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Definitions

• the present invention provides a novel method for producing pluripotent mammalian cell lines and differentiated cells, tissues and organs derived therefrom.
• the subject pluripotent cells will be produced by gynogenetic or androgenetic means, i.e., methods wherein the pluripotent cells are derived from oocytes containing only DNA of male or female origin and therefore will comprise all female-derived or male- derived DNA.
• the oocytes will comprise male or female- derived DNA of primate origin, e.g. human.
• a period of DNA synthesis occurs before the two sets of chromosomes condense and come together on the mitotic spindle of the first cleavage division. Fertilization is completed with the restoration of the diploid complement of chromosomes in the nuclei of the two-cell embryo.
• oocytes containing DNA of all male or female origin may because activated and result in the production of an embryo. Typically such embryo does not develop into an offspring, but rather stops developing fairly early in embryogenesis.
• the activation of oocytes and development of embryos that comprise DNA of all male or female origin is typically effected as a means of studying embryogenesis.
• the activation of oocytes containing DNA of all female origin, without any contribution from the male gamete, and the production of an embryo therefrom is known as "parthenogenesis.” This method has been used by many researchers as a means for studying embryogenesis in vitro.
• Parthenogenesis is a type of gynogenesis. Gynogenesis broadly is defined as the phenomena wherein an oocyte containing all female DNA becomes activated and produces an embryo. Gynogenesis includes parthenogenesis as well as activation methods wherein the spermatozoa activates the oocyte to complete meiosis, but fails to contribute any genetic material to the resulting embryo. As in parthenogenesis, the activated oocyte does not contain DNA of male origin. However, unlike parthenogenesis, however, the male gamete does make a contribution, i.e., it stimulates oocyte activation. Androgenesis can be considered to be the opposite of gynogenesis. This refers to the production and activation of an oocyte containing DNA of entirely male origin, and the development of an embryo therefrom.
• parthenogenetically activated oocytes may give rise to a variety of aberrations that occur during the completion of meiosis, which may result in the production of embryos of different genetic constitutions. It is known that artificial activation of mammalian oocytes, including oocytes containing DNA of all male or female origin, can be induced by a wide variety of physical and chemical stimuli. Examples of such methods are listed in the Table below. TABLE 1
• parthenogenetic oocytes have been well reported in the literature.
• Ware et al, Gamete Research, 22:265-275 (1989) teach the ability of bovine oocytes to undergo parthenogenetic activation using Ca ⁇ , Mg ++ -H + ionophore (A23187) or electric shock.
• Yang et al, Soc. Study Reprod., 46: 117 (1992) teaches activation of bovine follicular oocytes using cycloheximide and electric pulse treatment.
• Graham C. F. in Biol. Rev., 49:399-422 (1979) describes then existing methods for activating parthenogenetic mammalian embryos. Further, Matthew H.
• ES cells pluripotent or embryonic stem cells
• ES or pluripotent cell lines ES cells are extremely desirable because of their pluripotency which allows them to give rise to any differentiated cell type.
• ES cells are useful for the production of chimeric animals and as an in vitro model for differentiation studies, especially the study of genes involved in early development.
• Methods for deriving embryonic stem (ES) cell lines in vitro from early preimplantation mouse embryos are well known. (See, e.g., Evans et al., Nature, 29: 154-156 (1981); Martin, Proc. Natl. Acad.
• ES cells can be passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells (Evans et al., Id.) or a differentiation inhibiting source (Smith et al., ev. Biol. , 121 : 1 -9 ( 1987)) is present.
• a feeder layer of fibroblast cells Evans et al., Id.
• a differentiation inhibiting source Smith et al., ev. Biol. , 121 : 1 -9 ( 1987)
• ES cells have been previously reported to possess numerous applications. For example, it has been reported that ES cells can be used as an in vitro model for differentiation, especially for the study of genes which are involved in the regulation of early development. Mouse ES cells can give rise to germline chimeras when intro- prised into preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al., Nature, 309:255-256 (1984)). In view of their ability to transfer their genome to the next generation, ES cells have potential utility for germline manipulation of livestock animals by using ES cells with or without a desired genetic modification.
• nuclei from like preimplantation livestock embryos support the development of enucleated oocytes to term (Smith et al., Biol. Reprod., 40:1027- 1035 (1989); and Keefer et al., Biol. Reprod., 50:935-939 (1994)).
• This is in contrast to nuclei from mouse embryos which beyond the eight-cell stage after transfer reportedly do not support the development of enucleated oocytes (Cheong et al, Biol. Reprod., 48:958 (1993)). Therefore, ES cells from livestock animals are highly desirable because they may provide a potential source of totipotent donor nuclei, genetically manipulated or otherwise, for nuclear transfer procedures.
• Biotech, 6(1): 1-14 (1995) discloses the isolation of embryonic cell lines from porcine blastocysts. These cells are stably maintained in mouse embryonic fibroblast feeder layers without the use of conditioned medium, and reportedly differentiate into several different cell types during culture.
• Van Stekelenburg-Hamers et al. Mol. Reprod. Dev., 40:444-454 (1995), reported the isolation and characterization of purportedly permanent cell lines from inner cell mass cells of bovine blastocysts.
• the authors isolated and cultured ICMs from 8 or 9 day bovine blastocysts under different conditions to determine which feeder cells and culture media are most efficient in supporting the attachment and outgrowth of bovine ICM cells. They concluded that the attachment and outgrowth of cultured ICM cells is enhanced by the use of STO (mouse fibroblast) feeder cells (instead of bovine uterus epithelial cells) and by the use of charcoal-stripped serum (rather than normal serum) to supplement the culture medium. Van Stekelenburg et al reported, however, that their cell lines resembled epithelial cells more than pluripotent ICM cells.
• pluripotent cells and cell lines i.e., ES cells. More specifically, it is an object of the invention to provide a method for obtaining pluripotent cells or cell lines that comprise female-derived or male-derived DNA.
• pluripotent cells or cell lines that comprise primate female-derived or male- derived DNA, male or female DNA of human origin.
• pluripotent cells or cell lines that are isogenic to male or female donors, e.g., a human donor.
• pluripotent cells that comprise all female-derived or male-derived DNA for the production of differentiated cells, tissues and organs.
• pluripotent cells or cell lines preferably human pluripotent cells or cell lines
• primate preferably human, differentiated cells, tissues or organs by inducing differentiation of pluripotent cells or cell lines that comprise DNA of all mate or female origin.
• pluripotent cells that comprise all female-derived or male-derived DNA for the production of chimeric animals.
• a feeder layer e.g., fetal fibroblasts.
• Another object of the invention is to produce pluripotent cells that comprise a desired DNA modification. This will be effected by producing oocytes containing DNA of all male or female origin, wherein said DNA comprises a desired modification; activating said oocytes under conditions that give rise to an embryo having a discernible trophectoderm and inner cell mass; and culturing cells from said inner cell mass or the entire cell mass under conditions that inhibit differentiation and maintain said cells in a pluripotent state that contain the desired DNA modification.
• Still another object of the invention is to produce differentiated cells or tissues that contain a desired DNA modification comprising: (i) producing oocytes that contain DNA of all male or female origin, which DNA comprises at least one modification;
• differentiated cells containing said at least one DNA modification can be effected, e.g., by altering the cell culture procedure, e.g., by addition of growth factors that promote differentiation; by removal of feeder layers, by injection into a suitable animal, e.g., SCID mouse, whereby differentiated cells and/or tissues arise from said cells in vivo; or by the use thereof to produce a chimeric animal or fetus that contains differentiated cells that express the genotype of said male or female DNA, which comprises at least one DNA modification.
• a suitable animal e.g., SCID mouse
• Still another object of the invention is to provide cell cultures comprising pluripotent cells which optionally may be genetically modified, wherein said pluripotent cells are derived from embryos produced by androgenetic or gynogenetic methods.
• Yet another object of the invention is to provide differentiated cells and tissues derived from pluripotent cells which are themselves derived from embryos produced by androgenetic or gynogenetic methods.
• Still another object of the invention is to use said pluripotent cells, or differentiated cells derived therefrom, which optionally may be genetically modified for cell and gene therapy, tissue and organ transplantation, or for the study of embryogenesis and differentiation. For example, the effect of specific DNA modifications on embryonic development or the production of specific types of differentiated cells can be effected.
• Yet another object of the invention is to culture pluripotent cells and cell lines produced according to the invention with different combinations of hormones, cytokines, and growth factors and ratios thereof that induce the differentiation of the subject pluripotent cells into particular differentiated cell types. It is yet another object of the invention to provide cloned animals, having a particular genotype, by use of pluripotent cells, or differentiated cells provided therefrom, produced according to the invention as nuclear donors for nuclear transfer.
• the pluripotent cells or differentiated cells derived therefrom will be obtained by activation of an oocyte or blastomers that contains only DNA of human male or human female origin, which DNA is optionally genetically modified to produce an embryo containing a discernible trophectoderm and inner cells mass, and wherein the inner cell mass or a portion thereof is cultured under conditions that maintain said cells in a pluripotent undifferentiated state; and which pluripotent cells give rise to differentiated cell types in vivo (e.g., in a SCID mouse), or when cultured under appropriate conditions.
• an oocyte or blastomers that contains only DNA of human male or human female origin, which DNA is optionally genetically modified to produce an embryo containing a discernible trophectoderm and inner cells mass, and wherein the inner cell mass or a portion thereof is cultured under conditions that maintain said cells in a pluripotent undifferentiated state; and which pluripotent cells give rise to differentiated cell types in vivo (e.g., in
• Figure 1 (labeled 31-11): Embryoid body photographed at 100X magnification. Formed from explant plated directly on culture dish (no mouse fetal fibroblast feeder layer). Original colony (1023981-3) plated from a blastocyst activated previously. Light is deflected showing lipid content of cells.
• Figure 2 (labeled 31-18): Embryoid body photographed at 100X magnification. Formed from explant plated directly on culture dish (no mouse fetal fibroblast feeder layer). Original colony (1023981-3) plated from a blastocyst activated a week prior.
• Figure 3 (labeled 31-25): Embryoid body photographed at 100X magnification. Formed from explant plated directly on culture dish (no mouse fetal fibroblast feeder layer). Original colony (1023981-3) plated from a blastocyst activated week prior.
• Figure 4 (labeled 32-1): Edge of explanted stem cell colony photographed at 40X magnification.
• Original colony (1023981-3) plated from blastocyst activated week prior.
• Stem cell colony is top left and mouse fetal fibroblast feeder layer is bottom right.
• Figure 5 (labeled 32-3): Edge of explanted stem cell colony photographed at 100X magnification. Original colony (1023981-3) plated from blastocyst activated week prior. Stem cell colony is top left and mouse fetal fibroblast feeder layer is bottom right.
• Figure 6 (labeled 32-5): Center of explanted stem cell colony photographed at
• Figure 7 (labeled 32-12): Center of explanted stem cell colony photographed at 200X magnification. Original colony plated (1023981-3) from blastocyst activated week prior.
• Figure 8 (labeled 35-6): Edge of explanted stem cell colony photographed at 40X magnification. Original colony (0106992-2) plated from blastocyst activated week prior. Stem cell colony is top right. Mouse fetal fibroblast feeder layer is bottom left. Light is deflected showing difference in lipid content between the cells of the stem cell colony and the mouse fibroblast feeder layer.
• Figure 9 (labeled 35-8): Edge of explanted stem cell colony photographed at 40X magnification. Original colony (0106992-2) plated from blastocyst activated week prior. Stem cell colony is on the top. Mouse fetal fibroblast feeder layer is on the bottom. Light is deflected showing difference in lipid content between the cells of the stem cell colony and the mouse fibroblast feeder layer.
• Figure 10 (labeled 35-19): Edge of explanted stem cell colony photographed at 200X magnification. Original colony (0106992-2) plated from blastocyst activated week prior. Stem cell colony is on the left. Mouse fetal fibroblast feeder layer is on the right. Photograph shows differentiation of the cells at the edge of the stem cell colony.
• Figure 11 shows three monkey blastocysts on day 8 of development.
• Figure 12 shows a cell line (Cyno 1) obtained from one of the three monkey blastocytsts shown in Figure 11, before immunosurgery.
• Figure 13 shows the Cyno 1 cell line after immunosurgery.
• Figure 14 shows the Cyno 1 cell line 5 days after plating on a fibroblast feeder layer.
• Figure 15 shows a pluripotent cell line (referred to as Cyno 1) derived from a parthogenetically activated Cynomolgous monkey oocyte, growing on a mouse fibroblast layer.
• the feeder cells exhibit morphological characteristics of pluripotent cells, e.g. small nuclear cytoplasmic ratios and detectable cytoplasmic granules.
• Figure 16 shows RT-PCR results demonstrating differentiation of multiple somatic cell types in differentiating PPSCs: Brachy: Brachyury (T) protein, MYH2: Skeletal myosin Heavy Polypeptide 2, Enolase: Human neuron-specific Enolase 2, SHH: Human homolog of Sonic Hedgehog.
• Fig. 17 shows RT-PCR results using mRNA from monkey fibroblasts with or without reverse transcriptase and PPSCs with or without reverse transcriptase. A PCR product was detected of the predicted length only in the monkey fibroblasts cells.
• Gynogenesis - in the present invention refers to the production of an embryo containing a discernible trophectoderm and inner cell mass that results upon activation of a cell, preferably an oocyte, or other embryonic cell type, containing mammalian DNA of all female origin, preferably human female origin, e.g., human or non-human primate oocyte DNA.
• mammalian DNA may be genetically modified, e.g., by insertion, deletion or substitution of at least one DNA sequence, or may be unmodified.
• the DNA may be modified by the insertion or deletion of desired coding sequences, or sequences that promote or inhibit embryogenesis.
• Gynogenesis is inclusive of parthenogenesis which is defined below. It also includes activation methods wherein the sperm or a factor derived therefrom initiates or participates in activation, but the spermatozoal DNA does not contribute to the DNA in the activated oocyte.
• Androgenesis - in the present invention refers to the production of an embryo containing a discernible trophectoderm and inner cell mass that results upon activation of an oocyte or other embryonic cell type, e.g., blastomere, that contains DNA of all male origin, e.g., human spermatozoal DNA.
• said DNA of all male origin may be genetically modified, e.g., by the addition, deletion, or substitution of at least one DNA sequence (as described above with respect to gynogenesis).
• parthenogenesis refers to the process by which activation of the oocyte occurs in the absence of sperm penetration.
• parthenogenesis refers to the development of an early stage embryo comprising trophectoderm and inner cell mass that is obtained by activation of an oocyte or embryonic cell, e.g., blastomere, comprising DNA of all female origin.
• Pluripotent cell - in the present invention refers to a cell derived from a embryo produced by activation of a cell containing DNA of all female or male origin that can be maintained in vitro for prolonged, theoretically indefinite period of time in an undifferentiated state, that can give rise to different differentiated tissue types, i.e., ectoderm, mesoderm, and endoderm.
• the pluripotent state of said cells is preferably maintained by culturing inner cell mass or cells derived from the inner cell mass of an embryo produced by androgenetic or gynogenetic methods under appropriate conditions, preferably by culturing on a fibroblast feeder layer or another feeder layer or culture that includes leukemia inhibitory factor.
• the pluripotent state of such cultured cells can be confirmed by various methods, e.g., (I) confirming the expression of markers characteristic of pluripotent cells; (ii) production of chimeric animals that contain cells that express the genotype of said pluripotent cells; (iii) injection of cells into animals, e.g., SCID mice, with the production of different differentiated cell types in vivo; and (iv) observation of the differentiation of said cells (e.g., when cultured in the absence of feeder layer or LIF) into embryoid bodies and other differentiated cell types in vitro.
• various methods e.g., (I) confirming the expression of markers characteristic of pluripotent cells; (ii) production of chimeric animals that contain cells that express the genotype of said pluripotent cells; (iii) injection of cells into animals, e.g., SCID mice, with the production of different differentiated cell types in vivo; and (iv) observation of the differentiation of said cells (e.g.,
• Diploid cell - in the present invention typically refers to a cell, e.g., an oocyte or blastomere, having a diploid DNA content of all male or female origin.
• Haploid cell - in the present invention typically refers to a cell, e.g., an oocyte or blastomere having a haploid DNA content, wherein the haploid DNA is of all male or female origin.
• activation refers to methods whereby a cell containing DNA of all male or female origin is induced to develop into an embryo that has a discernible inner cell mass and trophectoderm, which is useful for producing pluripotent cells but which is itself incapable of developing into a viable offspring.
• activation is preferably effected under one of the following conditions: (I) conditions that do not cause second polar body extrusion; (ii) conditions that cause polar body extrusion but wherein polar body extrusion is inhibited; or (iii) conditions that inhibit first cell division of haploid oocyte.
• the present invention includes activation of oocytes or blastomeres that have been transplanted with two male or two female haploid nuclei.
• Metaphase II - stage of cell development wherein the DNA content of a cell consists of a haploid number of chromosomes with each chromosome represented by two chromatids.
• Embryo - in the present invention this typically refers to an embryo that results upon activation of a cell, e.g., oocyte or other embryonic cells containing DNA of all male or female origin, which optionally may be modified, that comprises a discernible trophectoderm and inner cell mass, which cannot give rise to a viable offspring and wherein the DNA is of all male or female origin.
• the inner cell mass or cells contained therein are useful for the production of pluripotent cells as defined previously.
• Inner cell mass - inner portion of an embryo which gives rise to fetal tissues are used to provide a continuous source of pluripotent cells in vitro.
• the inner cell mass refers to the inner portion of the embryo that results from androgenesis or gynogenesis, i.e., embryos that result upon activation of cells containing DNA of all male or female origin.
• DNA will be human DNA, e.g., human oocyte or spermatozoal DNA, which optionally has been genetically modified.
• Trophectoderm other portion of early stage embryo which gives rise to placental tissues.
• the trophectoderm is that of an embryo that results from androgenesis or gynogenesis, i.e., embryos that result from activation of cells that contain DNA of all male or female origin, e.g., human oocyte or spermatozoan.
• Differentiated cell - a non-embryonic cell that possesses a particular differentiated, i.e., non-embryonic state.
• the three earliest differentiated cell types are endoderm, mesoderm and ectoderm.
• the present invention provides novel methods for obtaining pluripotent cells and cell lines, wherein the DNA is derived from a single individual, i.e., male or female, by androgenesis or gynogenesis.
• immature oocytes eggs
• the oocytes then arrest at metaphase II.
• metaphase II the DNA content of the cell consists of a haploid number of chromosomes, each represented by two chromatids. Normally, the oocyte is ovulated at this stage and fertilized by the sperm.
• the sperm initiates the completion of meiosis in a process called activation.
• activation the pairs of chromatids separate, the second polar body is extruded, and the oocyte retains a haploid number of chromosomes, each with one chromatid.
• the sperm contributes the other haploid complement of chromosomes to make a full diploid cell with single chromatids.
• the chromosomes then progress through DNA synthesis during the first cell cycle. These cells then develop into embryos.
• embryos are developed by artificial activation of cells, typically mammalian oocytes or blastomeres containing DNA of all male or female origin.
• cells typically mammalian oocytes or blastomeres containing DNA of all male or female origin.
• Such methods include physical methods, e.g., mechanical methods such as pricking, manipulation or oocytes in culture, thermal methods such as cooling and heating, repeated electric pulses, enzymatic treatments, such as trypsin, pronase, hyaluronidase, osmotic treatments, ionic treatments such as with divalent cations and calcium ionophores, the use of anaesthetics such as ether, ethanol, tetracaine, lignocaine, procaine, phenothiazine, tranquilizers such as thioridazine, trifluoperazine, fluphenazine, chlorpromazine, the use of protein synthesis inhibitors such as cycloheximide, puromycin, the use of phosphorylation inhibitors, e.g., protein kinase inhibitors such as DMAP, combinations thereof, as well as other methods.
• physical methods e.g., mechanical methods such as pricking, manipulation or oocytes in
• a mammalian cell preferably in metaphase II, typically an oocyte or blastomere comprising DNA of all male or female origin is artificially activated by a known means for effecting artificial activation of oocytes or nuclear transfer fusions.
• the cell may extrude the second polar body or retain the second polar body.
• a mammalian cell preferably in metaphase II, e.g., a mammalian oocyte containing haploid content of DNA of all male or female origin is activated by an activation procedure that does not result in second polar body extrusion.
• This can be effected by various methods including the use of a phosphorylation inhibitor such as DMAP or by use of a micro filament inhibitor such as cytochalasin B, C or D, or a combination thereof.
• a phosphorylation inhibitor such as DMAP
• a micro filament inhibitor such as cytochalasin B, C or D, or a combination thereof.
• a haploid cell preferably in metaphase II, typically an oocyte or blastomere that comprises all male or female DNA, will be activated under conditions that result in the production of an embryo having a discernible trophectoderm and inner cell mass, but wherein the first cleavage event is prevented, thereby resulting in a diploid cell which develops into an embryo that cannot give rise to an offspring.
• the invention includes the embodiment wherein an enucleated cell, e.g., mammalian oocyte or blastomere, or other mammalian cytoplast, is transplanted with two male or female haploid nuclei, e.g., derived from oocytes or sperm, and is activated by an appropriate activation procedure to produce an embryo containing a discernible trophectoderm and inner cell mass which is incapable of giving rise to an offspring.
• the inner cell mass or cells derived therefrom are useful for obtaining pluripotent cells which may be maintained for prolonged periods in tissue culture.
• the activated cell e.g., oocyte, which is diploid
• the activated cell is allowed to develop into an embryo that comprises a trophectoderm and an inner cell mass.
• This can be effected using known methods and culture media that facilitate blastocyst development. Examples thereof are disclosed in our earlier patent application U.S. Patent No. 5,945,577, and have been well reported in the literature.
• Culture media suitable for culturing and maturation of embryos are well known and include Ham's F-10 +10% fetal calf serum, Tissue Culture Medium, 199 (TCM-199) + 10% fetal calf serum, Tyrodes - Albumin - Lactate - Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eaglets and Whitten's media, and CR1 medium.
• a preferred medium is for bovine embryos TCM-199 with Earl salts, 10% fetal calf serum, 0.2 mM Na pyruvate and 50 ⁇ g/ml gentamycin sulfate.
• a preferred medium for culturing pig embryos is NCSU23.
• Preferred medium for culturing primate embryos include modified Ham's F-10 medium (Gibco, Catalog No. 430- 1200 EB) supplemented with 1 ml/L synthetic serum replacement (SSR-2, Medl-Cult Denmark), and 10 mg/ml HSA; 80% Dulbecco's modified Eaglet's medium (DMEA, no pyruvate, high glucose formulation, Gibco BRL) with 20% fetal bovine serum, 0.1 mM B-mercaptoethanol, and 1% non-essential amino acid stock, and by methods and medium disclosed in Jones et al, Human Reprod.
• modified Ham's F-10 medium Gibco, Catalog No. 430- 1200 EB
• SSR-2 synthetic serum replacement
• HSA HSA
• DMEA Dulbecco's modified Eaglet's medium
• fetal bovine serum fetal bovine serum
• 0.1 mM B-mercaptoethanol 1% non-essential amino acid stock
• Activation by electrical pulses a. Place eggs in mannitol media containing 100 ⁇ M CaCL 2 b. Deliver three pulses of 1.0 kVcm "1 for 20 ⁇ second, each pulse 22 minutes apart. c. Move oocytes to culture media containing 5 ⁇ g/ml of cytochalasin B for three hours.
• Activation by exposure with ethanol followed by cytochalasin and cycloheximide a. Place oocytes in 7% ethanol for one minute. b. Move oocytes to culture media containing 5 ⁇ g/ml of cytochalasin B and 5 ⁇ g/ml of cycloheximide for five hours. c. Rinse four times and place in culture.
• Activation by microinjection of adenophostin a. Inject oocytes with 10 to 20 picoliters of a solution containing 10 ⁇ M of adenophostin. b. Place oocytes in culture.
• sperm factor 7. Activation by microinjection of sperm factor a. Inject oocytes with 10 to 20 picoliters of sperm factor isolated either from primates, pigs, bovine, sheep, goat, horse, mice, rat, rabbit or hamster. b. Place eggs in culture.
• the cells of the inner cell mass are then used to produce the desired pluripotent cell lines. This can be accomplished by transferring cells derived from the inner cell mass or the entire inner cell mass onto a culture that inhibits differentiation. This is preferably effected by transferring said inner cell mass cells onto a feeder layer that inhibits differentiation, e.g., fibroblasts or epithelial cells, such as fibroblasts derived from murines, ungulates, chickens, such as mouse or rat fibroblasts, 570 and SI-m220 feeder cells, BRL cells, etc., or other cells that produce LIF.
• a feeder layer that inhibits differentiation e.g., fibroblasts or epithelial cells, such as fibroblasts derived from murines, ungulates, chickens, such as mouse or rat fibroblasts, 570 and SI-m220 feeder cells, BRL cells, etc., or other cells that produce LIF.
• the inner cell mass cells are cultured on mouse fetal fibroblast cells or other cells which produce leukemia inhibitory factor, or in the presence of leukemia inhibitory factor. Culturing will be effected under conditions that maintain said cells in an undifferentiated, pluripotent state, for prolonged periods, theoretically indefinitely. Suitable conditions for culturing pluripotent cells, specifically pluripotent cells derived from ungulate inner cell mass are also described in our earlier patent U.S. Patent No. 5,945,577, as well as U.S. Patent No. 5,905,042, both of which are incorporated by reference herein in their entirety. As noted, the subject invention will give rise to pluripotent cells having DNA that is exclusively of male or female origin, which may be used to produce different differentiated cell types.
• such exclusively male or female DNA will be genetically modified before or after activation of the cell containing same, e.g., human oocyte.
• pluripotent cells are obtained that comprise a desired DNA modification, e.g., contain a desired coding sequence.
• the novelty of the invention is in the production of pluripotent cells that contain either female- or male-derived DNA. To the inventors' knowledge, this has not been reported for any species. Considerable work has been done in the mouse with pluripotent and totipotent embryonic stem cells but these have all been derived from either normal embryonic cells or primordial germ cells. Each of these cell types has both paternal and maternal DNA. Work has also been done with embryos containing either male-derived or female-derived DNA.
• donor cells for nuclear transfer, e.g. by the methods reported in U.S. Patent 5,945,577 or patents and patent applications by the Roshn Institute.
• these methods differ in that the ACT cloning methods utilize proliferating donor cells, e.g. mammalian somatic cells, such as fibroblasts, whereas the Roshn Institute methods utilize quiescent donor cells
• the plu ⁇ potent state of the cells produced by the present invention can be confirmed by various methods
• the cells can be tested for the presence or absence of characte ⁇ stic ES cell markers.
• characte ⁇ stic ES cell markers include SSEA-4, SSEA-3, TRA-1-60 and TRA-1-81 and are known in the art
• pluripotency can be confirmed by injecting the cells into a suitable animal, e.g., a SCID mouse, and observing the production of differentiated cells and tissues.
• a suitable animal e.g., a SCID mouse
• Still another method of confirming plu ⁇ potency is using the subject plu ⁇ potent cells to generate chime ⁇ c animals and observing the cont ⁇ bution of the introduced cells to different cell types. Methods for producing chime ⁇ c animals are well known in the art and are desc ⁇ bed in our related applications, incorporated by reference herein
• Yet another method of cultunng plu ⁇ potency is to observe their differentiation into embryoid bodies and other differentiated cell types when cultured under conditions that favor differentiation (e.g., removal of fibroblast feeder layers).
• the resultant pluripotent cells and cell lines preferably human pluripotent cells and cell lines, which are derived from DNA of entirely male or female original, have numerous therapeutic and diagnostic applications. Most especially, such pluripotent cells may be used for cell transplantation therapies or gene therapy (if genetically modified). Human ES cells have application in the treatment of numerous disease conditions.
• mouse embryonic stem (ES) cells are capable of differentiating into almost any cell type, e.g., hematopoietic stem cells. Therefore, human or other mammalian pluripotent (ES) cells produced according to the invention should possess similar differentiation capacity.
• the pluripotent cells according to the invention will be induced to differentiate to obtain the desired cell types according to known methods.
• human ES cells produced according to the invention may be induced to differentiate into hematopoietic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, etc., by culturing such cells in differentiation medium and under conditions which provide for cell differentiation.
• hematopoietic stem cells from an embryonic cell line by subjecting stem cells to an induction procedure comprising initially culturing aggregates of such cells in a suspension culture medium lacking retinoic acid followed by culturing in the same medium containing retinoic acid, followed by transferral of cell aggregates to a substrate which provides for cell attachment.
• one skilled in the art may culture the subject ES cells, including genetically engineered or transgenic ES cells, to obtain desired differentiated cell types, e.g., neural cells, muscle cells, hematopoietic cells, etc.
• desired differentiated cell types e.g., neural cells, muscle cells, hematopoietic cells, etc.
• Pluripotent cells produced by the invention may be used to obtain any desired differentiated cell type.
• Therapeutic usages of differentiated human cells are unparalleled.
• human hematopoietic stem cells may be used in medical treatments requiring bone marrow transplantation. Such procedures are used to treat many diseases, e.g., late stage cancers such as ovarian cancer and leukemia, as well as diseases that compromise the immune system, such as AIDS.
• Hematopoietic stem cells can be obtained, e.g., by incorporating male or female DNA derived from a male or female cancer or AIDS patient with an enucleated oocyte, obtaining pluripotent cells as described above, and culturing such cells under conditions which favor differentiation, until hematopoietic stem cells are obtained.
• Such hematopoietic cells may be used in the treatment of diseases including cancer and AIDS.
• the subject pluripotent cells may be used to treat a patient with a neurological disorder by culturing such cells under differentiation conditions that produce neural cell lines.
• Specific diseases treatable by transplantation of such human neural cells include, by way of example, Parkinson's disease, Alzheimer's disease, ALS and cerebral palsy, among others.
• Parkinson's disease it has been demonstrated that transplanted fetal brain neural cells make the proper connections with surrounding cells and produce dopamine. This can result in long- term reversal of Parkinson's disease symptoms.
• the great advantage of the subject invention is that it provides an essentially limitless supply of pluripotent, preferably pluripotent human cells that can be used to produce differentiated cells suitable for transplantation.
• pluripotent preferably pluripotent human cells that can be used to produce differentiated cells suitable for transplantation.
• Such cells should alleviate the significant problem associated with current transplantation methods, i.e., rejection of the transplanted tissue which may occur because of host-vs-graft or graft-vs-host rejection.
• rejection is prevented or reduced by the administration of anti -rejection drugs such as cyclosporin.
• anti -rejection drugs such as cyclosporin.
• such drugs have significant adverse side-effects, e.g., immunosuppression, carcinogenic properties, as well as being very expensive.
• the present invention should eliminate, or at least greatly reduce, the need for anti -rejection drugs.
• diseases and conditions treatable by cell therapy include, by way of example, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, i.e., hypercholesterolemia, heart diseases, cartilage replacement, burns, foot ulcers, gastrointestinal diseases, vascular diseases, kidney disease, urinary tract disease, and aging related diseases and conditions.
• This methodology can be used to replace defective genes, e.g., defective immune system genes, cystic fibrosis genes, or to introduce genes which result in the expression of therapeutically beneficial proteins such as growth factors, lymphokines, cytokines, enzymes, etc.
• the gene encoding brain derived growth factor may be introduced into human pluripotent cells produced according to the invention, the cells differentiated into neural cells and the cells transplanted into a Parkinson's patient to retard the loss of neural cells during such disease.
• BDNF BDNF-derived neurotrophic factor
• astrocytes have been transfected with BDNF gene using retroviral vectors, and the cells grafted into a rat model of Parkinson's disease (Yoshimoto et al., Brain Research, 691 :25-36, (1995)).
• Genes which may be introduced into the subject pluripotent cells include, by way of example, epidermal growth factor, basic fibroblast growth factor, glial derived neurotrophic growth factor, insulin-like growth factor (I and II), neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokine genes (interleukins, interferons, colony stimulating factors, tumor necrosis factors (alpha and beta), etc.), genes encoding therapeutic enzymes, etc.
• the present invention also includes the use of non-human cells in the treatment of human diseases.
• non-human primate pluripotent cells produced according to the invention should be useful for treatment of human disease conditions where cell, tissue or organ transplantation is warranted (given the phylogenetic closeness of primates and humans (immunogenicity should be less of a concern.)
• pluripotent cells and differentiated cells derived therefrom produced according to the present invention can be used within the same species (autologous, syngenic or allografts) or across species (xenografts).
• brain cells derived from bovine or porcine pluripotent cells may be used to treat Parkinson's disease.
• the subject pluripotent ES cells preferably human cells
• the subject pluripotent ES cells may be used as an in vitro model of differentiation, in particular for the study of genes which are involved in the regulation of early development.
• differentiated cell tissues and organs produced using the subject ES cells may be used in drug studies.
• the subject ES cells or differentiated cells derived therefrom may be used as nuclear donors for the production of other ES cells and cell colonies, or in the case of non-human cells, for the production of cloned animals.
• pluripotent cells obtained according to the invention may be used to identify proteins and genes that are involved in embryogenesis. This can be effected e.g. by differential expression, i.e. by comparing mRNA's that are expressed in pluripotent cells provided according to the invention to mRNAs that are expressed as these cells differentiate in to different cell types, e.g., neural cells, myocardiocytes, other muscle cells, skin cells, etc. Thereby, it may be possible to determine what genes are involved in differentiation of specific cell types.
• pluripotent cell lines produced according to the invention to cuctails of different growth factors so as to identify conditions that induce the production and proliferation of desired cell types.
• the example describes one method of producing pluripotent cells by androgenetic or gynogenetic activation and production of embryos.
• this example describes the production of pluripotent cells from parthenogenetically activated bovine oocytes (gynogenetic activation of bovine oocytes containing all female DNA effected using ionomycin and DMAP).
• this procedure resulted in the production of an embryo having a discernible trophectoderm and inner cell mass, the inner cell mass of which, when cultured on a mouse fetal fibroblast feeder layer gave rise to pluripotent cells which produce differentiated cells (See especially Figure 10).
• TL Hepes + 1 mg/ml BSA (fraction V), pH to 7.2-7.4. Filter, label and store at 4°C in a capped container.
• Mouse fetal fibroblasts are gamma irradiated for five minutes and plated at 1 x 10 6 cells per ml of ALPHA-MEM culture media. Leave overnight or until cells are plated and change media to desired culture media.
• Bovine oocytes were stripped eighteen hours post maturation using 0.01% solution of hyluronidase ( 1 mg/ml) contained in TL Hepes media. Afterward, the stripped oocytes are rinsed using HECM Hepes or TL Hepes. The resultant stripped, rinsed oocytes are then stored or used directly for activation. Storage can be effected, e.g, in ACM at 37 °C and five percent CO 2 until activation. Preferably, the ACM is equilibrated at 37 °C and five percent CO 2 for about two to three hours before usage for oocyte storage.
• Oocyte Activation An appropriate activation protocol is used that results in the production of embryos having a discernible trophectoderm and inner cell mass. As previously disclosed, various methods can be used.
• the inventors elected to effect activation by placing oocytes (prepared as above) in Zl media containing 5 ⁇ M of ionomycin for four minutes.
• This media was prepared by diluting 2 ⁇ L of 5mM ionomycin in 2 ml of Zl medium.
• the oocytes were washed using HECM Hepes, and are then incubated in DMAP/ ACM medium for three to four hours.
• This medium was prepared by dilution of 5 ⁇ L of 200 mM DMAP in 500 ⁇ l of ACM, with ACM DMAP preferably being equilibrated at 37°C and five percent CO 2 in air for two to three hours prior to usage.
• oocytes were washed four times in HECM
• washed oocytes were then placed in ACM on a mouse fibroblast feeder layer prepared as described above.
• the ACM media was again equilibrated at 37°C and five percent CO 2 in air for two to three hours prior to use.
• blastocysts having a discernible trophectoderm and inner cell mass (See Figures 1 through 10).
• the blastocysts were dissected using 30 gauge/ 1 inch needles and placed on mouse fetal fibroblast feeder layers in ALPHA-MEM tissue culture medium. The cells were incubated thereon at 37 °C and five percent CO 2 for one week.
• Figure 10 shows a pluripotent cell colony produced from a blastocyst produced by gynogenetic (parthenogenic) activation as described above, resulted in a pluripotent (stem cell) colony with differentiated cells being observed at the edge of the colony.
• Cynomolgous Monkey (Macaca fascicularis) were superovulated using a single injection of 1000 IU of pregnant mare's serum gonadtrophin (PMSG) and 500 IU of human chorionic gonadoprophin (hCG) four days later.
• PMSG pregnant mare's serum gonadtrophin
• hCG human chorionic gonadoprophin
• 2- Ovaries were retrieved by laparotomy and oocytes dissected from the follicles and placed in maturation media 36 to 48 hrs after (hCG).
• Maturation media consisted of medium-199 (Gibco BRL) with Earle's balanced salt solution supplemented with 20% fetal bovine serum, 10 IU/ml of PMSG, 10 IU/ml of hCG, 0.05 mg/ml of penicillin G and 0.075 mg/ml of steptomycin sulfate (Hong, 1999).
• metaphase II eggs were placed in 10 micromoles of Ionomycin followed incubation in 200 mM 6-DMAP (dimethylaminopurine) for 3 to 4 hrs.
• Embryo culture 4- Embryo culture. Commercially available embryo culture media 'Cooks' was used (modified SOF). Embryos were cultured with a co-culture of mitotically inactivated mouse embryonic fibroblasts as feeder layer. 5- Isolation of inner cell mass a- Upon development to blastocyst, embryos were placed in a buffered solution of
• ICM Inner Cell Mass
• Figure 15 shows the Cyno 1 cell line growing on top of a mouse fibroblast feeder layer. These cells show typical morphology of pluripotent-embryonic-cells such as small nuclear cytoplasmic ratio and the presence of cytoplasmic granules. These cells can be maintained in an undifferentiated state for a period of months. This is evidenced by screening of such cells after prolonged culturing for the expression of a cell marker characteristic of undifferentiated cells, Alkaline Phosphatase. As expected, cells were positive on passage 3 and on passage 5.
• transcripts of a predicted size for the mesodermally-derived transcripts brachyury and skeletal muscle myosin heavy polypeptide 2 were observed.
• the transcript sonic hedgehog essential for endoderm development was observed.
• the neuron-specific ectoderm marker enolase was observed as well as keratin (not shown) as markers of ectodermally derived cells.
• parthogenetic PPSCs To establish that the imprinting status of parthogenetic PPSCs is different than that of di-parental PPSCs we looked at the expression of several imprinted genes. Genes that are mono-allelically expressed from the paternal allele, would not be expected to be expressed in parthogenetic cells, as these cells are derived exclusively from the maternal genome.
• the Snrpn gene is mono-allelically expressed from the paternal allele in mouse blastocyst inner cell mass [Szabo,PE and Mann, JR; Genes & Development 9:3097-3108 (1995)].

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