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WO2006108284A1 - Procedes pour la derivation de cellules germinales a partir de cellules souches de peau - Google Patents

Procedes pour la derivation de cellules germinales a partir de cellules souches de peau Download PDF

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WO2006108284A1
WO2006108284A1 PCT/CA2006/000558 CA2006000558W WO2006108284A1 WO 2006108284 A1 WO2006108284 A1 WO 2006108284A1 CA 2006000558 W CA2006000558 W CA 2006000558W WO 2006108284 A1 WO2006108284 A1 WO 2006108284A1
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cells
oocyte
germ
stem cells
cell
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Julang Li
Paul W. Dyce
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University of Guelph
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University of Guelph
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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/24—Genital tract cells, non-germinal cells from gonads
    • C12N2502/243—Cells of the female genital tract, non-germinal ovarian cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/09—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells
    • C12N2506/094—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells from keratinocytes

Definitions

  • the invention is directed to purified populations of stem cells and their use in methods for the derivation of germ cells. More specifically the invention is directed to methods for the isolation and differentiation of skin stem cells into germ cells (oocytes) and purif ed populations of germ cells so derived. The invention also encompasses compositions of germ cells so produced and uses of such germ cells as a source for a variety of therapies and reproductive technologies.
  • Stem cell research is a rapidly advancing field working to uncover how a single cell develops into an organism and how damaged cells are replaced with healthy cells. It is hoped that this knowledge will lead to the development of cell-based therapies for tissue repair and possibly for the replacement of whole organs.
  • Stem cells offer the possibility of a renewable source of replacement cells and tissues to treat a myriad of diseases, conditions, and disabilities including Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, rheumatoid arthritis and fertility conditions.
  • Stem cells may also be used to develop specific populations of cells for further research and drug development and for use in therapeutic cloning.
  • stem cells and in particular, pluripotent stem cells capable of giving rise to any cell in the body.
  • one of the unanswered questions in mammalian developmental biology is when and where the fate of the germ cell is specified.
  • Mechanisms that govern differentiation of tissues must be uncovered to produce tissue-specific cell populations from undifferentiated embryonic stem cells.
  • Hwang et al. (Science VoI 303, 12 March 2004, pgs 1669-1674) describe the derivation of a pluripotent embryonic stem cell line derived from a cloned blastocyst.
  • H ⁇ bner et al. (Science, VoI 300, 23 May 2003, pgs 1251-1256) describe the isolation of mouse embryonic stem cells and their manupulation into oocytes.
  • U.S. 2005/0015824 describes methods for derivation of germ cells and oocytes therefrom where germ cells are derived from embryoid bodies of embryonic stem cells transformed with a GC-Oct4-GFP transgene.
  • Stem cells have been isolated from fetal porcine skin and were found to differentiate into neurons, astrocytes and adipocytes in vitro (Dyce, Zhu et al., 2004).
  • pluripotent stem cells in more non-invasive manners and from different sources to use in research and for different clinical therapies. More specifically, it is desirable to develop methods by which skin derived stem cells can be differentiated into oocytes. Furthermore, it is desirable to develop new methods of using such isolated stem cells to derive germ cells such as oocytes that can be used in a variety of research and therapeutic methods.
  • the present invention is directed to the isolation of stem cells from a skin source and the differentiation of such isolated skin stem cells into germ cells.
  • the skin stem cells are isolated from the skin of a mammalian fetus and the skin stem cells are then subjected to conditions to differentiate the isolated stem cells into oocytes.
  • the invention provides isolated cultures of stem cells derived from skin sources.
  • the invention also provides methods of differentiating such isolated skin stem cells into oocytes and other ovarian cells.
  • the invention further provides isolated cultures of such derived oocytes and various methods of using these oocytes in a variety of therapies.
  • According to an aspect of the present invention is a method for generating germ cells from skin derived stem cells.
  • a method for generating germ cells from skin stem cells comprising culturing said skin stem cells in a medium comprising follicular fluid for a time sufficient for said cells to express molecular markers associated with germ cell formation, further culturing said cells expressing said markers of germ cell differentiation for a time sufficient and under conditions for the formation of germ cells.
  • the method further comprises the isolation of the germ cells so produced.
  • According to another aspect of the present invention is a method for deriving germ cells from skin stem cells, the method comprising:
  • a method for deriving oocytes from skin stem cells comprising:
  • According to another aspect of the present invention is a method for deriving germ cells from skin stem cells, the method comprising:
  • molecular alteration selected from the group consisting of expression of c-mos, ZPA, ZPC and miosis marker spc3;
  • a method for deriving oocytes from skin stem cells comprising:
  • identifying morphological alterations and/or molecular alterations in said cells associated with oocyte formation said molecular alteration selected from the group consisting of expression of c-mos, ZPA, ZPC and meiosis marker scp3; and
  • composition of differentiating skin stem cells comprising a population of differentiating skin stem cells in a medium comprising follicular fluid.
  • composition of colony-like structures comprising differentiating skin stem cells, said composition comprising follicular fluid.
  • compositions of differentiating skin stem cells expressing a marker selected from the group consisting of c- kit, Oct 4, BMP15, Fragilis, Stella and Vasa wherein said composition comprises a medium containing follicular fluid.
  • the oocytes derived from skin-derived stem cells may be used in methods for tissue therapy and infertility, bypassing the problem of immuno- rejection from using allotransplantation.
  • a method for therapeutic cloning of tissues for the treatment of disease in a patient comprising: a) obtaining an oocyte derived from skin stem cells; b) transferring a somatic cell nucleus from said patient into said oocyte after enucleating the same to form a chimeric oocyte; activate the said chimeric oocyte into an embryo with either electrical pulse or chemical method c) culturing said embryo under conditions suitable to induce blastocyst formation; d) isolating embryonic stem cells from said blastocyst; e) exposing said cells to a culture medium which induces the stem cells to differentiate into a desired cell type; f) culturing the cells of step e) for a suitable time period to generate an effective amount of cells of said desired cell type; and g) optionally isolating the cells of step f).
  • a further aspect of the present invention is a method for screening test compounds for toxicity or teratogenic potential comprising : a) providing an oocyte derived from skin stem cells; b) exposing said oocyte to different amounts of said test compound; c) culturing said oocyte under conditions that promote blastocyst formation; and d) determining the effect, if any, of said test compound on the ability of said oocyte to form a blastocyst or determining the effect if any on gene expression pattern of early embryo development.
  • According to yet another aspect of the present invention is a method for preparing a non-human embryo capable of developing into a live-born animal, comprising : a) obtaining an oocyte derived from skin stem cells; b) transferring a somatic cell nucleus with desired genetic modification into said oocyte after enucleating the same to form a chimeric oocyte; and activate the said chimeric oocyte into an "embryo" with either electrical pulse or chemical method c) culturing said chimeric oocyte under conditions which result in the establishment of a non-human embryo.
  • the method further comprises embryo transfer into the uterus of recipient to allow development to term.
  • the isolated skin stem cells may be genetically altered and then stimulated to differentiate to produce germ cells that have incorporated the genetic alteration therein, these genetically altered oocytes can then be used in any of the methods of the present invention.
  • Figures 1A-1D show the expression of germ cell markers in skin sphere cells during induced differentiation.
  • Figure IA shows immunocytochemistry analysis showing the absence of c-kit expression at day 0 and its expression during differentiation.
  • Figure IB shows RT-PCR gel pictures indicating that Oct 4 mRNA was present in the skin sphere cells and that the expression was turned off at the early stage (day 4) of differentiation and re- expressed again from day 10 of differentiation, with the same expression pattern being observed in BMP15.
  • Figure 1C shows vasa expression detected at day 20 and 40 days of differentiation. DO is the day before differentiation and D4, DlO, D20 and D40 represent 4, 10, 20 and 40 days, respectively of differentiation. HPRT expression was used as a house keeping gene control. Neg, is PCR without RT on day 40 samples.
  • Figure ID shows skin- derived sphere cells isolated from new born pig.
  • Figure 2 show images during different time periods of differentiation.
  • Figure 2A is at 20 days of differentiation showing some colony-like structures are formed.
  • Figure 2B shows a colony that is composed of both Oct4 positive cells surrounded by Oct4 negative cells.
  • Figures 2C and 2D show that at about day 25 some colonies gradually detach from the rest of the culture and the bottom of the culture dish, suspended in the culture as aggregates.
  • Figures 3A-3C are micrographs showing the expelling of a large cell from the aggregate after transfer to oocyte growth medium and then transferred to oocyte growth medium.
  • Figure 3B shows that a large cell appeared to have a zona pellucida-like membrane and in Figure 3C a large cells reaching about 80-100 ⁇ M in diameter. These large cells were individually selected and grouped for RNA isolation.
  • Figure 3D shows RT-PCR demonstrating that the large cells express miosis marker spc3, c-mos and also express ZPA and ZPC.
  • Histone 2 was used as a house-keeping gene control for loading.
  • Figures 4A and 4B show the production of estradiol and progesterone by skin sphere cells during induced differentiation at the indicated times of induced differentiation, culture media was collected and ELISA performed to determine estradiol and progesterone levels (cells were cultured in oocyte growth medium in the absence of follicular fluid, DO represents the culture medium alone as a control). Data represent the mean ⁇ SE of five experiments.
  • Figure 4C shows the expression of aromatase, p450cl7, StAR and HPRT (as a housekeeping gene control) during induced differentiation by RT-PCR.
  • DO represents the day before differentiation and D4, DlO, D20 and D40 represent 4, 10, 20 and 40 days, respectively of differentiation.
  • Neg is PCR without RT on day 40 samples.
  • Figure 4D is immunocytochemistry showing the expression of P450SCC (left) and P450Arom (right) at day 40 of differentiation. Cells were counterstained with Hoechst to show nucleus.
  • Figure 5A shows the expression of the FSH receptor during induced germ cell formation and FSH.
  • Immunocytochemistry pictures show the expression of FSH receptor at day 20 and 40. Cells were counterstained with Hoechst to show nucleus.
  • Figure 5B shows levels of estradiol at day 50 of induced differentiation, where the skin cells were cultured for 24 hrs in the absence and presence (lOng/ml) of FSH. Culture media was collected and ELISA was performed for analysis of estradiol levels. Data represents the mean ⁇ SE of three experiments. P* ⁇ 0.05.
  • Figure 6A-6L are images showing the development of preimplantation embryo from oocyte-like cells.
  • the arrow points to a two cell stage embryo 24 hrs after IVF.
  • Figure 6B shows the same embryo becoming a four cell embryo 24 hrs later.
  • Figure 6C is Hoechst staining showing there is only one nucleus in the large cell.
  • Figures 6D and 6E show two and four cell embryo from oocyte like cell after chemical inactivation.
  • Figure 6F is Hoechst nuclear staining of a pathernogenetic embryo spontaneously generated in culture.
  • Figure 6G and 6K show examples of blastocyst embryo like structure generated from oocyte like cells.
  • Figure 6L shows a pathernogenetic embryo generated from natural oocyte which is morphologically similar to 6K.
  • Figure 7 shows the expression of Oct4 in morula (B) and early blastocyst (D) stage pathernogenetic embryos.
  • Figure 7A and 7C show the nuclear staining of the respective embryos.
  • Figure 8A-D show the expression of germ cell markers and generation of oocyte- cumulus-like complexes during induced skin sphere cell differentiation.
  • 8A Photomicrograph of a RT-PCR gel showing the expression of Oct 4, GDF 9b, DAZL and Vasa mRNA in skin sphere cells before and during induced differentiation.
  • DO the day before differentiation
  • D20 and D40 represent: 4, 10, 20, 40 days of differentiation, respectively.
  • Neg PCR without RT on the day 40 samples; expression of HPRT, a housekeeping gene, was used as control. The expression of these genes in the ovary was shown as a positive control.
  • the threshold cycle (Ct) of the PCR amplification (mean ⁇ SE) in this set of experiments ranged from 28 ⁇ 0.6 - 38 ⁇ 0.6; 8B: Some colony-like structures formed at day 20 of differentiation. 8C & 8D: At approximately day 30-40, some colonies gradually detached from each other and from the growth surface, and formed spherical aggregates in suspended cultures. Scale bars: b, c: 200 ⁇ m; d: 50 ⁇ m.
  • Figure 9A-S show the expression of oocyte markers and formation of meiosis-like morphology by oocyte-like cells.
  • 9A Phase contrast micrograph showing the expulsion of a "large cell” (arrow pointed) from the aggregate.
  • 9B A group of oocyte-like cells with an apparent zona pellucida-like membrane.
  • 9C An oocyte-like large cell.
  • 9D A bright field picture of a "large cell”, its nuclear staining.
  • 9E, and 9F Immunocytochemistry showing the detection of ZPC protein in this cell.
  • 9G bright field picture of a natural oocyte isolated from ovarian follicle, its nuclear staining 9H, and 91: immunocytochemistry showing the detection of ZPC protein in this oocyte.
  • 9J The large oocyte-like cells were individually picked and grouped as 10 cells for RNA isolation and RT-PCR. Transcripts of the meiosis marker Scp3, c-mos, ZPA, ZPC, Vasa, and Oct4 were detected in these large cells (60 - 100 ⁇ m). Histone 2 (H2A), a house-keeping gene, was used as a control. The threshold cycle (Ct) of the PCR amplification (mean ⁇ SE) in this set of experiments ranged from 27 ⁇ 0.7 - 36+1.2. k: A group of germ-cell- like cells isolated from day 32 of differentiation, arrows point to the cells that are the size stained in 9L.
  • 9L Depicts a representative Scp3 positive (green) cell counterstained with DAPI (blue).
  • 9M Germ cells were isolated from day 45 gestation fetus, arrows point to the cells that are the size stained in 9N.
  • 9N A representative Scp3 positive (green) germ cell counterstained with DAPI (blue).
  • 90 & 9P Depict orcein stained oocyte-like cells with a germinal vesicle (GV; arrow pointed) like structure.
  • 9Q An orcein stained natural pig oocyte (control), arrow points to its GV.
  • Panel 9S shows a representative orcein stained natural oocyte (control) demonstrating metaphase II plate (black arrow) and polar body (white arrow).
  • Figure lOA-0 show the steroid production and regulation by gonadotropin during induced differentiation of skin-derived cells.
  • 1OA & 1OB Cells were cultured in oocyte growth medium in the absence of follicular fluid, spent media were collected at the indicated time of induced differentiation, and levels of estradiol and progesterone were determined by ELISA. Data represents mean ⁇ SE of seven experiments.
  • 1OC Expression of p450 ArOm , p450 c i 7 , StAR and HPRT as determined by RT-PCR during induced differentiation and in the ovary (positive control). DO, D4, DlO and D40 represent the day before, 4, 10, 40 days of differentiation, respectively. Neg represents signals obtained with day 40 samples following PCR without RT.
  • the Ct values of the PCR amplification ranged from 28 ⁇ 0.6 - 38 ⁇ 0.5 in this set of experiments; 10D-10H: A phase contrast bright field image of a group of cell aggregates in the suspension culture at day 40 (10D). a representative image of immunocytochemical localization of p450 ArO m (10E) and Oct4 (10G) of an aggregate.
  • 1OF depicts the overlay image of p450 Ar o m (red) and Oct4 (green), counterstained with DAPI (blue) to show nucleus
  • (10H) is an overlay image of negative controls showing an aggregate where primary antibodies were omitted
  • i-m Depicts the natural oocyte-cumulus complex isolated from ⁇ 3 mm small follicles (101; bright field image) and immunocytochemistry images for p450 Arom (10J), Oct4 (10L), overlay image (10K) of p450 Arom (red) and Oct4 (green), counterstained with DAPI (blue), and an overlay image of negative controls showing an oocyte-cumulus complex where primary antibodies were omitted (10M).
  • n Expression of FSH receptor as determined by RT-PCR.
  • DO, DlO, D20 and D40 represent the day before, 10, 20, 40 days of differentiation, respectively. Neg represents signals obtained with day 40 samples following PCR without RT.
  • 10O At day 50 of induced differentiation, skin stem cells were cultured for 24 hr in the absence and presence of FSH (100 ng/ml). Spent media were collected and estradiol levels were determined by ELISA. Data represent the mean ⁇ SE of three experiments. * P ⁇ 0.05. Scale bar: d-h: and j-m: 50 ⁇ m, i: 100 ⁇ m.
  • Figure 11A-N shows the development of structures resembling preimplantation embryo from oocyte-like cells.
  • HA A phase contrast micrograph and nuclear staining (HB) of a blastocyst-like structure spontaneously developed in culture from an oocyte-like cell.
  • HC Another example of embryo-like structures generated from oocyte-like cells. Structure in HC exhibits morphological characteristics similar to a parthenogenetic embryo generated from natural oocyte (HD). Expression of Oct4 in blastocyst-like structures (11G,J) generated from oocyte-like cells, with HE and HH showing their bright field pictures, HF and HI showing their respective nuclear staining.
  • HK A bright field image of a parthenogenetic blastocyst-stage embryo (control) generated from natural oocyte, its nuclear staining (HL), and Oct4 staining (HM) depicted.
  • HN RT-PCR results showing the expression of ZPA, ZPC, Scp3, Vasa, GDF9b in the oocyte-like large cells was turned off after they developed into blastocyst-like structures.
  • Oct4 is expressed in both of the oocyte -like large cells and blastocyst-like structures.
  • Histone 2 (H2A) a house-keeping gene, was used as a control. "O”: oocyte-like large cells, "B”: blastocyst-like structures.
  • the expected sizes of the PCR products are: ZPA: 123 bp, ZPC: 202bp, Scp3: 197 bp, Vasa: 165 bp, GDF9b: 227 bp, Oct 4: 160 bp, H2A: 226 bp.
  • Figure 12A is a micrograft showing skin-derived sphere cells isolated from a fetal mouse at 15 days of gestation.
  • Figure 12B is a micrograft showing skin-derived sphere cells from postnatal mice.
  • FIG. 13A-B shows Oct4 mRNA expression in fetal mice skin-derived skin stem cells before and during porcine follicular fluid induced differentiation as determined by RT-PCR.
  • 13A shows a representative gel image of the RT-PCR products.
  • 13B shows real time PCR data on Oct4 expression analyzed with the 2 " ⁇ Ct method using HPRT for normalization. The Ct value for each gene was determined at a threshold of 30 fluorescence units. Data is the mean ⁇ SEM of three experiments. Un: undifferentiated group, Dl, D4, DlO: samples from Kay 1 (24 hours), Day 4 and Day 10 differentiation, respectively. Neg : negative control, using RNA isolated from oviduct.
  • Figure 14A-B shows GDF9b mRNA expression in fetal mice skin-derived skin stem cells before and during porcine follicular fluid induced differentiation as determined by RT- PCR.
  • 14A shows a representative gel image of the RT-PCR products.
  • 14B shows real time PCR data on GDF9b expression analyzed with the 2 ' ⁇ Ct method using HPRT for normalization. The Ct value for each gene was determined at a threshold of 30 fluorescence units. Data is the mean ⁇ SEM of three experiments. Un: undifferentiated group, Dl, D4, DlO: samples from Kay 1 (24 hours), Day 4 and Day 10 differentiation, respectively. Neg : negative control, using RNA isolated from oviduct.
  • Figure 15A-B shows DALZ mRNA expression in fetal mice skin-derived skin stem cells before and during porcine follicular fluid induced differentiation as determined by RT-PCR.
  • HA shows a representative gel image of the RT-PCR products.
  • 15B shows real time PCR data on DALZ expression analyzed with the 2 " ⁇ Ct method using HPRT for normalization. The Ct value for each gene was determined at a threshold of 30 fluorescence units. Data is the mean ⁇ SEM of three experiments. Un: undifferentiated group, Dl, D4, DlO: samples from Kay 1 (24 hours), Day 4 and Day 10 differentiation, respectively. Neg : negative control, using RNA isolated from oviduct.
  • FIG. 16A-B shows Stella mRNA expression in fetal mice skin-derived skin stem cells before and during porcine follicular fluid induced differentiation as determined by RT- PCR.
  • 16A shows a representative gel image of the RT-PCR products.
  • 16B shows real time PCR data on Stella expression analyzed with the 2 " ⁇ Ct method using HPRT for normalization. The Ct value for each gene was determined at a threshold of 30 fluorescence units. Data is the mean ⁇ SEM of three experiments. Un: undifferentiated group, Dl, D4, DlO: samples from Kay 1 (24 hours), Day 4 and Day 10 differentiation, respectively. Neg : negative control, using RNA isolated from oviduct.
  • FIG. 17A-B shows Vasa mRNA expression in fetal mice skin-derived skin stem cells before and during porcine follicular fluid induced differentiation as determined by RT-PCR.
  • 17A shows a representative gel image of the RT-PCR products.
  • 17B shows real time PCR data on Vasa expression analyzed with the 2 " ⁇ Ct method using HPRT for normalization. The Ct value for each gene was determined at a threshold of 30 fluorescence units. Data is the mean ⁇ SEM of three experiments. Un: undifferentiated group, Dl, D4, DlO: samples from Kay 1 (24 hours), Day 4 and Day 10 differentiation, respectively. Neg: negative control, using RNA isolated from oviduct.
  • Figure 18A-B shows Fragilis mRNA expression in fetal mice skin-derived skin stem cells before and during porcine follicular fluid induced differentiation as determined by RT- PCR.
  • 14A shows a representative gel image of the RT-PCR products.
  • 18B shows real time PCR data on Fragilis expression analyzed with the 2 ' ⁇ Ct method using HPRT for normalization. The Ct value for each gene was determined at a threshold of 30 fluorescence units. Data is the mean ⁇ SEM of three experiments. Un: undifferentiated group, Dl, D4, DlO: samples from Kay 1 (24 hours), Day 4 and Day 10 differentiation, respectively. Neg: negative control, using RNA isolated from oviduct.
  • Figure 19A-C is an immunocytochemistry analysis.
  • Figure 19A shows the absence of Oct 4 (green) and Aromertase (red) expression at early staged induced differentiation (24 hours).
  • Figures 19B/C show the expression aromertase and Oct4 at later stage of differentiation (D4, DlO for aromertase and DlO for Oct4).
  • Figure 19C shows that an Oct4 (green) positive cell surrounded by aromertase expressing cells (red), cells were counterstained with DAPI (blue) to show nucleus. Scale bar: 50 ⁇ m.
  • Figure 20A/B shows the generation of oocyte-like large cell from fetal mouse skin- derived stem cell.
  • 2OA is a bright field picture of an oocyte-like cell collected from day 12 of differentiation.
  • 2OB is immunocytochemistry showing the detection of ZPC protein in this cell. Scale bar 25 ⁇ m.
  • the present invention for the first time demonstrates that skin stem cells from fetal tissue have germline potential.
  • the invention provides isolated stem cells from a skin source, i.e. from mammalian skin that are differentiated into germ cells such as oocytes.
  • the invention provides isolated cultures of stem cells derived from skin sources.
  • the invention also provides methods of differentiating such isolated skin stem cells under conditions giving rise to germ cells, i.e. oocytes with the use of follicular fluid.
  • the invention further provides isolated cultures of such derived oocytes and various methods of using these oocytes in a variety of therapies and in research.
  • the invention also provides oocytes for fertilization.
  • the ability to generate oocytes from skin-derived cells offers new possibilities for tissue therapy and infertility, bypassing problems of immuno-rejection from using allotransplantation. Furthermore, the method of the invention is advantageous because obtaining stem cells from a skin source that can be differentiated to obtain oocytes or other germ cells is less invasive and more readily acceptable and less contraversial than using embryonic stem cells obtained from an embryo for example.
  • the invention has several potential uses including but not limited to: assisted reproductive technology such as providing oocytes; source of therapeutic stem cell lines; source of oocytes for nuclear transfer and therapeutic cloning (Hwang et al., 2004); source of oocytes for nuclear transplantation cloning of non-human animals; provide germ cell lines of animals that can be genetically manipulated for breed improvement; and using germ cell lines for human fertility studies.
  • assisted reproductive technology such as providing oocytes
  • source of therapeutic stem cell lines such as providing oocytes
  • source of oocytes for nuclear transfer and therapeutic cloning Hwang et al., 2004
  • source of oocytes for nuclear transplantation cloning of non-human animals provide germ cell lines of animals that can be genetically manipulated for breed improvement
  • germ cell lines for human fertility studies including but not limited to: assisted reproductive technology such as providing oocytes; source of therapeutic stem cell lines; source of oocytes for nuclear transfer and therapeutic cloning (Hwang et al., 2004); source of
  • a stem cell is an unspecialized cell that gives rise to a specific specialized cell, such as a blood cell. More specifically, stem cells are defined (Gilbert, (1994) Developmental Biology, 4th Eld. Sinauer Associates, Inc. Sunderland, Mass., p. 354) as cells that are "capable of extensive proliferation, creating more stem cells (self-renewal) as well as more differentiated cellular progeny.” These characteristics can be referred to as stem cell capabilities.
  • An ovarian follicle is a cavity in the ovary containing a maturing ovum surrounded by its encasing cells.
  • An oocyte is a cell from which an egg or ovum develops by meiosis; a female gametocyte.
  • a zygote refers to a fertilized one-cell embryo (fertilized ovum).
  • a germ cell refers to an ovum or a sperm cell or one of its developmental precursors. Germ cells are haploid and have only one set of chromosomes.
  • Totipotent is the ability of a cell, such as an egg, to give rise to unlike cells and thus to develop into or generate a new organism or part.
  • a totipotent cell can give rise to all of the cells of an animal when it is utilized in a procedure for developing an embryo from one or more nuclear transfer steps.
  • a blastocyst is the modified blastula stage of mammalian embryos, consisting of the inner cell mass and a thin trophoblast layer enclosing the blastocoel. Also called a blastodermic vesicle.
  • a follicle refers to a more or less spherical mass of cells usually containing a cavity.
  • Ovar an follicles comprise egg cells and the corona radiata.
  • An embryoid body is a three dimensional structure that forms from differentiated embryonic stem cells.
  • proliferation is in reference to cells, in particular, a group of cells that can increase in their number over a period of time.
  • differentiation refers to the developmental process whereby cells assume a specialized phenotype, i.e., acquire one or more characteristics or functions distinct from other cell types.
  • the differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway. In many but not all tissues, the process of differentiation is coupled with exit from the cell cycle, in these cases, the cells lose or greatly restrict their capacity to proliferate when they differentiate.
  • the term isolated may refer to a cell that is mechanically 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.
  • embryo or embryonic may refer to a developing cell mass that has not implanted into an uterine membrane of a maternal host.
  • 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.
  • An "embryonic cell” is isolated from and/or has arisen from an embryo. An embryo can represent multiple stages of cell development.
  • 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
  • an embryo having a blastocoel can be referred to as a blastocyst.
  • fetus may refer to a developing cell mass that has implanted into the uterine membrane of a maternal host.
  • a fetus can include such defining features as a genital ridge, for example.
  • a genital ridge is a feature easily identified by a person of ordinary skill in the art, and is a recognizable feature in fetuses of most animal species.
  • a fetal cell is any cell isolated from and/or has arisen from a fetus or derived from a fetus, including amniotic cells.
  • skin spheres (Figure ID) were first disassociated into single sphere cells and induced to attach to the bottom of the culture dish. These were then specifically induced to form germ cells in conditioned medium containing 5% porcine follicular fluid.
  • the receptor kinase c-kit is expressed in the ovarian follicle developing from the primordial to the mature stages (Manova, Huang et al. 1993).
  • Oct 4 is previously known to be primarily expressed in pluripotent lineages and is specifically expressed in cells participating in the generation of the germline lineage from day 8.5 onward in mice (Scholer, Dressier et al. 1990).
  • Growth differentiation factor-9b (GDF-9b; also known as bone morphogenetic protein 15; BMP 15), a member of the transforming growth factor ⁇ (TGFI3) superfamily is an oocyte-specific factor required for normal folliculogenesis (Dube, Wang et al. 1998).
  • Vasa expression starts at the post-migratory stage and is restricted to primordial germ cells during embryogenesis and germ cells undergoing gametogenic processes until the post-meiotic stage (Toyooka, Tsunekawa et al. 2000).
  • immunocytochemistry and/or RT-PCR were performed.
  • C-kit expression was not detectable in the skin sphere cells before differentiation. Approximately 10 days after induced differentiation in the conditioned medium, approximately 2% of cells were positive for c-kit staining. The percentage of c-kit positive cells increased to 9% and 25% by day 20 and 40 of differentiation, respectively (Figure IA).
  • Oct 4 mRNA transcript was present in the skin sphere cells as detected by RT-PCR.
  • oocytes are surrounded by the zona pellucida (ZP), an extracellular matrix comprising of three glycoproteins (ZPA, ZPB, ZPC) and highly homologous within mammalian species (for review see (Dunbar, Avery et al. 1994).
  • ZP zona pellucida
  • ZPA three glycoproteins
  • ZPB three glycoproteins
  • ZPC three glycoproteins
  • haploid gametes are generated from diploid parental cells via meiosis.
  • a complex series of events occurs, including chromosome pairing and synaptonemal complex (SC) formation.
  • SC synaptonemal complex
  • prophase I the recombinations occur at high frequency, which are usually required for proper chromosome segregation and essential for introducing genetic variation (reviewed in (Roeder 1997).
  • the synaptonemal complex protein SCP3 is a component of the synaptonemal complex; a meiosis-specific protein structure essential for synapsis of homologous chromosomes (Parra, Viera et al. 2004). In mice, SCP3 starts to be expressed at ⁇ 13.5 dpc in oocytes, consistent with the entry into prophase of meiosis (Di Carlo, Travia et al. 2000). As shown in Figure 3D, c-mos, ZPA, ZPC, and meiosis marker spc3 were all expressed in these oocye- like large cells, and in ovarian tissue (positive control), but not in oviductal tissue (negative control.
  • Ovarian estrogen production is the hallmark of preovulatory follicular development (review by Hillier, Whitelaw et al. 1994). If the cell aggregates are follicle-like structures, they should contain granulosa and theca cells, in addition to oocytes. In ovarian follicles, androgen precursors are produced by theca cells, and aromatized into estrogen in granulosa cells (reviewed in (Richards and Hedin 1988). In addition, granulosa cells also produce progesterone (Morley, Whitfield et al. 1992).
  • Ovarian steroid hormone synthesis is accomplished by the coordinated action of cytochrome P450 17 ⁇ - hydroxylase/17-20 lyase (P450 c i7), cytochrome p450 aromatase (P450 ar o m ), steroidogenic acute regulatory protein (StAR), and P450-linked side chain cleaving enzyme (P450 S sc)-
  • RNA was isolated from cells at day 0, 10, and 40 of differentiation. RT-PCR was performed to determine the expression of these enzymes during induced differentiation. P450 arom was not expressed in the skin sphere cells. Its transcript was detectable starting from day 40 of inducted differentiation.
  • FIG. 4C shows the results from the immunocytochemistry study, confirming the expression of P450 arom at the protein level and showing the expression of P450 S sc at day 40 of differentiation.
  • FSH follicular stimulating hormone
  • FSH receptor expression was assessed via immuno-cytochemistry. FSH receptor expression was undetectable before differentiation. The expression was evident at day 20 of differentiation, and increased with further differentiation ( Figure 5A). When added to differentiating culture, FSH significantly stimulated estradiol production ( Figure 5B), indicating that the differentiating cells are functionally responding to the stimulation by the gonadotropin.
  • Figure 6F shows the nuclear straining of a blastocyst stage embryo from a "large cell", the number of nuclei in this embryo is comparable to those of porcine pathernogenetic embryos generated from authentic oocyte (data not shown).
  • Figures 6GHIJK shows a few examples of blastocysts generated from individual "large cell”.
  • Figure 6L illustrates a pathernogenetic embryo generated from a natural oocyte. To study if these pathernogenetic embryo express gene that is consistent with early embryo development, immunocytochemstry was performed using antibody against Oct4.
  • Figure 7 shows that Oct 4 is expressed in the morula and blastocyst. The expression pattern of Oct 4 in the blastocyst is consistent with previous study showing this gene is both inner cell mass and trophoblast of the pig (Kirchhof, Carnwath et al. 2000).
  • Follicular fluid contains factors that are secreted from granulosa cells, theca cells, as well as oocytes in the follicles.
  • factors are present in follicular fluid such as for example, GDF9, GDF9b (BMP15; (McNatty, Moore et al. 2004) and SCF (Tanikawa, Harada et al. 2000) and basic fibroblast growth factor (Malamitsi-Puchner, Sarandakou et al. 2003).
  • hormones such as FSH, estrogen, and leptin are also present in follicular fluid.
  • follicular fluid can be used to differentiate skin stem cells to enter into the germ cell path.
  • the invention was repeated to detect additional germ cell markers ( Figures 8-11) confirming differentiation of the cells.
  • skin- derived stem cells were induced to form germ cells in medium supplemented with 5% porcine follicular fluid.
  • all three independent batches used in the experiment were double filtered (0.22 ⁇ M pore size).
  • Oct 4 mRNA was expressed in the skin sphere cells, the expression was turned off by day 4 of differentiation and resumed again from day 10 of differentiation and thereafter (Fig. 8A).
  • the expression pattern of GDF9b (BMP15) was similar to that of Oct 4 (Fig. 8A).
  • DAZL transcript was absent before differentiation, and was expressed from day 10 on of induced differentiation (Fig. 8A). Vasa expression was undetectable until day 20 of differentiation and thereafter (Fig. 8A). Morphologically, some colony-like structures started to form in the culture from approximately day 20 of differentiation (Fig. 8B). At around day 30 to 40 of culture, some of the colonies gradually detached from the growth surface, and formed suspended cell aggregates. Another 10 to 15 days later, a subpopulation of the aggregates appeared to have a large cell in the center; a morphology resembling the cumulus-oocyte complex within a follicle (Fig. 8C, 8D, data described with reference to figure 1).
  • RT-PCR revealed that P450 arom was not expressed in the stem cells, but was detectable starting from day 40 of induced differentiation. Similar to the expression pattern observed for Oct 4 and GDF9b, the p450 cl7 transcript was present before differentiation. Its expression was turned off at an early stage of differentiation and on again at day 40 during differentiation. StAR was constitutively expressed irrespective of the stage of differentiation (Fig. 10C).
  • Figure 1OE confirms the expression of p450 ara m at the protein level in a cell aggregate. A subpopulation of Oct4 expressing cells was surrounded by these p450 arom positive but Oct4 negative cells (Fig. 10F). This cell distribution pattern is similar to that of an ovarian oocyte-cumulus complex in which a germ cell is surrounded by Oct 4-negative and p450 arOm positive granulosa cells ( Figure 10K).
  • FSH receptor expression was undetectable before, and at the early stage of differentiation, but was evident at day 40 of differentiation (Fig. UN).
  • FSH significantly stimulated estradiol production (Fig. 110), indicating that the differentiating cells are functionally responding to the gonadotropin stimulation.
  • Figure 12C shows another example of a blastocyst-like structure generated from a "large cell”.
  • Figure 12D illustrates a parthenogenetic embryo generated from a natural oocyte, showing a structure similar to that in Figure 12C.
  • RT-PCR indicated that while Oct4 was expressed in both of the oocyte -like large cells and blastocyst-like structures, the expression of ZPA, ZPC, SCP3, Vasa and GDF9b in the oocyte-like large cells was turned off after they developed into blastocyst-like structures (Fig. 12N). This data suggests that expression of at least this set of genes has been reprogrammed during the transition from oocyte-like cells to blastocyst-like structures.
  • the invention was also carried out using skin-derived sphere cells isolated from fetal and new born mice (Figure 12A, 12B). Briefly, the germ cell markers (Oct4, GDF9b, DAZL, Stella, Vasa, and Fragillis) that are consistent with germ cell development were detected in culture before and during inducted differentiation ( Figures 13 to 18). With the exception of vasa, which seems to be expressed in all the time points tested, the expression of most of the markers was turned off at a time point during inducted differentiation and re-expressed again later (Figure 19A-C). The down-regulation period may represent the transition from stem cell to germ cell.
  • the germ cell markers Oct4, GDF9b, DAZL, Stella, Vasa, and Fragillis
  • the present invention is first to demonstrate the use of skin stem cells to generate germ cells.
  • skin cells taken from the skin of fetal porcine as well as fetal mouse could be differentiated into germ cells, i.e. oocytes.
  • pigs and mice were used, it is understood however, that the method of the invention may be applicable to mammals in general and most preferably humans.
  • skin cells may be obtained from both female and male species of mammals.
  • the skin cells may be derived any time from about mid gestation up until after birth and still be induced to differentiate into oocytes in vitro.
  • skin stem cells may be isolated anytime during gestation at the first presence of skin tissue in any mammal and also taken after birth.
  • skin cells are derived from about 35 to about 50 days of gestation and still in other aspects, skin cells may be derived from about 35 to about 45 days of gestation in pigs and in mice.
  • Any source of mammalian follicular fluid may be used in the method of the invention to differentiate the skin derived stem cells.
  • follicular fluid matched to the same subject is used, in other aspects different sources of the same species may be used.
  • follicular fluid from a different mammalian species to that from which the skin stem cells are derived may be used in the method.
  • any fluid i.e.
  • follicular -type fluid that is provided to contain several of the factors secreted by granulosa cells, theca cells and oocytes in the follicles may be used in the method of the invention so long as it may induce differentiation of the skin stem cells as can be determined by one of skill in the art following the examples provided herein.
  • the amount of follicular fluid provided in the conditioned medium in which the skin stem cells are cultured and induced to differentiate may vary as is understood by one of skill in the art. In aspects this may range from up to about 15 mis (%v/v) or more and any range up to about 15 mis (%v/v).
  • this may be up to about 10 mis (%v/v) and still in other aspects may be up to about 5 mis (%v/v).
  • the amount of time that is required for differentiation in the conditioned medium containing follicular fluid may also vary as is understood by one of skill in the art and can be readily determined following the examples provided herein by determination of certain expression factors.
  • skin stem cells can be induced to differentiate into germ cells, that is, oocytes. Such oocytes may further be fertilized to produce a zygote. It is also understood that the method of the invention is useful for the generation of various ovarian cells such as granulosa and theca cells in the ovary as it was demonstrated that the differentiating cells produced estradiol and progesterone. These steroid hormone producing cells could potentially be used for autologous tissue therapy for women in menopause.
  • the current invention demonstrates that somatic stem cells from later stages of fetal development also have the intrinsic machinery to develop into oocytes.
  • the present invention offers additional clinical advantages: inducing stem cells from skin to differentiate into oocytes either for the purpose of assistant reproduction or tissue therapy could potentially bypass the need to destroy natural embryos and avoid immuno-rejection of using allotransplantation.
  • the oocytes obtained by the method of the invention may be used for screening various test compounds for toxicity or teratogenic potential. As such test compounds may also be screened for their effect on the vitality (ability of living, growing and developing and whether it is normal, without defect) of such oocytes (into a blastocyst for example).
  • the oocyte(s) are exposed to various amounts of a test compound and a control. After a time period of culturing under suitable conditions to induce blastocyst formation, the effect if any of the test compound can be determined on blastocyst formation or any other desired parameter.
  • the present invention can be used to generate oocytes to facilitate the production of cloned non-human animals which possess genetically desired phenotypic traits and thus has great use in the field of animal husbandry and in veterinary medicine.
  • skin stem cells are obtained and germ cells, i.e. oocytes derived in accordance with the methods described herein.
  • oocytes derived in accordance with the methods described herein.
  • one of skill in the art may generate non-human animals and embryos using the oocytes provided by the method of the present invention.
  • the generated oocytes of the invention may also be used to treat infertility.
  • Infertility may be the result of defective oocytes present in the female.
  • functional oocytes may be generated for use in in vitro fertilization and in other methods for the treatment of infertility particularly in animals.
  • the oocytes derived from the method of the invention may be enucleated and a somatic cell nucleus from the infertile female may be then transferred into the enucleated oocyte.
  • the isolated skin stem cells may first be genetically modiFied as is understood by one of skill in the art.
  • the genetically modified skin stem cells can then be induced to become a germ cell, i.e. oocyte which can be used in an in vitro fertilization procedure to produce a embryo.
  • the embryo would be transfered to the uterus of recipient to allow develop to term.
  • the enucleated oocytes are cultured to form blastocysts from which stem cells may then be isolated and passaged to develop desired tissues such as for example but not limited to liver, pancreas, muscle and nervous tissue.
  • desired tissues such as for example but not limited to liver, pancreas, muscle and nervous tissue.
  • Cells were isolated as previously described (Dyce, Zhu et al. 2004) from day 35-45 gestation porcine fetal skin. Cells were passaged 1-2 times prior to induce differentiation.
  • fetuses were removed from the uterus of 35-45 day pregnant sows and fetal skin from the back was carefully dissected free from other tissue and cut into 1-2 mm 2 pieces. These pieces were washed three times in Hank's balanced salt solution (HBSS), and digested with 0.2% trypsin for 35 minutes at 37 0 C, followed by 0.1% DNAase for 1 minute at room temperature. Tissue pieces were washed with HBSS x2, DMEM-F12 x3 (1 : 1), and mechanically dissociated via vortexing and pipetting in 2 ml of medium.
  • HBSS Hank's balanced salt solution
  • the cell suspension was poured tough a 40 ⁇ M strainer (Falcon), centrifuged, and resuspended in stem cell medium: DMEM-F12 (1 : 1) containing antibiotics, B-27 (Gibco), 20 ng/ml EGF (Sigma) and 40 ng/ml bFGF (Sigma).
  • Cells were cultured in 100 mm tissue culture dishes (Sarstedt) in a 37 0 C, 5% CO 2 tissue-culture incubator. Floating spheres were formed following 24-48 hrs of culture. To passage floating cell spheres, medium containing spheres was centrifuged and the pellet was gently dissociated using a large bore pipette.
  • RT-PCR on differentiated cultures was performed as previously described (Dyce et al. 2004).
  • RT-PCR on individual large cells or blast-like cells was performed by freezing them in 7 ⁇ l lysis buffer containing 14 U porcine RNase inhibitor (Amersham) and 5 mM DTT (Invitrogen) at -80 degrees Celsius until use. Cells were then lysed by boiling for one minute and vortexing for 2 minutes and then stored on ice. The samples were then DNase treated and RT-PCR was carried out as previously described (Zhu, Craig et al. 2004).
  • Real-Time PCR was carried out on a Smart Cycler (Cepheid) using Quantitect SYBR green PCR kit (Qiagen). 2.5ul of DNase treated cDNA was added to 12.5 ⁇ l SYBR green mix and 0.3 ⁇ M each of forward and reverse primers. Primers and expected product size are shown in table 1. All products were sequenced to confirm identity.
  • Skin sphere cells were dissociated and plated on poly-D lysine/laminin (Sigma) coated culture dishes in differentiation medium as previously described with modifications.
  • Differentiation medium consisted of DMEM supplemented with, 5% FBS, 5% porcine follicular fluid, 0.23 imM sodium pyruvate, 0.1 mM non-essential amino acids (Gibco), 2 mM L-glutamine (Gibco), and 0.1 mM ⁇ -mercaptoethanol. Cells were maintained in this culture by replacing half the medium every 3-4 days. Following aggregate formation, medium was removed by centrifugation, aggregates were re-plated in fresh conditioned medium and cultured a further 10-14 days.
  • Immunoctochemistry was performed as previously described with modifications (Craig, Zhu et al. 2005). Briefly, cells were washed twice with PBS and fixed in 4% paraformaldehyde in PBS for 20 minutes. Cells were then washed three times in PBS with 0.1% tween20, and incubated for 20 minutes in PBS with 1% triton-X. Cells were then blocked for 3 hours in PBS with 5% BSA (PBS-B).
  • Cells were then incubated with primary antibody, 1/500 anti-Oct4 (Santa Cruz Biotechnology), 1/100 anti-Aromatase (Acris), 1/500 anti-SCC (Chemicon International), 1/500 anti-FSHr (Novus Biologicals), 1/600 anti-C-kit (BD Bioscience), for 2 hours followed by washing in PBS-B and incubating with 1/250 phycoerythrin (PE)-conjugated goat anti-rabbit IgG or 1/500 FITC-conjugated rat anti- mouse IgG for 1 hour at room temperature.
  • primary antibody 1/500 anti-Oct4 (Santa Cruz Biotechnology), 1/100 anti-Aromatase (Acris), 1/500 anti-SCC (Chemicon International), 1/500 anti-FSHr (Novus Biologicals), 1/600 anti-C-kit (BD Bioscience)
  • DAPI 4'-6-Diamidino-2-phenylindole
  • Oocytes and oocyte-like large cells were fixed in fixing solution (25% acetic acid plus 75% ethanol) for 12 hours at room temperature. Orcein staining was performed by incubating these oocytes in 1% acetic orcein for 30 minutes. The cells were then mounted with mounting medium (20% acetic acid plus 20% glycerin) and the glass cover was sealed with nail polish.
  • Example 7 Porcine primordial germ-cell isolation
  • Primordial germ cells were isolated as described (Shim et al., 1998, Theiogenology 49, 521-528). Briefly, sows at day 45 of gestation were killed and fetuses were isolated from the uterus. Genital ridges were gently lifted and isolated from the mesonephros and dorsal wall of the embryo using fine forceps. The isolated genital ridges were washed once with PBS and incubated in 0.02% EDTA solution for 20 min at room temperature. After incubation, the primordial germ cells were dissociatated by gentle disruption of the genital ridge using fine forceps. Supernatant containing the released cells was collected and centrifuged at 80Og for 5 min. Cells were then fixed in 4% paraformaldehyde until Immunocytochemistry was performed.
  • Oocyte maturation and IVF was performed as previously described (Craig, Zhu et al. 2005). Briefly, in vitro oocyte maturation (IVM) was performed as follows. Cell aggregates were washed three times with maturation medium [TCM199 (Gibco) supplemented with 5 IU/ml FSH (Sioux Biochemicals), 5 IU/ml LH (Sioux Biochemicals), 0.1 mg/ml Cysteine (Sigma), 10 ng/ml EGF (Sigma)] and incubated at 38.5°C for 42-44 hrs.
  • TCM199 Gibco
  • FSH 5 IU/ml FSH
  • 5 IU/ml LH (Sioux Biochemicals)
  • Cysteine Sigma
  • EGF EGF
  • Matured oocytes were washed three times in PBS containing 10% FBS, and once in fertilization medium containing 60 ⁇ M glucose, 34 ⁇ M sodium citrate, 12.4 ⁇ M EDTA, 17 ⁇ M citric acid, 54 ⁇ M trizma base and 3 ⁇ M caffeine-sodium benzoate (Sigma). The semen was collected from a boar, washed twice in PBS containing 10% FBS, and resuspended in fertilization medium. Approximately 50 oocytes were incubated in NCSU-23 (as described by (Lee, Kim et al. 2003) with sperm at 38.5 0 C for 10 min. Oocytes and attached sperm were then transferred to NCSU-23 supplemented with 4 mg/ml BSA and covered with light mineral oil. Embryos were cultured for 6-7 days in vitro to reach the blastocyst stage.
  • Aggregates removed from the differentiation culture were plated in 24 well dishes in fresh growth medium. Aggregates were maintained in culture for 15 days by replacing half the medium every 5 days. Medium was collected at day 10 and 15 of culture and assayed for hormone presence using ELISA.
  • Estradiol present in the cell culture medium was analyzed using an enzyme immunoassay kit for estradiol (Oxford Biomedical Research EA 70) according to the manufacturer protocol.
  • Progesterone present in the cell culture medium was analyzed using an enzyme immunoassay kit for progesterone (Oxford Biomedical Research EA 74) according to the manufacturer protocol.
  • Example 12 Methods of preparing non-human embryos for use in research and for fertility In vitro fertilization and embryo culture
  • Matured oocytes were washed three times in PBS containing 10% FBS, and once in fertil zation medium containing 60 ⁇ M glucose, 34 ⁇ M sodium citrate, 12.4 ⁇ M EDTA, 17 ⁇ M citric acid, 54 ⁇ M trizma base and 3 ⁇ M caffeine-sodium benzoate (Sigma). The semen was collected from a boar, washed twice in PBS containing 10% FBS, and re-suspended in fertilization medium. Approximately 50 oocytes were incubated in NCSU-23 as described by Lee, K., et al. 2003) with sperm at 38.5 0 C for 10 min. Oocytes and attached sperm were then transferred to NCSU-23 supplemented with 4 mg/ml BSA and covered with light mineral oil. Embryos were cultured for 6-7 days in vitro to reach the blastocyst stage.
  • SCP3 is expressed by female and male primordial germ cells of the mouse embryo.

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Abstract

L’invention est une population de cellules souches de peau à partir desquelles des cellules germinales sont dérivées. Plus particulièrement l’invention est orientée vers des procédés pour l’isolation et la différenciation des cellules souches de peau en des cellules germinales (ovocytes) et des populations purifiées des cellules germinales ainsi dérivées. L’invention comprend aussi des compositions de cellules germinales ainsi produites et les utilisations de telles cellules germinales comme source pour divers traitements et technologies reproductives.
PCT/CA2006/000558 2005-04-12 2006-04-12 Procedes pour la derivation de cellules germinales a partir de cellules souches de peau Ceased WO2006108284A1 (fr)

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WO2014205042A1 (fr) * 2013-06-20 2014-12-24 Elwha Llc Amélioration rapide d'animaux
US9681615B2 (en) 2013-06-20 2017-06-20 Elwha Llc Rapid breeding of plants
WO2025017354A1 (fr) * 2023-07-14 2025-01-23 Id Medical Group Australia Pty Ltd Population isolée de cellules autologues dérivées à partir de deux sources enrichie en cellules souches mésenchymateuses (csm) et procédés d'obtention et d'utilisation de celle-ci

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2022847A1 (fr) * 2007-08-07 2009-02-11 Irma Virant-Klun Cellules souches pluripotentes, procédés pour leur isolation et leur utilisation, et milieux de culture
WO2009019010A1 (fr) * 2007-08-07 2009-02-12 VIRANT-KLUN Irma Cellules souches de fluide folliculaire, procédés permettant de les isoler et leur utilisation, et milieux de culture
WO2014205042A1 (fr) * 2013-06-20 2014-12-24 Elwha Llc Amélioration rapide d'animaux
US9681615B2 (en) 2013-06-20 2017-06-20 Elwha Llc Rapid breeding of plants
WO2025017354A1 (fr) * 2023-07-14 2025-01-23 Id Medical Group Australia Pty Ltd Population isolée de cellules autologues dérivées à partir de deux sources enrichie en cellules souches mésenchymateuses (csm) et procédés d'obtention et d'utilisation de celle-ci

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