FIELD OF THE INVENTION
The present invention relates to methods for “de-differentiating” and/or altering the life-span of desired recipient cells, preferably human somatic cells. These methods have application especially in the context of cell therapies and the production of genetically modified cells.
BACKGROUND OF THE INVENTION
Nuclear transfer first gained acceptance in the 1960's with amphibian nuclear transplantation. (Diberardino, M. A. 1980, “Genetic stability and modulation of metazoan nuclei transplanted into eggs and ooctyes”, Differentiation, 17-17-30; Diberardino, M. A., N. J. Hoffner and L. D. Etkin, 1984; “Activation of dormant genes in specialized cells”, Science, 224:946-952; Prather, R. S. and Robl, J. M., 1991, “Cloning by nuclear transfer and splitting in laboratory and domestic animal embryos”, In: Animal Applications of Research in Mammalian Development, R. A. Pederson, A. McLaren and N. First (ed.), Cold Spring Harbor Laboratory Press.) Nuclear transfer was initially conducted in amphibians in part because of the relatively large size of the amphibian oocyte relative to that of mammals. The results of these experiments indicated to those skilled in the art that the degree of differentiation of the donor nucleus was greatly instrumental, if not determinative, as to whether a recipient oocyte containing such cell or nucleus could effectively reprogram said nucleus and produce a viable embryo. (Diberardino, M. A., N. J. Hoffner and L. D. Etkin, 1984, “Activation of dormant genes in specialized cells.”, Science, 224:946-952; Prather, R. S. and Robl, J. M., 1991, “Cloning by nuclear transfer and splitting in laboratory and domestic animal embryos”, In: Animal Applications of Research in Mammalian Development, R. A. Pederson, A. McLaren and N. First (ed.), Cold Spring Harbor Laboratory Press).
Much later, in the mid 1980s, after microsurgical techniques had been perfected, researchers investigated whether nuclear transfer could be extrapolated to mammals. The first procedures for cloning cattle were reported by Robl et al (Robl, J. M., R. Prather, F. Barnes, W. Eyestone, D. Northey, B. Gilligan and N. L. First, 1987, “Nuclear transplantation in bovine embryos”, J. Anim. Sci., 64:642-647). In fact, Dr. Robl's lab was the first to clone a rabbit by nuclear transfer using donor nuclei from earlier embryonic cells (Stice, S. L. and Robl, J. M., 1988, “Nuclear reprogramming in nuclear transplant rabbit embryos”, Biol. Reprod., 39:657-664). Also, using similar techniques, bovines (Prather, R. S., F L. Barnes, M L. Sims, Robl, J. M., W. H. Eyestone and N. L. First, 1987, “Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte”, Biol. Reprod., 37:859-866) and sheep (Willadsen, S. M., 1986, “Nuclear transplantation in sheep embryos”, Nature, (Lond) 320:63-65), and putatively porcines (Prather, R. S., M. M. Sims and N. L. First, 1989, “Nuclear transplantation in pig embryos”, Biol. Reprod., 41:414), were cloned by the transplantation of the cell or nucleus of very early embryos into enucleated oocytes.
In the early 1990s, the possibility of producing nuclear transfer embryos with donor nuclei obtained from progressively more differentiated cells was investigated. The initial results of these experiments suggested that when an embryo progresses to the blastocyst stage (the embryonic stage where the first two distinct cell lineages appear) that the efficiency of nuclear transfer decreases dramatically (Collas, P. and J. M. Robl, 1991, “Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos”, Biol. Reprod., 45:455-465). For example, it was found that trophectodermal cells (the cells that form the placenta) did not support development of the nuclear fusion to the blastocyst stage. (Collas, P. and J. M. Robl, 1991, “Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos”, Biol. Reprod., 45:455-465). By contrast, inner cell mass cells (cells which form both somatic and germ line cells) were found to support a low rate of development to the blastocyst stage with some offspring obtained. (Collas P, Barnes F L, “Nuclear transplantation by microinjection of inner cell mass and granulosa cell nuclei”, Mol Reprod Devel., 1994, 38:264-267) Moreover, further work suggested that inner cell mass cells which were cultured for a short period of time could support the development to term. (Sims M, First NL, “Production of calves by transfer of nuclei from cultured inner cell mass cells”, Proc Natl Acad Sci, 1994, 91:6143-6147)
Based on these results, it was the overwhelming opinion of those skilled in the art at that time that observations made with amphibian nuclear transfer experiments would likely be observed in mammals. That is to say, it was widely regarded by researchers working in the area of cloning in the early 1990's that once a cell becomes committed to a particular somatic cell lineage that its nucleus irreversibly loses its ability to become “reprogrammed”, i.e., to support full term development when used as a nuclear donor for nuclear transfer. While the exact molecular explanation for the apparent inability of somatic cells to be effectively reprogrammed was unknown, it was hypothesized to be the result of changes in DNA methylation, histone acetylation and factors controlling transitions in chromatin structure that occur during cell differentiation. Moreover, it was believed that these cellular changes could not be reversed.
Therefore, it was quite astounding that in 1998, the Roslin Institute reported that cells committed to somatic cell lineage could support embryo development when used as nuclear transfer donors. Equally astounding, and more commercially significant, the production of transgenic cattle which were produced by nuclear transfer using transgenic fibroblast donor cells was reported shortly thereafter by scientists working at the University of Massachusetts and Advanced Cell Technology.
Also, recently two calves were reportedly produced at the Ishikawa Prefecture Livestock Research Centre in Japan from oviduct cells collected from a cow at slaughter. (Hadfield, P. and A. Coghlan, “Premature birth repeats the Dolly mixture”, New Scientist, Jul. 11, 1998) Further, Jean-Paul Renard from INRA in France reported the production of a calf using muscle cells from a fetus. (MacKenzie, D. and P. Cohen, 1998, “A French calf answers some of the questions about cloning”, New Scientist, March 21.) Also, David Wells from New Zealand reported the production of a calf using fibroblast donor cells obtained from an adult cow. (Wells, D. N., 1998, “Cloning symposium: Reprogramming Cell Fate—Transgenesis and Cloning,” Monash Medical Center, Melbourne, Australia, April 15-16)
Differentiated cells have also reportedly been successfully used as nuclear transfer donors to produce cloned mice. (Wakayama T, Perry ACF, Zucconi M, Johnsoal KR, Yanagimachi R., “Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei”, Nature, 1998, 394:369-374.)
Still further, an experiment by researchers at the University of Massachusetts and Advanced Cell Technology was recently reported in a lead story in the New York Times, January 1999, wherein a nuclear transfer fusion embryo was produced by the insertion of an adult differentiated cell (cell obtained from the cheek of an adult human donor) into an enucleated bovine oocyte. Thus, it would appear, based on these results, that at least under some conditions differentiated cells can be reprogrammed or de-differentiated.
Related thereto, it was also recently reported in the popular press that cytoplasm transferred from oocyte of a young female donor “rejuvenated” an oocyte of an older woman, such that it was competent for reproduction.
However, it would be beneficial if methods could be developed for converting differentiated cells to embryonic cell types, without the need for cloning, and the production of embryos, especially given their potential for use in nuclear transfer and for producing different differentiated cell types for therapeutic use. Also, it would be beneficial if the cellular materials responsible for de-differentiation and reprogramming of differentiated cells could be identified and produced by recombinant methods, thereby improving the efficiency of cellular reprogramming.
OBJECTS OF THE INVENTION
Therefore, it is an object of the invention to provide novel methods for “de-differentiating” and/or altering the life-span of desired cells.
It is a more specific object of the invention to provide a novel method for “de-differentiating” and/or altering the life-span of a desired differentiated cell by introducing the cell or cell nucleus with cytoplasm and then transplanting the de-differentiated nucleus into a surrogate cytoplast such as from an ES cell of a less differentiated cell, preferably an oocyte or blastomere, or another embryonic cell type.
It is another object of the invention to alter the life-span and/or to de-differentiate desired cells, typically mammalian differentiated cells, prior, concurrent, or subsequent to genetic modification.
It is another object of the invention to provide an improved method of cell therapy wherein the improvement comprises administering cells which have been de-differentiated or have an altered life-span by the introduction of cytoplasm obtained from a cell of a less or undifferentiated state, preferably an oocyte or blastomere or placing nuclei from said somatic cell into a solution containing an extract of the oocyte or blastomere embryo, or ES cell or purified proteins from the same.
It is still another object of the invention to identify the component or components in oocyte cytoplasm responsible for de-differentiation and/or alteration of cell life-span, e.g., by fractionation or subtractive hybridization, i.e. fractionation of protein, RNA or DNA.
It is still another object of the invention to provide a novel method of therapy, especially of the skin, by administering a therapeutically effective amount of cytoplasm obtained from a substantially undifferentiated or undifferentiated cell, preferably an oocyte or blastomere, or the purified active components of the same.
It is another object of the invention to provide novel compositions for therapeutic, dermatologic and/or cosmetic usage that contain cytoplasm derived from substantially undifferentiated or undifferentiated cells, preferably an oocyte or blastomere, or purified active components of same.
It is another object of the invention to provide cells for use in cell therapy which have been “de-differentiated” or have an altered life-span by the introduction of cytoplasm from a substantially undifferentiated or undifferentiated cell, preferably an oocyte or blastomere, or purified active components of same.
It is still another object of the invention to provide an improved method of cloning via nuclear transfer wherein the improvement comprises using as the donor cell or nucleus a cell which has been de-differentiated and/or has had its life-span altered by the introduction of cytoplasm from a substantially undifferentiated or undifferentiated cell, or purified active components of same, or cross-species NT where the purified active component is expressed to facilitate reprogramming.
It is another object of the invention to rejuvenate nuclei isolated from desired differentiated cells by contacting same with cytoplasm from oocytes, blastomeres, ES, or other embryonic cell types.
It is another object of the invention to provide screening assays to identify proteins, or nucleic acid sequences that are released from differentiated cell nuclei upon contacting with cytoplasm, or fractions derived from oocyte cytoplasm from oocytes, blastomeres, ES cells or other embryonic cell types, that are involved in all reprogramming.
It is another specific object of the invention to provide screening assays, e.g. differential or subtractive hybridization to identify mRNAs that expressed in oocyte cytoplasm or in embryonic cell types that are involved in cell programming.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides novel methods for producing cells, preferably mammalian cells and, most preferably, human cells that have been de-differentiated and/or which have an altered (increased) life-span by the juxtaposition of the donor cell with cytoplasm from an undifferentiated or substantially undifferentiated cell, preferably an oocyte or blastomere, or another embryonic cell type. In a particularly preferred embodiment, the present invention will be used to produce cells in a more primitive state, especially embryonic stem cells or inner cell mass cells.
The resultant cells are useful in gene and cell therapies, and as donor cells or nuclei for use in nuclear transfer.
“Ooctye”—In the present invention, this refers to any oocyte, preferably a mammalian oocyte, that develops from an oogonium and, following meiosis, becomes a mature ovum.
“Metaphase II ooctye”—The preferred stage of maturation of oocytes used for nuclear transfer (First and Prather, Differentiation, 48:1-8). At this stage, the oocyte is sufficiently “prepared” to treat an introduced donor cell or nucleus as it does a fertilizing sperm.
“Donor Cell”—In the present invention, this refers to a cell wherein some or all of its cytoplasm is transferred to another cell (“recipient cell”). The donor cell is typically a primitive or embryonic cell type, preferably an oocyte, blastomere, or inner cell mass cell.
“Recipient Cell”—This refers to a cell into which all or part of the cytoplasm of a donor cell, wherein such donor cell is of a more primitive cell type relative to the recipient cell, is transferred. This transfer can be accomplished by different methods, e.g., microinjection or by contacting donor cells with liposomal encapsulated cytoplasm or enucleating the donor cell and incubating with cytoplasmic extract. Typically, the donor cell is an oocyte, blastomere or inner cell mass cell, and the recipient cell is a somatic cell, preferably a human somatic cell.
“Blastomere”—Embryonic, substantially undifferentiated cells contained in blastocyst stage embryos.
“Embryonic cell or embryonic cell type”—In the present invention, this will refer to any cell, e.g., oocyte, blastomere, embryonic stem cell, inner cell mass cell, or primordial germ cell, wherein the introduction of cytoplasm therefrom into a differentiated cell, e.g., human somatic cell in tissue culture, results in de-differentiation and/or lengthening of the life-span of such differentiated cell.
“Cell having altered life-span”—In the present invention this refers to the change in cell life-span (lengthening) that results when cytoplasm of a more primitive or less differentiated cell type, e.g., an embryonic cell or embryonic cell type, e.g., oocyte or blastomere, is introduced into a desired differentiated cell, e.g., a cultured human somatic cell.
“Embryonic stem cell (ES cell)”—In the present invention this refers to an undifferentiated cell that has the potential to develop into an entire organism, i.e., a cell that is able to propagate indefinitely, maintaining its undifferentiated state and, when induced to differentiate, be capable of giving rise to any cell type of the body.
“Nuclear Transfer”—Introduction of cell or nuclear DNA of donor cell into enucleated oocyte which cell or nucleus and oocyte are then fused to produce a nuclear transfer fusion or nucleus fusion embryo. This NT fusion may be used to produce a cloned embryo or offspring or to produce ES cells.
“Telomerase”—A ribonucleoprotein (RNP) particle and polymerase that uses a portion of its internal RNA moiety as a template for telomere repeat DNA synthesis (U.S. Pat. No. 5,583,016; Yu et al, Nature, 344:126 (1990); Singer and Gottschling, Science, 266:404 (1004); Autexier and Greider, Genes Develop., 8:563 (1994); Gilley et al, Genes Develop., 9:2214 (1995); McEachern and Blackburn, Nature, 367:403 (1995); Blackburn, Ann. Rev. Biochem., 61:113 (1992); Greider, Ann Rev. Biochem., 65:337 (1996).) The activity of this enzyme depends upon both its RNA and protein components to circumvent the problems presented by end replication by using RNA (i.e., as opposed to DNA) to template the synthesis of telomeric DNA. Telomerases extend the G strand of telomeric DNA. A combination of factors, including telomerase processivity, frequency of action at individual telomeres, and the rate of degradation of telomeric DNA, contribute to the size of the telomeres (i.e., whether they are lengthened, shortened, or maintained at a certain size). In vitro telomerases may be extremely processive, with the Tetrahymena telomerase adding an average of approximately 500 bases to the G strand primer before dissociation of the enzyme (Greider, Mol. Cell. Biol., 114572 (1991).)
“Genetically modified or altered”—In the present invention this refers to cells that contain one or more modifications in their genomic DNA, e.g., additions, substitutions and/or deletions.
“De-differentiation”—In the present invention, this refers to the changes in a differentiated cell, e.g., human somatic cell in tissue culture, that result upon introduction of cytoplasm from a more primitive, less differentiated cell type, e.g., an oocyte or other embryonic cell.
“Totipotent”—In the present invention this refers to a cell that gives rise to all of the cells in a developing body, such as an embryo, fetus, an animal. The term “totipotent” can also refer to a cell that gives rise to all of the cells in an animal. A totipotent cell can give rise to all of the cells of a developing cell mass when it is utilized in a procedure for creating an embryo from one or more nuclear transfer steps. An animal may be an animal that functions ex utero. An animal can exist, for example, as a live born animal. Totipotent cells may also be used to generate incomplete animals such as those useful for organ harvesting, e.g., having genetic modifications to eliminate growth of a head such as by manipulation of a homeotic gene.
“Ungulate”—In the present invention this refers to a four-legged animal having hooves. In other preferred embodiments, the ungulate is selected from the group consisting of domestic or wild representatives of bovids, ovids, cervids, suids, equids, and camelids. Examples of such representatives are cows or bulls, bison, buffalo, sheep, big-horn sheep, horses, ponies, donkeys, mule, deer, elk, caribou, goat, water buffalo, camels, llama, alpaca, and pigs. Especially preferred in the bovine species are Bos Taurus, Bos Indicus, and Bos buffaloes cows or bulls.
“Immortalized” or “permanent” cell—These terms as used in the present invention in reference to cells can refer to cells that have exceeded the Hayflick limit. The Hayflick limit can be defined as the number of cell divisions that occur before a cell line becomes senescent. Hayflick set this limit to approximately 60 divisions for most non-immortalized cells. See, e.g., Hayflick and Moorhead, 1971, Exp. Cell. Res., 25:585-621; and Hayflick, 1965, Exp. Cell Research, 37:614-636, incorporated herein by reference in their entireties, including all figures, tables and drawings. Therefore, an immortalized cell line can be distinguished from non-immortalized cell lines if the cells in the cell line are able to undergo more than 60 divisions. If the cells of a cell line are able to undergo more than 60 cell divisions, the cell line is an immortalized or permanent cell line. The immortalized cells of the invention are preferably able to undergo more than 70 divisions, are more preferably able to undergo more than 90 divisions, and are most preferably able to undergo more than 90 cell divisions.
Typically, immortalized or permanent cells can be distinguished from non-immortalized and non-permanent cells on the basis that immortalized and permanent cells can be passaged at densities lower than those of non-immortalized cells. Specifically, immortalized cells can be grown to confluence (e.g., when a cell monolayer spreads across an entire plate) when plating conditions do not allow physical contact between the cells. Hence, immortalized cells can be distinguished from non-immortalized cells when cells are plated at cell densities where the cells do not physically contact one another.
“Culture”—In the present invention this term refers to one or more cells that are static or undergoing cell division in a liquid medium. Nearly any type of cell can be placed in cell culture conditions. Cells may be cultured in suspension and/or in monolayers with one or more substantially similar cells. Cells may be cultured in suspension and/or in monolayers with heterogeneous population cells. The term heterogeneous as utilized in the previous sentence can relate to any cell characteristics, such as cell type and cell cycle stage, for example. Cells may be cultured in suspension and/or in monolayers with feeder cells.
“Feeder Cells”—This refers to cells grown in co-culture with other cells. Feeder cells include, e.g., fibroblasts, fetal cells, oviductal cells, and may provide a source of peptides, polypeptides, electrical signals, organic molecules (e.g., steroids), nucleic acid molecules, growth factors, cytokines, and metabolic nutrients to cells co-cultured therewith. Some cells require feeder cells to be grown in tissue culture.
“Reprogram”—This term as used in the present invention refers to materials and methods that can convert a differentiated cell into a less differentiated, more primitive cell type, e.g., an embryonic stem cell.
“Embryo”—In the present invention this refers to a developing cell mass that has not implanted into the uterine membrane of a maternal host. Hence, the term “embryo” as used herein can refer to a fertilized oocyte, a cybrid (defined herein), a pre-blastocyst stage developing cell mass, and/or any other developing cell mass that is at a stage of development prior to implantation into the uterine membrane of a maternal host. Embryos of the invention may not display a genital ridge. Hence, an “embryonic cell” is isolated from and/or has arisen from an embryo.
“Fetus”—In the present invention refers to a developing cell mass that has implanted into the uterine membrane of a maternal host. A fetus can include such defining features as a genital ridge, for example. A genital ridge is a feature easily identified by a person of ordinary skill in the art and is a recognizable feature in fetuses of most animal species.
“Fetal cell”—as used herein can refer to any cell isolated from and/or has arisen from a fetus or derived from a fetus.
“Non-fetal cell”—refers to a cell that is not derived or isolated from a fetus.
“Senescence”—In the present invention this refers to the characteristic slowing of growth of non-immortal somatic cells in tissue culture after cells have been maintained in culture for a prolonged period. Non-immortal cells characteristically have a defined life-span before they become senescent and die. The present invention alleviates or prevents senescence by the introduction of cytoplasm from a donor cell, typically an oocyte or blastomere, into a recipient cell, e.g., a cultured human somatic cell.