% to about 15%, or from about 1% to about 10%, or from about 1% to about 5%. If a serum substitute or serum replacement is used as the calcium source, these can be added to the medium according to the manufacturer's suggested protocol. Examples of serum substitutes include but are not limited to commercially available substitutes such as Ultroser™ from Pall Corporation, milk or milk fractions such as but not limited to nonfat dry milk filtrate.

[0077] The range of Ca⁺² concentration used in the embodiments of the present invention can vary according to cell type. In one embodiment, the concentration of Ca⁺² in the medium used in the methods of the present invention is from 0.1 mM to 10.0 mM. In more specific embodiments, the concentration of Ca⁺² in the medium used in the methods of the present invention can be from about 0.2 mM to about 8 mM, from about 0.4 mM to about 7 mM, from about 0.5 mM to about 5 mM, from about 0.8 mM to about 4 mM, from about 1.0 mM to about 3 mM, from about 1.2 mM to about 2.8 mM, from about 1.4 mM to about 2.6 mM and from about 1.5 mM to about 2.5 mM.

[0078] The methods of the present invention comprise inhibiting rho associated coiled-coil protein kinase (ROCK) in the culture. Rho kinase belongs to the Rho GTPase family of proteins, which includes the Rho, Racl and Cdc42 kinases. One of the best characterized effector molecule of Rho is ROCK, which is a serine/threonine kinase that binds to the GTP-bound form of Rho. The catalytic kinase domain of ROCK, which comprises conserved motifs characteristic of serine/threonine kinases, is found at the N-terminus. ROCK proteins also have a central coiled-coil domain, which includes a Rho-binding domain (RBD). The C- terminus is made up of a pleckstrin-homology (PH) domain with an internal cysteine -rich domain. The coiled-coil domain is thought to interact with other a- helical proteins. The RBD, located within the coiled-coil domain, interacts only with activated Rho GTPases, including RhoA, RhoB, and RhoC. The pH domain is thought to interact with lipid mediators such as arachidonic acid and sphingosylphosphorylcholine, and may play a role in protein localization. Interaction of the pH domain and RBD with the kinase domain results in an auto- inhibitory loop. In addition, the kinase domain is involved in binding to RhoE, which is a negative regulator of ROCK activity.

[0079] The ROCK family currently consists of two members, ROCK1 (also known as ROKfi or pl60ROCK) and ROCK2 (also known as ROKa). ROCK1 is about 1354 amino acids in length and ROCK2 is a bout 1388 amino acids in length. The amino acid sequences of human ROCK1 and human ROCK2 are well known. For example, the amino acid sequence of ROCK 1 and ROCK2 can be found at UniProt Knowledgebase (UniProtKB) Accession Number Q13464 and 075116, respectively. The nucleotide sequences of human ROCK1 and ROCK2 can be found at GenBank Accession Number NM_005406.2 and NM_004850, respectively. The nucleotide and amino acid sequences of ROCK1 and ROCK2 proteins from a variety of animals are also well-known and can be found in both the UniProt and GenBank databases.

[0080] Although both ROCK isoforms are ubiquitously expressed in tissues, they exhibit differing intensities in some tissues. For example, ROCK2 is more prevalent in brain and skeletal muscle, while ROCK1 is more abundant in liver, testes and kidney. Both isoforms are expressed in vascular smooth muscle and heart. In the resting state, both ROCK1 and ROCK2 are primarily cytosolic, but are translocated to the membrane upon Rho activation. ROCK activity is regulated by several different mechanisms, thus Rho-dependent ROCK activation is highly cell-type dependent, ranging from changes in contractility, cell permeability, migration and proliferation to apoptosis. At least 20 ROCK su bstrates have been identified. See Hu and Lee, Expert Opin. Ther. Targets 9:715-736 (2005) and Loirand et al, Cir. Res. 98:322-334 (2006) and Riento and Ridley, Nat. Rev. Mol. Cell Biol. 4:446- 456 (2003) all of which are incorporated by reference.

[0081] The role of ROCK in regulating apoptotic signaling is highly cell-type dependent and stimulus dependent. On the other hand, ROCK has also been associated with mediating cell-survival signals in vitro and in vivo. A ROCK-mediated pro-survival effect has been reported in epithelial cells, cancer cells and endothelial cells, as well as in other cell types. In airway epithelial cells, inhibition with Y- 27632 or HA 1077 (also known as fasudil) induces membrane ruffling, loss of actin stress fibers and apoptosis (Moore et al., Am. J. Respir. Cell Mol. Biol. 30:379-387, 2004).

[0082] Rho/ROCK activation may also play a pro-survival role during oxidative stress-induced intestinal epithelial cell injury (Song et al., Am. J. Physiol. Cell Physiol. 290:C1469-1476, 2006). ROCK has also been associated with pro-survival events in thyroid cancer cells (Zhong et al., Endocrinology 144:3852-3859, 2003), glioma cells (Rattan et al, J. Neurosci. Res. 83:243-255, 2006), human umbilical vein endothelial cells (Li et al., J. Biol. Chem. 277:15309-15316, 2002), hepatic stelate cells (Ikeda et al., Am. J. Physiol. Gastrointest. Liver Physiol. 285:G880-886, 2003) and human

neuroblastoma cells (De Sarno et al., Brain Res. 1041: 112-115, 2005). Evidence of ROCK playing a pro-survival role has also been reported in vivo, for example in vascular smooth muscle cells (Shibata et al, Circulation 103:284-289, 2001) and spinal motor neurons (Kobayashi et al, J. Neurosci.

24:3480-3488, 2004).

[0083] As used herein, inhibiting ROCK can mean to reduce the activity, function or expression of at least one of ROCKl or ROCK2. The activity, function or expression may be completely suppressed, i.e., no activity, function or expression, or the activity, function or expression may simply be lower in treated versus untreated cells. In general, ROCK phosphorylates LIM kinase and myosin light chain (MLC) phosphatase after being activated through binding of GTP-bound Rho. One embodiment of the present invention thus involves blocking the upstream pathway of ROCKl and/or ROCK2, for example GTP-bound Rho, such that ROCKl and/or ROCK2 is not activated or its activity is reduced over untreated cells. Other upstream effectors include but are not limited to, integrins, growth factor receptors, including but not limited to, TGF-beta and EGFR, cadherins, G protein coupled receptors and the like. Another embodiment of the present invention thus involves blocking the activity, function or expression of downstream effector molecules of activated ROCKl and/or ROCK2 such that ROCKl and/or ROCK2 can not propagate any signal or can only propagate a reduced signal over untreated cells. Downstream effectors include but are not limited to, vimentin, LIMK, Myosin light chain kinase, NHE1, cofilin and the like.

[0084] The methods of the present invention comprise inhibiting ROCK while culturing the KE or NKE cells, in particular normal KE or NKE cells. In one embodiment, inhibiting ROCK is accomplished by addition of a ROCK inhibitor to the culture medium. In this embodiment where a ROCK inhibitor is added to culture medium. [0085] Examples of ROCK inhibitors include but are not limited to Y-27632, HA1100, HA1077 and GSK429286, the structures of which are depicted in PCT Application No. WO 2012/065067, which is incorporated by reference. These compounds are well known and commercially available.

Additional small molecule Rho kinase inhibitors include but are not limited to those described in PCT Publication Nos. WO 03/059913, WO 03/064397, WO 05/003101, WO 04/112719, WO 03/062225 and WO 03/062227, and described in U.S. Patent Nos. 7,217,722 and 7,199,147, and U.S. Patent Application Publication Nos. 2003/0220357, 2006/0241127, 2005/0182040 and 2005/0197328, the contents of all of which are incorporated by reference.

[0086] Another way of inhibiting ROCK kinase would be through the use of RNA interference (RNAi). RNAi techniques are well known and rely on double-stranded RNA (dsRNA), where one stand of the dsRNA corresponds to the coding strand of the mRNA that codes for ROCK1, and the other strand is complementary to the first strand. The requirements of optimal RNAi species for a given nucleotide sequence are well-known or can be readily ascertained given the state of the art. For example, it is known that optimal dsRNA is about 20-25nt in length, with a 2 base overhand on the 3' end of each strand of the dsRNA, often referred to as short interfering RNAs (siRNA). Of course, other well- known configurations such as short hairpin RNA (shRNA) may also work. shRNAs are one continuous RNA strand where a portion is self-complementary such that the molecule is double-stranded in at least one portion. It is believed that the cell processed shRNA into siRNA. The term RNAi molecule, as used herein, is any double stranded double-stranded RNA (dsRNA), where one stand of the dsRNA corresponds to the coding strand of the mRNA that codes for the target gene to be silenced, and the other strand is complementary to the first strand.

[0087] Accordingly, one embodiment of the present invention involves the use of at least one RNAi molecule and/or at least one antisense molecule, to inhibit the activity of ROCK. In one specific embodiment, the RNAi molecule and/or antisense molecule is specific towards ROCK1. In another embodiment, the RNAi molecule or antisense molecule is specific towards ROCK2. In yet another embodiment, the RNAi molecule and/or antisense molecule is specific towards both ROCK1 and ROCK2. In still another embodiment, at least two RNAi molecules and/or antisense molecules are used, where one is specific towards ROCK1 and the other is specific towards ROCK2.

[0088] The RNAi molecules and/or antisense molecules may be part of the cell culture by simply soaking the cells with the naked RNAi molecules and/or antisense molecules as has been reported Clemens, J.C., et al., PNAS, 97(12):6499-6503 (2000), which is incorporated by reference. The RNAi molecules and/or antisense molecules may also be part of a complex, such as a liposomal complex that can be used to insert RNAi molecules or antisense/molecules into the cells. [0089] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged ds NA molecules to form a stable complex. The positively charged dsRNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et at., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0090] Liposomes that are pH-sensitive or negatively-charged entrap dsRNA rather than complex with it. Since both the dsRNA and the lipid are similarly charged, repulsion rather than complex formation occurs. The dsRNA is thus entrapped in the aqueous interior of these liposomes. pH- sensitive liposomes have been used, for example, to deliver dsRNA encoding the thymidine kinase gene to cell monolayers in culture (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274). One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyi phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyi phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. Liposomes that include nucleic acids have been described, for example, in WO 96/40062, U.S. Pat. No. 5,264,221, U.S. Pat. No. 5,665,710 and Love et al., WO 97/04787 all of which are incorporated by reference.

[0091] Another type of liposome, a transfersome, is a highly deformable lipid aggregate which is attractive for drug delivery vehicles. (Cevc et al., 1998, Biochim Biophys Acta. 1368(2): 201-15.) Transfersomes may be described as lipid droplets which are so highly deformable that they can penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, for example, they are shape adaptive, self-repairing, frequently reach their targets without fragmenting, and often self-loading. Transfersomes can be made, for example, by adding surface edge-activators, usually surfactants, to a standard liposomal composition.

[0092] Another way ROCK1 and/or ROCK2 RNAi can gain access to the cells in the methods of the present invention is through the use of DNA expression vectors that encode the RNAi molecules and/or antisense molecules. Certain embodiments can utilize only one vector, for example when the RNAi molecule is a shRNA, or when opposing promoters are placed on either side there of the coding sequence for the RNAi molecule. Thus "inhibiting the activity of ROCK" includes the use of DNA that, when transcribed, can block the activity, function or production of ROCK. The liposomal delivery systems described above are one way in which the DNA encoding an RNAi and/or antisense can enter the cell.

[0093] Alternatively, the DNA encoding an RNAi and/or antisense can be prepared in a viral vector system that has the capability of entering into cells. These are well-known in the art and include Madzak et al., J. Gen. Virol., 73: 1533-36 (1992) (papovavirus SV40); Berkner et al., Curr. Top.

Microbiol. Immunol., 158: 39-61 (1992) (adenovirus); Moss et al., Curr. Top. Microbiol. Immunol., 158: 25-38 (1992) (vaccinia virus); Muzyczka, Curr. Top. Microbiol. Immunol., 158: 97-123 (1992) (adeno-associated virus); Margulskee, Curr. Top. Microbiol. Immunol., 158: 67-93 (1992) (herpes simplex virus (ISV) and Epstein-Barr virus (HBV)); Miller, Curr. Top. Microbiol. Immunol., 158: 1-24 (1992) (retrovirus); Brandyopadhyay et al., Mol. Cell. Biol., 4: 749-754 (1984) (retrovirus); Miller et al., Nature, 357: 455-450 (1992) (retrovirus); Anderson, Science, 256: 808-813 (1992) (retrovirus); C. Hofmann et al., Proc. Natl. Acad. Sci. USA, 1995; 92, pp. 10099-10103 (baculovirus).

[0094] In another embodiment, ROCK 1 and/or 2 are inhibited using genetic manipulation techniques, such as, but not limited to, transgenic techniques involving either knockout or dominant negative constructs. Such constructs are disclosed in Khyrul, W., et al., J. Biol. Chem., 279(52):54131- 54139 (2004), which is incorporated by reference herein.

[0095] As mentioned above, one embodiment of blocking ROCK would be to individually or collectively block or inhibit the upstream or downstream effectors molecules of ROCK using any of the methods described herein, such as but not limited to small molecule inhibitors, RNAi techniques, antisense techniques and/or genetic manipulation. Accordingly, any upstream effectors that could be inhibited include but are not limited to, integrins, growth factor receptors, including but not limited to, TGF-beta and EGFR, cadherins, G protein coupled receptors and the like. In addition, any downstream effectors that could be inhibited include but are not limited to, vimentin, LIMK, Myosin light chain kinase, NHE1, cofilin and the like.

[0096] After culturing in the conditions of the present invention, the cells may be removed from these conditions and placed in a cell culture environment where the environment is not the conditions described herein. Any combination of one, two, three or four of: IL6, EfnA5, the calcium source and the ROCK inhibitor may be absent in the subsequent environment. As used herein, a "subsequent environment" when used in connection with a cell culture environment is a cell culture environment in which at least one of the IL6, EfnA5, the calcium source and the ROCK inhibitor is absent. In one embodiment, the ROCK inhibitor, the calcium source or the IL6 or EfnA5 are absent in the subsequent environment. In another embodiment, the IL6 and/or EfnA5 and ROCK inhibitor are absent from the subsequent environment. In another embodiment, the IL6 and/or EfnA5 and calcium source are absent from the subsequent environment. In another embodiment, the calcium source and ROCK inhibitor are absent from the subsequent environment. In another embodiment, the IL6 and/or EfnA5, ROCK inhibitor and calcium source are a bsent from the subsequent environment.

[0097] In one embodiment, the subsequent environment to the KE or NKE cells, the late passage KE or NKE cells and/or the conditionally immortalized KE or NKE cells is an environment that promotes differentiation of the cells. The subsequent environment may be an in vivo environment that is similar or identical to the organ from which the cells were originally derived, i.e., an autologous implant. For example, hepatocytes that have been cultured according to the methods of the present invention can be reintroduced into the liver of the subject from which the cells were initially biopsied or isolated.

[0098] The subsequent environment may be an in vitro environment that is that more closely resembles the biochemical or physiological properties of the organ from which the cells were originally derived once placed in this subsequent environment. The subsequent environment may also be a "synthetic environment" such that factors known to promote differentiation in vitro are added to the cell culture. For example, late passage liver epithelial cells, once placed in a subsequent environment that is designed to promote differentiation of the cells, may begin to form clusters and/or express proteins that resemble mature liver epithelial cells.

[0099] In one embodiment, KE or NKE cells, the late passage KE or NKE cells andmor the conditionally immortalized KE or NKE cells are placed into a su bsequent environment that is specific to stimulate differentiation of cells into the cells of the organ from which the cells were originally derived. For example, conditionally immortalized prostate epithelial cells can be removed from the conditions of the present invention and placed into culture conditions designed to promote differentiation of prostate cells. Various environments for culturing epithelial cells are detailed in Culture of Epithelial Cells (Ian Freshney and Mary G. Freshney, Eds. Wiley-Liss, Inc.) (2^nd Ed. 2002), which is incorporated by reference.

[00100] Alternatively, the cells can be seeded in a subsequent environment into or onto a natural or synthetic three-dimensional cell culture surfaces. One non-limiting example of a three-dimensional surface is a Matrigel^e-coated culture surface. Other three dimensional culture environments include surfaces comprising collagen gel and/or a synthetic biopolymeric material in any configuration, such as but not limited to a hydrogel. Of course, a variety of three dimensional culture surfaces may be used simultaneously with the methods the present invention. If a three-dimensional culture environment is used, the feeder cells may or may not be used as well. These three-dimensional cell culture surface environments may or may not promote differentiation.

[00101] In one embodiment, KE or NKE cells, the late passage KE or NKE cells and or the conditionally immortalized NKE cells can be genetically modified to express a protein of interest. The genetic modification of the cells would not be a modification designed to immortalize the cells, such as the insertion of a viral protein. Rather, the genetic modification of the cells would be designed to, for example, insert a transgene that codes for a protein.

[00102]The methods by which the transgenes are introduced into the cells are standard methods known from the literature for in vitro transfer of DNA into mammalian cells, such as electroporation; calcium phosphate precipitation or methods based on receptor-mediated endocytosis, disclosed in WO 93/07283, which is incorporated by reference. Other methods and materials for inserting a gene of interest into cells are disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory Press, Third Edition (2001), which is incorporated by reference.

[00103] A wide variety of genes of interest can be expressed in the KE or NKE cells, the late passage KE or NKE cells and or the conditionally immortalized KE or NKE cells. These genes of interest include, but are not limited to, sequences encoding toxins, clotting factors, enzymes, prodrug converting enzymes, antigens which stimulate immune responses, tumor necrosis factors, cytokines, and various proteins with therapeutic applications (e.g., growth hormones and regulatory factors).

[00104] After transfecting the KE or NKE cells, the late passage KE or NKE cells and/or the conditionally immortalized KE or NKE cells of the present invention, these cells that were successfully transfected can be selected for using a markers that are well known in the art. After selection of the successfully transfected cells, the genetically modified KE or NKE cells, the late passage KE or NKE cells and/or the conditionally immortalized KE or NKE cells of the present invention can be cultured using the cell culture techniques of the present invention to produce a population of genetically modified KE or NKE cells, late passage KE or NKE cells and/or conditionally immortalized KE or NKE cells. These cells can su bsequently be collected and placed into a subsequent environment as described above, including but not limited to being placed back into the subject, i.e., an autologous implant. [00105]The present invention is also directed to methods of identifying candidate treatments for a subject in need of treatments for which the subject has a condition marked by the presence of abnormal or diseased KE or NKE cells. Such conditions marked by the presence of abnormal or diseased KE or NKE cells include but are not limited to neoplasias, a hyperplasias or malignant tumors or benign tumors. The methods comprising obtaining a sample of the abnormal KE or NKE cells from the subject and culturing the abnormal KE or NKE cells according to any of the culture methods of the present invention to produce an in vitro population of abnormal KE or NKE cells. For example, CTCs may be isolated from the organism's circulation, and the methods of the present invention may be utilized to obtain a sufficient number of cells for further analysis, such as but not limited to, phenotypically or genetically characterizing the cells. Once a sufficient number of abnormal or diseased KE or NKE cells are obtained, these cells can also be assayed to determine a response profile, which can be used to identify a candidate treatment for the subject.

[00106] A response profile, as used herein, is a collection of one or more data points that would indicate, e.g., to a clinician, the likelihood that a particular treatment will produce a desired response in the abnormal KE or NKE cells if they were in an in vivo setting. A "response" as used in connection with a response profile may or may not be either cell death by any means (necrosis, toxicity, apoptosis etc) or a reduction of the growth rate of the abnormal cells. The response profile need not predict a response with 100% accuracy. A response profile can be a single data point or it can be a collection of data.

[00107] Any method can be used to identify or determine the response profile of a given population of abnormal KE or NKE cells. For example, the response profile may be assessed by sequencing at least part of the DNA or RNA that is isolated from the abnormal cells. This may be particularly useful when it is suspected that a virus, e.g., human papilloma virus (HPV), human immunodeficiency virus (HIV) may be causing the a bnormal condition. It is not necessary that all of the DNA/RNA be sequenced to provide at least one data point for the response profile. For example, using well- known techniques involving polymerase chain reaction (PCR), it would currently be a matter of simple procedure to use PCR primers with sequences specific for the DNA/RNA suspected of being present, e.g., HPV or HIV, in a PCR reaction to determine if a product is made. If no detectable product is generated after the PCR reaction using specific primers, it may be possible to conclude that the portion of the virus for which the PCR primers are specific may not be present. Likewise, determining the absence of a particular DNA/RNA sequence could also be a data point in a response profile. In this manner, the DNA or RNA is "sequenced" for the purposes of the present invention, although the precise sequence is not determined for the entire DNA/RNA sequence isolated from the cells. Thus, "sequencing" as used herein may or may not result in generating the entire nucleotide sequence of the isolated DNA/ NA. Other methods can also be used to determine the sequence of the isolated DNA/RNA such as, but not limited to Southern blots, Northern blots, RT- PCR, automated sequencing and the like. Methods of sequencing DNA/RNA are well known in the art and need not be repeated herein.

[00108] Similarly, the response profile may be assessed by identifying the presence or absence of at least a portion of one mRNA that may be produced in the abnormal KE or NKE cells in vitro. Like determining the sequence of the DNA/RNA above, the precise sequence of the mRNA need not be determined for the entire mRNA isolated from the cells. Methods that can also be used to determine the presence or absence of the sequence of the isolated mRNA include but are not limited to Northern blots, RT-PCR, automated sequencing and the like. Methods of identifying the presence or a bsence of the at least one mRNA are well known in the art and need not be repeated herein.

[00109] Similarly, the response profile may be assessed by identifying the presence or absence of at least a portion of one protein that may be produced in the abnormal KE or NKE cells in vitro. Like determining the sequence of the DNA/RNA above, the precise amino acid sequence of the present or absent protein need not be determined for the entire protein. Methods that can also be used to determine the presence or absence of the sequence of the isolated protein include but are not limited to Western blots, immunohistochemical methods, ELISA methods, and the like. Methods of identifying the presence or absence of the at least one protein are well known in the art and need not be repeated herein. The presence or absence of a protein, e.g., a receptor, may indicate that the cells are susceptible to a particular treatment that may, for example, result in cell death.

[00110]The response profile may be assessed by subjecting the abnormal KE or NKE cells in vitro to a chemotherapeutic agent and determining the response of the cells to the chemotherapeutic agent. As used herein, a chemotherapeutic agent is not limited to traditional cancer treatments but is used to indicate a therapeutic treatment of any kind using a chemical entity. In one embodiment, the response to the therapeutic agent can be assessed by determining the therapeutic index of the agent on the cells. Determining the therapeutic index is common in the art and is simply the ratio of the LD50/EC50, with the LD₅₀ representing the median lethal dose and the EC₅₀ representing the half maximal dose of the agent on the cells. Other methods to assess a response to the agent include but are not limited to determining dose response curves, cell survival curves and the like. In one embodiment, the agent that is used to determine the response of the abnormal KE or NKE cells to the agent can be the same or a different agent that is later administered to the subject. [00111]The present invention is also directed to methods of identifying an abnormal KE or NKE cell in a subject. These methods comprise culturing at least one candidate abnormal KE or NKE cell isolated from the subject according to the cell culture methods of the present invention. Once the KE or NKE cells, the late passage KE or NKE cells and or the conditionally immortalized KE or NKE cells have been expanded, a tissue origin profile can be determined for the cells to determine the likely tissue of origin of the candidate abnormal KE or NKE cells. At least one feature of the KE or NKE cells, the late passage KE or NKE cells and or the conditionally immortalized KE or NKE cells can be compared to the same feature of normal KE or NKE cells that are obtained from the same tissue as that of the determined tissue origin profile of the candidate abnormal KE or NKE cells. Any difference between abnormal or diseased cells and normal cells can be used, including but not limited to, cell growth characteristics, for example, colony formation on a cell surface, Matrigel™ or other three-dimensional surface. Other means of determining differences between diseased and normal cells include, but are not limited to, assessing the proteomic profile of the cells, assessing the metabolomic profile of the cells, assessing the genomic profile, and/or using other biological assays that will highlight a difference between diseased or abnormal cells and normal cells. A detected difference in the candidate abnormal KE or NKE cells and the normal KE or NKE cells would indicate that the candidate abnormal KE or NKE cells are abnormal compared to normal KE or NKE cells.

[00112]The same methods that are used to assess a response profile can be used to assess a tissue origin profile. For example, the candidate abnormal cells can be assayed for mRNA transcript production, protein expression and tissue origin can also be assessed visually through histological evaluation. Methods of assessing a tissue origin profile also include immunohistochemical staining. Once a likely tissue of origin has been established for the candidate abnormal cells, the cells can be assayed for at least one feature of normal cells from the same tissue. For example, if a candidate abnormal cell has been identified as originating from mammary tissue, these cells can be assayed for the BRCAl and/or BRCA2 mutation, overexpression of the HER-2/neu growth factor receptor and the like. If the candidate abnormal cell has the BRCAl mutation, the cell can then be confirmed as being an abnormal mammary cell. The invention is not limited to the types of assays used to identify the tissue of origin; nor is the invention limited to the types of assays used to determine differences in normal and potentially abnormal cells. The cell culture methods of the present invention enable the identification of these cells by providing methods for expanding the isolated cells.

[00113]The present invention also provides kits for culturing KE or NKE cells and/or generating conditionally immortalized KE or NKE cells. The kits can include culture vessels, culture media in wet or dry form and/or individual media components such as serum or some other calcium source. The kit may or may not include frozen feeder cells, other chemicals, such as trypsin, for passaging cells, etc.

Examples

[00114] Factors that are present in conditioned medium (CM) produced by irradiated murine fibroblasts may be important for conditional immortalization of primary human epithelial cells. These factors were identified by the analysis of irradiated vs. non-irradiated CM using antibody arrays, as well as analysis of signaling pathways induced in the epithelial cells. Human ectocervical cells (HECs) were cultured and studied and the proliferation of HECs was examined to assess the effects of CM and various factors.

F Medium for Primary Cell Culture

[00115] Stock solutions were made for the preparation of the F-medium. Hydrocortisone/EGF MIX (1000X): Dissolve hydrocortisone (Sigma # H-0888) in 100% ethanol at 0.5 mg/ml. Mix 1 ml of this with 19 ml DM EM containing 2.5 μg EGF. (GIBCO supplement kit for keratinocyte serum-free medium includes a 2.5 μg vial of recombinant human EGF.) Store sterile 1.1 ml aliquots at -20°C (hydrocortisone = 25 μg/ml; EGF = 0.125 μg/ml). Insulin (5mg/ml): ): Dissolve 100 mg insulin (Sigma # 1-5500) in 20 ml distilled water containing 200 μΙ glacial acetic acid. Filter sterilize and store 1.1 ml aliquots at -20°C. Cholera Toxin (11.7 μΜ): Dissolve 1 mg vial of cholera toxin (Sigma # C-3012) in 1 ml distilled water and filter sterilize. Stable at 4°C for about 1 year. Complete DMEM (lx): 500 ml DMEM (GIBCO # 11965-092) + 50 ml fetal bovine serum (GIBCO # 16140-071) + 5.5 ml 100X glutamine (GIBCO # 25030-081) + 5.5 ml 100X pen/strep (GIBCO # 15140-122). Sterile. Store at 4°C. F-12 nutient mix (lx): GIBCO # 11765-054. Sterile. Store at 4°C. Gentamicin (10 mg/ml): GIBCO # 15710-064. Sterile. Stable at room temperature. Fungizone (250 μg/ml): Fisher Scientific #

BP264550. Store sterile 1.1 ml aliquots at -20°C.

[00116]The F-medium was prepared from the stock solutions according to Table 1. Table 1

COMPONENT 500 ml 1000 ml

Complete DMEM 373 ml 746 ml

F12 nutrient mix 125 ml 250 ml

Hydrocortisone/EGF mix 0.5 ml 1.0 ml

Insulin 0.5 ml 1.0 ml Fungizone 0.5 ml 1.0 ml

Gentamicin 0.5 ml 1.0 ml

Cholera toxin 4.3 μΙ 8.6 μΙ

[00117] The components were mixed, filtered with 0.2 μιη sterile filter a nd stored at 4°C. Proliferation of HECs under various culture conditions

[00118] H ECs were cultured with F-medium (as control) and CM from medium or medium after lysing. J2 murine fibroblasts (J2) were cultured in F-medium and the factor(s) responsible for producing CM were released in F-medium as J2 disintegrate due to apoptosis. NI H 3T3 murine fibroblasts (3T3) were cultured in F-medium and the conditioning factors were released when cells were lysed in F-medium by means of u ltrasonic disruption. The results are shown in Fig. 1. The proliferation of H ECs was measured. There are at least two conditioning factors: one that does not require irradiation (present in I - J2 CM and IR- 3T3 lysates), and one that does (additional level of activity in IR+ J2 CM and IR+ 3T3 lysates).

[00119] HECs were cultured with different CM a nd the proliferation of HECs was measured. CM contained one or more radiation-independent particulate factors (> 80S pellet from IR- and I R+ sonicated cells resuspended in fresh F-medium) and one or more radiation-induced solu ble factors (< 80S supernatant from I R+ cells only). The particulate factor(s) induced enhanced proliferation, but the solu ble factor(s) showed little activity in the a bsence of the particulate fraction. The com bination of solu ble and particulate factors ind uced the highest level of HEC proliferation. The results are shown in Fig. 2.

Screening of effective factors

[00120] Phorphoproteins were screened by antibody array in HECs cultured in keratinockte growth medium (KGM), conditioned F-medium (CM), or in co-culture with irradiated J2 cells with or without (+/-) the ROCK kinase inhibitor, Y27632. The results (fold change) are shown in Ta ble 2.

[00121] Ta ble 2 - Phosphoprotein screen

AMPKctl 12.0 24.0 - (T183)

E K1/2 10.1 4.3 2.2

(T202, Y204/T185, Y187)

HSP60 5.5 21.0 -

(total protein)

JNK1/2/3 5.1 5.0 -

(T183, Y185/T221, Y223) p53 4.6 3.0 4.2 (S46)

CREB 4.4 6.2 - (S133) p53 3.7 6.3 3.3 (S15) p53 3.2 44.1 2.5 (S392)

GSK-3a/ 2.8 - - (S21/S9)

RSK1/2/3 2.7 3.6 - (S380/S386/S377)

Hck 2.5 - - (Y411)

C-Jun 2.1 4.0 - (S63)

MSK1/2 - 2.2 - (S376/S360)

FAK - -7.0 - (Y397)

Akt 1/2/3 - - (S473)

PRAS40 - -2.3 - (T246)

HSP27

[00122] In Table 2, only changes > 2-fold are shown. FYJ2 = cells in F-medium, co-cultured with irradiated J2 cells and the ROCK inhibitor Y27632; CMY = cells in conditioned F-medium with the ROCK inhibitor Y27632; KGM = keratinocyte growth medium; FJ2 = cells in F-medium, co-cultured with irradiated J2 cells without the ROCK inhibitor Y27632. Positive (no sign) = more active; negative (minus sign) = less active.

[00123] Receptor tyrosine kinases (RTKs) were screened for activity (tyrosine phosphorylation) by antibody array in HECs cultured in KGM, CM, or in co-culture with irradiated J2 cells with or without (+/-) the ROCK kinase inhibitor, Y27632. The results (fold change) are shown in Table 3.

Table 3

-2.7

FGFR-1 -■^"> ά

Fit-3

Insulin R -10.8

Mer -8,6

Axl -8.5

FGFR-3 -3.5

ErbB2 -8 1

Tie-2

EphB3 -4.6

ALK

EphAlO

VEGFR-3

EGFR

RYK ^*■¾ "ί

[00124] In Table 3, only changes > 2-fold are shown. FYJ2 = cells in F-medium, co-cultured with irradiated J2 cells and the ROCK inhibitor Y27632; CMY = cells in conditioned F-medium with the ROCK inhibitor Y27632; KGM = keratinocyte growth medium; FJ2 = cells in F-medium, co-cultured with irradiated J2 cells without the ROCK inhibitor Y27632. Positive (no sign) = more active; negative (minus sign) = less active.

[00125]The ephrin receptor A7 was strongly activated by irradiated J2 cells in the RTK screen. Fig. 3 shows representative photographs of cultured HECs with CM and various factors. In CM+Y27632, HECs grew in colonies and were smaller than in F-medium + Y27632. Dimeric recombinant Efn A5, chimera with Fc domain of mouse IgG (the Fc domain allows for disulfide-based dimerization), induced colonies and small cells in F-medium + Y27632. Cultures with His-tagged Efn A5 were monomeric and showed normal cell size.

[00126] Fig. 4 shows proliferation of cultured HECs with CM or different amounts of Fc-Efn A5 (Fc- A5). Maximal proliferation of HECs in F-medium + Y27632 was induced by an Efn A5 decamer. Protein A (pA) can bind five Efn A5-Fc dimers (Fc-A5). The activity of 20 ng/ml Fc-A5 was increased by pre-binding to pA at a 2.5:1 molar ratio (0.8 ng/ml) and further increased by pre-binding at a 5: 1 molar ratio (1.6 ng/ml). Proliferation of HECs was increased using 40 ng/ml Fc-A5 and 3.3 ng/ml pA (also a 5: 1 molar ratio).

[00127] The protein levels of a num ber of factors were screened in irradiated vs. non-irradiated J2 CM. Fig. 5 shows only the factors with more tha n 2 fold change as a result of irradiation. Positive: increase; negative: decrease.

[00128] Fig. 6 is a schematic graph showing the activation cascade by inflammatory cytokines. The phosphoprotein array shows activation of J NK 1/2., c-Jun and p38 MAPK and supports the idea that at least one of the radiation-induced solu ble factors is an inflammatory cytokine, for example I L6 and I L11.

[00129] FIG. 7 is a schematic graph showing the activation cascade by I L-6. Independent Western blots showed that Janus kinase 1 (JAKl), Janus kinase 2 (JAK2) and signal transd ucer and activator of transcription 3 (STAT3) were activated during conditional reprogramming, thereby showing activation of the JAK/STAT pathway during conditional reprogramming of H ECs and implicating IL-6 as a radiation-induced solu ble conditioning factor. The results are shown in Fig. 8.

Proliferation of HECs under various culture conditions

[00130] Fig. 9 shows the relative proliferation when cultured with different factors. Fig. 9 shows the results of H EC proliferation assay in F-medium containing 10 μΜ Y27623 (all bars), 40 ng/ml Fc-Efn A5 pre-bound to 3.3 ng/ml protein A (5: 1 molar ratio) and 0-50 ng/ml I L-6. 10 ng/ml I L-6 was optimal under these cond itions for H EC proliferation.

[00131] Fig. 10 shows proliferation of H ECs was stimulated by CM made from irradiated human foreskin fibroblasts (H FF), especially when supplemented with lysates of the irradiated HFFs resuspended in the volume of F-medium in which they were cultured (+ IX lysate), or in half as much medium (+ 2X lysate). To generate a renewa ble source of human fibroblasts, the H FFs were immortalized by introduction of the gene encoding the hTE T su bunit of human telomerase.

Claims

What is Claimed is:

1. A method of continuously culturing epithelial cells, the method comprising a) culturing the epithelial cells in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, and b) inhibiting the activity of ho kinase (ROCK) in the epithelial cells during culturing.

2. The method of claim 1, wherein the epithelial cells are non-keratinocyte epithelial (NKE) cells.

3. The method of claim 2, wherein the NKE cells are primary cells.

4. The method of claim 2, wherein the NKE cells are not primary cells.

5. The method of claim 2, wherein the NKE cells are tumor cells.

6. The method of any of claims 2-5, wherein the NKE cells are selected from the group

consisting of squamous cells, columnar cells, adenomatous cells and transitional epithelial cells.

7. The method of claim 5, wherein the NKE cells are selected from the group consisting of prostate cells, mammary cells, hepatocytes, pancreatic islet cells, pulmonary epithelial cells, kidney cells, bladder cells, stomach epithelial cells, large intestinal epithelial cells, small intestinal epithelial cells, urethral epithelial cells, testicular epithelial cells, ovarian epithelial cells, thyroid cells, parathyroid cells, adrenal cells, thymus cells, gall bladder cells and pituitary cells.

8. The method of claim 1, wherein the epithelial cells are keratinocyte epithelial (KE) cells.

9. The method of claim 8, wherein the KE cells are primary cells.

10. The method of claim 8, wherein the KE cells are not primary cells.

11. The method of claim 8, wherein the KE cells are tumor cells.

12. The method of any of claims 1-11, wherein the calcium-containing medium comprises serum or a serum replacement.

13. The method of any of claims 1-12, wherein the epithelial cells are not co-cultured with feeder cells.

14. The method of any of claims 1-12, wherein the epithelial cells are not cultured in feeder cell- conditioned media.

15. The method of any of claims 1-14, wherein inhibiting the activity of Rho kinase (ROCK) in the epithelial cells comprises inhibiting Rho kinase (ROCK 1), Rho kinase 2 (ROCK 2) or both.

16. The method of any of claim 1-15, wherein inhibiting the activity of ROCK comprises culturing the epithelial cells in the presence of a small molecule ROCK inhibitor.

17. The method of claim 16, wherein the small molecule ROCK inhibitor is selected from the group consisting of Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

18. The method of any of claims 1-15, wherein inhibiting the activity of ROCK comprises

culturing the non-keratinocyte epithelial cells in the presence of an RNA interference (RNAi) molecule specific for ROCK 1, ROCK 2 or both.

19. The method of any of claims 1-18, further comprising culturing the epithelial cells in the presence of one or more of the compounds selected from the group consisting of granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), epiregulin (ERG), betacellulin (BC), lnterleukin-11 (IL11), a collagen and heparin-binding EGF-like growth factor (HB-EGF).

20. A method of stimulating growth of epithelial cells, the method comprising a) culturing the epithelial cells in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, and b) inhibiting the activity of Rho kinase (ROCK) in the epithelial cells during culturing. whereby culturing the epithelial cells while inhibiting the activity of the Rho kinase will stimulate the growth of the epithelial cells.

21. The method of claim 20, wherein the epithelial cells are non-keratinocyte epithelial (NKE) cells.

22. The method of claim 21, wherein the NKE cells are primary cells.

23. The method of claim 21, wherein the NKE cells are not primary cells.

24. The method of claim 21, wherein the NKE cells are tumor cells.

25. The method of any of claims 21-24, wherein the NKE cells are selected from the group

consisting of squamous cells, columnar cells, adenomatous cells and transitional epithelial cells.

26. The method of claim 24, wherein the NKE cells are selected from the group consisting of prostate cells, mammary cells, hepatocytes, pancreatic islet cells, pulmonary epithelial cells, kidney cells, bladder cells, stomach epithelial cells, large intestinal epithelial cells, small intestinal epithelial cells, urethral epithelial cells, testicular epithelial cells, ovarian epithelial cells, thyroid cells, parathyroid cells, adrenal cells, thymus cells, gall bladder cells and pituitary cells.

27. The method of claim 20, wherein the epithelial cells are keratinocyte epithelial (KE) cells.

28. The method of claim 27, wherein the KE cells are primary cells.

29. The method of claim 27, wherein the KE cells are not primary cells.

30. The method of claim 27, wherein the KE cells are tumor cells.

31. The method of any of claims 20-30, wherein the calcium-containing medium comprises serum or a serum replacement.

32. The method of any of claims 20-31, wherein the epithelial cells are not co-cultured with feeder cells.

33. The method of any of claims 20-31, wherein the epithelial cells are not cultured in feeder cell-conditioned media.

34. The method of any of claims 20-33, wherein inhibiting the activity of Rho kinase (ROCK) in the epithelial cells comprises inhibiting Rho kinase (ROCK 1), Rho kinase 2 (ROCK 2) or both.

35. The method of any of claim 20-34, wherein inhibiting the activity of ROCK comprises

culturing the epithelial cells in the presence of a small molecule ROCK inhibitor.

36. The method of claim 35, wherein the small molecule ROCK inhibitor is selected from the group consisting of Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

37. The method of any of claims 20-34, wherein inhibiting the activity of ROCK comprises

culturing the non-keratinocyte epithelial cells in the presence of an RNA interference (RNAi) molecule specific for ROCK 1, ROCK 2 or both.

38. The method of any of claims 20-37, further comprising culturing the epithelial cells in the presence of one or more of the compounds selected from the group consisting of granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), epiregulin (ERG), betacellulin (BC), lnterleukin-11 (IL11), a collagen and heparin-binding EGF-like growth factor (HB-EGF).

39. A method of identifying a candidate treatment for a su bject 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 a sample of the abnormal epithelial cells from the subject, b) culturing the epithelial cells in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, and at least one Rho kinase (ROCK) inhibitor, to produce a population of abnormal epithelial cells in vitro, c) determining a response profile of at least a portion of the abnormal epithelial cells in vitro, and d) identifying a candidate treatment for the subject based on the determined response profile.

40. The method of claim 39, wherein the abnormal epithelial cells are non-keratinocyte

epithelial (NKE) cells.

41. The method of claim 40, wherein the abnormal NKE cells are primary cells.

42. The method of claim 40, wherein the abnormal NKE cells are not primary cells.

43. The method of claim 40, wherein the abnormal NKE cells are tumor cells.

44. The method of any of claims 40-43, wherein the abnormal NKE cells are selected from the group consisting of squamous cells, columnar cells, adenomatous cells and transitional epithelial cells.

45. The method of claim 43, wherein the abnormal NKE cells are selected from the group

consisting of prostate cells, mammary cells, hepatocytes, pancreatic islet cells, pulmonary epithelial cells, kidney cells, bladder cells, stomach epithelial cells, large intestinal epithelial cells, small intestinal epithelial cells, urethral epithelial cells, testicular epithelial cells, ovarian epithelial cells, thyroid cells, parathyroid cells, adrenal cells, thymus cells, gall bladder cells and pituitary cells.

46. The method of claim 39, wherein the abnormal epithelial cells are keratinocyte epithelial (KE) cells.

47. The method of claim 46, wherein the abnormal KE cells are primary cells.

48. The method of claim 46, wherein the abnormal KE cells are not primary cells.

49. The method of claim 46, wherein the abnormal KE cells are tumor cells.

50. The method of any of claims 39-49, wherein the calcium-containing medium comprises serum or a serum replacement.

51. The method of any of claims 39-50, wherein the abnormal epithelial cells are not co-cultured with feeder cells.

52. The method of any of claims 39-50, wherein the abnormal epithelial cells are not cultured in feeder cell-conditioned media.

53. The method of any of claims 39-52, wherein inhibiting the activity of ho kinase (ROCK) in the epithelial cells comprises inhibiting Rho kinase (ROCK 1), Rho kinase 2 (ROCK 2) or both.

54. The method of any of claim 39-53, wherein inhibiting the activity of ROCK comprises

culturing the epithelial cells in the presence of a small molecule ROCK inhibitor.

The method of claim 54, wherein the small molecule ROCK inhibitor is selected fro group consisting of Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

56. The method of any of claims 39-53, wherein inhibiting the activity of ROCK comprises culturing the non-keratinocyte epithelial cells in the presence of an RNA interference (RNAi) molecule specific for ROCK 1, ROCK 2 or both.

57. The method of any of claims 39-53, further comprising culturing the epithelial cells in the presence of one or more of the compounds selected from the group consisting of granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), epiregulin (ERG), betacellulin (BC), lnterleukin-11 (IL11), a collagen and heparin-binding EGF-like growth factor (HB-EGF).

58. A method of administering autologous target epithelial cells to a subject in need of

additional target epithelial cells, the method comprising a) obtaining a sample of target epithelial cells from the subject, b) culturing the target epithelial cells in a calcium-containing medium with lnterleukin-6 (IL6) or ephrin A5 (EfnA5), or both, and at least one Rho kinase (ROCK) inhibitor, to produce a population of abnormal epithelial cells in vitro, c) collecting the population of target autologous epithelial cells in vitro, and d) administering the collection of target autologous epithelial cells to the subject in need of autologous epithelial cells.

59. The method of claim 58, wherein the target epithelial cells are non-keratinocyte epithelial (NKE) cells.

60. The method of claim 59, wherein the target NKE cells are primary cells.

61. The method of claim 59, wherein the target NKE cells are not primary cells.

62. The method of any of claims 59-61, wherein the target NKE cells are selected from the group consisting of squamous cells, columnar cells, adenomatous cells and transitional epithelial cells.

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

64. The method of claim 58, wherein the target epithelial cells are keratinocyte epithelial (KE) cells.

65. The method of claim 64, wherein the target KE cells are primary cells.

66. The method of claim 64, wherein the target KE cells are not primary cells.

67. The method of any of claims 58-66, wherein the calcium-containing medium comprises serum or a serum replacement.

68. The method of any of claims 57-67, wherein the epithelial cells are not co-cultured with feeder cells.

69. The method of any of claims 58-67, wherein the target epithelial cells are not cultured in feeder cell-conditioned media.

70. The method of any of claims 58-69, wherein inhibiting the activity of Rho kinase (ROCK) in the epithelial cells comprises inhibiting Rho kinase (ROCK 1), Rho kinase 2 (ROCK 2) or both.

71. The method of any of claim 58-70, wherein inhibiting the activity of ROCK comprises

culturing the epithelial cells in the presence of a small molecule ROCK inhibitor.

72. The method of claim 71, wherein the small molecule ROCK inhibitor is selected from the group consisting of Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

73. The method of any of claims 58-70, wherein inhibiting the activity of ROCK comprises

culturing the non-keratinocyte epithelial cells in the presence of an RNA interference (RNAi) molecule specific for ROCK 1, ROCK 2 or both.

74. The method of any of claims 58-73, further comprising culturing the epithelial cells in the presence of one or more of the compounds selected from the group consisting of granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), epiregulin (ERG), betacellulin (BC), lnterleukin-11 (IL11), a collagen and heparin-binding EGF-like growth factor (HB-EGF).