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WO2009005844A1 - Procédés permettant une spermatogenèse de mammifère femelle et une oogenèse de mammifère mâle à l'aide d'une nanobiologie synthétique - Google Patents

Procédés permettant une spermatogenèse de mammifère femelle et une oogenèse de mammifère mâle à l'aide d'une nanobiologie synthétique Download PDF

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WO2009005844A1
WO2009005844A1 PCT/US2008/050000 US2008050000W WO2009005844A1 WO 2009005844 A1 WO2009005844 A1 WO 2009005844A1 US 2008050000 W US2008050000 W US 2008050000W WO 2009005844 A1 WO2009005844 A1 WO 2009005844A1
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cells
female
sperm
male
germ
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Gregory Aharonian
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Gregory Aharonian
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos

Definitions

  • the present invention relates generally to methods for mammalian reproduction. More specifically, the present invention includes methods for developing sperm containing a female's chromosomes, or developing eggs containing a male's chromosomes, and the sperm or eggs so produced. More particularly, the present invention includes methods for transplanting genetically-altered female germ and stem cells into functional, but sterilized (artificial), male testes to produce female sperm; and methods for cultivating genetically-altered male germ and stem cells to produce functional male eggs. The invention has applications in the fields of genetics, medicine and animal husbandry.
  • gametogenesis refers in general to spermatogenesis and oogenesis.
  • gametogenesis refers in general to spermatogenesis and oogenesis.
  • Germ line cellular transformations e.g., using artificial SRY-negative Y chromosomes
  • Other methods disclosed herein provide "male eggs" (oogenesis).
  • Producing female sperm requires re-engineering processes for production of sperm in males by compensating for differences in genotype and imprinting between males and females.
  • the production of sperm in males comprises the following stages:
  • Spermatogenesis mitotic divisions and imprinting of spermatogonia into spermatocytes
  • Spermatogenesis meiotic divisions and imprinting of spermatocytes into spermatid
  • Spermiogenesis transformation of tailless spermatids into sperm
  • PPCs Primordial germ cells
  • the PGCs migrate towards the genital ridge where they continue to undergo a period of growth and proliferation.
  • the medulla of the genital ridge eventually gives rise to the seminiferous cords, which lengthen and become extensively folded to form the seminiferous tubules.
  • Migratory primitive germ cells that have reached these medullary cords termed gonocytes, then move to a position adjacent to the basal lamina of the cords and enter a nonproliferative growth phase.
  • the gonocytes mitotically differentiate to form primitive spermatogonia, which are progenitors to the eventual sperm.
  • spermatogenesis and spermiogenesis occur as germ cells move through the testis and supporting cells in the testis needed to produce sperm, in particular Sertoli and Leydig cells.
  • Sertoli cells have receptors for follicle stimulating hormone (FSH) and testosterone, important regulators of spermatogenesis. Hormonal interactions between these supporting cells, non-testis organs (hypothalamus and pituitary glands), and the male germ stem cells regulate the growth of sperm.
  • FSH follicle stimulating hormone
  • testosterone important regulators of spermatogenesis. Hormonal interactions between these supporting cells, non-testis organs (hypothalamus and pituitary glands), and the male germ stem cells regulate the growth of sperm.
  • FSH follicle stimulating hormone
  • hormone follicle stimulating hormone
  • testosterone important regulators of spermatogenesis. Hormonal interactions between these supporting cells, non-testis organs (hypothalamus and pituitary glands), and the male germ stem
  • the gonads of XY and XX individuals do not exhibit detectable morphological differences and are essentially female.
  • both male and female embryos have a double set of sex ducts.
  • primordial germ cells in the cortex begin to degenerate while the testis cords in the center of the gonad continue to develop into seminiferous tubules.
  • the gonad is thus converted into a testis.
  • the central testis cords disappear, while the oocytes in the peripheral cortex survive and become surrounded by follicle cells; the gonad is developing into an ovary. Either event is triggered by the migration of primordial germ cells to the gonad.
  • Mammalian embryos are initially "sexless", with the default pathway being ovarian differentiation (glibly, men from ovaries, not women from ribs).
  • the DSS (dosage-sensitive sex reversal) locus also suggests being female as the default case for mammals, with SRY proposed to override one or more genes in DSS, and with two copies of DSS overwhelming SRY, causing male -to-female sex reversal [JIM98].
  • epithelial somatic cells of the bipotent gonadal primordium differentiate into Sertoli cells, and this in turn triggers testis development [MCL91 ] .
  • Imprinting is an epigenetic, gamete-of-origin-, and therefore parent-of-origin-,dependent modification of the genome, i.e., changes in DNA function without changes in DNA sequences.
  • all humans have pairs of genes, one on each chromosome inherited from each parent, that are both expressed in cells.
  • One class of imprinted genes are those that are parentally imprinted. When an autosomal gene at an imprintable location on a chromosome passes through gametogenesis of one sex, its ability to be expressed is unaffected. However, when this gene passes through gametogenesis of the opposite sex, it becomes inactivated - it cannot be expressed.
  • imprinted Such an inactivated gene is termed "imprinted". Approximately one percent of human genes are imprinted. Some imprinted genes control fetal growth (such as the IGF2/H19 pair), as evidenced by gynogenetic embryos (no male imprints) having poor placental development but normal embryonic development, while androgenetic embryos (no female imprints) having the reverse.
  • fetal growth such as the IGF2/H19 pair
  • gynogenetic embryos no male imprints
  • androgenetic embryos no female imprints having the reverse.
  • the results of parental imprinting have been long recognized by phenotypical effects, as far back as 3000 years by mule breeders in Asia Minor.
  • Other epigenetic modifications include histone modifications and use of non-coding RNAs, but imprinting dominates.
  • Epigenetics can be viewed as an evolutionary gift from our sexually liberal female placental ancestors, who survived better by always having generally healthy babies (averaged sized embryos) from multiple fathers (who survived better by having their children start out as larger embryos at the expense of imperiling future offspring from other fathers).
  • a second class of imprinted genes involves the entire X chromosome in women, where early in development, one X chromosome in each cell of a woman is randomly imprinted ("X-inactivation").
  • Stem cells live for only a short time and so must be replaced periodically. Since most cells lose their mitotic potential in the course of terminal differentiation, they are not able to create their replacements. Stem cells, however, possess two fundamental properties: the ability to self-renew and the ability to produce numerous differentiated progeny. Stem cells can be classified into two types according to how they self-renew: stereotypic and populational. Stereotypic stem cells self-renew strictly by asymmetric divisions that produce a daughter stem cell and a differentiated daughter cell. Populational stem cells divide symmetrically to produce two daughter cells each of which has an equal probability of differentiating. In mammals, it has yet to be determined which type of behavior germ stem cells exhibit, though germ cells are "ageless” as compared to somatic cells in that their chromosome ends are maintained by high levels of telomerase.
  • hematopoietic stem cells There are a variety of families of stem cells, including hematopoietic, mesenchymal, epithelial and germinal.
  • the differentiation pathways for hematopoietic stem cells start with a pluripotent stem cell and ends with a variety of blood system cells such as red blood cells (erythrocytes), white blood cells (monocytes), and platelets.
  • the technique of bone marrow transfer is based on the goal of transplanting hematopoietic stem cells from one animal to another (or in cases of chemotherapy, removing bone marrow, applying chemotherapy (which can damage bone marrow), and returning the bone marrow back after treatment).
  • the spermatogonia in male testis and the oogonia in the female ovaries, both of which are derived from primordial germ stem cells in the embryo.
  • the terminal differentiation pathways of oogonia and spermatogonia in mammals are similar until the final stages of terminal differentiation. Indeed the similarities are quite interesting. For example, some of the stages of meiotic division for sperm can take place if the spermatogonia are inserted into a female egg [SAS98].
  • the first pathway suggested included the production of chimeras, i.e., fusing two embryos (having either two mothers and two fathers, two mothers and one father, or two fathers and one mother - achieved in mice, suggested for human homosexual procreation in [SIL97]).
  • chimeras i.e., fusing two embryos (having either two mothers and two fathers, two mothers and one father, or two fathers and one mother - achieved in mice, suggested for human homosexual procreation in [SIL97]).
  • problems with this procedure including the use and discarding of multiple embryos, the physiological "patchiness" of resulting chimeras (think of Calico cats), the politics of abortion, etc. and the presence of multiple male chromosomes in a child with two mothers.
  • Klinefelter men i.e. men who have a 47,XXY genotype
  • stem cells are removed from a male's testes. After the male's testes are sterilized to kill off all remaining defective stem cells, the stem cells are reintroduced into the testes where they thrive and produce sperm. Such stem cells can be altered in vitro to correct gene defects before reintroduction into the testes.
  • Brinster and Zimmerman suggested transplanting female cells into the testes [BRN93], but failed to address the problems of which female cells to transplant, how to transform female imprinting patterns into male imprinting patterns (a problem which plagues cloning), and how to compensate for the missing Y chromosome.
  • the latter failure is crucial since some Y-linked genes in spermatogenic cells are essential for the spermatogenesis process (such as the nuclear-expressed RNA-Binding Motif Y (RBMY) genes), a process which is disrupted where the female cells have no Y chromosome, a disruption unacceptable for any clinical/reproductive use of female sperm for humans that is subject to governmental regulations.
  • West and Cibelli propose a method of cloning animals from adult animals, where the cloned offspring are of the opposite sex of the adult animal, by adding/deleting X and Y chromosomes from the cells of the adult animal before using nuclear transfer cloning techniques [WES02].
  • Their technique fails to be useful for humans because of the imprinting problems associated with cloning, and the unacceptable ethical problems of creating a new human who decades later can supply sperm or eggs to his/her even older "parent".
  • mice embryonic stem cells of either sex could start the process of becoming both sperm and eggs, but also failed to report results on compensating for mission gametogenesis genes or switching imprinting patterns, and they also failed to research the use of testicular transplantation [SCHOS] 5 [SCHOS] 5 [SCHOO].
  • SCHOS testicular transplantation
  • the present invention provides methods for creating female sperm.
  • the methods of the invention include: creating female germ cells (e.g., via cloning and/or retrodifferentiating female stem cells to make them more pluripotent); expanding in vitro the number of such cells; transdifferentiating these cells to facilitate the expression of spermatogenesis factors (for example, using synthetic or artificial Y chromosomes); transplanting the resulting cells into a sterilized testes or artificial testicular environment; and allowing the transplanted cells to develop into competent spermatid nuclei or biologically functioning sperm.
  • a second female's egg is fertilized using these spermatids or sperm, or the first female's egg is fertilized with her own sperm for self-fertilization, an alternative to cloning by nuclear transfer.
  • the present invention also provides methods for creating male eggs.
  • the methods of the invention include: transdifferentiating adult male germ cells to facilitate development into an egg (for example, adding an extra X chromosome); transplanting the resulting cells into an artificial ovarian environment; and allowing the transplanted cells to develop into competent eggs.
  • a second male's sperm is used to fertilize such male eggs, or the first male's egg is fertilized with his own sperm for self-fertilization, an alternative to cloning by nuclear transfer.
  • stem cells non-germ stem cells
  • stem cells with the appropriate modifier, such as “hematopoietic stem cells”.
  • Primordial, primitive and differentiated germ cells such as oogonia and spermatogonia will be referred to as “germ cells” or “germinal cells”, which are often referred to in the medical literature somewhat confusingly as either “germ cells” or “stem cells”.
  • spermatogenesis refers to the entire process of transforming germ cells into sperm, reflecting the general use of that term.
  • spermatogenesis refers to the mitotic or meiotic process of germ cell to spermatid conversion (spermatogonia -> [mitosis] -> primary spermatocytes ⁇ diploid ⁇ -> [meiosis] -> secondary spermatocytes ⁇ haploid ⁇ -> [meiosis] -> spermatid), and "spermiogenesis” to refer to the process of spermatid to sperm conversion which occurs in the context of close association with Sertoli cells to facilitate natural delivery of the paternal genome.
  • any one or more of these medical technologies can be used within the spirit of the methods disclosed herein, as can any such medical technologies developed during the lifetime of this patent.
  • the present invention provides methods for creating sperm cells having chromosomes directly derived from an adult female's cells (as opposed to sperm cells for a male that have an X chromosome from his mother).
  • the present invention provides methods for creating egg cells having chromosomes directly derived from an adult male's cells (as opposed to egg cells for a female that have an X chromosome from her father).
  • a diploid germ cell from a female is provided.
  • the diploid germ cell can be derived using methods and materials known to those of skill in the cloning and developmental biology arts, including, but not limited to, the following methods reviewed herein below: extraction from the ovarian surface epithelium, generation from non-germ stem cells (or other adult cells) which either are retrodifferentiated and then differentiated, by cloning, parthenogenesis or transdifferentiation.
  • spermatogenic capacity is then provided to the female germ cell using one or more of the following techniques: testicular drug delivery, gene therapy vectors, natural Y chromosomes, and/or mammalian artificial chromosomes.
  • testicular drug delivery gene therapy vectors
  • natural Y chromosomes and/or mammalian artificial chromosomes.
  • the spermatogenically augmented female germ cells so produced are then prepared and transplanted under conditions effective to insure that the female germ cells are imprinted as are male germ cells and X-inactivated where and when necessary.
  • the resulting female spermatogonia are transplanted into a sterilized male testis or artificial testicular environment (e.g., in vitro spermatogenesis) to provide for the production of sperm carrying the chromosomes of the female from whom the female germ cell was derived.
  • a sterilized male testis or artificial testicular environment e.g., in vitro spermatogenesis
  • stem cells of lower animal forms such as the medusa which can be retrodifferentiated
  • mammalian stem cells are less naturally programmable.
  • the ability to so reprogram stem cells creates a multiplicity of methods for treating diseases, fighting aging, replenishing cells of organs (including cosmetic uses such as breast augmentation), and enhancing physiological performance (imagine athletes being able to transplant the stem cells of world champion athletes into their muscles).
  • the retrodifferentiated stem cells will later be differentiated to become germ cells, stem cells as uncommitted as possible are desired.
  • stem cells There are multiple sources for such cells: obtaining germ cells from the ovarian surface, obtaining stem cells from menstrual blood, preserving fetal stem cells, in vitro retrodifferentiating stem cells to form embryo-like stem cells, and extracting cells from the inner cell mass of a genetically identical embryo derived by cloning or parthenogenesis.
  • female germ cells are provided by extraction from the ovarian surface epithelium (OSE) of an adult woman. It has long been assumed that oogenesis does not occur in adult mammals: that females are born with all of these eggs they will ever possess, with such haploid oocytes having undergone partial meiosis before going into arrest. Thus the need for the methods disclosed herein to create adult female diploid germ cells, using complex techniques such as cloning or stem cell transdifferentiation. Such techniques, however, can be simplified if not eliminated by extraction of such diploid germ cells from the ovarian surface epithelium (OSE) of an adult woman.
  • OSE ovarian surface epithelium
  • Such extracted OSE germ cells can then have their female imprinting patterns adjusted to male imprinting patterns, and any adult oocyte-specific markers can be removed and replaced in vitro with any needed pre-spermatogenesis male germ cell markers. Such cells can then be compensated for any needed Y chromosome spermatogenesis genes, and cultivated in vivo or in vitro to produce sperm. Such cellular modifications can be applied to female stem cells prepared using the following techniques as well.
  • Sample approaches include using centrifugation to separate maternal and a variety of fetal cells (U.S. Patent 5,641,622), and/or where the isolated fetal cells are cryopreserved (U.S. Patent 5,192,553). Capturing and saving such cells of a newly born girl eliminates the germ cell creation steps of the methods disclosed herein, at the cost of having to store such cells until the girl becomes an adult.
  • Cloning to produce embyronic stem cells requires the use of embryos. Such use can be avoided by creating embryo-like stem cells from adult cells by retrodifferentiation.
  • One method of making a population of mammalian pluripotent embryonic stem cells is by incubating a population of diploid spermatogenic cells in a composition comprising a growth enhancing amount of basic fibroblast growth factor, leukemia inhibitory factor, membrane associated steel factor, and soluble steel factor.
  • these techniques are extended to non-germ stem cells to be applicable to female non-germ stem cells, since the motivation of this aspect of the methods disclosed herein is the lack of adult female diploid germ stem cells.
  • Such extension can be accomplished using methods and materials familiar to those persons having ordinary skill in the art: for example, in 2007, scientists reported converting adult skin cells (fibroblasts) into germline-competent stem cells [SKN07], first achieving so for mice and then for humans.
  • Stem cells acquired/prepared through fetal cell preservation or embryonic-like stem cell induction will need to have their imprinting erased and X chromosomes activated (see below).
  • mammalian cloning techniques can be used to recreate such cells, with the opportunity to obtain germ stem cells that have the requisite cell surface markers that recognize the Sertoli cell surface to which germ cells bind during spermatogenesis.
  • One such marker protein is the mitochondrial capsule selenoprotein encoded by the GPX-4 gene on chromosome 19.
  • Such markers can be induced on the surface of non-germ embryonic stem cells acquired by other methods (such as embryonic-like stem cell induction) by activating the corresponding gene(s).
  • One approach for cloning is that of nuclear transfer [CAM97] developed at the Roslin Institute, a method for reconstituting an animal embryo by transferring the nucleus from a quiescent donor cell into a suitable recipient cell.
  • the donor cell is quiescent, in that it is caused to exit from the growth and division cycle at Gl and to arrest in the GO state. Nuclear transfer may take place by cell fusion.
  • One report of higher cloning success rates in mice [WAK98] was by transferring cumulus nuclei from a female ovary into an enucleated female oocyte.
  • the reconstituted embryo then acts as a source of stem cells to be artificially differentiated into primordial germ cells or naturally developed (primordial germ cells are detectable in the epithelium of an extraembryonic membrane, the yolk sac, during weeks one to three after fertilization). While initially certain cells types were preferred for the transfer, recent research is greatly expanding which cells can be used (see, e.g., U.S. Patent 6,011,197).
  • primordial germ cells are found in the yolk sac when the embryo is about three weeks old (in humans). Such germ cells migrate by weeks four to five onto the genital ridge, after which they trigger gonad development. Prior to entry into the genital ridge, development of male and female germ cells is indistinguishable. Extraction and expansion of the migrating germ cells at the fourth week provides a source of germ cells (see U.S. Patent 5,744,347) for in vitro cultivation and/or alteration to compensate for the Y chromosome, and can be accomplished by those having ordinary skill in he art. For example, Gearhart et al.
  • female diploid germ cells are obtained by cloning an adult woman and isolating and culturing germ cells from the embryo at 7 to 9 weeks post-cloning [VLl], between the time that the primordial germ cells start migrating out of the yolk sac and the time that the germ cells are colonizing the genital ridge.
  • VLl 7 to 9 weeks post-cloning
  • female diploid germ cells are obtained by cloning an adult woman, isolating embryonic stem cells from the clone, and transforming the stem cells into embryonic germ cells [VI.2], which at least in mice, were cultured in vitro sufficiently competently as haploid pre-sperm cells to allow successful fertilization via ICSI [VI.3, and related VI.6].
  • the second method has the ethical advantage over the first method by making earlier use of the embryo, while requiring more effort (such as using growth factors) to insure that the germ cells have the proper germ cell markers [VI.4] and imprint erasures. In recent years, many published studies have shown the viability of so converting embryonic stem cells [VI.5].
  • the embryonic stem cells are obtained via parthenogenesis, where an egg of an adult woman is chemically or electrically "tricked” into becoming an embryo. While such embryos never result in a successful pregnancy (the male epigenetic information is missing), such parthenogenetic embryos can serve as a source of embryonic stem cells.
  • Embyonic stem cell induction and cloning, and stem cell transdifferentiation, methods raise the problem of epigenetic reprogramming of the original adult cell, as multiple studies have reported the abnormal phenotypes of clones for the few cases where cloned embryos managed to be born all. However, it is also observed that progeny of cloned animals have normal phenotypes, since their DNA inherited from a cloned parent were normally reprogrammed during gametogenesis in the parent, similar to the reprogramming experienced by the females cells in the methods disclosed herein when injected into the male testicles.
  • stem cell retro- (or trans-) differentiation Some of the steps for stem cell retro- (or trans-) differentiation, and/or gene therapy for such cells, have low efficiency rates. Those cells successfully altered are cultivated in vitro to produce larger numbers of candidate cells for other alterations.
  • a growing industry is forming around the need for stem and germ cell expansion, for example a system for selective clonogenic expansion of relatively undifferentiated cells, including, without limitation: (a) a tube containing a plurality of beads of a size which permits a plurality of the undifferentiated cells to grow thereon, the beads bearing on their surfaces a selective binding molecule which binds to a surface antigen present on the relatively undifferentiated cells, wherein the antigen is not present on the surfaces of the relatively differentiated cells; (b) means for continuously providing nutrients to the relatively undifferentiated cells growing on the beads, wherein the nutrients are delivered via a fluid which flows through the tube and past the beads; and (c) means for continuously harvesting relatively undifferentiated cells downstream of the beads (PC
  • combinations of the above-described techniques are used to create an adult female diploid germ cell.
  • these techniques are insufficient to enable female spermatogenesis.
  • Genes on the male's Y chromosome still have to be accounted for, since diploid female germ cells are similar to germ cells from males that have deleted Y chromosomal fertility loci as seen in some forms of azoospermia.
  • diploid female germ cells are similar to germ cells from males that have deleted Y chromosomal fertility loci as seen in some forms of azoospermia.
  • a trivial transfer of diploid oogonia to a (sterilized) testis would achieve, by itself, dysfunctional sperm unlikely to satisfy regulatory concerns.
  • the present invention further provides for addressing this critical deficiency.
  • Yq genes/clusters on the distal part of the Y chromosome
  • TSPY DEL97
  • DAZ also referred to as DAZ/SPGY
  • RBM/YRRM RBM/YRRM
  • AZF Azoospermia Factor
  • one such gene (or gene cluster) for spermatogenesis in humans is DAZ (Deleted in Azoospermia), located in intervals 6E and/or 6D of the distal portion of the long arm of the Y chromosome.
  • the DAZ gene (or cluster, as several copies have been detected in interval 6) is approximately 3.1 kb in size, and encodes a 366 amino acid protein homologous in certain domains to several RNA binding proteins, the DAZ protein restricted to late spermatids and sperm. Alterations (such as deletions) in DAZ typically result in reduced sperm counts.
  • RBMY RNA-Binding Motif Y
  • RBMYl RNA-Binding Motif Y
  • RBMY is a multiple copy gene
  • RBMY homologs on the X chromosomes and autosomes don't compensate for the loss of some copies of the RBMY genes (with the homologs binding differently), a fatal problem for human female germ cells that don't have any RBMY genes (complete deletion, similar to the complete sterility of the rare 46,XX men with a normal male phenotype [ABUOl]).
  • RBMY deletion illustrates an important difference between spermatogenesis in humans and mice, since for mouse germ cells, RBMY-deficiency does not lead to sterility but to abnormal sperm development. [SKR07].
  • Stem cells transplanted from females necessarily lack the needed Y chromosome spermatogenic genes, and thus cannot be developed into normal sperm without the additional intervention provided by the methods of the present invention.
  • Genes such as the RBMY genes, or the products they express should be provided in the testicular environment along with the female stem cells for female spermatogenesis.
  • the primary spermatocytes undergo the two meiotic divisions, the resulting spermatids have haploid chromosomes.
  • Half of these spermatids have only the X chromosome and yet continue to develop into mature sperm, due to cytoplasmic bridge connections between X and Y spermatids.
  • Spermatogenic factors are thus needed to help, at least, the initial retrodifferentiated/transdifferentiated stem cell develop to the primary spermatocyte stage.
  • the methods of the present invention include using one or more of the at least four methods known for providing spermatogenic factors: incorporation of (parts of a) natural Y chromosome or of an artificial chromosome with some Y chromosome genes into the female stem cells, testicular drug delivery, gene therapy treatment of the female stem cells, and mixing in tagged male spermatogonia.
  • Non-testicular-originating proteins that contribute to spermatogenesis such as follicle-stimulating hormone and luteinizing hormone, required by and present in both male and females, need not be compensated for necessarily, although in some embodiments, they are provided by administering promoters such as gonadotropin-releasing hormone stimulatory agonist (see, e.g., PCT Publication WO 97/22625).
  • the methods of the present invention require adding an X chromosome into male cells, preferably by adding a natural X chromosome.
  • compensation of XX germ cells for necessary Y chromosome genes includes preparing a sample of human Y chromosomes by bulk isolation flow cytometry, or micro-cell mediated chromosome transfer (MMCT - further discussed below), to be incorporated into the germ cells.
  • the Y chromosomes can be obtained from the cells of a SRY -negative man or woman (for example, in 2000, German scientists reported on a healthy mother/daughter pair [ROTOO] whose genotype was SRY-negative 47,XXY).
  • XY sperm are to be produced (some 47-XXY non-mosaic Klinefelter men produce a small number of XY sperm [GUT97]), creating XXY offspring (saving future generations from having to add a Y chromosome).
  • the Y chromosomes can be obtained from cells of any XY male, for example, the Y chromosome of a woman's family member (such as a brother or father).
  • Minichromosomes are naturally occurring chromosomes that have sections of genes deleted or have been fragmented (U.S. Patent 5,288,625 and U.S. Patent 4,464,472).
  • An alternative to using artificial Y chromosomes based as described below is to use mini- Y-chromosomes created by deleting from existing Y chromosomes those genes that do not contribute to spermatogenesis, for example, using telomere directed chromosome breakage to create a mini-Y-chromosome [HEL96] or a mini-X-chromosome [FAR95].
  • MMCT is preferred for adding a copy of a male's X chromosome to his cells.
  • MAC mammalian artificial chromosome
  • a mammalian artificial chromosome is a non-integrating DNA construct that can replicate autonomously and be stably maintained alongside endogenous chromosomes, with a fully functional mammalian centromere, replication initiation sites, and mammalian telomeres.
  • the base DNA is engineered from naturally occurring neutral DNA sequences with no genetic information, and plasmid vectors are used to insert heterologous genes at specific integration sites on the artificial chromosome arms [HAD97, HADOO].
  • MACs can be incorporated into cells such as germ and stem cells.
  • Bacteriophage lambda clones may contain up to 22 kilobases(kb); cosmid clones up to 40 kb. DNAs of this size are routinely introduced in the production of transgenic animals.
  • the large capacity cloning vectors include bacteriophage Pl, Pl artificial chromosomes (PACs), cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and mammalian artificial chromosomes. Of these, YACs and MACs have several distinct advantages.
  • the maximum insert size that can be contained in YACs and MACs are multiple megabases, a region too large to be cloned intact with traditional bacterial cloning systems. This large insert size permits the study of intact genes, multigenic loci, distant regulatory sequences and higher order genomic structure in the context of their native sequences. Decisions need not be made regarding the regulatory relevance of sequences or distances between genes and control elements, as must be done in the synthesis of mini-transgenes in which sequences must be pared.
  • site-specific mutagenesis can be readily and efficiently performed in vivo with the homologous recombination system of the host cell instead of recombinant DNA technology.
  • Point mutations, deletions, insertions and replacements can easily be introduced into a YAC or MAC without producing unwanted alterations.
  • a Pl can maintain up to 100 kb, much less than the maximum capacity of YACs and MACs.
  • PACs and BACs have cloning capacities up to 350 kb, but performing homologous recombination to introduce mutations has not been demonstrated, making the process of mutagenesis more tedious.
  • the preferred vector for the methods disclosed herein are MACs since YACs don't replicate in mammalian cells. However modified YACs containing human centromeres and telomeres seem to function as MACs [IKE98][OKA98].
  • the spermatogenesis/ AZF regions are a subset of 24 megabases of euchromatin and 30 megabases of heterochromatin in the Y chromosome.
  • the heterochromatin is entirely composed of, and the euchromatin is largely composed of, repeated non-coding DNA sequences. If spermatogenesis can occur with all of the coding and only one third of these non-coding DNA sequences, only about 20 megabases of Y chromosome DNA need to be carried in the artificial chromosome, which is the current carrying capacity of at least one (patented) mammalian artificial chromosome (U.S. 6,743,967). At a minimum, (multiple copies of) the 10 to 20 Y chromosome protein- coding genes with no X chromosome homologue are incorporated into the artificial chromosome (except for SRY).
  • MACs can be introduced into cells by a variety of methods: electroporation, direct uptake (such as by calcium phosphate precipitation), uptake of isolated chromosomes by lipofection [LOR99], by microcell fusion, microinjection or by lipid-mediated carrier systems.
  • Methods of specific targeted incorporation of heterologous genes into the MAC are available without extraneous random integration into the genome of recipient cells, such as the use of homology targeting vectors.
  • the heterologous gene of interest is subcloned into a targeting vector which contains nucleic acid sequences that are homologous to nucleotides present in the MAC.
  • the vector is then introduced into cells containing the artificial chromosome for specific site-directed integration into the MAC through a recombination event at sites of homology between the vector and the chromosome.
  • the homology targeting vectors may also contain selectable markers for ease of identifying cells that have incorporated the vector into the MAC as well as lethal selection genes that are expressed only upon external integration of the vector into the recipient cell genome.
  • Two such homology targeting vectors are lambdaCF-7 and p-lambdaCF-7-DTA [HAD97, HADOO], commercialized by the Canadian company Chromos Molecular Systems under the tradename ACE System.
  • the ACE product line consists of the Platform ACE artificial chromosome, the ACE Targeting Vector for loading genes onto the artificial chromosome, and ACE Integrase, an enzyme for allowing multiple rounds of loading.
  • One Chromos patent U.S. 6,743,967 claims a capacity of about 20 megabases, which is the size of the Y chromosome with only one-third of the non-coding sequences in the heterochromatin and euchromatin, and the coding sequences of the euchromatin.
  • One advantage of the large size of ACEs is that they can be purified using flow cytometry.
  • Still other types of mammalian artificial chromosomes can be used in accordance with the invention, such as described in, e.g., U.S. Patent Applications 2007/0004002 and 2003/0064509.
  • Y chromosomes can be tagged with a reporter gene, such as for a fluorescent protein, to ease the sorting out of any Y sperm produced.
  • patent 6,936,469 which is incorporated herein by reference, discloses one way to incorporate the Y chromosomes into germ cells by mixing the Y chromosomes with a cationic lipid/polymer delivery agent (to neutralize the charge of the Y chromosome), and then putting the mixture in contact with the germ cells.
  • a cationic lipid/polymer delivery agent to neutralize the charge of the Y chromosome
  • the resulting 47,XXY female germ cells are very similar to germ cells of non-mosaic 47,XXY Klinefelter men who can produce sperm, with the male (and thus female equivalents) cells undergoing mitosis and meiosis as is, or with some of the 47,XXY cells undergoing trisomy-rescue during initial stages of mitosis to form 46,XY cells as seen in normal men.
  • natural and artificial X chromosomes (and mixtures thereof) can be prepared, and added to male diploid germ cells using these in vitro techniques (with trisomy-rescue induced to convert the 47,XXY male cells to more female-like 46,XX cells).
  • MMCT micro-cell mediated chromosome transfer
  • cells from which the Y chromosomes are to be obtained are treated with the drugs colcemid and colchicines, which arrests the cells in the metaphase stage, with prolonged exposure interfering with mitotic spindle formation, and causing the cells to multinucleate and form nuclear membranes around individual or small numbers of chromosomes.
  • the treated cells are centrifuged in the presence of cytochalasin B, which disrupts microfilaments and prevents cells from returning in interphase, leading to micro-cell extrusion.
  • the micro-cells are collected and filtered though membranes with pore sizes as small as 3 micrometers, to select for micro-cells containing a single chromosome.
  • Sorting out of Y-chromosome-only micro-cells can occur at this stage, with the advantage of allowing genes to be added to the sole Y chromosome that have been found useful for transforming adult somatic cells into embryonic-like stem cells.
  • these micro- cells are fused to recipient female diploid germ cells, typically by adding phytohemagglutinin-P (PHA- P) to cause cell agglutination and polyethylene glycol (PEG) to dissolve cell membranes.
  • PHA- P phytohemagglutinin-P
  • PEG polyethylene glycol
  • genes on artificial chromosomes may not be imprinted in the same ways as genes on natural chromosomes.
  • testicular drug delivery techniques are used to provide compensation for any required missing Y chromosome genes.
  • testicularly-acting drug species can be site-specifically and sustainedly delivered to the testes (for the methods disclosed herein, the testes of the volunteer host male) by administering a pharmacologically effective amount of the target drug species tethered to a reduced, blood-testis barrier penetrating lipoidal from of a dihydropyridine-pyridium salt-type redox carrier, e.g., 1,4-dihydrotrigonelline.
  • the target drug species can be some of the proteins coded by spermatogenic genes such as DAZ.
  • GDNF glial cell line-derived neurotrophic factor
  • any missing spermatogenic genes or gene products are compensated for using gene therapy methods.
  • Such methods will be familiar to persons having ordinary kill in the art.
  • U.S. Patents 5,695,935, 5,871,920, and 6,020,476 describe methods to extract a gene from a DNA sample from a male and detect if the gene is altered. Such methods enable an unaltered gene from another male, or a portion thereof encoding a functional protein, to be inserted into cells in which the functional protein is expressed and/or secreted to remedy the deficiency caused by the absence of the native gene in male (or female) cells.
  • the necessary gene products are provided directly instead of by genetic transcription. Since healthy male spermatogonia already have present the necessary spermatogenic genes, they could be used to express proteins that do not remain internal to the spermatogonia.
  • a sample of male spermatogonia is removed from a male testis before sterilization, and tagged, for example, by adding a fluorescent probe such as Hoechst 33342. These tagged spermatogonia are mixed in with the altered female cells, and the entire mass is transplanted back into the testis. Sperm would be produced, some from the female's cells and some from the male's cells. Using conventional cell filtering techniques, those sperm that are fluorescent are be easily identified and removed, leaving only sperm with chromosomes from the female.
  • Imprinting is one type of process involving the epigenome, i.e., modifications to genes that change the functioning of the gene without changing the DNA sequences that comprises the gene. At least three types of epigenetic modifications have been observed: imprinting via DNA methylation of cytosine residues in CpG dinucleotides (forming differentially methylated regions - DMRs), modification of core histones (a "histone code”), and gene silencing due to non-coding RNAs.
  • Imprinting There are three types of imprinting modifications: 1) in women, the random total inactivation of one X chromosome in each cell ("X-inactivation"); 2) in men and women, random inactivation of some autosomal genes; and 3) in men and women, modification of a small percentage ( 100 to 200) of one of each of a pair of autosomal genes depending on the person's sex. Sex-specific imprinting is imparted in the germ line, where inherited maternal and paternal imprinting is erased and new imprinting established according to the individual's sex. [0077] Imprinting partly is a process of functional inactivation, with inactive regions of chromatin having altered methylation patterns at the 5-position of cytosine.
  • DNA methyltransferases are the only known mammalian enzymes that catalyze the formation of 5-methyl cytosine (i.e., adding a methyl group CH 3 " to a cytosine); and thus play a key role in establishing and maintaining methylation patterns during male spermatogenesis, as seen in measurements of substantial levels of DNA methyltransferase mRNA and protein expression in mitotic types A and B spermatogonia, spermatocytes and round spermatids [JUE94]. This is consistent with the presence of DNA methyltransferase during the frequent cell divisions of spermatogonia, where methylation patterns are maintained on each daughter strand of DNA following semiconservative DNA replication. Such DNA methyltransferases also help maintain methylation patterns during DNA replication for somatic cell divisions.
  • spermatogenic female germ cells formed as described above have their imprints erased, and are then imprinted as male germ cells.
  • Many imprints are the result of gene site-specific DNA methylation, so that before male imprints can be made for female cells (or female imprints for male cells), the old imprints have to be erased, e.g., actively using DNA demethylases or passively during replication if methylation is interfered with (e.g., to remove any DNMTl that typically maintains methylation patterns during replication), and/or by using histone -modifying enzymes.
  • Kerjean [II.3] The results of Ker jeans [II.3] suggest that some human male germ cell imprints remain erased until imprinting occurs in adult spermatogenesis.
  • Male germ cells undergo mitosis starting from the fetal stage until death.
  • Male germ cells undergo meiosis primarily post-puberty, with about 80% of the germ cells in the adult testis being in the (post) meiotic stages.
  • Imprinting of the male genome starts during fetal stages of mitosis, with the bulk of germ-cell specific methylation patterns being acquired prior to the type A spermatogonia stage.
  • Any epigenetic mitotic/meiotic modifications to male germ cells in the testes that occur post-puberty e.g., histone-to-protamine exchange, methylation of the maternally- expressed non-coding RNA gene H19 [SOL98], etc. will also happen to altered female germ cells when transplanted into a post-puberty testicular environment (or in vitro equivalents).
  • male germ line imprinting may be a waste of energy (e.g., for any imprinted genes only active in somatic cells), and may not need to be fully replicated, since paternal imprints in the paternal pronucleus are quickly erased shortly after fertilization at the zygote stage, and experience additional erasure as the embryo undergoes two waves of demethylation.
  • Kerjean reports that the imprinted gene MEST/PEG1 remains unimprinted throughout spermatogenesis even until the mature spermatozoa stage.
  • Any imprinting establishment that occurs from the fetal period to pre-puberty can be achieved, for example, using the mitotic steps of procedures for in vitro conversion of male embryonic stem cells into embryonic germ cells to similarly cultivate, in vitro, female diploid germ cells that have been reset epigenetic ally, before the female germ cells undergo testicular transplantation Chuma and colleagues [CHU05] report on extracting mouse primordial germ cells and transplanting them into a natural testicular environment to produce sperm resulting in normal fertile offspring, supporting the idea that epigenetic modifications in the male germ line during mitotic stages of spermatogenesis pre-puberty do not to be exactly established, if at all, but rather are amenable to in vitro engineering.
  • the largest cluster of imprinted genes is the X chromosome itself.
  • X chromosome In mammalian females with two X chromosomes (one inherited from a male), one of the X chromosomes is inactivated in all somatic cells, except during some stages of oogenesis.
  • the sole X chromosome In males, the sole X chromosome is always active, except during the latter- and post-stages of spermatogenesis. At the beginning, mitotic, states of spermatogenesis, both of the X and Y chromosomes are transcriptionally active.
  • female diploid germ cells either with an XXY genotype or a trisomy-rescued XY genotype, prepared, for example, by either cloning, stem cell transdifferentiation or ovarian germ cell extraction, have any inactivated X chromosomes reactivated before entry into the mitotic stages of spermatogenesis.
  • fibroblast stem cells can be reprogrammed to a state highly similar to embryonic stem cells, and that with certain transcription factors (Oct4, Sox2, cMyc, Klf4), any inactivated X chromosomes are reactivated [MAH07].
  • Any similar combination of transcription factors can be used herein to reactivate X chromosomes in female diploid germ cells.
  • the fully activated female germ cells will have a complement of somatic histones (i.e., the histone coding of the egg and the histone coding due to replacement of protamines in the sperm with maternally deposited histones soon after fertilization), to then undergo chromatin remodeling during spermatogenesis.
  • somatic histones i.e., the histone coding of the egg and the histone coding due to replacement of protamines in the sperm with maternally deposited histones soon after fertilization
  • testes are immunologically privileged site for allografts [HED83] due to Sertoli cell secretions (cells which are now being used to produce localized immune suppression to minimize rejection during transplantation (U.S. Patent 5,830,460).
  • Sertoli cell secretions cells which are now being used to produce localized immune suppression to minimize rejection during transplantation (U.S. Patent 5,830,460).
  • Animal experiments have shown that most male -to-female grafts (such as skin) are rejected (due in part to H-Y antigens coded by gene(s) on the Y chromosome), whereas transplants made in other sex combinations (for example female -to-male) more often succeed.
  • immunosuppresive drugs can minimize transplant rejection, along with temporary treatment in the thymus of the male host to induce tolerance.
  • Female stem cells transplanted into the testes can survive the months needed for spermatogenesis without being disrupted by the male's immune system rejecting the transplanted cells.
  • a first testicular environment for female spermatogenesis is due to Brinster and Zimmerman
  • a second testicular environment uses in vitro techniques, for example, by culturing germ cells in vitro by contacting said cells with non-tumorigenic Sertoli, Leydig and peritubular cells [MIL95], or cultivating sperm in vitro in a culture broth augmented with spermatogenesis factors such as GDNF and LIF (for example, U.S. patent application 2006/0265774) [SHI06].
  • MIL95 non-tumorigenic Sertoli, Leydig and peritubular cells
  • SHI06 spermatogenesis factors
  • a variety of research efforts are directed towards creating artificial organs (for example, U.S. Patent 5,541,107). Using a combination of these techniques, an in vitro testicular environment could be prepared, eliminating the need for a male's sterilized testis.
  • HUE98 in vitro environment
  • rat germinal cells in cultures of whole cell population from rat seminiferous tubules
  • round spermatids can then be used to fertilize an oocyte, with all events during spermiogenesis, such as somatic and testis-specific histones being replaced by protoamines, thus being somewhat optional.
  • In vivo spermatogenesis is preferred over in vitro spermatogenesis.
  • Testicular transplantation (in vivo) techniques are preferable to in vitro techniques for culturing germ cells into sperm cells, for at least three reasons. First, chromosome imprinting occurs naturally in the testicular environment, as opposed to having to be engineered in vitro. Second, signaling between somatic and germ cells occurs naturally in the testicular environment, as opposed to having to be engineered in vitro.
  • Testicular transplantation methods are also preferable so that required modifications to sperm are done naturally (e.g., addition of sperm proteins necessary for fertilization, such as PAWP, and one protein that tags sperm mitochondria for destruction - ubiquitin), as opposed to having to engineer the modifications in vitro.
  • testicular transplantation methods are preferable so that telomerase- mediated telomere maintenance and elongation occurs naturally during the (pachtene) stages of meiosis.
  • testicular transplantation methods are preferable so that any preferential nuclear positioning of sperm chromosomes occurs naturally, for later unpacking of the sperm chromosomes after fertilization.
  • a third testicular environment is possible by cross-species spermatogenesis, for example, achieving rat spermatogenesis using mouse testes [CLO96].
  • Germ cells from transgenic rats were transplanted into recipient immunodeficient nude mice previously treated with busulfan. In one animal, a ratio of one rat sperm to 39 mouse sperm subsequently occurred among cells present in the epididymides.
  • Sperm of the two species were normal and distinctive in head and tail morphology (indicating sperm morphology is inherent to the germ cell), though it has yet to be determined if cell surface glycoproteins that the rat sperm acquire in the mouse epididymides will allow fertilization with a rat egg.
  • Rat spermatogenesis in mouse testis implies contact between rat germ cells and mouse Sertoli cells with cellular junctions, and contacts between the rat germ cells and mouse somatic supporting cells. This is surprising, given that the timing of the differentiation process from stem cell spermatogonia to the mature form, are unique and species specific, and suggests that xenogenic spermatogenesis is likely possible for other species, in particular between the primates [MCL98]. Since rats and mice are believed to have diverged between about 10-11 million years ago, which is twice as long as the separation of men and chimpanzees, then it is likely, for example, for human sperm to be formed in chimpanzee testes (in particular transmitochondrial chimpanzees).
  • the present invention provides methods for making "male eggs". Since adult males have diploid germ cells in their testes, there is no need to recreate such diploid cells. Similar to the procedures above that isolate Y chromosomes from adult male cells (and then incorporate them into female diploid germ cells), the first step for creating male eggs is to isolate X chromosomes from an adult male, and add the X chromosomes to the adult male's germ cells, for example, using the chromosome addition techniques of U.S. patent 6,936,469 or similar techniques such as micro-cell mediated chromosome transfer.
  • the germ cells are treated in vitro to erase male imprinting patterns and sperm cell markers so that the male cells have the characteristics of embryonic germ cells, making them more amenable to being imprinted similarly to female germ cells during oogenesis.
  • the male cells can then be increased in number via in vitro mitotic divisions. Some of the resulting male cells will undergo trisomy -rescue, losing the Y chromosome, resulting in germ cells with a 46,XX genotype, the same genotype found in normal female germ cells.
  • RNA interference can be used to block the effects of the RBMY gene during oogenesis.
  • Co-pending patent application 11/831,402 filed 31 July 2007 (incorporated in its entirety herein by reference), discloses business methods for patenting applications of new science, such as same-sex procreation, for the manufacturing of entertainment products.
  • the science of female sperm and male eggs as disclosed herein provides much structure and functionality to use female sperm and/or male eggs as a plot element when such cells are signified with audio and/or visual signals in an entertainment article of manufacture fixed on a medium. Such signals function to physically transform the human mind.
  • MPEP Rule 608 (Eighth Edition, Sept. 2007), citing CF .R. 1.3, states that "If during the course of examination of a patent application, an examiner notes the use of language that could be deemed offensive to any race, religion, sex, ethnic group or nationality, he or she should object to the use of the language as failing to comply with the Rules of Practice.” As many Americans will find the idea of, let alone the discussion of, same-sex procreation to be religiously offensive (since it implies that phrases such as "Honor your mother and father" are not the words of any omniscient god or gods), disclosed herein are arguments on the relative non-offensiveness of same-sex procreation.
  • MPEP Rule 608 a violates Constitutional guarantees of Freedom of Speech, and b) violates Constitutional guarantees of Due Process public notice (the phrase "could be deemed” is unconstitutionally vague, as is the use of "obvious” in 35 U.S. C. 103).
  • MMCT Microcell-mediated chromosome transfer
  • HSD97 "Artificial chromosomes, uses thereof, and methods for preparing artificial chromosomes", Hadlaczky and Szalay, PCT WO97/40183 (Oct 1997)
  • stem cell is a stem cell
  • Hall, Cell, v33 (May 1983) 11
  • Kee K et al. "Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells", Stem Cells Dec. 2006 Dec;15(6):831-7; see also Chen S et al., “Self-renewal of embryonic stem cells by a small molecule", PNAS. 2006 14 Nov;103(46): 17266-71

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Abstract

L'invention porte sur des procédés permettant de produire un sperme femelle par incorporation de chromosomes d'une femelle dans une cellule de sperme par l'intermédiaire de procédés de spermatogenèse essentiellement naturels. Les procédés comprennent le transplant de cellules souches femelles (altérées) ou de cellules germinales clonées (altérées) dans des testicules stérilisés d'un mâle. Des cellules souches/germinales femelles diploïdes sont altérées de deux façons -transdifférenciation pour faciliter l'expression de facteurs de spermatogenèse (par exemple, à l'aide de chromosomes artificiels) et/ou rétrodifférenciation pour augmenter la pluripotence et les effacements d'imprégnation. Les cellules altérées sont transplantées dans des testicules mâles (artificiels) pour se développer en spermes, qui sont utilisés pour fertiliser un œuf d'une seconde femelle ou un œuf de la femelle initiale. L'invention porte également sur des procédés permettant de produire des œufs mâles par addition d'un chromosome X supplémentaire à des cellules germinales d'un mâle adulte et mise en culture des cellules germinales in vitro.
PCT/US2008/050000 2007-07-02 2008-01-01 Procédés permettant une spermatogenèse de mammifère femelle et une oogenèse de mammifère mâle à l'aide d'une nanobiologie synthétique WO2009005844A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6215039B1 (en) * 1991-12-06 2001-04-10 The Trustees Of The University Of Pennsylvania Repopulation of testicular seminiferous tubules with foreign cells, corresponding resultant germ cells, and corresponding resultant animals and progeny
US20030232430A1 (en) * 2001-11-26 2003-12-18 Advanced Cell Technology Methods for making and using reprogrammed human somatic cell nuclei and autologous and isogenic human stem cells
US20050273870A1 (en) * 1999-10-14 2005-12-08 University Of Massachusetts Preparation and selection of donor cells for nuclear transplantation
US20060294603A1 (en) * 2001-02-23 2006-12-28 Forsberg Eric J Cloning of transgenic animals comprising artificial chromosomes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6215039B1 (en) * 1991-12-06 2001-04-10 The Trustees Of The University Of Pennsylvania Repopulation of testicular seminiferous tubules with foreign cells, corresponding resultant germ cells, and corresponding resultant animals and progeny
US20050273870A1 (en) * 1999-10-14 2005-12-08 University Of Massachusetts Preparation and selection of donor cells for nuclear transplantation
US20060294603A1 (en) * 2001-02-23 2006-12-28 Forsberg Eric J Cloning of transgenic animals comprising artificial chromosomes
US20030232430A1 (en) * 2001-11-26 2003-12-18 Advanced Cell Technology Methods for making and using reprogrammed human somatic cell nuclei and autologous and isogenic human stem cells

Non-Patent Citations (1)

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Title
NAYERNIA K. ET AL.: "Derivation of male germ cells from bone marrow stem cells", LAB. INVEST., vol. 86, no. 7, July 2006 (2006-07-01), pages 654 - 663, XP003013476 *

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