| PATENT NUMBER | This data is not available for free |
| PATENT TITLE |
Methods for altering cell fate |
| PATENT ABSTRACT | The invention provides methods for altering the expression profile of a cell to convert the cell from one cell type to a desired cell type. These reprogrammed cells may be used in a variety of medical applications for treating a mammal in need of a particular cell type |
| PATENT INVENTORS | This data is not available for free |
| PATENT FILE DATE | December 10, 2001 |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of reprogramming a cell, said method comprising the steps of: (a) incubating a nucleus from a donor cell with a reprogramming media under conditions that allow the removal of a factor from said nucleus or the addition of a factor from said reprogramming media to said nucleus; and (b) inserting said nucleus or a chromatin mass formed from said nucleus into a recipient cell or cytoplast, thereby forming a reprogrammed cell. 2. A method of reprogramming a cell, said method comprising the steps of: (a) incubating a chromatin mass from a donor cell with a reprogramming media under conditions that allow the removal of a factor from said chromatin mass or the addition of a factor from said reprogramming media to said chromatin mass; and (b) inserting said chromatin mass or a nucleus formed from said chromatin mass into a recipient cell or cytoplast, thereby forming a reprogrammed cell. 3. A method of reprogramming a cell, said method comprising incubating a permeabilized cell with a reprogramming media under conditions that allow the removal of a factor from the nucleus or chromatin mass of said permeabilized cell or the addition of a factor from said reprogramming media to said nucleus or chromatin mass, thereby forming a reprogrammed cell. 4. A cell produced using the method of claim 1, 2, or 3, wherein said cell expresses a combination of two or more endogenous mRNA molecules or endogenous proteins that is not expressed in a naturally-occurring cell. 5. A cell that expresses a T-cell receptor or IL-2 and one or more fibroblast-specific proteins. 6. A cell that expresses a neurofilament protein and one or more fibroblast-specific proteins. 7. A cell that expresses the neurofilament protein NF200 and is immortalized. 8. A cell that expresses Oct4 or alkaline phosphatase and one or more fibroblast-specific proteins. 9. A cell that expresses one or more fibroblast-specific proteins and grows in aggregates, forms colonies, or forms embryoid bodies. 10. A method of treating or preventing a disease, disorder, or condition in a mammal, said method comprising the steps of: (a) incubating a nucleus from a donor cell with a reprogramming media under conditions that allow the removal of a factor from said nucleus or the addition of a factor from said reprogramming media to said nucleus; (b) inserting said nucleus or a chromatin mass formed from said nucleus into a recipient cell or cytoplast, thereby forming a reprogrammed cell; and (d) administering said reprogrammed cell to a mammal in need of said cell type. 11. A method of treating or preventing a disease, disorder, or condition in a mammal, said method comprising the steps of: (a) incubating a chromatin mass from a donor cell with a reprogramming media under conditions that allow the removal of a factor from said chromatin mass or the addition of a factor from said reprogramming media to said chromatin mass; (b) inserting said chromatin mass or a nucleus formed from said chromatin mass into a recipient cell or cytoplast, thereby forming a reprogrammed cell; and (d) administering said reprogrammed cell to a mammal in need of said cell type. 12. A method of treating or preventing a disease, disorder, or condition in a mammal, said method comprising the steps of: (a) incubating a permeabilized cell with a reprogramming media under conditions that allow the removal of a factor from the nucleus or chromatin mass of said permeabilized cell or the addition of a factor from said reprogramming media to said nucleus or chromatin mass, thereby forming a reprogrammed cell; and (b) administering said reprogrammed cell to a mammal in need of said cell type. 13. The method of claim 3 or 12, wherein said reprogramming media is an interphase reprogramming media or a mitotic reprogramming media. 14. The method of claim 1 or 10, wherein said nucleus remains membrane-bounded and the chromatin in said nucleus does not condense during incubation with said reprogramming media. 15. The method of claim 1 or 10, wherein a chromatin mass is formed from incubation of said nucleus in said reprogramming media. 16. The method of claim 1 or 10, wherein said chromatin mass is incubated in an interphase reprogramming media under conditions that allow a nucleus to be formed from said chromatin mass and said reformed nucleus is inserted into said recipient cell or said recipient cytoplast. 17. The method of claim 3 or 12, wherein said reprogrammed cell is incubated under conditions that allow the membrane of said reprogrammed cell to reseal. 18. The method of any one of claims 1-3 or 10-12, wherein at least 5 mRNA or protein molecules are expressed in said reprogrammed cell that are not expressed in said donor cell or said permeabilized cell. 19. The method of claims 18, wherein said 5 mRNA or protein molecules are specific for a cell type of interest. 20. The method of any one of claims 1-3 or 10-12, wherein at least 5 mRNA or protein molecules are expressed in said donor cell or said permeabilized cell that are not expressed in said reprogrammed cell. 21. The method of any one of claims 1-3 or 10-12, wherein said donor cell or said permeabilized cell is an interphase or mitotic cell. 22. The method of any one of claims 1-3 or 10-12, wherein said donor cell, said permeabilized cell, said recipient cell, said recipient cytoplast, or said reprogrammed cell is an epithelial cell, neural cell, epidermal cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, B-cell, Jurak cell, T-cell, erythrocyte, macrophage, monocyte, fibroblast, muscle cell, embryonic stem cell, or adult stem cell. 23. The method of claim 1, 3, 10, or 12, wherein said donor cell or said permeabilized cell is a B-cell, Jurak cell, or fibroblast and said reprogrammed cell is a T-cell. 24. The method of claim 3 or 12, wherein said recipient cell or said cytoplast is an undifferentiated cell. 25. The method of claim 3 or 12, wherein said reprogramming media is a cell extract. 26. The method of any one of claims 10-12 wherein said donor cell, said permeabilized cell, said recipient cell, or said recipient cytoplast is from a human. 27. The method of any one of claims 10-12, wherein said disease, disorder, or condition is a neurological, endocrine, structural, skeletal, vascular, urinary, digestive, integumentary, blood, immune, autoimmune, inflammatory, or muscular disease, disorder, or condition. 28. A method for measuring endogenous alkaline phosphatase protein in a cell, nucleus, chromatin mass, cell lysate, or in vitro sample, said method comprising the steps of: (a) contacting a solid support with a test sample from cell, nucleus, chromatin mass, cell lysate, or in vitro sample and with a reference sample, wherein said test sample has a known protein concentration or is derived from a known number of cells, and wherein said reference sample has a known level of alkaline phosphatase protein or activity; (b) measuring the level of endogenous alkaline phosphatase protein or activity in said test sample; and (c) comparing said level of endogenous alkaline phosphatase protein or activity in said test sample with the level of alkaline phosphatase protein or activity in said reference sample, thereby determining the level of alkaline phosphatase protein in said cell, nucleus, chromatin mass, cell lysate, or in vitro sample. 29. The method of claim 28, wherein said solid support is a membrane or plastic surface. 30. The method of claim 28, wherein said cell is a stem cell. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION [0002] In general, the invention features methods for converting cells into a desired cell type and methods for administering these reprogrammed cells to a mammal for the treatment or prevention of disease. [0003] Despite having essentially the same genome, different classes of somatic cells in a particular mammal have distinctive phenotypes due to the different combinations of genes that they express. These different expression profiles allow cells to perform certain functions, such as the secretion of a hormone or cartilage. [0004] Because many diseases and injuries are caused by damage to a particular class of cells, methods are needed to produce cells of a desired cell type that may be used to replace these damaged cells. Preferably, these replacement cells have the same genotype as the damaged cells. SUMMARY OF THE INVENTION [0005] The purpose of the present invention is to provide methods for altering the characteristics or functions of cells. In particular, these methods involve incubating a nucleus or chromatin mass from a donor cell with a reprogramming media (e.g., a cell extract) under conditions that allow nuclear or cytoplasmic components such as transcription factors to be added to, or removed from, the nucleus or chromatin mass. Preferably, the added transcription factors promote the expression of mRNA or protein molecules found in cells of the desired cell type, and the removal of transcription factors that would otherwise promote expression of mRNA or protein molecules found in the donor cell. If desired, the chromatin mass may then be incubated in an interphase reprogramming media (e.g., an interphase cell extract) to reform a nucleus that incorporates desired factors from either reprogramming media. Then, the nucleus or chromatin mass is inserted into a recipient cell or cytoplast, forming a reprogrammed cell of the desired cell type. In a related method, a permeabilized cell is incubated with a reprogramming media (e.g., a cell extract) to allow the addition or removal of factors from the cell, and then the plasma membrane of the permeabilized cell is resealed to enclose the desired factors and restore the membrane integrity of the cell. If desired, the steps of any of these methods may be repeated one or more times or different reprogramming methods may be performed sequentially to increase the extent of reprogramming, resulting in a greater alteration of the mRNA and protein expression profile in the reprogrammed cell. Furthermore, reprogramming medias may be made representing combinations of cell functions (e.g., medias containing extracts or factors from multiple cell types) to produce unique reprogrammed cells possessing characteristics of multiple cell types. [0006] Accordingly, in a first aspect, the invention provides a method of reprogramming a cell. This method involves incubating a nucleus with a reprogramming media (e.g., a cell extract) under conditions that allow the removal of a factor from the nucleus or the addition of a factor to the nucleus. Then the nucleus or a chromatin mass formed from incubation of the nucleus in the reprogramming media is inserted into a recipient cell or cytoplast, thereby forming a reprogrammed cell. In one preferred embodiment, the nucleus is incubated with an interphase reprogramming media (e.g., an interphase cell extract). Preferably, the nucleus remains membrane-bounded, and the chromosomes in the nucleus do not condense during incubation with this interphase reprogramming media. In another preferred embodiment, a chromatin mass is formed from incubation of the nucleus in a mitotic reprogramming media (e.g., a mitotic extract). Preferably, this chromatin mass is then incubated in an interphase reprogramming media under conditions that allow a nucleus to reform, and the reformed nucleus is inserted into the recipient cell or cytoplast. [0007] In a related aspect, the invention provides another method of reprogramming a cell. This method involves incubating a chromatin mass with a reprogramming media (e.g., a cell extract) under conditions that allow the removal of a factor from the chromatin mass or the addition of a factor to the chromatin mass. Then the chromatin mass or nucleus formed from incubation of the chromatin mass in a reprogramming media (e.g., an interphase extract) is inserted into a recipient cell or cytoplast, thereby forming a reprogrammed cell. In one preferred embodiment, the chromatin mass is generated by incubating a nucleus from a donor cell in a detergent and salt solution, in a protein kinase solution, or in a mitotic reprogramming media in the presence or absence of an antibody to NuMA or to another protein of the nucleus. In another preferred embodiment, the chromatin mass is isolated from mitotic cells. [0008] In another related aspect, the invention provides yet another method of reprogramming a cell. This method involves incubating a permeabilized cell with a reprogramming media (e.g., a cell extract) under conditions that allow the removal of a factor from the nucleus or chromatin mass of the permeabilized cell or the addition of a factor to the nucleus or chromatin mass, thereby forming a reprogrammed cell. In one preferred embodiment, the permeabilized cell is incubated with an interphase reprogramming media (e.g., an interphase cell extract). Preferably, the nucleus in the permeabilized cell remains membrane-bounded, and the chromosomes in the nucleus do not condense during incubation with this interphase reprogramming media. In another preferred embodiment, a chromatin mass is formed from incubation of the permeabilized cell in a mitotic reprogramming media. In yet another preferred embodiment, the reprogrammed cell is incubated under conditions that allow the membrane of the reprogrammed cell to reseal. If desired, the permeabilized cell may be formed by incubating an intact cell with a detergent, such as digitonin, or a bacterial toxin, such as Streptolysin O. [0009] The invention also provides reprogrammed cells generated using any method of the invention or a combination of methods of the invention. These cells are useful for the treatment or prevention of a disease due to a deficiency in a particular cell type. Additionally, reprogrammed cells that express two or more mRNA molecules or proteins that are each specific for a certain cell type may have novel combinations of phenotypes and activities that are useful for the treatment of disease. For example, cells that maintain the ability of the donor cell to divide and gain the ability to form a functional T-cell receptor or a functional neurofilament are useful for the generation of multiple T-cells or neurons for therapeutic applications. Once transplanted into a subject, these cells may maintain the ability to divide, thereby reducing the dose or dosing frequency of the transplant cells that is required to treat, prevent, or stabilize a disease. The characterization of these cells may also result in the identification of proteins involved in the regulation of gene expression. [0010] In one such aspect, the invention features a cell that expresses a combination of two or more endogenous mRNA molecules or endogenous proteins that is not expressed by a naturally-occurring cell. In a related aspect, the invention features a cell that expresses a combination of two or more endogenous mRNA molecules or endogenous proteins at a level that is at least 10, 20, 50, 75, or 100 fold greater than the expression level of the corresponding mRNA molecules or proteins in any naturally-occurring cell. In preferred embodiments of the above aspects, the cell expresses a combination of 5, 10, 25, 50, 75, 100, 150, 300, or more endogenous mRNA molecules or endogenous proteins that is not expressed by a naturally-occurring cell. In another preferred embodiment, the cell expresses 1, 3, 5, 10, 25, 50, 100, or more endogenous mRNA molecules or endogenous proteins that are specific for one cell type and expresses 1, 3, 5, 10, 25, 50, 100, or more endogenous mRNA molecules or endogenous proteins that are specific for another cell type. In other preferred embodiments, the cell has a combination of 2, 5, 10, 25, 50, 75, 100, 150, 300, or more activities or phenotypes that are not exhibited in a naturally-occurring. In yet other preferred embodiments, the cell is able to divide or is immortalized and expresses a neuronal protein such as the NF200 neurofilament protein or any other protein expressed by differentiated neurons. In still other preferred embodiments, the cell is able to divide or is immortalized and expresses IL-2, an IL-2 receptor, a T-cell receptor, CD3, CD4 and CD8, CD45 tyrosine phosphatase, or any other protein expressed in hematopoietic cells. In yet another embodiment, the cell is formed from the reprogramming of a donor fibroblast cell, nucleus, or chromatin mass, and the reprogrammed cell expresses one or more cytoskeleton proteins such as an integrin at a level that is at least 25, 50, 75, 90, or 95% lower that the corresponding level in the donor fibroblast under the same conditions. In another embodiment, a reprogrammed cell formed from a donor fibroblast or liver cell (e.g., a hepatocyte) expresses IL-2, a neurofilament protein, a T-cell receptor, Oct4, or insulin. [0011] In a related aspect, the invention provides a cell that expresses a T-cell specific protein (e.g., T-cell receptor protein, IL-2 receptor, CD3, CD4, or CD8) and one or more fibroblast-specific proteins. Preferably, stimulation of the cell with an antigen or an antiCD3 antibody induces the expression of the .alpha.-chain of the IL-2 receptor. In another aspect, the invention provides a cell that expresses a hematopoietic-specific protein (e.g., CD45 typrosine phosphatase) and one or more fibroblast-specific proteins. In another related aspect, the invention provides a cell that expresses a neuron-specific protein (e.g., a neurofilament protein such as NF200) or forms neurites and expresses one or more fibroblast-specific proteins. In still another aspect, the invention provides a cell that expresses a neurofilament protein (e.g., NF200) or forms neurites and is immortalized. In yet another aspect, the invention provides a cell that expresses a stem cell-specific protein (e.g., Oct4) or alkaline phosphatase and one or more fibroblast-specific proteins. In still another aspect, the invention provides a cell that expresses one or more fibroblast-specific proteins and grows in aggregates, forms colonies, or forms embryoid bodies. Preferred fibroblast-specific proteins include cell adhesion molecules that e.g., promote anchoring of one or more reprogrammed cells to a site of interest in a host patient. Fibroblast-specific growth factors (e.g., the FGF family of proteins) are other exemplary fibroblast-specific proteins. [0012] These methods for reprogramming cells are useful for the generation of cells of a desired cell type, for example, for medical applications. Accordingly, the invention also provides methods for the treatment or prevention of disease in a mammal that include administering a reprogrammed cell to the mammal. [0013] In one such method, the invention features a procedure for treating or preventing a disease, disorder, or condition in a mammal. This method involves incubating a nucleus from a donor cell with a reprogramming media (e.g., a cell extract) under conditions that allow the removal of a factor from the nucleus or the addition of a factor to the nucleus. The nucleus or a chromatin mass formed from the nucleus is inserted into a recipient cell or cytoplast, thereby forming a reprogrammed cell. The reprogrammed cell is then administered to the mammal in need of the cell type. In one preferred embodiment, the nucleus is incubated with an interphase reprogramming media. Preferably, the nucleus remains membrane-bounded, and the chromosomes in the nucleus do not condense during incubation with this interphase reprogramming media. In another preferred embodiment, a chromatin mass is formed from incubation of the nucleus in a mitotic reprogramming media. Preferably, this chromatin mass is then incubated in an interphase reprogramming media under conditions that allow a nucleus to be formed from the chromatin mass, and the reformed nucleus is inserted into the recipient cell or cytoplast. Preferably, the donor cell is from the mammal (for example, a human) in need of the cell type. Examples of diseases, disorders, or conditions that may be treated or prevented include neurological, endocrine, structural, skeletal, vascular, urinary, digestive, integumentary, blood, immune, auto-immune, inflammatory, endocrine, kidney, bladder, cardiovascular, cancer, circulatory, digestive, hematopoeitic, and muscular diseases, disorders, and conditions. In addition, reprogrammed cells may be used for reconstructive applications, such as for repairing or replacing tissues or organs. [0014] In a related aspect, the invention provides another method of treating or preventing a disease, disorder, or condition in a mammal (for example, a human). This method involves incubating a chromatin mass from a donor cell with a reprogramming media (e.g., a cell extract) under conditions that allow the removal of a factor from the chromatin mass or the addition of a factor to the chromatin mass. The chromatin mass or a nucleus formed from incubating the chromatin mass in an interphase reprogramming media is inserted into a recipient cell or cytoplast, thereby forming a reprogrammed cell. In one preferred embodiment, the chromatin mass used in this method is generated by incubating a nucleus from a donor cell in a detergent and salt solution, in a protein kinase solution, or in a mitotic reprogramming media in the presence or absence of an antibody to NuMA. In another preferred embodiment, the chromatin mass is isolated from mitotic cells. The reprogrammed cell is then administered to a mammal in need of the cell type. Preferably, the donor cell is from the recipient mammal. Examples of diseases, disorders, or conditions that may be treated or prevented include neurological, endocrine, structural, skeletal, vascular, urinary, digestive, integumentary, blood, immune, auto-immune, inflammatory, endocrine, kidney, bladder, cardiovascular, cancer, circulatory, digestive, hematopoeitic, and muscular diseases, disorders, and conditions. In addition, reprogrammed cells may be used for reconstructive applications, such as for repairing or replacing tissues or organs. [0015] In still another related aspect, the invention provides another method of treating or preventing a disease, disorder, or condition in a mammal (for example, a human) that involves incubating a permeabilized cell with a reprogramming media (e.g., a cell extract) under conditions that allow the removal of a factor from the nucleus or chromatin mass of the permeabilized cell or the addition of a factor to the nucleus or chromatin mass. The reprogrammed cell formed from this step is administered to a mammal in need of that cell type. In one preferred embodiment, the permeabilized cell is incubated with an interphase reprogramming media. Preferably, the nucleus in the permeabilized cell remains membrane-bounded, and the chromosomes in the nucleus do not condense during incubation with the interphase reprogramming media. In another preferred embodiment, a chromatin mass is formed from incubation of the permeabilized cell in a mitotic reprogramming media. In yet another preferred embodiment, the reprogrammed cell is incubated under conditions that allow the membrane of the reprogrammed cell to reseal prior to being administered to the mammal. Preferably, the permeabilized cell is from the mammal in need of that cell type. In another preferred embodiment, the permeabilized cell is formed by incubating an intact cell with a detergent, such as digitonin, or a bacterial toxin, such as Streptolysin 0. Examples of diseases, disorders, or conditions that may be treated or prevented include neurological, endocrine, structural, skeletal, vascular, urinary, digestive, integumentary, blood, immune, auto-immune, inflammatory, endocrine, kidney, bladder, cardiovascular, cancer, circulatory, digestive, hematopoeitic, and muscular diseases, disorders, and conditions. In addition, reprogrammed cells may be used for reconstructive applications, such as for repairing or replacing tissues or organs. [0016] The invention also provides methods for measuring an endogenous activity (e.g., an endogenous enzymatic activity) or an endogenous protein in a cell, nucleus, chromatin mass, cell lysate, or in vitro sample. In one such aspect, the method involves contacting a solid support with a test sample from a cell, nucleus, chromatin mass, cell lysate, or in vitro sample, and with a reference sample. The test sample has an endogenous activity of interest that is naturally found in the test sample (e.g., luciferase activity or phosphatase activity, e.g., alkaline phosphatase activity), and the test sample has a known protein concentration or is derived from a known number of cells. The reference sample has a known level of the activity of interest (e.g., luciferase activity or phosphatase activity) or a known amount of naturally-occurring or recombinant protein having the activity. The level of luciferase or phosphatase activity in the test sample is measured and compared to the level of luciferase or phosphatase activity in the reference sample, thereby determining the level of luciferase or phosphatase activity in the cell, nucleus, chromatin mass, cell lysate, or in vitro sample. In one preferred embodiment, the luciferase or phosphatase activity is performed by a naturally-occurring protein encoded by an endogenous nucleic acid under the control of an endogenous promoter. This method may also be used to measure any other endogenous activity of interest. In another preferred embodiment, the activity is specific for one cell type or specific for a family of related cell types. In various embodiments, the activity of interest is the chemical alteration of one or more substrates to form a product. Preferably, either one of the substrates or one of the products is detectable. Detectable labels are well known in the art and include, without limitation, radioactive labels (e.g., isotopes such as .sup.32P or .sup.35S) and nonradioactive labels (e.g., chemiluminescent labels or fluorescent labels, e.g., fluorescein). [0017] In a related aspect, the invention provides a method for measuring the level of an endogenous protein in a cell, nucleus, chromatin mass, cell lysate, or in vitro sample. This method involves contacting a solid support with a test sample from a cell, nucleus, chromatin mass, cell lysate, or in vitro sample, and with a reference sample. The test sample has an endogenous, detectable protein of interest (e.g., luciferase, alkaline phosphatase, or Oct4) that is naturally found in the test sample, and the test sample has a known protein concentration or is derived from a known number of cells. The reference sample has a known amount of the protein of interest, e.g., naturally-occurring or recombinant luciferase, alkaline phosphatase, or Oct4 protein. The signal from the luciferase, alkaline phosphatase, or Oct4 protein in the test sample is measured and compared to the signal from the corresponding protein in the reference sample, thereby determining the amount of luciferase, alkaline phosphatase, or Oct4 protein in the cell, nucleus, chromatin mass, cell lysate, or in vitro sample. This method may also be used to measure the level of any other endogenous protein of interest. In one preferred embodiment, the protein of interest is encoded by an endogenous nucleic acid under the control of an endogenous promoter. In a preferred embodiment, the protein of interest is specific for one cell type or specific for a family of related cell types. In various embodiments, the protein of interest has a detectable label or binds another molecule (e.g., an antibody) with a detectable label. Exemplary detectable labels include radioactive labels (e.g., isotopes such as .sup.32P or .sup.35S) and nonradioactive labels (e.g., chemiluminescent labels or fluorescent labels, e.g., fluorescein). [0018] In preferred embodiments for the above methods of measuring the level of an activity or protein of interest, the solid support is contacted with multiple reference samples, each with a different level of activity or a different amount of the protein of interest. According to this embodiment, a standard curve may be generated from the reference samples and used to determine the level of activity or the amount of the protein of interest in the test sample. In various embodiments, the cell is a stem cell such as an embryonic stem cell or an adult stem cell from brain, blood, bone marrow, pancreas, liver, skin, or any other organ or tissue. In other embodiments, the cell has been exposed to an extract from a stem cell. In yet other embodiments, the test sample is a from a nuclear or cytoplasmic cell extract. Useful solid supports include any rigid or semi-rigid surface that may be contacted with the sample. The support can be any porous or nonporous water insoluble material, including, without limitation, membranes, filters, chips, slides, fibers, beads, gels, tubing, strips, plates, rods, polymers, particles, microparticles, capillaries, and plastic surfaces. If desired, the support can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the samples are contacted. [0019] In preferred embodiments of various aspects of the invention, at least 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300, or more mRNA or protein molecules are expressed in the reprogrammed cell that are not expressed in the donor or permeabilized cell. In another preferred embodiment, the number of mRNA or protein molecules that are expressed in the reprogrammed cell, but not expressed in the donor or permeabilized cell, is between 1 and 5, 5 and 10, 10 and 25, 25 and 50, 50 and 75, 75 and 100, 100 and 150, 150 and 200, or 200 and 300, inclusive. Preferably, at least 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300, or more mRNA or protein molecules are expressed in the donor or permeabilized cell that are not expressed in the reprogrammed cell. In yet another preferred embodiment, the number of mRNA or protein molecules that are expressed in the donor or permeabilized cell, but not expressed in the reprogrammed cell, is between 1 and 5, 5 and 10, 10 and 25, 25 and 50, 50 and 75, 75 and 100, 100 and 150, 150 and 200, or 200 and 300, inclusive. Preferably, the mRNA or protein molecules are specific for the cell type of the donor, permeabilized, or reprogrammed cell, such that the molecules are only expressed in cells of that particular cell type. In still another preferred embodiment, these mRNA or protein molecules are expressed in both the donor cell (i.e., the donor or permeabilized starting cell) and the reprogrammed cell, but the expression levels in these cells differ by at least 2, 5, 10, or 20-fold, as measured using standard assays (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). In other embodiments, the expression of one or more cytoskeleton proteins such as integrins is decreased by at least at least 2, 5, 10, or 20-fold compared to the donor fibroblast cell. In yet other embodiments, the reprogrammed cell expresses a neurofilament protein, T-cell receptor protein, IL-2, IL-2 receptor, insulin, or Oct4 at a level that is at least 2, 5, 10, or 20-fold greater that the corresponding level in the donor or permeabilized cell. [0020] In other preferred embodiments, the size of the donor or permeabilized cell differs from that of the reprogrammed cell by at least 10, 20, 30, 50, 75, or 100%, as measured using standard methods. In another preferred embodiment, the volume of cytoplasm in the donor or permeabilized cell differs from that in the reprogrammed cell by at least 10, 20, 30, 50, 75, or 100%, based on standard methods. In yet another preferred embodiment, the reprogrammed cell has gained or lost an activity relative to the donor or permeabilized cell, such as secretion of a particular hormone, extracellular matrix component, or antibody. In another embodiment, the reprogrammed cell has gained the ability to produce and secrete an interleukin such as IL-2, gained the ability to form a neurofilament, neurite, or axon, or gained the ability to form an embryoid body. In another embodiment, cell has gained the ability to express a T-cell receptor or IL-2 receptor or to produce insulin. Preferably, the .beta.-chain of the IL-2 receptor is expressed constitutively, and the .alpha.-chain is expressed upon activation (e.g., by stimulation with an anti-CD3 antibody or by presentation of an antigen). In other embodiments, a reprogrammed cell such as a stem cell or fibroblast has gained the ability to contract, resembling a contracting muscle cell or beating heart cell. [0021] In still other preferred embodiments, the reprogramming media is an interphase reprogramming media, such as an extract formed from cells synchronized in one or more of the following phases of the cell cycle: G.sub.0, G.sub.1, S, or G.sub.2 phase. In another preferred embodiment, the reprogramming media is an extract formed from cells synchronized in mitosis or from unsynchronized cells. Preferably, the reprogramming media is an extract from the cell type one wishes the donor or permeabilized cell to become, or the reprogramming media is a solution containing factors specific for the cell type one wishes the donor or permeabilized cell to become. Examples of cells that may be used to generate extracts to reprogram cells into stem cells include embryonic stem cells and adult stem cells from brain, blood, bone marrow, pancreas, liver, skin, or any other organ or tissue. Preferably, the donor or permeabilized cell is an interphase or mitotic somatic cell. In another preferred embodiment, the reprogramming media is modified by the enrichment or depletion of a factor, such as a DNA methyltransferase, histone deacetylase, histone, nuclear lamin, transcription factor, activator, repressor, growth factor, hormone, or cytokine. The reprogramming media may or may not contain exogenous nucleotides. In other preferred embodiments, a chromatin mass in a reprogramming media or formed in a permeabilized cell is contacted with a vector having a nucleic acid encoding a gene of interest under conditions that allow homologous recombination between the nucleic acid in the vector and the corresponding nucleic acid in the genome of the chromatin mass, resulting in the alteration of the genome of the chromatin mass. Due to the lack of an intact plasma membrane and the lack of a nuclear membrane, a chromatin mass in a permeabilized cell may be easier to genetically modify than a naturally-occuring cell. Preferably, the chromatin mass or nucleus is purified from the reprogramming media prior to insertion into the recipient cell or cytoplast, or the reprogrammed cell is purified prior to administration into the mammal. Preferably, the donor or permeabilized cell is haploid (DNA content of n), diploid (2n), or tetraploid (4n), and the recipient cell is hypodiploid (DNA content of less than 2n), haploid, or enucleated. [0022] Preferred donor cells, permeabilized cells, recipient cells, reprogrammed cells, and sources of cytoplasts include differentiated cells, such as epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-cells, T-cells, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells; and undifferentiated cells, such as embryonic or adult stem cells. In another preferred embodiment, the donor or permeabilized cell is a differentiated cell, and the reprogrammed cell is a differentiated cell of another cell type. In yet another preferred embodiment, the donor or permeabilized cell is an undifferentiated cell, and the reprogrammed cell is a differentiated cell. In still another preferred embodiment, the donor or permeabilized cell is a differentiated cell, and the reprogrammed cell is an undifferentiated cell. If desired, an undifferentiated reprogrammed cell may be induced to differentiate into a desired cell type in vitro using standard methods, such as by exposure to certain growth factors, hormones, interleukins, cytokines, or other cells. In another preferred embodiment, the undifferentiated reprogrammed cell differentiates into a desired cell type in vivo after administration to a mammal. In yet another preferred embodiment, the donor or permeabilized cell is a B-cell, Jurkat cell, endothelial cell, epithelial cell, or fibroblast, and the reprogrammed cell is a T-cell. It is also contemplated that the nucleus or chromatin mass may be inserted into a recipient cell or cytoplast of the desired cell type or of the same cell type as the donor or permeabilized cell. In still another preferred embodiment, the donor cell, permeabilized cell, recipient cell, or recipient cytoplast is from a human or non-human mammal. In yet another preferred embodiment, the donor nucleus or chromatin mass is from a transgenic cell or mammal or contains a mutation not found in the donor cell or not found in a naturally-occurring cell. The donor or permeabilized cell can be non-immortilized or naturally, spontaneously, or genetically immortilized. The donor cell, permeabilized cell, recipient cell, or cytoplast can be from a source of any age, such as an embryo, fetus, youth, or adult mammal. Cells from younger sources may have acquired fewer spontaneous mutations and may have a longer life-span in vitro or after transplantation in vivo. [0023] Preferably, a disease-causing mutation in a regulatory region, promoter, untranslated region, or coding region of a gene in a donor nucleus or chromatin mass is modified to replace the mutant sequence with a sequence that is not associated with the disease. Alternatively, a nucleic acid is inserted into the donor nucleus or chromatin mass that includes a promoter operably-linked to a sequence of the gene that does not contain a mutation associated with a disease. Preferably, the sequence of the gene is substantially identical to that of a naturally-occurring gene that does not contain a polymorphism or mutation associated with a disease. Examples of mutations that may be rescued using these methods include mutations in the cystic fibrosis gene; mutations associated with Dunningan's disease such as the R482W, R482Q, and R584H mutations in the lamin A gene; and mutations associated with the autosomal-dominant form of Emery Deyfuss muscular dystrophy such as the R249Q, R453W, and Q6STOP mutations in the lamin A gene. In the Q6STOP mutation, the codon for Gln6 is mutated to a stop codon. [0024] Preferred transgenic donor nuclei, chromosomes, or chromatin masses encode a heterologous MHC Class I protein having an amino acid sequence substantially identical to the sequence of an MHC Class I protein found in the mammal to whom the reprogrammed cells will be administered for therapeutic applications. Alternatively, the donor nuclei or chromatin masses may encode a heterologous MHC Class 1 protein having an amino acid sequence substantially identical to the sequence of an MHC Class 1 protein found in another mammal of the same genus or species as the recipient mammal. Reprogrammed cells that express such MHC proteins are less likely to elicit an adverse immune response when administered to the mammal. Other preferred donor nuclei or chromatin masses are modified to express a heterologous protein that inhibits the complement pathway of the recipient mammal, such as the human complement inhibitor CD59 or the human complement regulator decay accelerating factor (h-DAF) (see, for example, Ramirez et al, Transplantation 15:989-998, 2000; Costa et al., Xenotransplantation 6:6-16, 1999). In yet another preferred embodiment, the donor nucleus or chromatin mass has a mutation that reduces or eliminates the expression or activity of a galactosyltransferase, such as alpha(1,3)-galactosyltransferase (Tearle et al., Transplantation 61:13-19, 1996; Sandrin, Immunol. Rev. 141:169-190, 1994; Costa et al, Xenotransplantation 6:6-16, 1999). This enzyme modifies cell surface molecules with a carbohydrate that elicits an adverse immune response when cells expressing this galactose alpha(1,3)-galactose epitope are administered to humans. Thus, reprogrammed cells that have a lower level of expression of this epitope may have a lower incidence of rejection by the recipient mammal. [0025] With respect to the therapeutic methods of the invention, it is not intended that the administration of reprogrammed cells to a mammal be limited to a particular mode of administration, dosage, or frequency of dosing; the present invention contemplates all modes of administration, including intramuscular, intravenous, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide a dose adequate to prevent or treat a disease. Preferably, the cells are administered to the mammal from which the donor or permeabilized cell is obtained. Alternatively, the donor or permeabilized cell may be obtained from a different donor mammal of the same or a different genus or species as the recipient mammal. Examples of preferred donor mammals include humans, cows, sheep, big-horn sheep, goats, buffalos, antelopes, oxen, horses, donkeys, mule, deer, elk, caribou, water buffalo, camels, llama, alpaca, rabbits, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, and primates such as monkeys. The cells may be administered to the mammal in a single dose or multiple doses. When multiple doses are administered, the doses may be separated from one another by, for example, one week, one month, one year, or ten years. One or more growth factors, hormones, interleukins, cytokines, or other cells may also be administered before, during, or after administration of the cells to further bias them towards a particular cell type. Additionally, one or more immunosuppressive agents, such as cyclosporin, may be administered to inhibit rejection of the transplanted cells. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. [0026] As used herein, by "chromatin mass" is meant more than one chromosome not enclosed by a membrane. Preferably, the chromatin mass contains all of the chromosomes of a cell. A chromatin mass containing condensed chromosomes may be formed by exposure of a nucleus to a mitotic reprogramming media (e.g., a mitotic extract), or a chromatin mass may be isolated from mitotic cells as described herein. Alternatively, a chromatin mass containing decondensed or partially condensed chromosomes may be generated by exposure of a nucleus to one of the following, as described herein: a mitotic reprogramming media (e.g., a mitotic extract) in the presence of an anti-NuMA antibody, a detergent and salt solution, or a protein kinase solution. [0027] A chromatin mass may be formed naturally or artificially induced. An exemplary naturally-occurring chromatin mass includes a set of metaphase chromosomes, which are partially or maximally condensed chromosomes that are not surrounded by a membrane and that are found in, or isolated from, a mitotic cell. Preferably, the metaphase chromosomes are discrete chromosomes that are not physically touching each other. Exemplary artificially induced chromatin masses are formed from exposure to a reprogramming media, such as a solution containing factors that promote chromosome condensation, a mitotic extract, a detergent and salt solution, or a protein kinase solution. Artificially induced chromatin masses may contain discrete chromosomes that are not physically touching each other or may contain two or more chromosomes that are in physical contact. [0028] If desired, the level of chromosome condensation may be determined using standard methods by measuring the intensity of staining with the DNA stain, DAPI. As chromosomes condense, this staining intensity increases. Thus, the staining intensity of the chromosomes may be compared to the staining intensity for decondensed chromosomes in interphase (designated 0% condensed) and maximally condensed chromosomes in mitosis (designated 100% condensed). Based on this comparison, the percent of maximal condensation may be determined. Preferred condensed chromatin masses are at least 50, 60, 70, 80, 90, or 100% condensed. Preferred decondensed or partially condensed chromatin masses are less than 50, 40, 30, 20, or 10% condensed. [0029] By "nucleus" is meant a membrane-bounded organelle containing most or all of the DNA of a cell. The DNA is packaged into chromosomes in a decondensed form. Preferably, the membrane encapsulating the DNA includes one or two lipid bilayers or has nucleoporins. [0030] By "donor cell" is meant a cell from which a nucleus or chromatin mass is derived. [0031] By "cytoplast" is meant a membrane-enclosed cytoplasm. Preferably, the cytoplast does not contain a nucleus, chromatin mass, or chromosome. Cytoplasts may be formed using standard procedures. For example, cytoplasts may be derived from nucleated or enucleated cells. Alternatively, cytoplasts may be generated using methods that do not require an intact cell to be used as the source of the cytoplasm or as the source of the membrane. In one such method, cytoplasts are produced by the formation of a membrane in the presence of cytoplasm under conditions that allow encapsulation of the cytoplasm by the membrane. [0032] By "permeabilization" is meant the formation of pores in the plasma membrane or the partial or complete removal of the plasma membrane. [0033] By "reprogramming media" is meant a solution that allows the removal of a factor from a nucleus, chromatin mass, or chromosome or the addition of a factor from the solution to the nucleus, chromatin mass, or chromosome. Preferably, the addition or removal of a factor increases or decreases the level of expression of an mRNA or protein in the donor cell, chromatin mass, or nucleus or in a cell containing the reprogrammed chromatin mass or nucleus. In another embodiment, incubating a permeabilized cell, chromatin mass, or nucleus in the reprogramming media alters a phenotype of the permeabilized cell or a cell containing the reprogrammed chromatin mass or nucleus relative to the phenotype of the donor cell. In yet another embodiment, incubating a permeabilized cell, chromatin mass, or nucleus in the reprogramming media causes the permeabilized cell or a cell containing the reprogrammed chromatin mass or nucleus to gain or loss an activity relative to the donor cell. [0034] Exemplary reprogramming medias include solutions, such as buffers, that do not contain biological molecules such as proteins or nucleic acids. Such solutions are useful for the removal of one or more factors from a nucleus, chromatin mass, or chromosome. Other preferred reprogramming medias are extracts, such as cellular extracts from cell nuclei, cell cytoplasm, or a combination thereof. Yet other reprogramming medias are solutions or extracts to which one or more naturally-occurring or recombinant factors (e.g., nucleic acids or proteins such as DNA methyltransferases, histone deacetylases, histones, nuclear lamins, transcription factors, activators, repressors, growth factors, hormones, or cytokines) have been added, or extracts from which one or more factors have been removed. Still other reprogramming medias include detergent and salt solutions and protein kinase solutions. In some embodiments, the reprogramming media contains an anti-NuMA antibody. By "interphase reprogramming media" is meant a media (e.g., an interphase cell extract) that induces chromatin decondensation and nuclear envelope formation. By "mitotic reprogramming media" is meant a media (e.g., a mitotic cell extract) that induces chromatin condensation and nuclear envelope breakdown. If desired, multiple reprogramming media may be used simultaneously or sequentially to reprogram a donor cell, nucleus, or chromatin mass. [0035] By "addition of a factor" is meant the binding of a factor to chromatin, a chromosome, or a component of the nuclear envelope, such as the nuclear membrane or nuclear matrix. Alternatively, the factor is imported into the nucleus so that it is bounded or encapsulated by the nuclear envelope. Preferably, the amount of factor that is bound to a chromosome or located in the nucleus increases by at least 25, 50, 75, 100, 200, or 500%. [0036] By "removal of factor" is meant the dissociation of a factor from chromatin, a chromosome, or a component of the nuclear envelope, such as the nuclear membrane or nuclear matrix. Alternatively, the factor is exported out of the nucleus so that it is no longer bounded or encapsulated by the nuclear envelope. Preferably, the amount of factor that is bound to a chromosome or located in the nucleus decreases by at least 25, 50, 75, 100, 200, or 500%. [0037] By "enrichment or depletion of a factor" is meant the addition or removal of a naturally-occurring or recombinant factor by at least 20, 40, 60, 80, or 100% of the amount of the factor originally present in the reprogramming media. Alternatively, a naturally-occurring or recombinant factor that is not naturally present in the reprogramming media may be added. Preferred factors include proteins such as DNA methyltransferases, histone deacetylases, histones, nuclear lamins, transcription factors, activators, repressors, growth factors, cytokines, and hormones; membrane vesicles; and [0038] is organelles. In one preferred embodiment, the factor is purified prior to being added to the reprogramming media, as described below. Alternatively, one of the purification methods described below may be used to remove an undesired factor from the reprogramming media. [0039] By "purified" is meant separated from other components that naturally accompany it. Typically, a factor is substantially pure when it is at least 50%, by weight, free from proteins, antibodies, and naturally-occurring organic molecules with which it is naturally associated. Preferably, the factor is at least 75%, more preferably, at least 90%, and most preferably, at least 99%, by weight, pure. A substantially pure factor may be obtained by chemical synthesis, separation of the factor from natural sources, or production of the factor in a recombinant host cell that does not naturally produce the factor. Proteins, vesicles, chromosomes, nuclei, and other organelles may be purified by one skilled in the art using standard techniques such as those described by Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). The factor is preferably at least 2, 5, or 10 times as pure as the starting material, as measured using polyacrylamide gel electrophoresis, column chromatography, optical density, HPLC analysis, or western analysis (Ausubel et al., supra). Preferred methods of purification include immunoprecipitation, column chromatography such as immunoaffinity chromatography, magnetic bead immunoaffinity purification, and panning with a plate-bound antibody. [0040] By "mRNA or protein specific for one cell type" is meant an mRNA or protein that is expressed in one cell type at a level that is at least 10, 20, 50, 75, or 100 fold greater than the expression level in all other cell types. Preferably, the mRNA or protein is only expressed in one cell type. [0041] By "mutation" is meant an alteration in a naturally-occurring or reference nucleic acid sequence, such as an insertion, deletion, frameshift mutation, silent mutation, nonsense mutation, or missense mutation. Preferably, the amino acid sequence encoded by the nucleic acid sequence has at least one amino acid alteration from a naturally-occurring sequence. Examples of recombinant DNA techniques for altering the genomic sequence of a cell, embryo, fetus, or mammal include inserting a DNA sequence from another organism (e.g., a human) into the genome, deleting one or more DNA sequences, and introducing one or more base mutations (e.g., site-directed or random mutations) into a target DNA sequence. Examples of methods for producing these modifications include retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, homologous recombination, gene targeting, transposable elements, and any other method for introducing foreign DNA. All of these techniques are well known to those skilled in the art of molecular biology (see, for example, Ausubel et al., supra). Chromatin masses, chromosomes, and nuclei from transgenic cells, tissues, organs, or mammals containing modified DNA may be used in the methods of the invention. [0042] By "substantially identical" is meant having a sequence that is at least 60, 70, 80, 90, or 100% identical to that of another sequence or to a naturally-occurring sequence. Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. [0043] By "immortilized" is meant capable of undergoing at least 25, 50, 75, 90, or 95% more cell divisions than a naturally-occurring control cell of the same cell type, genus, and species as the immortalized cell or than the donor cell from which the immortalized cell was derived. Preferably, an immortalized cell is capable of undergoing at least 2, 5, 10, or 20-fold more cell divisions than the control cell. More preferably, the immortalized cell is capable of undergoing an unlimited number of cell divisions. Examples of immortalized cells include cells that naturally acquire a mutation in vivo or in vitro that alters their normal growth-regulating process. Other preferred immortalized cells include hybridoma cells which are generated using standard techniques for fusion of a myeloma with a B-cell (Mocikat, J. Immunol. Methods 225:185-189, 1999; Jonak et al., Hum. Antibodies Hybridomas 3:177-185, 1992; Srikumaran et al., Science 220:522, 1983). Still other preferred immortalized cells include cells that have been genetically modified to express an oncogene, such as ras, myc, abl, bcl2, or neu, or that have been infected with a transforming DNA or RNA virus, such as Epstein Barr virus or SV40 virus (Kumar et al., Immunol. Lett. 65:153-159, 1999; Knight et al., Proc. Nat. Acad. Sci. USA 85:3130-3134, 1988; Shammah et al., J. Immunol. Methods 160-19-25, 1993; Gustafsson and Hinkula, Hum. Antibodies Hybridomas 5:98-104, 1994; Kataoka et al., Differentiation 62:201-211, 1997; Chatelut et al., Scand. J. Immunol. 48:659-666, 1998). Cells can also be genetically modified to express the telomerase gene (Roques et al., Cancer Res. 61:8405-8507, 2001). [0044] By "non-immortilized" is meant not immortalized as described above. [0045] The present invention provides a number of advantages related to the alteration of cell fate. For example, these methods may be generally applied to produce cells of any desired cell type. Because these methods involve incubating a nucleus, a chromatin mass, or a permeabilized cell in a reprogramming media (e.g., a cell extract) to allow reprogramming, the efficiency of reprogramming may be enhanced by adding factors to the reprogramming media that facilitate reprogramming or by removing factors that inhibit reprogramming. These reprogrammed cells may be transplanted into mammals for the treatment or prevention of conditions involving damage or deficiency of a particular cell type. If desired, the reprogrammed cells may be manipulated using standard molecular biology techniques to correct a disease-causing mutation before administering the cells to a recipient mammal. [0046] Other features and advantages of the invention will be apparent from the following detailed description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS [0047] The application file contains drawings executed in color (FIGS. 13, 14A, 14B, 14C, 15, and 17A). Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee. [0048] FIG. 1 is an illustration of a gel showing the amount of IL-2 mRNA synthesized by human T-cells stimulated with anti-CD3 antibodies for the indicated lengths of time. Lanes "30c," "60c," and "120c" refer to mock-stimulated T-cells. [0049] FIG. 2 is an illustration of nuclei purified from resting T-cells. [0050] FIG. 3A is the immunofluorescence analysis of nuclear uptake and binding of the T-cell specific transcription factor NFAT in cell-free reprogramming extracts. For this assay, nuclei purified from resting T-cells ("Input" nuclei) were incubated with reprogramming extract from stimulated T-cells for 30 minutes. [0051] FIG. 3B in an illustration of an immunoblot showing the nuclear uptake of NFAT, c-Jun/AP1,NF.kappa.B, Oct1, and MAP kinase (Erk1 and Erk2). Input nuclei ("Input") and nuclei incubated in either a stimulated extract ("SE"), a control extract prepared from unstimulated T-cells (denoted "USE" for unstimulated extract), or a stimulated extract containing a monoclonal antibody against nucleoporins ("SE+mAb414") were analyzed. [0052] FIG. 3C is a bar graph showing the percentage of transcription factors bound to DNA, reported as the mean .+-.the standard deviation. [0053] FIG. 4A is an illustration of an immunoblot showing the DNA-binding of the chromatin remodeling SWI/SNF complex in T-cell nuclei exposed to a buffer control, an unstimulated extract, or a stimulated extract for 30 minutes and sedimented through sucrose. [0054] FIG. 4B is a graph showing the percentage of DNA bound and unbound SWI/SNF complex in nuclei incubated in extract for the indicated lengths of time, as assessed using a nuclear retention assay. The SWI/SNF complex was visualized on Western blots using anti-BRG1 antibodies. [0055] FIG. 4C is an illustration of an immunoblot showing the nuclear retention of the SWI/SNF complex in stimulated T-cells in vivo. For this assay, T-cells were stimulated with anti-CD3 antibodies for 30 minutes, and the soluble and DNA bound fractions of the SWI/SNF complex were assessed as described for FIG. 4A. [0056] FIG. 4D is an illustration of an immunoblot showing hyperacetylation of the IL-2 gene of T-cell nuclei in vivo and in vitro. An MNAse-soluble chromatin fraction was prepared from resting and anti-CD3-stimulated T-cells. The immunoblot shows the binding of an IL-2 probe and a control .beta.-actin probe to DNA from the immunoprecipitate of the chromatin fraction with an anti-acetylated histone H4 (H4ac) probe ("bound") and the binding to DNA in the supernatant ("unbound") fractions. [0057] FIG. 5A is a gel showing the synthesis of IL-2 mRNA in cell-free extracts. Resting T-cell nuclei ("Input nuclei") were incubated for 30 minutes at 30.degree. C. in an unstimulated extract or a stimulated extract. As controls, nuclei were incubated in a stimulated extract containing either 100 .mu.g/ml RNAse A, 100 .mu.g/ml DNAse I, mAb414 antibodies, or the lectin WGA. [0058] FIG. 5B is a gel showing that IL-2 mRNA production in vitro is dependent on PolI transcription. Nuclei were exposed for 30 minutes to a stimulated extract containing increasing concentrations of the RNA PollI inhibitor actinomycin D (0, 5, 10, 50, 100 and 500 nM), and IL-2 mRNA synthesis was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). [0059] FIG. 5C is an illustration of a gel showing that IL-2 mRNA production was induced in nuclei exposed to a T-cell extract treated with anti-CD3 antibodies but not induced by exposure to extracts treated with anti-CD3 antibodies from the B-cell line Reh, human 293T fibroblasts, or HeLa cells. The arrows in the illustration point to the 467 base pair IL-2 RT-PCR product. [0060] FIG. 6A is the immunofluorescence analysis showing nuclear uptake and binding of the T-cell specific transcription factor NFAT by nuclei from the B-cell line Reh, 293T fibroblasts, or HeLa cells that have been incubated with the stimulated T-cell extract, as described for FIG. 3A. [0061] FIG. 6B in an illustration of an immunoblot showing the nuclear uptake of NFAT, c-Jun/AP1, and NF.kappa.B by these nuclei, as described for FIG. 3B. [0062] FIG. 7 is a picture of a gel showing RT-PCR analysis of IL-2 mRNA synthesis in human peripheral blood T-cells stimulated with anti-CD3 antibodies. At indicated time points, cells were lysed, and total RNA was isolated for RT-PCR analysis using IL2-specific primers. Mock (H.sub.2O)-stimulated cells were analyzed at 30, 60, and 120 minute time points ("30c," "60c," "120c") [0063] FIGS. 8A-8D are pictures and graphs that illustrate nuclear uptake and chromatin binding of transcriptional activators of the IL-2 gene in stimulated T-cell extract. [0064] FIG. 8A is a picture of nuclei purified from quiescent T-cells ("Input nuclei") and incubated in stimulated extract for 30 minutes. Nuclear integrity was assessed by phase contrast microscopy and membrane labeling with 10 .mu.g/ml of the lipophilic dye DiOC.sub.6 (bar, 10 .mu.m). [0065] FIG. 8B is a picture of an immunoblot showing the relative levels of NFAT, AP-1, NF.kappa.B, Oct-1, Erk,and an exogenous BSA-NLS conjugate in input resting T-cell nuclei ("Input nuclei"), input unstimulated extract, and input stimulated extract. Nuclear uptake of these factors was examined in nuclei exposed to stimulated extract, unstimulated extract, or stimulated extract containing the nuclear pore blocking antibody mAb414. Blots were also probed using anti-histone H4 antibodies as a gel loading control. [0066] FIG. 8C is a graph of the immunoblotting analysis of intranuclear anchoring of imported transcription factors in nuclei exposed to unstimulated extract or stimulated extract. Intranuclear anchoring was assessed using a nuclear retention assay and immunoblotting analysis of Triton X-100 insoluble ("bound") fractions. The percentage (mean .+-.SD) of bound factors was determined by densitometric analysis of duplicate blots. [0067] FIG. 8D is a picture of immunoblotting analysis of nuclear matrix ("Mtx") and chromatin ("Chr") fractions prepared from nuclei exposed to stimulated extract. NuMA and RNA Pol IIo were used as markers of the nuclear matrix and chromatin, respectively. Anti-NuMA antibodies and anti-Pol IIo mAb CC3 were gifts from D. Compton (Dartmouth Medical School, Hanover, N.H., USA) and M. Vincent (Universite Laval, Quebec, Canada), respectively. [0068] FIGS. 9A-9E illustrate chromatin remodeling in resting T-cell nuclei exposed to a stimulated T-cell extract. [0069] FIG. 9A is a gel showing the anchoring of the chromatin remodeling complex SWI/SNF in nuclei exposed to stimulated extract. Purified quiescent T-cell nuclei ("Input nuclei") were incubated in cell lysis buffer ("Buffer"), unstimulated extract, or stimulated extract for 30 minutes, and sedimented through sucrose. Free and bound SWI/SNF fractions were assessed using the nuclear retention assay and immunoblotting with anti-BRG1 antibodies. [0070] FIG. 9B is a graph of the percentage of bound SWI/SNF, as determined using the nuclear retention assay during incubation of nuclei in stimulated extract or unstimulated extract. [0071] FIG. 9C is a gel showing nuclear retention of SWI/SNF in cultured stimulated T-cells. T-cells were stimulated with anti-CD3 antibodies and after 30 minutes, intranuclear bound and free fractions of SWI/SNF were assessed as in FIG. 9A. [0072] FIG. 9D is a graph of the ATPase activity of the SWI/SNF complex. After incubation of resting T-cell nuclei in stimulated extract or unstimulated extract for 30 minutes, SWI/SNF was immunoprecipitated from nuclear lysates using anti-BRG1 antibodies and hydrolysis of 1 nM exogenous ATP ("ATP+") by the immune precipitate ("BRG1-IP") was determined in a luminometric assay. Control precipitations were carried out using pre-immune IgGs ("Pre-I IgG"). Top rows show an anti-BRG1 immunoblot of the BRG1-IPs. Where indicated, BRG1-IP was treated with 100 .mu.g/ml DNAse I for 15 minutes prior to the assay ("DNAse+"). Relative ATPase activities are expressed as mean (.+-.SD) relative light units (RLU) in the assay subtracted to the RLU of the control pre-immune precipitate (RLU=2,700, set to zero). [0073] FIG. 9E is a gel showing the hyperacetylation of the IL-2 promoter in T-cell nuclei in culture and in vitro. Microccocal nuclease-soluble chromatin ("Input") was prepared from unstimulated ("Unstim.") and anti-CD3-stimulated ("Stim.") T-cells. AcH4 was immunoprecipitated, and DNA isolated from anti-acH4 precipitates ("Bound") and supernatant ("Unbound") fractions. DNA was dot-blotted and hybridized with an IL-2 promoter-specific probe and a control .beta.-actin probe. [0074] FIGS. 10A-10D are gels showing the transcription of IL-2 mRNA by T-cell and non-T-cell nuclei in cell-free extracts. [0075] FIG. 10A is a gel showing transcription by resting T-cell nuclei ("Input nuclei") incubated in stimulated extract ("Nuclei-SE") or unstimulated extract ("Nuclei-USE"). As controls, nuclei were incubated in stimulated extract containing either 100 .mu.g/ml RNAse A, 100 .mu.g/ml DNAse I, mAb414, or wheat germ agglutinin ("WGA"). At the end of the incubation, total RNA was isolated from the reaction mix, and 15 ng was used as the template for RT-PCR using IL-2-specific primers. Input stimulated extract and stimulated extract containing 1.2 .mu.g total RNA isolated from IL-2 producing T-cells ("Pos. control SE") were analyzed as controls. [0076] FIG. 10B is a picture of a gel showing that IL-2 mRNA synthesis in vitro is RNA Pol II-dependent. Resting T-cell nuclei were exposed to stimulated extract containing 0, 5, 10, 50, 100 or 500 nM of the RNA Pol II inhibitor, actinomycin D. IL-2 mRNA synthesis was analyzed by RT-PCR at the end of incubation. [0077] FIG. 10C is a gel showing transcription by nuclei from primary HUVEC cells, NT2 cells, and Hela cells that were reprogrammed for 2 hours in stimulated extract or unstimulated extract. As controls, nuclei were incubated in stimulated extract containing either 100 .mu.g/ml RNAse A, mAb414, or 50 nM actinomycin D ("ActD"). Total RNA was isolated, and IL-2 RNA synthesis was examined by RT-PCR. [0078] FIG. 10D is a picture of a gel showing transcription by resting T-cell nuclei that were incubated for two hours in stimulated extract, or in extracts from 293T, HeLa, or Bjab cells, all prepared after treating each cell type with anti-CD3 and cross-linking antibodies to mimic T-cell stimulation. IL-2 mRNA synthesis in each extract was analyzed by RT-PCR. [0079] FIGS. 11A-11C demonstrate the nuclear import and chromatin binding of transcriptional activators of the IL-2 gene in 293T fibroblast nuclei exposed to stimulated T-cell extract. [0080] FIG. 11A is a picture of nuclei purified from 293T fibroblasts (0 min) and incubated in stimulated T-cell extract. Uptake of NFAT was examined by immunofluorescence (bar, 10 .mu.m). [0081] FIG. 11B is a picture of immunoblotting analysis showing nuclear uptake of NFAT, AP-1, NF.kappa.B, Oct-1, and BSA-NLS in input 293T nuclei and 293T nuclei exposed to either stimulated T-cell extract, unstimulated T-cell extract, or stimulated T-cell extract and mAb414. Anti-histone H4 antibodies were used as a loading control. [0082] FIG. 11C is a picture of an immunoblot showing nuclear matrix ("Mtx") and chromatin ("Chr") fractions prepared from 293T nuclei treated with stimulated T-cell extract. [0083] FIGS. 12A-2E demonstrate chromatin remodeling and activation of the IL-2 gene in stimulated T-cell extract. [0084] FIG. 12A is a gel showing intranucleuear anchoring of the human SWI/SNF complex. In the left section of the immunoblot, nuclei were isolated from resting ("-") or anti-CD3-stimulated (".alpha.-CD3") T-cells, and intranuclear free and bound SWI/SNF assessed by immunoblotting of detergent-soluble and insoluble nuclear fractions using anti-BRG1 antibodies. In the right section of the immunoblot, free and bound SWI/SNF fractions were visualized in 293T nuclei incubated in stimulated T-cell extract and sedimented through sucrose. [0085] FIG. 12B is a graph of the percent of bound SWI/SNF in 293T nuclei exposed to stimulated T-cell extract or unstimulated T-cell extract, based on densitometric analysis of duplicate blots. [0086] FIG. 12C is a graph of the ATPase activity of the SWI/SNF complex. Following exposure of 293T nuclei to stimulated T-cell extract or unstimulated T-cell extract, SWI/SNF was immunoprecipitated from nuclear lysates using anti-BRG1 antibodies and hydrolysis of 1 nM exogenous ATP ("ATP+") by the immune precipitate ("BRG1-IP") was determined in a luminometric assay. Control precipitations were carried out using pre-immune IgGs ("Pre-I IgG"). ATP levels are expressed as mean (.+-.SD) relative light units (RLU). Elevated ATP levels reflect low ATPase activity of the BRG1-IP. [0087] FIG. 12D is an immunoblot showing the hyperacetylation of the IL-2 locus in 293T nuclei. MNase-soluble chromatin was prepared from input 293T nuclei ("Input N"), and the nuclei were exposed to unstimulated T-cell extract or stimulated T-cell extract. Acetylated H4 was immunoprecipitated, and DNA was isolated from anti-acH4 precipitate ("Bound") and supernatant ("Unbound") fractions. DNA was dot-blotted and hybridized to an IL-2 probe (top rows) and a control .beta.-actin probe (bottom rows). FIG. 12 is a picture of a set of gels showing transcription of the IL-2 gene. Nuclei from 293T, NT2, and resting T-cells were incubated for two hours in unstimulated T-cell extract ("Nuclei/UTE") or stimulated T-cell extract ("Nuclei/STE"). As controls, nuclei were incubated in stimulated T-cell extract containing either 100 .mu.g/ml RNAse A, mAb414, or 50 nM actinomycin D ("ActD"). RNA was isolated from the reaction mix, and IL-2 transcription was examined by RT-PCR. [0088] FIG. 13 is a graph demonstrating that 293T fibroblasts reprogrammed in the Jurkat-TAg extract display altered gene expression. Relative mRNA levels in 293T-cells incubated in stimulated Jurkat-TAg extract or in control 293T extract were compared using a cytokine gene expression array. Bars represent fold increase or decrease in transcription of indicated genes in Jurkat extract-treated cells, measured as the ratio of reprogrammed/control probe hybridization signal strength. Genes with an over two-fold increase or decrease in expression level are shown. Different color backgrounds separate distinct gene groups. [0089] FIGS. 14A-14C demonstrate that 293T fibroblasts reprogrammed in Jurkat-TAg extract exhibit hematopoietic cell markers and function. [0090] FIG. 14A is a picture of 293T-cells exposed to a control 293T extract (top row) or a stimulated Jurkat-TAg extract (middle row), and Jurkat-TAg cells (bottom row) and analyzed by immunofluorescence using indicated FITC-conjugated antibodies. CD3, CD4, CD8 were detected at four days post-reprogramming; CD45 was detected at 11 days post-reprogramming. The C.gamma. subunit of PKA ("PKA-C.gamma.") was examined as a positive control. [0091] FIG. 14B is a picture of the immunofluorescence analysis of the cells using anti-TCR.alpha..beta. antibodies at 11 days post-reprogramming. DNA was labeled with propidium iodide. [0092] FIG. 14C is a picture of each cell type stimulated with anti-CD3 antibodies and PMA for 24 hours, starting at two days post-reprogramming. Stimulated and unstimulated cells were analyzed by immunofluorescence using anti-IL-2R.beta. (green) and anti-IL-2R.alpha. (red) antibodies. Stimulation of Jurkat cells and Jurkat extract-treated 293T fibroblasts elicited IL-2-R.alpha. synthesis. DNA (blue) was labeled with Hoechst 33342. Merged images are shown (bars, 10 .mu.m). [0093] FIG. 15 is a picture of the immunofluorescence analysis of 293T fibroblasts reprogrammed in NT2 extract, demonstrating that the reprogrammed cells express the neurofilament protein NF-200. NT2 or control 293T extract-treated fibroblasts were examined by immunofluorescence using anti-NF200 antibodies at 15 days post-reprogramming. DNA was labeled with Hoechst 33342 (bar, 10 .mu.m). [0094] FIG. 16A is a set of pictures showing the morphology of NIH3T3 fibroblasts and mouse ES cells. [0095] FIG. 16B is a set of pictures showing the morphology of NIH3T3 fibroblasts reprogrammed in mouse embryonic stem cell extract, fibroblasts mock-reprogrammed in NIH3T3 extract, and intact NIH3T3 cells exposed to ES cell extract. [0096] Phase contrast micrographs are shown (bars, 20 .mu.m). [0097] FIGS. 17A and 17B are pictures of the immunofluorescence and immunoblotting analysis, respectively, of Oct4 in NIH3T3 cells, embryonic stem cells and NIH3T3 fibroblasts reprogrammed in embryonic stem cell extract ("NIH/ES ext."; day-4 post-reprogramming). NIH3T3 cells exposed to a control NIH3T3 cell extract do not express Oct4 ("NIH/NIH ext."). [0098] FIG. 18 is a picture of a membrane showing the detection of alkaline phosphatase activity in mouse embryonic stem cells. The top row contains lysates of NIH3T3 cells, and the bottom row contains lysates of embryonic stem cells. Two and 6 lysate were applied onto the membrane (protein concentration is 20 .mu.g/.mu.l). [0099] FIG. 19 is a picture of a membrane showing the detection of alkaline phosphatase activity in NIH3T3 cells reprogrammed in the embryonic stem cell extract. DETAILED DESCRIPTION [0100] We have developed novel methods of reprogramming cells by exposing them or their genetic material to a reprogramming media (e.g., a cell extract). This reprogramming refers to decreasing or eliminating the expression of genes specific for the donor cell or increasing the expression of genes specific for another cell type. For example, we have shown that incubation of nuclei from resting T-cells, B-cells, or fibroblasts in an extract from stimulated T-cells results in migration of a T-cell specific transcription factor from the extract into the nuclei. Additionally, the reprogramming of nuclei from resting T-cells, fibroblasts, endothelial (HUVEC), differentiated epithelial (HeLa), and neuronal precursor (NT2) cells induced expression of the IL-2 gene, a gene that is otherwise repressed by the nuclei. Reprogramming of resting T-cell and fibroblast nuclei also induced hyperacetylation of the IL-2 gene and intranuclear anchoring of a chromatin remodeling complex. Thus, reprogramming media such as extracts may be used to alter the expression profile of the genetic material of a donor cell such that it resembles that of the cells used to prepare the reprogramming media. [0101] The methods for reprogramming a cell that are described herein may be used to convert a cell into another cell-type that is closely related by origin or character. For example, members of the connective-tissue family, such as fibroblasts, smooth muscle cells, osteoblasts, adiopocytes, and chrondrocytes, may be interconverted using these methods. Additionally, hepatocytes may be converted into insulin-producing B-cells because both of these cell types express many of the same genes. Alternatively, a cell may be converted into a desired cell type that is distantly related to the donor cell and thus shares few or no characteristics or functions with the donor cell. [0102] In one such reprogramming method, a nucleus from an interphase donor cell is incubated in a reprogramming media prepared from interphase cells (e.g., an interphase cell extract) under conditions that allow export of factors, such as transcription regulatory factors, from the nucleus and the import of factors from the reprogramming media into the nucleus. The nucleus is then inserted into a recipient cell or cytoplast, forming a reprogrammed cell. Preferably, the cells used to prepare the interphase reprogramming media are the cell type one wishes the reprogrammed cell to become. Due to the different factors in the nucleus of the reprogrammed cell compared to that of the donor cell, the reprogrammed cell expresses a different set of mRNA and protein molecules and thus has a different phenotype than that of the donor cell. To achieve optimum reprogramming efficiency, 2, 3, 5, or more rounds of reprogramming may be performed. The reprogrammed cells may also be cultured under conditions promoting sustained changes in cell function. For example, the cells may be cultured with additional components such as antigens, interleukins, growth factors, cytokines, or other cells. The reprogrammed cells can also be transplanted into a host animal or patient, in the organ where they are supposed to function. Local environment cues may facilitate reprogramming. [0103] In a related method, the nucleus from a donor interphase cell is incubated in a mitotic reprogramming media (e.g., a mitotic cell extract), a detergent and salt solution, or a protein kinase solution to promote nuclear envelope dissolution and possibly chromatin condensation, forming a chromatin mass. This nuclear envelope breakdown and chromatin condensation facilitate the release of factors from the chromatin mass. Alternatively, a chromatin mass may be isolated from a donor mitotic cell. In one embodiment of this method, the chromatin mass is inserted into a recipient cell or cytoplast of the desired cell type. After this nuclear transfer, a nucleus is reformed from the donor chromatin mass. Additionally, desired factors from the cytoplasm of the recipient cell or cytoplast migrate into the nucleus and bind the exogenous chromosomes, resulting in the expression of desired genes by the reprogrammed cell. To promote the sustained expression of the desired genes, 2, 3, 5, or more rounds of reprogramming may be performed, and the reprogrammed cells may also be cultured with additional components such as antigens, interleukins, growth factors, cytokines, or other cells. [0104] In another embodiment of this method, the chromatin mass is first incubated in an interphase reprogramming media (e.g., an interphase cell extract) as described above to further promote the release of undesirable factors from the chromatin mass and the binding of desirable factors from the interphase reprogramming media to the chromatin mass. The incubation in the interphase reprogramming media also results in the formation of a nuclear membrane, encapsulating the chromatin mass and desired factors from the reprogramming media. The reformed nucleus is then inserted into a recipient cell or cytoplast of the desired cell type or of any other cell type. [0105] As an alternative to isolating nuclei or chromatin masses from donor cells for subsequent incubation in a reprogramming media, donor cells may be gently permeabilized and incubated in the reprogramming media (e.g., a cell extract). Permeabilization of the plasma membrane allows factors to enter and leave the cell. The cells may either be incubated in an interphase reprogramming media to allow the nucleus to remain membrane-bounded or with a mitotic reprogramming media to allow the dissolution of the nuclear membrane and formation of a chromatin mass. After incubation in the reprogramming media, the plasma membrane may be resealed, trapping desired factors from the reprogramming media inside the cell. If desired, this reprogramming method can be repeated 1, 2, 3, 4, 5, or more times. For example, after the resealed cell is cultured for a certain length of time (e.g., after 2 days, 7 days, 14 days, 3 weeks, 4 weeks, 8 weeks, or longer) in the presence or absence of factors such as antigens, interleukins, growth factors, cytokines, or other cells to promote reprogramming, the cells are permeabilized and subjected to an additional round of reprogramming. Additional cycles of reprogramming may result in more stable and heritable epigenetic changes and in prolonged expression of the phenotype or proteins of interest from the reprogrammed cells. [0106] This whole cell reprogramming method was utilized to reprogram permeabilized, human fibroblast cells using an extract from the lymphoblastic leukemia cell line, Jurkat-TAg (hereafter referred to as Jurkat), resulting in the remodeling of chromatin, activation of lymphoid-specific genes, and establishment of a T-cell-specific activity. For example, T-cell-specific antigens such as the CD3-T-cell receptor (TCR) complex were expressed by the reprogrammed cells, and the IL-2 receptor was assembled in response to CD3-TCR stimulation of these cells. After exposure to an NT2 neuronal precursor cell extract, permeabilized fibroblasts expressed a neurofilament protein and extended neurite-like outgrowths in culture. Fibroblasts were also reprogrammed into cells resembling embryonic stem cells. [0107] Reprogrammed cells generated from these methods may be used to replace cells in a mammal in need of a particular cell type. The reprogramming methods may be used to either directly produce cells of the desired cell type or to produce undifferentiated cells which may be subsequently differentiated into the desired cell type. For example, stem cells may be differentiated in vitro by culturing them under the appropriate conditions or differentiated in vivo after administration to an appropriate region in a mammal. To optimize phenotypic and functional changes, reprogrammed cells can be transplanted into the organ (e.g., a heart) where they are intended to function in an animal model or in human patients shortly after reprogramming (e.g., after 1, 2, 3, 5, 7, or more days). Reprogrammed cells implanted in an organ may be reprogrammed to a greater extent than cells grown in culture prior to transplantation. Cells implanted in an animal organ may be removed from the organ and transplanted into a recipient mammal such as a human, or the animal organ may be transplanted into the recipient. [0108] To increase the length of time the cell, nuclei, or chromatin mass may be reprogrammed in vitro prior to administration to a mammal for the treatment of disease, the donor cell may be optionally modified by the transient transfection of a plasmid containing an oncogene flanked by loxP sites for the Cre recombinase and containing a nucleic acid encoding the Cre recombinase under the control of an inducible promoter (Cheng et al., Nucleic Acids Res. 28(24):E108, 2000). The insertion of this plasmid results in the controlled immortalization of the cell. After the cell is reprogrammed into the desired cell-type and is ready to be administered to a mammal, the loxP-oncogene-loxP cassette may be removed from the plasmid by the induction of the Cre recombinase which causes site-specific recombination and loss of the cassette from the plasmid. Due to the removal of the cassette containing the oncogene, the cell is no longer immortalized and may be administered to the mammal without causing the formation of a cancerous tumor. [0109] Examples of medical applications for these reprogrammed cells include the administration of neuronal cells to an appropriate area in the human nervous system to treat, prevent, or stabilize a neurological disease such as Alzheimer's disease, Parkinson's disease, Huntington's disease, or ALS; or a spinal cord injury. In particular, degenerating or injured neuronal cells may be replaced by the corresponding cells from a mammal. This transplantation method may also be used to treat, prevent, or stabilize autoimmune diseases including, but not limited to, insulin dependent diabetes mellitus, rheumatoid arthritis, pemphigus vulgaris, multiple sclerosis, and myasthenia gravis. In these procedures, the cells that are attacked by the recipient's own immune system may be replaced by transplanted cells. In particular, insulin-producing cells may be administered to the mammal for the treatment or prevention of diabetes, or oligodendroglial precursor cells may be transplanted for the treatment or prevention of multiple sclerosis. For the treatment or prevention of endocrine conditions, reprogrammed cells that produce a hormone, such as a growth factor, thyroid hormone, thyroid-stimulating hormone, parathyroid hormone, steroid, serotonin, epinephrine, or norepinephrine may be administered to a mammal. Additionally, reprogrammed epithelial cells may be administered to repair damage to the lining of a body cavity or organ, such as a lung, gut, exocrine gland, or urogenital tract. It is also contemplated that reprogrammed cells may be administered to a mammal to treat damage or deficiency of cells in an organ such as the bladder, brain, esophagus, fallopian tube, heart, intestines, gallbladder, kidney, liver, lung, ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, or uterus. [0110] Reprogrammed cells may also be combined with a matrix to form a tissue or organ in vitro or in vivo that may be used to repair or replace a tissue or organ in a recipient mammal. For example, reprogrammed cells may be cultured in vitro in the presence of a matrix to produce a tissue or organ of the urogenital system, such as the bladder, clitoris, corpus cavermosum, kidney, testis, ureter, uretal valve, or urethra, which may then be transplanted into a mammal (Atala, Curr. Opin. Urol. 9(6):517-526, 1999). In another transplant application, synthetic blood vessels are formed in vitro by culturing reprogrammed cells in the presence of an appropriate matrix, and then the vessels are transplanted into a mammal for the treatment or prevention of a cardiovascular or circulatory condition. For the generation of donor cartilage or bone tissue, reprogrammed cells such as chondrocytes or osteocytes are cultured in vitro in the presence of a matrix under conditions that allow the formation of cartilage or bone, and then the matrix containing the donor tissue is administered to a mammal. Alternatively, a mixture of the cells and a matrix may be administered to a mammal for the formation of the desired tissue in vivo. Preferably, the cells are attached to the surface of the matrix or encapsulated by the matrix. Examples of matrices that may be used for the formation of donor tissues or organs include collagen matrices, carbon fibers, polyvinyl alcohol sponges, acrylateamide sponges, fibrin-thrombin gels, hyaluronic acid-based polymers, and synthetic polymer matrices containing polyanhydride, polyorthoester, polyglycolic acid, or a combination thereof (see, for example, U.S. Pat. Nos. 4,846,835; 4,642,120; 5,786,217; and 5,041,138). [0111] These methods are described further below. It is noted that any of the methods described below can be performed with reprogramming media other than cell extracts. For example, a reprogramming media can be formed by adding one or more naturally-occurring or recombinant factors (e.g., nucleic acids or proteins such as DNA methyltransferases, histone deacetylases, histones, nuclear lamins, transcription factors, activators, repressors, growth factors, hormones, or cytokines) to a solution, such as a buffer. Preferably, one or more of the factors are specific for the cell type one wishes the donor cell to become. EXAMPLE 1 One-Step In Vitro Reprogramming Method [0112] In the following method for reprogramming cells, nuclei are isolated from interphase cells and incubated in an interphase reprogramming media (e.g., an interphase cell extract) under conditions that allow the addition of factors from the reprogramming media to the nuclei or the removal of factors from the nuclei. Preferably, the nuclei remain membrane-bounded during this incubation. The reprogrammed nuclei are then isolated from the reprogramming media and inserted into recipient cells or cytoplasts. [0113] Isolation of nuclei [0114] Preferably, cells from the subject who will receive the reprogrammed cells are used as the source of donor nuclei. However, cells from other members of the same species or members of a different species or genus may be used. As many as several million nuclei may be isolated from synchronized or unsynchronized cell populations in culture. The cell populations may be synchronized naturally or chemically. Preferably, at least 40, 60, 80, 90, or 100% of the cells in a population are arrested in interphase, such as in one or more of the following phases of the cell cycle: G.sub.0, G.sub.1, S, or G.sub.2, using standard procedures. [0115] To accomplish this, cells may be incubated, for example, in low serum, such as 5%, 2%, or 0% serum, for 1, 2, 3, or more days to increase the percentage of cells in G.sub.0 phase. To synchronize cells in G.sub.1, the cells may be grown to confluence as attached cells and then incubated in 0.5-1 .mu.g/ml nocodazole (Sigma Chemicals, St. Louis, Mo.) for 17-20 hours, as described previously (see, for example, Collas et al., 1999 and references therein). The flasks containing the attached cells are shaken vigorously by repeatedly tapping the flasks with one hand, resulting in the detachment of mitotic cells and G.sub.1 phase doublets. The G.sub.1 phase doublets are pairs of elongated cells at the end of the division process that are still connected by a thin bridge. Detached G.sub.1 phase doublets may be isolated from the media based on this characteristic doublet structure. The G.sub.1 phase doublets may remain attached or may divide into two separate cells after isolation. [0116] To increase the percentage of cells in S phase, the cells may be cultured in the presence of aphidicolin which inhibits DNA polymerase-.alpha. and thus inhibits DNA synthesis and arrests cells in S phase. Alternatively, cells may be incubated in the presence of excess thymidine. The resulting high intracellular concentration of thymidine relative to that of other nucleotides also inhibits DNA polymerase. [0117] Cells may be synchronized in G.sub.2 by incubating the cells in the presence of aphidicolin to arrest them in S phase and then washing the cells three times by repeated centrifugation and resuspension in phosphate buffered saline (PBS), as described herein. The cells are then incubated for a length of time sufficient for cells to enter G.sub.2 phase. For example, cells with a doubling time of approximately 24 hours, may be incubated for between 6 and 12 hours to allow them to enter G.sub.2 phase. For cells with shorter or longer doubling times, the incubation time may be adjusted accordingly. [0118] The synchronized or unsynchronized cells are harvested in PBS using standard procedures, and several washing steps are performed to transfer the cells from their original media into a hypotonic buffer (10 mM Hepes, pH 7.5, 2 mM MgCl.sub.2, 25 mM KCl, 1 mM DTT, 10 .mu.M aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10 .mu.M soybean trypsin inhibitor, and 100 .mu.M PMSF). For example, the cells may be washed with 50 ml of PBS and pelleted by centrifugation at 500.times. g for 10 minutes at 4.degree. C. The PBS supernatant is decanted, and the pelleted cells are resuspended in 50 ml of PBS and centrifuged, as described above. After this centrifugation, the pelleted cells are resuspended in 20-50 volumes of ice-cold hypotonic buffer and centrifuged at 500.times. g for 10 minutes at 4.degree. C. The supernatant is again discarded and approximately 20 volumes of hypotonic buffer are added to the cell pellet. The cells are carefully resuspended in this buffer and incubated on ice for at least one hour, resulting in the gradual swelling of the cells. [0119] To allow isolation of the nuclei from the cells, the cells are lysed using standard procedures. For example, 2-5 ml of the cell suspension may be transferred to a glass homogenizer and Dounce homogenized using an initial 10-20 strokes of a tight-fitting pestle. Alternatively, the cell suspension is homogenized using a motorized mixer (e.g., Ultraturrax). If desired, cell lysis may be monitored using phase contrast microscopy at 40-fold magnification. During this homogenization, the nuclei should remain intact and most or preferably all of the originally attached cytoplasmic components such as vesicles, organelles, and proteins should be released from the nuclei. If necessary, 1-20 .mu.g/ml of the cytoskeletal inhibitors, cytochalasin B or cytochalasin D, may be added to the aforementioned hypotonic buffer to facilitate this process. Homogenization is continued as long as necessary to lyse the cells and release cytoplasmic components from the nuclei. For some cell types, as many as 100, 150, or more strokes may be required. The lysate is then transferred into a 15 ml conical tube on ice, and the cell lysis procedure is repeated with the remainder of the suspension of swollen cells. Sucrose from a 2 M stock solution made in hypotonic buffer is added to the cell lysate, resulting in a final concentration of 250 mM sucrose. This solution is mixed by inversion, and the nuclei are pelleted by centrifugation at 400.times. g in a swing out rotor for 10 to 40 minutes at 4.degree. C. The supernatant is then discarded, and the pelleted nuclei are resuspended in 10-20 volumes of nuclear buffer (10 mM Hepes, pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 25 mM KCl, 1 mM DTT, 10 .mu.M aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10 .mu.M soybean trypsin inhibitor, and 100 .mu.M PMSF). The nuclei are sedimented and resuspended in 1-2 volumes of nuclear buffer, as described above. The freshly isolated nuclei may either be used immediately for in vitro reprogramming and nuclear transfer into recipient cells or cytoplasts as described below or stored for later use. For storage, the nuclei are diluted in nuclear buffer to a concentration of approximately 10.sup.6/ml. Glycerol (2.4 volumes of 100% glycerol) is added and mixed well by gentle pipetting. The suspension is aliquoted into 100-500 .mu.l volumes in 1.5-ml tubes on ice, immediately frozen in a methanol-dry ice bath, and stored at -80.degree. C. Prior to use, aliquots of the nuclei are thawed on ice or at room temperature. One volume of ice cold nuclear buffer is added, and the solution is centrifuged at 1,000.times. g for 15 minutes in a swing out rotor. The pelleted nuclei are resuspended in 100-500 .mu.l nuclear buffer and centrifuged as described above. The pelleted nuclei are then resuspended in a minimal volume of nuclear buffer and stored on ice until use. [0120] Preparation of the Reprogramming Extract [0121] Interphase cultured cells as harvested using standard methods and washed by centrifugation at 500.times. g for 10 minutes in a 10 ml conical tube at 4.degree. C. Preferably, the cells are of the desired cell type that one wishes the recipient cell or cytoplast to become. The supernatant is discarded, and the cell pellet is resuspended in a total volume of 50 ml of cold PBS. The cells are centrifuged at 500.times. g for 10 minutes at 4.degree. C. This washing step is repeated, and the cell pellet is resuspended in approximately 20 volumes of ice-cold interphase cell lysis buffer (20 mM Hepes, pH 8.2, 5 mM MgCl.sub.2, 1 mM DTT, 10 .mu.M aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10 .mu.M soybean trypsin inhibitor, 100 .mu.M PMSF, and optionally 20 .mu.g/ml cytochalasin B). The cells are sedimented by centrifugation at 800.times. g for 10 minutes at 4.degree. C. The supernatant is discarded, and the cell pellet is carefully resuspended in no more than one volume of interphase cell lysis buffer. The cells are incubated on ice for one hour to allow swelling of the cells. The cells are lysed by either sonication using a tip sonicator or Dounce homogenization using a glass mortar and pestle. Cell lysis is performed until at least 90% of the cells and nuclei are lysed, which may be assessed using phase contrast microscopy. The sonication time required to lyse at least 90% of the cells and nuclei may vary depending on the type of cell used to prepare the extract. [0122] The cell lysate is placed in a 1.5-ml centrifuge tube and centrifuged at 10,000 to 15,000.times. g for 15 minutes at 4.degree. C. using a table top centrifuge. The tubes are removed from the centrifuge and immediately placed on ice. The supernatant is carefully collected using a 200 .mu.l pipette tip, and the supernatant from several tubes is pooled and placed on ice. This supernatant is the "interphase cytoplasmic" or "IS15" extract. This cell extract may be aliquoted into 20 .mu.l volumes of extract per tube on ice and immediately flash-frozen on liquid nitrogen and stored at -80.degree. C. until use. Alternatively, the cell extract is placed in an ultracentrifuge tube on ice (e.g., fitted for an SW55 Ti rotor; Beckman). If necessary, the tube is overlayed with mineral oil to the top. The extract is centrifuged at 200,000.times. g for three hours at 4.degree. C. to sediment membrane vesicles contained in the IS 15 extract. At the end of centrifugation, the oil is discarded. The supernatant is carefully collected, pooled if necessary, and placed in a cold 1.5 ml tube on ice. This supernatant is referred to as "IS200" or "interphase cytosolic" extract. The extract is aliquoted and frozen as described for the IS15 extract. [0123] If desired, the extract can be enriched with additional nuclear factors. For example, nuclei can be purified from cells of the cell type from which the reprogramming extract is derived and lysed by sonication as described above. The nuclear factors are extracted by a 10-60 minute incubation in nuclear buffer containing NaCl or KCl at a concentration of 0.15-800 mM under agitation. The lysate is centrifuged to sediment unextractable components. The supernatant containing the extracted factors of interest is dialyzed to eliminate the NaCl or KCl. The dialyzed nuclear extract is aliquoted and stored frozen. This nuclear extract is added at various concentrations to the whole cell extract described above prior to adding the nuclei for reprogramming. [0124] As an alternative to a cell extract, a reprogramming media can also be formed by adding one or more naturally-occurring or recombinant factors (e.g., nucleic acids or proteins such as DNA methyltransferases, histone deacetylases, histones, nuclear lamins, transcription factors, activators, repressors, growth factors, hormones, or cytokines) to a solution, such as a buffer. Preferably, one or more of the factors are specific for the cell type one wishes the donor cell to become. [0125] Reprogramming of Nuclei in the Extract [0126] Either freshly isolated or thawed purified nuclei are resuspended in the reprogramming media described in the previous section at a concentration of 4,000-5,000 nuclei/.mu.l. An ATP generating system (1 mM ATP, 10 mM creatine phosphate, 25 .mu.g/ml creatine kinase) and 100 .mu.M GTP are added to the interphase extract to promote active uptake of nuclear components by the exogenous nuclei. The reaction is incubated at 30.degree. C. for up to two hours. Uptake of specific nuclear components may be monitored by immunofluorescence analysis of the nuclei, as shown in FIGS. 3A and 6A. [0127] Purification of Reprogrammed Nuclei out of the Extract [0128] The reprogrammed nuclei are centrifuged at 1,000.times. g for 10-30 minutes through a M sucrose cushion prepared in nuclear buffer at 4.degree. C. The nuclei are washed by resuspending them in 500 .mu.l cold nuclear buffer and centrifuging at 1,000.times. g for 10 minutes at 4.degree. C. The nuclei are resuspended in nuclear buffer and held on ice until use for nuclear transfer into the cytoplasm of recipient cells or cytoplasts. [0129] Enucleation of Recipient Cells [0130] Preferably, part or all of the DNA in the recipient cell is removed or inactivated. This destruction or removal of the DNA in the recipient cell prevents the genetic material of the cell from contributing to the characteristics and function of the reprogrammed cell. One method for destroying the nucleus of the cell is exposure to ultraviolet light (Gurdon, in Methods in Cell Biology, Xenopus Laevis:--Practical Uses in cell and Molecular Biology, Kay and Peng, eds., Academic Press, California, volume 36:pages 299-309, 1991). Alternatively, the nucleus may be surgically removed by any standard technique (see, for example, McGrath and Solter, Science 220:1300-1319, 1983). In one possible method, a needle is placed into the cell, and the nucleus is aspirated into the inner space of the needle. The needle may then be removed from the cell without rupturing the plasma membrane (U.S. Pat. Nos. 4,994,384 and 5,057,420). [0131] Introduction of Reprogrammed Nuclei into Recipient Cells or Cytoplasts [0132] The nuclei are introduced into recipient cells or cytoplasts of either the desired cell type or of any other cell type using standard methods, such as microinjection or electrofusion (see, for example, U.S. Pat. Nos. 4,997,384 and 5,945,577). The reconstituted cells are placed back in culture and allowed to recover, divide, and differentiate according to the reprogrammed pathway. Gene expression by the reprogrammed cells may be monitored using standard Northern analysis to measure expression of mRNA molecules, preferably mRNA molecules that are specific for the donor cell, recipient cell, or the desired cell type (Ausubel et al., supra). Expression of specific mRNA molecules may also be detected using standard reverse-transcription polymerase chain reaction (RT-PCR) assays with primers designed to specifically bind an mRNA molecule of interest. Alternatively, the expression of multiple cell specific mRNA molecules may be monitored using standard DNA chip technology with cDNA arrays (Marrack et al., Current Opinion in Immunology 12, 206-209, 2000; Harkin, Oncologist. 5:501-507, 2000; Pelizzari et al., Nucleic Acids Res. 2;28(22):4577-4581, 2000; Marx, Science 289(5485):1670-1672, 2000). The cells may be analyzed for a reduction in expression of genes specific for the cell type of the donor cell, recipient cell, or recipient cytoplast. Additionally, cells may be assayed for an increase in expression of genes specific for the desired cell type. Examples of mRNA molecules that are indicative of reprogramming to generate a stem cell include H-19, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, GCTM-2, Oct-4, Genesis, GCNF, GDF-3, and TDGF-1. Neural cell specific mRNA molecules include, but are not limited to, NGF, NF-H, NeuN, NSE, and CD1 lb. For analyzing the conversion to an adipocyte cell fate, expression of mRNA molecules such as leptin, PPAR.lambda.1, PPAR.lambda.2, SREBP1C, IR, and TNF.alpha. may be monitored. IGF-1and IR are indicative of insulin producing cells. Additionally, the cells may be analyzed for expression of particular proteins using standard Western or immunofluorescence analysis (Ausubel et al., supra). [0133] Examples of other characteristics of the reprogrammed cell that may be analyzed to determine whether it has been converted into the desired cell type include the size of the cell, cell morphology, ability to grow as an adherent cell, ability to grow as an attached cell, volume of cytoplasm, and location of a centrosome. The functions of the reprogrammed cells may also be tested, such as the ability of red blood cells to transport O.sub.2 and CO.sub.2, the ability of B-cells to make antibodies, and the ability of neutrophiles to phagocytose and destroy invading bacteria. Additionally, the production of lipids by adicotypes may be determined using standard microscopy to visualize lipid droplets in the cells. |
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