AU2005202687A1 - Methods of generating germ cells from stem cells and uses thereof - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Monash University Actual Inventor(s): Orly Lacham-Kaplan Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Meurr 3 AUSTRALIA Invention Title: METHODS OF GENERATING GERM CELLS FROM STEM CELLS AND USES THEREOF Our Ref 748077 POF Code: 119548/119548 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 6ooeq r 2 METHODS OF GENERATING GERM CELLS FROM STEM CELLS AND USES

STHEREOF

TECHNICAL FIELD

CO

\0 The present invention relates to the field of cell biology. More specifically, the invention Srelates to methods of generating germ cells from stem cells, and more particularly to S 10 methods of generating female germ cells from stem cells and uses thereof.

NBACKGROUND

Germ cells are responsible for transmitting genetic information and for reproducing totipotency. from generation to generation. Thus, methods to separate germ cells from gonadal tissue ovaries and testes) have been explored for the purpose of understanding the biologic events related to germ cell differentiation and proliferation and more importantly, in the hope of providing a treatment to many infertile individuals. However, the use of primordial germ cells and germ stem cells in transplantation studies to restore fertility has been initiated with varied degrees of success, leading scientists to push boundaries even further by using embryonic stem cells (ESC) as the starting point for germ cell differentiation.

Embryoid bodies established from ESC, consist of tissue lineages typical of the early embryo. Whilst the potential of ESC to generate all lineages of the embryo in vivo has been widely reported in the literature, there has been comparatively little data describing the derivation of germ cells from ESC in vitro. However, recent studies have now shown that mouse and human ESC appear to differentiate spontaneously into germ cell precursor cells in vitro, as identified by the expression of a variety of germ cell markers (Toyooka et al., 2003; Hubner et al., 2003; Geijsen et al., 2004; Clark et al, 2004). Moreover, when isolated, germ stem cells will progress in gamete differentiation towards putative sperm and oocytes (Hubner et al., 2003; Toyooka et al., 2003; Geijsen et al., 2004).

For instance, the report by Geijsen et al. (ibid) showed that the addition of retinoic acid to isolated EB resulted in manifestation of a cell population with positive SSEA-1 expression that stained positive for alkaline phosphatase, indicative of PGClike cells. The in vitro differentiation of germ cells identified by this group was confirmed W:\provisional\742767 Oocytes lestes\742767_speci_200605-final.doc 3 o by the positive expression of markers such as Oct3/4 Piwil2 and DAZL. The Sry gene, specific to the male germ cell, was also expressed in some of the cells. This Fspecification to a male germ-line was confirmed by the identification of upregulation of Sacrosin and heparin in the highly specified cells, suggesting that the default phenotype of female germ cells was suppressed in their cultured EB. The differentiated cells underwent limited meiosis to produce a small number of haploid cells and these cells were able to fertilize mature oocytes by intracytoplasmic injection, with approximately 00 of the embryos developing to the blastocyst stage in vitro.

This striking discovery has raised the possibility that sterility due to lack of germ cells could be treated in the future using stem cell therapy. However, the factors that drive the differentiation of ES cells into germ cells in vitro remain elusive.

It is therefore an aspect of the present invention to provide methods for the differentiation of stem cells toward primordial germ cells and putative gametes.

SUMMARY OF THE INVENTION In an aspect of the present invention, there is provided a method of generating a female germ cell from a stem cell, said method comprising exposing the stem cell to testicular tissue or conditioned media derived from a culture of testicular tissue.

In a preferred embodiment of the present invention, there is provided a method of generating a female germ cell from a stem cell, said method comprising exposing the stem cell to a conditioned media derived from a culture of testicular tissue. In a further preferred embodiment, the germ cell is an oocyte or an oogonium.

In another aspect of the present invention, there is provided a female germ cell generated obtained by the methods according to the present invention. Also provided in accordance with the present invention is a conditioned media derived from a culture of testicular tissue for use in generation female germ cells in accordance with the methods of the present invention.

Also in accordance with the present invention there is provided a method for generating an embryo from a female germ cell generated by a method as herein described. Preferably, the embryo is a non-human embryo.

W:\provisional\742767 Qocytes testes\742767_sped 200605-final doc An embryo, preferably a non-human embryo, and/or animal generated by the Smethod as hereinbefore described are also encompassed by the present invention.

In yet another aspect of the present invention, there is provided a method of screening for compounds that modulate the generation of a female germ cell from a Sstem cell in accordance with the methods of the present invention, the method oO \0 comprising exposing a stem cell to testicular tissue, or conditioned media derived from 0a culture of testicular tissue, in the presence of a test compound and assessing whether the test compound modulates the generation of the female germ cell from the stem cell and/or the growth and/or maturation of the female germ cell.

FIGURES

Figure 1 shows a culture of testicular cells (testicular cell culture; TCC) derived from new born testes at 10 days and at 45 days of culture. At 10 days of culture, germ cells (GC) appear semi-floating on a monolayer of cells as single cells or in pairs or groups. At 45 days no GC are evident and unique clusters appear within the cultures.

Figure 2 shows immunofluorescence staining of germ cells from TCC at 2 weeks of culture. Cells were stained for Oct 3/4 Mvh but rarely for c-kit (c2) Figure 3 shows monolayers of mouse embryonic stem (ES) cells grown in LIF-DMEM containing 10% FCS Disaggregated ES cells cultured in hanging drops in LIF-free DMEM result in the formation of embryoid bodies The edges of embryoid bodies (EB) become defined within 48 hr. The size of the EB is gradually increasing, as evidenced by a darkening of the centre of the EB.

Figure 4 shows RT-PCR analysis in the identification of germ cell markers expressed by mouse embryonic stem (ES) cells from 24 hr (ES1) and 48 hr (ES2), 48 hr embryoid bodies fibroblasts muscle cells and liver cells Except for Stella, all other markers were expressed in 24 hr ES cells. All germ cell markers were expressed in ES 48 hr old ES cells and EB. C-kit was also expressed in fibroblasts.

Figure 5 shows immunofluorescence staining of 48 hr EB grown in LIF-free DMEM.

Cells were stained for Oct 3/4 c-kit Mvh (c2) (x400) WMpromsional\742767 Oocytes testes\742767 speci_200605-final doc Figure 6 shows immunofluorescence staining of 120 hr EB grown in LIF-free DMEM.

SCells were stained for Oct 3/4 c-kit Mvh (c2) (x800) Figure 7 shows the generation of unique ovarian-like structures from 120 hr EB grown in TCC conditioned medium within 6-7 days of culture (Fig 7 a, These ovarian-like structures contain follicles In many of these follicles, "egg" like cells are evident NO Scale equal to Figure 8 shows putative eggs separated from ovarian structures. The oocytes are Ssurrounded by 2-3 layers of cells. When removed, the 'oocytes" were naked, with no evidence of zona pellucida surrounding them. Scale is equal to 2 Figure 9 shows the expression of Sry and Stra8 in ES cells, EB grown in LIF-free DMEM or TCC conditioned DMEM and germ cells. The genes were expressed in most ES cells and most of EB regardless of the culture medium used. Germ cells did not express the Stra 8 but expressed the Sry gene as expected. Muscle cells did not express any of the genes. ES cells from 24 hr culture; 48 h-old EB; 120 hold EB grown in TCC conditioned DMEM transferred to 0.5 ml to TCC conditioned DMEM; 120 h-old EB grown in TCC conditioned DMEM transferred to 0.5 ml LIFfree DMEM; 120 h-old EB grown in LIF-free DMEM transferred to 0.5 ml LIF-free DMEM; 120 h-old EB grown in LIF-free DMEM transferred to 0.5 ml TCC conditioned DMEM; ES cells in conditioned DMEM; ES cells in LIF-free DMEM; Germ cells from TCC; and (10) Muscle.

Figure 10 shows the expression of oocyte markers Fig a, Zpl, Zp2, Zp3 in putative ovaries obtained from EB-derived ES cells. Fig a and Zp3 were expressed in ovarian tissue obtained from 1 day old females and from putative ovaries developed in-vitro from ES cells Zpl and Zp 2 are not expressed in putative ovaries but are expressed in new born ovaries None of the oocyte markers were expressed in male gonads obtained from a new born male or by muscle cells (control).

DETAILED DESCRIPTION OF THE INVENTION WMprovisional\742767 Oocytes testes\742767-spe_200605.final doc b In an aspect of the present invention, there is provided a method of generating Sa female germ cell from a stem cell, said method comprising exposing the stem cell to Stesticular tissue or conditioned media derived from a culture of testicular tissue.

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S 5 Applicants have surprisingly found that a portion of stem cells cultured in the presence of testicular cells, or conditioned media derived therefrom, will differentiate into follicle-like structures comprising oocytes.

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Stem cells can be derived from a number of different sources, including, but not S 10 limited to, early embryos (blastocysts) created by in vitro fertilization; early embryos created by inserting the nucleus from an adult cell into an egg with its nucleus removed; immature germ cells; organs of an aborted fetus; blood cells of the umbilical cord at the time of birth; certain adult tissues g. bone marrow); and mature adult tissue cells which have been reprogrammed to behave like stem cells. The stem cell may be derived from a male or female donor, more preferably from a male donor. In a preferred embodiment, the stem cell is an embryonic stem (ES) cell, more preferably an ES cell derived from a human donor.

The term "female germ cell" as used herein can refer to a primordial female germ cell, a gonocyte, an oogonium or an oocyte. It can also denote a follicle-like structure from which oocytes can be derived.

For the purposes of the present invention, the term "embryo" or "embryonic" as used herein can refer to a developing cell mass that has not implanted into a uterine membrane of a maternal host. Hence, the term "embryo" as used herein can refer to a fertilized oocyte, a pre-blastocyst stage developing cell mass, and/or any other developing cell mass that is at a stage of development prior to implantation into an uterine membrane of a maternal host. Embryos of the invention may not display a genital ridge. An "embryonic cell" is isolated from and/or has arisen from an embryo.

An embryo can represent multiple stages of cell development. For example, a one cell embryo can be referred to as a zygote, a solid spherical mass of cells resulting from a cleaved embryo can be referred to as a morula, and an embryo having a blastocoel can be referred to as a blastocyst.

The term "embryonic stem cell" as used herein can refer to pluripotent or totipotent cells isolated from an embryo that are maintained in in vitro cell culture. Such cells are rapidly dividing cultured cells isolated from cultured embryos which retain in W:\provisionaI\742767 Oocytes testes\742767_speci_200605-final doc Sculture the ability to give rise, in vivo, to any or all cell types which comprise the adult animal, including the germ cells. Embryonic stem cells may be cultured with or without Sfeeder cells.

Embryonic stem cells can be established from embryonic cells isolated from embryos at any stage of development, including blastocyst stage embryos and preblastocyst stage embryos. Embryonic stem cells may have a rounded cell morphology 00 0 and may grow in rounded cell clumps on feeder layers. Embryonic stem cells are well Sknown to a person of ordinary skill in the art (see, WO 97/37009; Yang Anderson, 1992, Theriogenology 38: 315-335; Piedrahita et al., 1998, Biol. Reprod. 58: 1321-1329; Wianny et al., 1997, Biol. Reprod. 57: 756-764; Moore Piedrahita, 1997, c In Vitro Cell Biol. Anim. 33: 62-71; Moore, Piedrahita, 1996, Mol. Reprod. Dev. 139-144; Wheeler, 1994, Reprod. Fert. Dev. 6: 563-568; Hochereau- de Reviers Perreau, Reprod. Nutr. Dev. 33: 475-493; Strojek et al. 1990, Theriogenology 33: 901- 903; Piedrahita et al. 1990, Theriogenology 34: 879-901; and Evans et al. 1990, Theriogenology 33: 125-129).

ES cells can be maintained indefinitely without senescence in in vitro culture in an undifferentiated state in the presence of leukemia inhibitory factor (LIF) or on top of a layer of mitotically inactivated mouse embryonic fibroblasts (MEF). For example, murine ES cells remain undifferentiated when grown either on mitotically inactivated MEF or in the presence of relatively high concentrations of leukemia inhibitory factor (LIF), whereas human ES cells appear to remain undifferentiated only when grown on mitotically inactivated MEF.

In a preferred embodiment of the present invention, the stem cell creates an "embryoid body", which would be understood by those skilled in the art as generally referring to an intermediate stage during the differentiation of ES cells into all three different germ layers and their derivates. Preferably, embryoid bodies are prepared by the differentiation of ES cells in vitro in a so-called "hanging drop". Each drop may contain around 400 cells, which gradually multiply and coalesce in the bottom of the drop into an embryoid body. This embryoid body generally contains many different cell types that demonstrate distinct functions precursors to cardiomyocytes). Thus, in a preferred embodiment, the embryoid bodies form from ES cells in a way rather similar to differentiation in the blastocyst in vivo, whereby ES cells differentiate into an ectoderm and inner cell mass, followed by differentiation of the inner cell mass into W:\provisional\742767 Qocytes testes\742767_spe 200605-finaldoc 8 Sendoderm, the final cell mass containing endoderm, ectoderm and mesoderm. These Ccell types can then give rise to many different cell lineages, and finally tissues.

SThus, in a preferred embodiment of the present invention, there is provided a method of generating a female germ cell from an embryoid body, said method comprising exposing the embryoid body to testicular tissue or conditioned media r- derived from a culture of testicular tissue.

00

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O A "blastocyst" is a preimplantation embryo that develops from a morula. A in 10 blastocyst has an outer layer called the trophoblast that is required for implantation into the uterine epithelium and an inner cell mass that contains the embryonic stem cells and will give rise to the embryo proper. A blastocyst normally contains a blastocoel or a blastocoelic cavity.

The term "testicular tissue" as used herein refers to a cell (or a culture of testicular cells) that has been derived from a testis (or testes). A testicular cell can include, but is not limited to, a spermatogone, a spermatid, a primary and/or secondary spermatocyte, a spermatozoon, a Leydig cell, a Sertoli cell, a fibroblast, an epithelial cell, a cell of the connective tissue, and combinations thereof.

A testicular cell (or a plurality thereof) may be derived from the testis (or testes) by any number of means known to those skilled in the art. For example, a testicular cell may be isolated mechanically from a testis using an appropriate solution for dispersion or suspension Trypsin-EDTA). Such a solution will generally be a balanced salt solution normal saline, PBS, Hank's balanced salt solution, etc.), conveniently supplemented with fetal calf serum, BSA, normal goat serum, or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. The term "isolated" as used herein can refer to a cell that is separated from another group of cells. Examples of a group of cells are a developing cell mass, a cell culture, a cell line and an animal, or organ therefrom. It would be appreciated that the techniques employed to isolate a testicular cell is typically not unduly detrimental to the viability of the selected cells. A testicular cell may be derived from the testis of any number of species, including (but not limited to) human, primate, mouse, pig and horse. A testicular cell may also be derived from a testicular cell line maintained in vitro, whether that cell line is immortalised or not. For example, testicular cells derived from a testis (or testes) may be cultured in vitro over a solid support (such W:provisional\742767 Oocytes testes\742767-spec1200605-flnal.doc as a Petri-dish or flask) until confluent reaching 80% confluence). Testicular cells that have adhered to the solid support may then be placed into suspension by Sexposure to a solution of Trypsin-EDTA and replated at a lower cell density 1:3 or S1:2). This process may then be repeated as necessary during the proliferative phase of testicular cell growth in vitro.

The term "testicular tissue" can also refer to an extract of a testicular cell (or a 00 plurality thereof), such as a sample of homogenized testicular cells, whether the ihomogenization is performed mechanically or chemically chemical lysis).

The term "permanent" or "immortalized" as used herein in reference to cells can N refer to cells that may undergo cell division and double in cell numbers while cultured in an in vitro environment a multiple number of times until the cells terminate. A permanent cell line may double over 10 times before a significant number of cells terminate in culture. Preferably, a permanent cell line may double over 20 times or over times before a significant number of-cells terminate in culture. More preferably, a permanent cell line may double over 40 times or 50 times before a significant number of cells terminate in culture. Most preferably, a permanent cell line may double over times before a significant number of cells die in culture.

Testicular tissue is preferably isolated from a testis or testes derived from a mouse between the ages of embryonic day 12 to post-natal day 14. Even more preferably, the testis (or testes) is isolated from a 1 day-old mouse.

It would generally be understood by those skilled in the art that conditions which support the differentiation of a stem cell into a female germ cell according to the methods of the present invention will vary depending on the type of stem cell being used. Suitable culture media includes (but is not limited to) Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1% non-essential amino acids and 1% antibiotic/antimycotic solution.

Alternatively, a stem cell is cultured in the presence of testicular cells which have been cultured in vitro for a period in which active testicular germ cell proliferation is evident. Preferably, this is about 4 days to about 28 days. The present inventors have shown that testicular cells cultured during this period provide optimal conditions for the generation of female germ cells from embryonic stem cells exposed thereto, particularly where there is evidence of testicular germ cell proliferation. For instance, W:'provisional\742767 Oocytes testes\742767-spe_200605-final doc 0 the present inventors have found that during the period of about 4 days to about 28 Sdays, cultured testicular cells derived from a 1 day-old mouse testis will show signs of Smaximal germ cell proliferation between about 4 days and about 28 days following Sinitial plating, as evidenced by increasing cell numbers. Typically, germ cells at this stage will "float" away from the testicular cells adhered to the solid support culture flask, Petri-dish, etc), particularly when the solid support is agitated.

00 I The female germ cell generated by the method of the present invention Sis preferably an oocyte, more preferably an oogonium. The present inventors have S 10 surprisingly found that stem cells exposed to testicular tissue derived from the testis will Sdifferentiate into follicle-like ovarian structures containing female germ cells oocytes, oogonia). The female germ cell generated by the method of the present invention can be identified by any number of means known to those skilled in the art, including an analysis of surface markers Oct3/4, Mvh and c-kit) by immunohistochemical techniques using antibodies immunospecific for the surface markers. The female germ cell generated by the method of the present invention can also be identified by the analysis of gene expression Oct3/4, Mvh, c-kit, Stella, DAZL, Figa, ZP1, ZP2, ZP3 and Stra8) using polymerase chain reaction (PCR) amplification and/or in situ hybridization. A combination of the aforementioned techniques may also be used to identify a female germ cell generated by the methods according to the present invention. In a preferred embodiment, the female germ cell generated by the method of the present invention is an oogonium or an oocyte which express germ cell markers Oct3/4, c-kit, Stella, DAZL, Mvh, Figa, ZP1, ZP2, ZP3 and Stra8.

In a preferred embodiment of the present invention, testicular cells may be isolated from the testis (or testes) and plated as a cell suspension onto a culture flask, Petri dish or the like, and cultured under conditions that support the growth (e.g.

proliferation) of the gonadal cells in vitro Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1% non-essential amino acids and 1% antibiotic/antimycotic solution.

The term "culture", "cultured" and the like, as used herein in reference to cells, can refer to one or more cells that are undergoing cell division or not undergoing cell division in an in vitro environment. An in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example. Specific examples of suitable in vitro environments for cell cultures W:provisiona\742767 Qocytes testesN742767_speci_200605.fnal.doc 11 are described in Culture of Animal Cells: a manual of basic techniques sup. rd edition), 1994, R. I. Freshney (ed. Wiley-Liss, Inc.; Cells: a laboratory manual (vol.1), F1998, D. L. Spector, R. D. Goldman, L. A. Leinwand Cold Spring Harbor SLaboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J.

S 5 Morgan John Wiley and Sons, Ltd.

The term "suspension" as used herein can refer to cell culture conditions in 00oO cells are not attached to a solid support. Cells proliferating in suspension can be stirred while proliferating using apparatus well known to those skilled in the art, such as S 10 magnetic stirrers.

The term "plated" or "plating" as used herein in reference to cells can refer to establishing cell cultures in vitro. For example, cells can be diluted in cell culture media and then added to a cell culture plate, dish, or flask. Cell culture plates are commonly known to a person of ordinary skill in the art. Cells may be plated at a variety of concentrations and/or cell densities.

The term "proliferation" as used herein, in reference to a cell or a group of cells, can refer to a cell, or group of cells, that can increase in number over a period of time through mitosis).

In another aspect of the present invention, there is provided a method of generating a female germ cell from a stem cell, said method comprising exposing the stem cell to conditioned media derived from a culture of testicular tissue.

In a preferred embodiment, the conditioned media is any suitable culture media that is capable of supporting the differentiation of a stem cell into a germ cell as hereinbefore described. For example, the conditioned media can be Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1% nonessential amino acids and 1% antibiotic/antimycotic solution. In a preferred embodiment, the conditioned media is derived from a culture of testicular cells cultured for a period in which germ cell proliferation is evident, most preferably for a period of about 4 days to about 28 days after initial plating of testicular cells. The conditioned media may be used in the methods of the present invention immediately, or stored under appropriate conditions for future use.

W:\provisiona\742767 Oocytes testes742767ped200605-final.doc 12 0 The conditioned media is generally capable of supporting the differentiation of Sthe stem cell into a female germ cell without further modification. Alternatively, the Fconditioned media may be modified by adding one or more supplements or removing Sone or more of its constituents so as to modify the differentiation of a stem cell into a female germ cell according to the methods of the present invention.

Thus, in another aspect of the present invention there is provided a conditioned 00 IDmedia for use in generating a female germ cell from a stem cell, said conditioned

(N

media derived from a culture of testicular tissue.

(Ni S 0In a further aspect of the present invention, there is provided a culture of testicular tissue for use in generating a female germ cell from a stem cell by a method of the present invention.

In yet another aspect of the present invention, there is provided a female germ cell oocyte, oogonium) generated by the methods of the present invention.

Female germ cells described herein will have many uses in the art, including the treatment of infertility, therapeutic cloning and reproductive cloning.

Whilst the methods of the present invention may be performed in vitro, they can also be performed in vivo. For example, testicular tissue a population of testicular cells) may be administered to an individual systemically via the blood stream or directly into tissue), whereby the stem cells of the individual are then exposed to the administered testicular tissue and, as a consequence, differentiate into female germ cells. For example, testicular cells administered to the individual may be in suspension in an appropriate media or buffer as hereinbefore described, or incorporated into natural or artificial scaffolding matrix) that is capable of supporting the growth of the testicular cells.

In a preferred embodiment of the present invention, testicular tissue cells derived from a testis/testes, whether in suspension or in a suitable matrix), can be implanted by injection or any other suitable means) into an ovary of a female subject, wherein stem cells subsequently exposed to the implanted testicular tissue differentiate into viable female germ cells oocytes, oogonia). In another preferred embodiment of the present invention, stem cells isolated from an individual may be implanted by injection, or any other suitable means, into the testis (or testes), whereby subsequent exposure of the stem cells to the testicular tissue in vivo would result in the W:provisionaI\742767 Oocytes+ testes\742767 spect_200605-final.doc 13 0 generation of female germ cells. That is, the male subject to which the stem cells are administered acts as a surrogate for the generation of female germ cells in accordance Swith the present invention.

The differentiation of the stem cells into female germ cells in vivo may occur without further intervention via factors within the extracellular milieu). Alternatively, testicular tissue may be co-administered to an individual with compounds that promote 00 O the differentiation of the stem cells into female germ cells in vivo, such as various Sgrowth factors or small molecules drugs) that are identified by the skilled addressee as promoting the differentiation of the stem cells into female germ cells.

SThe differentiation of a stem cell into a female germ cell in vivo may be particularly useful in treating an infertile female.

Preferably, the female germ cell is genetically modified. Gene therapy may be employed to modify the testicular cells and/or stem cells pertaining to the present invention. For example, gene therapy may be used to modify the cell's genome through the insertion, deletion, and/or mutation of nuclear DNA. The modification may be performed prior to or during the exposure of the stem cell to the gonadal cell.

The term "modified nuclear DNA" and the like, as used herein, can refer to a nuclear deoxyribonucleic acid sequence of a cell, embryo, fetus, or animal of the invention that has been manipulated by one or more recombinant DNA techniques.

Examples of recombinant DNA techniques are well known to a person of ordinary skill in the art, which can include inserting a DNA sequence from another organism a human organism) into target nuclear DNA, deleting one or more DNA sequences from target nuclear DNA, and introducing one or more base mutations sitedirected mutations) into target nuclear DNA. Cells with modified nuclear DNA can be referred to as "transgenic cells" or "chimeric cells" for the purposes of the invention.

Transgenic cells can be useful as materials for nuclear transfer cloning techniques provided herein. The phrase "modified nuclear DNA" may also encompass "corrective nucleic acid sequence(s)" which replace a mutated nucleic acid molecule with a nucleic acid encoding a biologically active, phenotypically normal polypeptide. The constructs utilized to generate modified nuclear DNA may optionally comprise a reporter gene encoding a detectable product.

As used herein, the terms "reporter", "reporter system", "reporter gene", or "reporter gene product" shall include an operative genetic system in which a nucleic W:\provisiona1\742767 Oocytes testes\742767 sped 200605-fnal~doc 14 0 acid comprises a gene that encodes a product that when expressed produces a Sreporter signal that is a readily measurable, by biological assay, immunoassay, Sradioimmunoassay, or by colorimetric, fluorogenic, chemiluminescent or other Smethods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the 00 O reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition S 10 signals, transcriptional termination signals and the like.

(The term "promoters" or "promoter" as used herein can refer to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product. A promoter is preferably linked operatively to an adjacent DNA sequence. A promoter typically increases an amount of recombinant product expressed from a DNA sequence as compared to an amount of the expressed recombinant product when no promoter exists. A promoter from one organism can be utilized to enhance recombinant product expression from a DNA sequence that originates from another organism. In addition, one promoter element can increase an amount of recombinant products expressed for multiple DNA sequences attached in tandem. Hence, one promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well-known to persons of ordinary skill in the art.

The term "enhancers" or "enhancer" as used herein can refer to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product. Enhancer elements are typically located upstream of a promoter element or can be located downstream of a coding DNA sequence a DNA sequence transcribed or translated into a recombinant product or products). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a DNA sequence that encodes recombinant product.

Enhancer elements can increase an amount of recombinant product expressed from a DNA sequence above increased expression afforded by a promoter element. Multiple enhancer elements are readily available to persons of ordinary skill in the art.

Uses of the Female Germ Cells Generated by the Methods of the Present Invention WMproisional\742767 Oocytes testes\742767_sped 200605flnal.doc O Female germ cells oocytes, oogonia) generated by the methods of the N present invention could be genetically transformed and induced to differentiate into Sdesired cell types, for use in the generation of tissues for Stherapeutic cloning. Such methods would obviate the need for harvesting oocytes and 5 would provide a continuous source for such cells. Additionally, the ethical concerns of harvesting human oocytes would be overcome by such a method.

00 N The ability to obtain female germ cells from stem cells according to the present 0 invention therefore provides a means to reproducibly generate replacement cells or 'n 10 tissues which are useful in therapeutic cloning protocols. Once female germ cells are Sobtained, they may be enucleated.

The term "enucleated" as used herein in reference to a female germ cell, such as an oocyte, typically refer to an oocyte which has had its nucleus or its chromosomes removed. Typically, a needle can be placed into an oocyte and the nucleus can be aspirated into the needle. The needle can be removed from the oocyte without rupturing the plasma membrane. This enucleation technique is well known to a person of ordinary skill in the art (see, U.S. Pat. No. 4,994, 384; U.S. Pat. No. 5,057, 420 and Willadsen, 1986, Nature 320: 63-65). An enucleated oocyte is preferably prepared from an oocyte that has been matured for greater than 24 hours, preferably matured for greater than 36 hours, more preferably matured for greater than 48 hours, and most preferably matured for about 53 hours.

The terms "maturation" and "matured" as used herein can refer to a process in which a female germ cell is incubated in a culture media in vitro. Maturation culture media can contain multiple types of components, including hormones and growth factors. Time of maturation can be determined from the time that an oocyte is placed in a maturation medium to the time that the oocyte is subject to a manipulation g., enucleation, nuclear transfer, fusion, and/or activation). Oocytes can be matured in multiple media well known to a person of ordinary skill in the art (see, e. Mattioli et al., 1989, Theriogenology 31: 1201-1207; Jolliff Prather, 1997, Biol. Reprod. 56: 544- 548; Funahashi Day, 1993, J. Reprod. Fert. 98: 179-185; Nagashima et al., 1997, Mol. Reprod. Dev. 38: 339-343; Abeydeera et al., 1998, Biol. Reprod. 58: 213-218; Funahashi et al., 1997, Biol. Reprod. 57: 49-53; and Sawai et al., 1997, Biol. Reprod.

57: 1-6).

W:\provisional\742767 Qocytes+ testes\742767 speci 200605-final.doc 16 0 Somatic cell nuclei are obtained from the patient to be treated and somatic cell transfer is then performed. If the patient to be treated has a genetic mutation that is Sundesirable, the somatic cells utilized for nuclear transfer may optionally be Stransformed with a "corrective nucleic acid sequence" which corrects underlying genetic defect. These cells are then further cultured to form blastocysts from which transgenic stem cells may then be isolated. Transgenic stem cells so obtained may also be optionally transfected at this point with a "corrective nucleic acid sequence".

00 s The stem cells so obtained are then passaged and exposed to a receptor ligand 0 cocktail to induce differentiation into the desired cell lineage as exemplified herein tr 10 below.

Tissues currently being developed from stem cells include, but are not limited to: blood vessels (Kocher, A. A. et al., Nature Med. (2001) 7: 430-436; Jackson, K. A.

et al. J. Clin. Invest. (2001) 107: 1395-1402), bone (Petite, H. et al, Nature Biotech.

(2000) 18: 959-963), cartilage (Johnstone, B. et al., Clin. Orthop. (1999) S156-S162), cornea (Tsai, R. J. et al. N. Eng. J. Med. (2000) 343: 86-93), dentin (Gronthos, S. et al. Proc. Natl. Acad. Sci. USA (2000) 97: 13625-13620), heart muscle (Klug, M. G. et al. J. Clin. Invest. (1996) 98: 216-224; review Boheler, K. R. et al. Cir. Res. (2002) 91: 189-201), liver (Lagasse, E. et al., Nature Med. (2000) 6: 1229-1234), pancreas (Soria, B. et al., Diabetes (2000) 49: 1-6; Ramiya, V. K. et al., Nature Med. (2000) 6: 278-282), nervous tissue (Bjorkland, Novaritis Found. Symp. (2000) 231: 7-15; Lee, S. H. et al., Nature Biotechnology, (2000) 18: 675-679; Kim, J. H. et al., Nature (2002) 418: 50-56), skeletal muscle (Gussoni, E. et al., Nature (1999) 401: 390-394), and skin (Pellegrini, G. et al., Transplantation (1999) 68: 868-879). Some of the tissues being generated from stem cells are described in further detail below.

The term "nuclear transfer" as used herein can refer to introducing a full complement of nuclear DNA from one cell to an enucleated cell. Nuclear transfer methods are well known to a person of ordinary skill in the art (see, Nagashima et al., 1997, Mol. Reprod. Dev. 48: 339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41: 414-419; Prather et al., 1990, Exp. Zool.

255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259: 257-266; and Terlouw et al., 1992, Theriogenology 37: 309. Nuclear transfer may be accomplished by using oocytes that are not surrounded by a zona pellucida.

The term "transgenic" animal or cell preferably refers to animals or cells whose genome has been subject to technical intervention including the addition, removal, or W:provisionaI\742767 Oocytes testes\742767 spe 200605-final.doc 17 0 modification of genetic information. The term "chimeric" preferably refers to an animal Sor cell whose genome has modified.

SThus, in accordance with another aspect of the present invention, there is provided a method for generating a non-human embryo, the method comprising: i) obtaining a female germ cell generated by a method according to the present invention; ii) transferring a somatic cell nucleus into the germ cell after enucleating the same to I form a chimeric oocyte; and iii) culturing said chimeric germ cell under conditions which O result in the establishment of a non-human embryo. This method may further comprise implanting the embryo into a recipient female so as to produce a fetus that undergoes full fetal development and parturition to generate a live born animal.

Reproductive Cloninq of Non-human Animals To date, sheep, cattle, mice and pigs have been cloned successfully. The ability to generate female germ cells such as oocytes and oogonia in accordance with the present invention facilitates the production of cloned non-human animals which possess genetically superior phenotypic traits.

In an exemplary approach, stem cells are obtained and germ cells derived in accordance with the methods set forth herein. Germ cells so derived are then induced to form either oocytes or spermatogonia.

Once oocytes are obtained they may be subjected to the methods set forth in the following patent documents: US Patent 6,258,998; US Patent 6,107,543; US Patent 6,147,276 and US Patent 6,215,014. Given the disclosures of the foregoing patents, the skilled person is readily able to generate non-human animals and embryos using the oocytes provided herein.

Treatment of Infertility Using the Female Germ Cells Generated by the Methods of the Present Invention Often, infertility is the result of defective oocytes present in the female. In accordance with the methods of the invention, it is now possible to create genetically identical functional oocytes for use in in vitro fertilization methods for the treatment of infertility. In such a method, human stem cells are obtained and oocytes derived as herein described.

W :provisiona1\742767 Occytes testes\742767-spe200605-finaljdoc Z Oocytes so obtained may then be enucleated and a somatic cell nucleus from Fthe infertile female transferred into the enucleated oocyte. Blastocysts are then Sobtained from which stem cells which are genetically identical to the infertile female are isolated. Such stem cells are then treated as described herein to generate a second generation of germ cells. The germ cells are subjected to culture conditions which promote the formation of oocytes which can then be used in in vitro fertilization 00 IDmethods. As mentioned previously, such methods eliminate the moral and ethical Sconcerns and practical difficulties associated with human oocyte harvesting.

(Ni S SFinally, it is known that gametes are exquisitely sensitive to many toxic compounds, and their damage has severe consequences for fertility and subsequent normal development. Thus, oocytes generated in accordance with the invention may be utilized in screening methods to determine the toxicity and/or teratogenic potential of test compound by assessing stem cell differentiation towards a female germ cell (e.g.

oocyte, oogonium), follicle growth, oocyte development and embryo formation in vitro.

Thus, in yet another aspect of the present invention there is provided a method of screening for compounds that modify the differentiation of a stem cell towards a female germ cell, the method comprising exposing a stem cell to a testicular cell, or conditioned media derived from a testicular cell culture, in the presence of a test compound and assessing whether the test compound alters the differentiation of the stem cell towards a female germ cell.

Methods which enhance follicular growth in vitro, or similar methods, in conjunction with cryopreservation of immature follicles, may also allow the follicle reserves present in the ovaries of a few females to re-establish populations of species currently under the threat of extinction. In this regard, it should be possible to stem cells from valuable livestock for the purpose of generating female germ cells in accordance with the methods of the present invention. Pure-bred embryos may therefore be generated to be gestated by genetically unrelated females. The embryos or the female germ cells could be stored frozen, which would facilitate their preservation and management.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters WAprovisiona1\742767 Oocytes testes\ 7 42767_spea200605&final doc 19 0 formed part of the prior art base or were common general knowledge in the field 0 Srelevant to the present invention as it existed in Australia before the priority date of g each claim of this application.

C 5 Throughout the description and claims of this specification the word "comprise", and variations of the word such as "comprising" and "comprises", are not intended to exclude other additives or components or integers or steps.

00

NO

SIt would also be well appreciated by one skilled in the art that the methods of S 10 treatment hereinbefore described could be used in any number of combinations with each other, or with other treatment regimes currently employed in the art.

Examples of the procedures used in the present invention will now be more fully described. It should be understood, however, that the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES

Example 1: Creation of Embryoid Bodies (EB) From Mouse Embryonic Stem Cells Mouse male ES cells (contribution of Dr. Peter Mountford, Stem Cell Sciences, Australia) were cultured on gelatine Sigma, Castle Hill, Australia) coated 50 ml plastic flasks (Falcon, Becton Dickinson, Lane Cove, Australia) in a Dulbecco's Modified Eagle Medium (DMEM; Gibco-Life Technologies, Mulgrave, Australia) supplemented with 10% fetal calf serum (FCS, Gibco- Life Technologies) and recombinant mouse leukaemia inhibitory factor (LIF; 10001U/ml, Chemicon, Boronia, Australia), 1% w/w non-essential amino acids (Gibco-Life Technologies), 1 mM pmercaptoethanol (Sigma) and 1% w/w penicillin/streptomycin (Gibco-Life Technologies). Flasks were incubated at 37 0 C in 5% CO 2 in air. Primary cultures were let to reach confluence before cells were lifted, split and replated. For this, flasks were washed once with protein free Dulbecco's phosphate buffer saline (D-PBS, Gibco- Life Technologies) before addition of 3 ml Trypsin-EDTA (Gibco- Life Technologies) solution. Flasks were placed on 37 0 C warm stage for 2-3 min. The solution containing the dispersed ES cells was aspirated and placed in 10 ml conical plastic tube W:'provisional742767 Oocytes testes\742767speci-20065-final.doc 0 containing 7 ml LIF-DMEM solution. The tubes were centrifuged for 3 min in 1000g.

The supernatant was removed and the cells resuspended in 5 ml LIF-DMEM from 0 which aliquots of 1 ml were placed in new gelatine coated flasks containing 6 ml LIF-

DMEM.

EB were created using the hanging drop method (Hopfl et al., 2004). Once secondary ES cell cultures reached confluence, cells were lifted as described before, 00 washed and resuspended in LIF-free DMEM supplemented with 10% FCS or in 0 testicular cell conditioned DMEM conditioned medium to a concentration of 100,000 'i 10 cells/ml. Twenty microliter drops of the suspension were placed on the lid of 10 cm plastic culture dish (Falcon). The lid was turned upside down and placed on the bottom part of the dish, which was filled with sterile water creating hanging drops. Dishes were incubated at 370C in 5% CO 2 in air. EBs were cultured for 48, 72 and 120 hr before transferred to 0.5 ml DMEM or testicular cell conditioned medium for up to 2 weeks.

Disassociated ES cell monolayers (Figure 3a) cultured in LIF-free DMEM in hanging drops for 24 hr formed a single cluster with some loose cells around it. By 48 hr the cluster almost doubled in size and formed a defined edge line with a darker centre (Figure 3b). A small gradual growth in size was evident from 72 hr to 120 hr with no further morphological changes observed in EBs at these time points.

Example 2: Testicular Cell Culture (TCC) Testicular cell cultures (TCC) were prepared from testicular tissue of 1 day old newborn F2 (C57BI x CBA, F1, parents) male mice. The testes of 10 males were removed from the body into Trypsin-EDTA solution. The tissue was torn to pieces using fine forceps and left in the Trypsin solution for 5 min. The suspension was collected into 15 ml plastic conical tube. The tube centrifuged for 300g for 3 min and the supernatant removed. One ml of Dulbecco's Phosphate Buffer saline (D-PBS; Gibco- Life Technologies) containing 10% FCS was added to the tube and the pellet mixed with the solution thoroughly. The mixture was left at room temperature for 10 min after which the top 0.8 ml were removed and placed in another conical tube. Additional 11 ml of DMEM medium supplemented with 10% FCS, 1% non-essential amino acids and 1% penicillin/streptomycin solution, were added to the cell suspension. The solution was mixed well before divided into 6 wells (2 ml in each) of a six well culture dish (Falcon). Once reached 80% confluence, established cultures were lifted using WAprovisional\742767 Oocytes testes\742767speci_20060O5final doc

I

0 Trypsin-EDTA as described for ES cells and either frozen or diluted 1:2 and replated Sinto new wells.

STesticular germ cell proliferation was evident in TCC 8-10 days from initiation.

The round cells were clustered together and easily disassociated from other cells when dishes were shaken. Germ cells differed in size and appeared as single cells or Sattached to each other forming pairs or rows (Figure la). By 30-35 days from initiation, 00 0 the number of germ cells was gradually reduced and by 40-45 days, floating germ cells 0 were rarely observed. Clusters of cells (Figure 1b) appeared within the cultures and N 10 they were no longer used for collecting conditioned medium. As identified by O immunofluorescence analyses, germ cells formed in TCC were positive to Oct 3/4 and to Mvh but very few cells were positive to c-kit (Figure 2).

Following treatment with Trypsin-EDTA, ES and testicular cells were washed with freezing solution for 3 min at 1000g. For ES cells the freezing solution was composed of 90% FCS and 10% Dimethyl Sulphoxide (DMSO; Sigma). For testicular cells the freezing solution was composed of 60% DMEM, 10% DMSO and 30% FCS.

Pellets were resuspended with the corresponding freezing solution in 1 ml cryovials (Nunc, Roskilde, Denmark). Vials were placed in -70 0 C for 48 hr before placed in liquid nitrogen storage tank. For thawing, vials were removed from the storage tank into a 370C water bath for 5 min. Thawed solutions were collected into 15 ml plastic conical tubes (Falcon) containing 4 ml of the corresponding culture medium. Tubes were centrifuged for 3 min at 1000g. The supernatant was removed and the pellets were resuspended in 1 ml culture medium. Additional 9 ml of culture medium were added to the tubes before the suspensions were placed in organ culture dishes or flasks (Falcon).

Example 3: Preparation of Conditioned Media derived from TCC Conditioned media was collected from established testicular cell cultures, 10-12 days after initiation of cultures. Conditioned media was collected only from cultures with obvious germ cell proliferation. TCC which did not show a substantial proliferation of germ cells within the 10 days were not used. The conditioned media was collected from established TCC every 3 days starting from 10 days after initiation. Conditioned media was collected, filtered and either stored at -20 0 C or used immediately.

W:'provisional\742767 Oocytes testes\742767sped_200605..inal doc

I

22 O Example 4: Generation of Female Germ Cells from Embryoid Bodies Following C Exposure to Conditioned Media Derived From TCC A. Cell Culture Experiments CN TCC and 48h-old ES cell cultures were analyzed by immunofluorescence for the expression of the germ cell markers Oct 3/4, Mvh and cKit. ES cell cultures were 00oO also analyzed by RT-PCR for the expression of the germ cell markers Oct 3/4, Mvh, ScKit, Stella and DAZL.

In a first series of experiments, EB were created from disassociated ES cells cultured in hanging drops of LIF-free-DMEM. EB at 48, 72 and 120 hr from initiation were then examined by immunofluorescence staining for the expression of the germ cell markers Oct 3/4, Mvh and cKit. In addition, 48 hr old EB were examined by RT- PCR for the expression of the germ cell markers Oct 3/4, Mvh, cKit Stella and DAZL.

Muscle and liver cells and fibroblasts were used as controls for the RT-PCR analyses.

In a second series of experiments, EB were created from disassociated ES cells as described previously and cultured in hanging drops of LIF-free DMEM or TCs conditioned DMEM. At 48, 72 and 120 hr, EBs were transferred into 0.5 ml LIF-free DMEM or TC conditioned DMEM resulting in 4 groups for each time point: a) EB growing in LIF-free DMEM drop followed by culture in 0.5 ml LIF-free

DMEM;

b) EB growing in TC conditioned DMEM drop followed by culture in 0.5 ml TC conditioned DMEM; c) EB growing in LIF-free DMEM drop followed by culture in 0.5 ml TC conditioned DMEM; and d) EB growing in TC conditioned DMEM drop followed by culture in 0.5 ml LIF-free

DMEM.

The EB from the different four groups were examined each day and morphological changes were recorded. In addition, ES cells from primary cultures were disassociated and cultured in LIF-free DMEM or TC conditioned DMEM for up to 3 weeks. The cultures were examined each day and morphological changes were recorded. The culture solution for ES cells and EB was replaced every 3 days.

WAprovisional\742767 Oocytes testes\742767 spec_200605-finaldoc V 23 O In a third series of experiments, EB were created in LIF-free DMEM or TC conditioned N DMEM drops for 120 hr before transferred to either 0.5 ml LIF-free DMEM or TC conditioned DMEM for 2 weeks. Cultures were monitored for morphological changes each day. After 2 weeks, EB from the 4 groups were collected and analyzed for the S 5 expression of the Sry and Stra8 genes, which are specific to male and female gonads, respectively. Muscle and male germ cells collected from TCC were used as control for the expression analyses by RT-PCR.

(00 O In a fourth series of experiments, EB were created in TC conditioned DMEM n 10 drops for 120 hr before transferred to TC conditioned DMEM for 2 weeks. Cultures O were monitored for morphological changes each day. After 2 weeks, EB analyzed for the expression of the Figa, ZP1, ZP2 and ZP3 genes, which are specific to oocytes.

Ovaries and testes of newborn fetuses were used as positive and negative control respectively. Muscle cells were used as control for the expression analyses by RT-

PCR.

B. Marker analyses by Immunofluorescence ES cells, EB and testicular cells were examined for the expression of Oct-3/4, ckit, and the mouse VASA homologue Mvh. The cultures were fixed using 100% methanol for 5 min followed by three washes in cold D-PBS for 5 min. Washed cultures were treated with blocking solution (D-PBS+10 FCS) for a minimum of 2 hr before washed with D-PBS and stained with first antibodies for Oct-3/4 (Santa Cruz Biotechnology, California, USA) c-kit (Santa Cruz Biotechnology), and Mvh (thankfully provided by Dr., Toshiaki Noce, Mitsubishi Kagaku Institute of Life Sciences, Japan) overnight at 4 0 C. For Oct-3/4 and Mvh analyses, antibodies were diluted and cells were washed in D-PBS contained 0.1% Triton X-100 (Sigma) to increase permeabilization of the cells' membrane. Antibodies were diluted according to manufacturer or provider instructions. Cultures were washed with D-PBS, or D-PBS with 0.1% Triton-X three times for 5 min and the fluorescent secondary antibody was added for 30 min during which cultures were kept at dark. Goat anti-rat-FITC (Green, Santa Cruz Biotechnology) with absorption and emission wave lengths of 494 nm and 519 nm, respectively, was used to identify c-kit. Goat anti-rabbit-Rhodamine IgG (Red, Santa Cruz Biotechnology) with absorption and emission wave lengths of 570 nm and 590 nm, respectively, was used to identify Oct-3/4 and Mvh. Cells were washed with cold D-PBS three times for 5 min followed by 1 hr incubation in D-PBS contained 0.1% Triton X-100 in dark. Stained cells were visualised under an Olympus 1X70 inverted W:\provisional\742767 Oocytes testes\742767_speci_200605-finaldoc 24 O fluorescent microscope (Olympus Optical Co., Tokyo, Japan) using the appropriate N excitation wavelengths filters. Cultures were washed in D-PBS and stained with 4'6- Diamidino-2-phenyiodole dilactate (DAPI; Roche Applied Science, Castle Hill, SAustralia) with absorption and emission wave lengths of 344 nm and 450 nm, C 5 respectively, dissolved (1:1000) in anti fade Vectashield mounting medium (Vector laboratories Inc., Burlingame, USA).

rO 00 N0 C. Marker analyses by RT-PCR n 10 Total RNA was isolated and purified from ES cells, EBs and Zin-40 mice 0fibroblasts liver and muscle tissues using the RNeasy Mini kit (Qiagen, Hillden, Germany). The RT-PCR was analysed in 1% agarose (Progen Biosciences, QLD, Australia) stained with ethidium bromide (Invitrogen, Carlsbad, CA, USA) and visualized under UV illumination, using the Superscript III One-Step RT-PCR system (Invitrogen). The PCR conditions were denaturation at 950C for 5 min followed by cycles through 950C for 30 sec, 55 0 C for 30 sec, 720C for 1 min and 72 0 C for 10 min.

The following specific primers were used for the amplification: Oct-3/4: sense 5' CTCGAACCACATCCTTCTCT and antisense

GTTCTCTTTGGAAAGGTGTTC.

c-kit: sense 5' CATGGCTGCATTCTGACAAATTCAC and antisense

CTCCATCGGTTACAAATACTGTAG.

Mvh: sense 5' CTAGAGCACAGCCCCATAGTTGAAAGAT and antisense 5' TGCAGATAAACACTGAAACAGGCTA.

Stella: sense 5' GAGATGGCTGCGCGTCCGGGA and antisense

CTCAGTGGCAGCCACAGGCCT.

DAZL: sense 5' CCACCACAGTTCCAGAGTGTTTGG and antisense

CTTGAGTAACAAGAGAGTTTCTCAG.

Stra8: sense 5' GCAACCAACCCAGTGATGATGG and antisense

CATCTGGTCCAACAGCCTCAG

WMprovisional\742767 Qocytes testes\742767_sped 200605-final doc 0 Sry: sense 5' TTACAGCCTGCAGTTGCCTC and antisense

SCATGGAACTGCTGCTCCTGG.

5 D. Results Similar to ES cells grown in LIF-DMEM on gelatin, ES cells in LIF-free DMEM or 00 \0 TC conditioned medium, attached to the plastic and formed small clusters by 5-7 days

(N

0from initiation. These however, did not continue to form unified confluent cultures but S 10 clumps, which were spread all over the dish. Clusters formed from ES cells cultured in TC conditioned medium showed a more distinctive round morphology.

All 48 and most 72 hr old EB grown in LIF-free DMEM drops and transferred into 0.5 ml LIF-free DMEM or TC conditioned DMEM, plated down on the plastic surface within 1 day. The 120 hr EB grown in LIF-free DMEM transferred to 0.5 ml LIFfree DMEM or TC conditioned DMEM plated down by 3-4 days after transfer. When plated down, peripheral cells of the EB formed monolayers that continued to spread around the centre of the EB. Within one week the central cells degenerated and the peripheral cells had varied morphological shapes. In a few occasions, clusters of cells had an obvious beating rhythm indicative of their differentiation into cardiac muscle cells.

Most 48 hr EB grown in TC conditioned DMEM drop, which were transferred to ml TC conditioned DMEM plated down on the plastic surface within 2 days.

However, most 72 and all 120 hr EB, grown in TC conditioned DMEM drop, which were transferred to 0.5 ml TC conditioned DMEM remained semi-floating with only few cells attached to the plastic surface. Within 6-7 days a unique morphological changes were evident in these EB. Follicle-like structures start forming within the entire EB resulting in the appearance of ovarian structure (Fig 7 a, b, c, The follicular structures were about 70-80(pm in diameter (Fig. 8) contained large cells ranging from 20 to 35pm in diameter with no zona pellucida. These putative oocytes were similar to growing oocytes in vivo, an early developmental stage during oogenesis. Similarly, most 120 hr EB grown in TC conditioned DMEM hanging drops, which were transferred into 0.5 ml LIF-free DMEM remained semi-floating and had similar morphological changes. Only few of the 72 hr and none of the 48 hr old EB grown in TC conditioned DMEM drop which were transferred to 0.5 ml LIF-free DMEM showed these morphological changes.

W:Xprovisional\742767 -Oocytes testes\742767 sped 200605-finaI.doc 26 0 These findings suggest that commitment of cells within EB to the germ line is more likely to happen if EBs are exposed to TCs conditioned DMEM drops for at least days.

ES cells obtained from 24 and 48 hr old cultures grown on gelatine in LIF- DMEM were positively stained to Oct-3/4 but were negative to Mvh and c-kit. However, positive expression for Oct-3/4, Mvh and c-kit in 24 and 48 hr old ES cell cultures was oO \0 identified by RT-PCR. Additional germ cell markers such as Stella and DAZL, were 0also expressed by ES cells as identified by RT-PCR (Figure 4).

Immunofluorescence staining of 48 and 72 hr EBs in LIF-free DMEM identified cells that are positive to Oct-3/4, Mvh and c-kit (Figure 5 and Figure Because of the tight structure of EBs a strong background staining was evident in all samples. RT-PCR on 48 hr EBs confirmed positive expression of all germ cell markers analysed (Figure 4).

As identified by RT-PCR, the Sry and Stra8 genes were expressed by ES cells and by EB regardless the medium they were cultured in (Figure As expected, Sry was expressed in male germ cells but not Stra8. Muscle cells expressed neither of the genes. In addition positive expression of the oocyte markers Figa, and ZP3 was also identified in putative ovaries produced from ES cells in-vitro. Expression of all oocyte markers examined was positive in ovarian tissue obtained from newborn females but negative in testicular tissue.

Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

W.Aprovisional\742767 Oocytes testes\742767speci-200 6-final.doc

Claims (15)

1. 2. A method of generating a female germ cell from a stem cell, said method 00 IN comprising exposing the stem cell to a conditioned media derived from a culture Sof testicular tissue. U'
2. 3. A method according to claim 1 or 2, wherein the testicular tissue comprises a cell derived from a testis, or a plurality thereof.
3. 4. A method according to claim 3, wherein the testicular tissue comprises a cell selected from the group including a spermatogone, a spermatid, a primary spermatocyte, a secondary spermatocyte, a spermatozoon, a Leydig cell, a Sertoli cell, a fibroblast, an epithelial cell, a cell of the connective tissue. A method according to claim 3 or 4, wherein the testicular cell is cultured for a period of about 4 days to about 28 days.
4. 6. A method according to any one of claims 3 to 5, wherein the testicular tissue is derived from a mouse of about embryonic day 12 to about post-natal day 14 in age.
5. 7. A method according to claim 6, wherein the testicular tissue is derived from a 1 day old mouse.
6. 8. A method according to claim 2, wherein the conditioned media is derived from a culture of testicular tissue cultured for a period during testicular germ cell proliferation.
7. 9. A method according to claim 8, wherein the conditioned media is derived from a culture of testicular tissue cultured for a period of about 4 days to about 28 days. WMprovisional\742767 Oocytes testes\742767-specd2008O5flnal doc 28 A method according to any one of claims 1 to 9, wherein the stem cell is selected from the group including a stem cell derived from an early embryo S(blastocyst) created by in vitro fertilization; a stem cell derived from an early Sembryo created by inserting the nucleus from an adult cell into an enucleated S 5 oocyte; an immature germ cell; a stem cell derived from an organ of an aborted fetus; a stem cell derived from umbilical cord blood; a stem cell derived from adult tissues; and a stem cell derived from a mature adult cells which has been 0o IN reprogrammed to behave like a stem cell. t' 10 11. A method according to any one of claims 1 to 10, wherein the stem cell is derived from a male donor. (N
8. 12. A method according to any one of claims 1 to 11, wherein the stem cell is an embryonic stem (ES) cell.
9. 13. A method according to claim 12, wherein the stem cell is a human ES cell.
10. 14. A method according to any one of claims 1 to 13, wherein the stem cell is an embryoid body. A method according to any one of claims 1 to 14, wherein the female germ cell is selected from the group including an ovarian follicle, a primordial female germ cell, a gonocyte, an oocyte and an oogonium.
11. 16. A method according to any one of claims 1 to 15, wherein the female germ cell expresses a marker selected from the group including Oct3/4, Mvh, c-kit, Stella, DAZL, Figa, ZP1, ZP2, ZP3 and Stra8.
12. 17. A conditioned media derived from a culture of testicular tissue for use in generating a female germ cell.
13. 18. Testicular tissue for use in generating a female germ cell.
14. 19. A female germ cell generated by a method according to any one of claims 1 to 16. An embryo generated from a female germ cell according to claim 19. WMprovisional\742767 Oocytes testes\742767-ped200605-flnaldoc c 21. A non-human embryo generated from a female germ cell according to claim 19. S22. A method of screening for compounds that modulate the generation of a female germ cell from a stem cell, said method comprising exposing a stem cell, in the presence of a test compound, to testicular tissue and assessing whether the test compound modulates the generation of the female germ cell from the stem 0 cell and/or the growth and/or maturation of the female germ cell. (N
15. 23. A method of screening for compounds that modulate the generation of a female 0germ cell from a stem cell, said method comprising exposing a stem cell, in the presence of a test compound, to a conditioned media derived from a culture of testicular tissue and assessing whether the test compound modulates the generation of the female germ cell from the stem cell and/or the growth and/or maturation of the female germ cell. DATED: 20 June, 2005 PHILLIPS ORMONDE FITZPATRICK Attorneys for: MONASH UNIVERSITY W:provisionaI\742767 Qocytes testes\742767-peci20065final doc