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    • 专利摘要:
    Method to obtain neural stem cells and neural progenitor cells from non-embryonic ovarian cortical tissue. The present invention relates to a method for obtaining neural stem cells and neural progenitor cells (ctn/cpn) from ovarian cortical tissue of mammalian prepubertal females, without the need to add induction and/or expansion factors to the culture medium. Neural lineage. With the method of the invention ctn/cpn are obtained after 6-21 days of incubation, improving the results by adding fsh to the culture medium. The ctn/cpn obtained by this method can be used in experimental models of in vitro neurogenesis, in targeted differentiation of ctn/cpn to generate various types of nerve cells of interest in regenerative medicine of the nervous system, with application to autologous transplantation in cellular therapy of neurodegenerative pathologies, as well as for the evaluation of neurotoxicity of various drugs, contaminants and chemical compounds. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2605655A1
    申请号:ES201601014
    申请日:2016-11-29
    公开日:2017-03-15
    发明作者:Rosa Ana PICAZO GONZÁLEZ；Belén SÁNCHEZ MALDONADO；Concepción ROJO SALVADOR；Pilar GARCÍA PALENCIA；Ángeles SÁNCHEZ PÉREZ；Pedro José ARANDA ESPINOSA；Teresa ENCINAS CEREZO；Juan Antonio GILABERT SANTOS；José María ROS RODRÍGUEZ；María Lourdes GALICIA GUERRERO
    申请人:Universidad Complutense de Madrid；
    IPC主号:
    专利说明:

    DESCRIPTION

    Method to obtain neural stem cells and neural progenitor cells from non-embryonic ovarian cortical tissue.
     5
    Technical sector

    The present invention falls within the biomedicine sector. More specifically, it refers to the isolation and culture of neural stem cells and neural progenitors (CTN / CPN) from ovarian cortical tissue culture. 10

    State of the art

    Neural stem cells (CTN) are multipotent cells that derive from embryonic stem cells or adult stem cells, after a process of specification that takes place both during embryonic development and, probably, in the postnatal period. In the course of neurogenesis, they experience intense proliferative activity followed by differentiation to generate different types of nerve cells. Although the CTNs are multipotent, the neural progenitor cells (NPCs) that derive directly from the CTNs, are compromised with the neural lineage, to which their differentiation potential is restricted. However, NPCs undergo numerous mitoses before definitively initiating differentiation towards the final cellular destination (Gage and Temple, 2013). Therefore, CTN / CPN have the capacity to generate all three types of nerve cells. when exposed to the action of appropriate factors and stimuli. 25

    CTN / CPN can generate spheroids spontaneously (Beck et al., 2011). The formation of spheroids, known in this case as neurospheres, is a consequence of the intense proliferative activity of these cells and the high level of surface adhesive protein expression, (reviewed by Achilli et al., 2012), so that 30 Each mitosis generates daughter cells that aggregate, or undergo aggregation without the need for previous mitosis. These spheroids can be integrated by CTN, CPN and nerve cell precursors whose presence depends largely on the conditions of the culture system. Of the transcription factors SOX2, Nanog and OCT4, (associated with pluripotentiality), they predominantly express SOX2 (Wang and 35 cols., 2012) and characteristic markers of the neural lineage, such as nestin, vimentin, PAX6, p75, among others ( Zhang and Jiao, 2015), in addition to showing intense proliferative activity.

    Currently, there are several procedures for obtaining CTN / CPN. First, they can be obtained from embryonic stem cells. that in culture are exposed to conditions of specification, expansion and subsequent differentiation to specific types of nerve cells (Lee et al., 2007; Naka et al., 2008). This approach involves obtaining and using preimplantation embryos, which adds complexity to animal experimentation, and ethical and legal impossibility in the human species.

    The need to overcome the ethical barriers related to the use of human embryos and to generate procedures of isolation, differentiation and autologous cell transplantation in programs of human regenerative medicine, opened a wide field of 50 research activity aimed at establishing procedures for induction of
    pluripotentiality in differentiated somatic cells. reprogramming of the cell genome. Takahashi and Yamanaka (2006) managed to reprogram murine fibroblasts to pluripotent cells, by transfection of the OCT4, SOX2, c-Myc and Klf genes. CTNs can be obtained from induced pluripotent cells, subjected to specification, expansion and differentiation procedures, similar to those applied to embryonic stem cells (Chambers et al., 2009; Koch et al., 2011). Although induction procedures have been optimized by different research groups, eliminating the expression of associated oncogenes, this approach adds even more complexity and collateral disadvantages due to the possible unwanted overexpression of transcripts related to reprogramming. 10

    As an alternative to these genome reprogramming procedures, chemical induction methods of pluripotentiality have been developed. Recently, a procedure has been described for obtaining CPN from embryonic fibroblasts in culture, under hypoxia conditions and exposed to the action of inhibitors of several signaling pathways (Cheng et al., 2014). In recent years, alternative pathways for chemical reprogramming of somatic cells have been investigated (Biswas and Jiang, 2016), as well as the direct conversion of somatic cells into nerve cells, after exposure to hypoxia conditions (Thier et al. , 2012) .These strategies require a wide contrast, before their application in cell therapy. twenty

    Finally, CTN / CPN can be isolated directly from neurogenic areas of the brain during embryonic development (Tropepe et al., 1999) and in the postnatal and adult period (Reynolds and Weiss, 1992), to be used in experimental models of In vitro development and neurodegenerative pathologies. 25

    For ethical or scientific-technical reasons, the previously mentioned experimental approaches are not entirely suitable for the establishment of autologous transplant models that provide a significant advance in regenerative medicine of the nervous system, for which it would be essential to obtain and use 30 adult stem cells of the individual. In relation to this strategy, research on neural derivation of adult stem cells from the connective tissue of skeletal muscle (Romero Ramos et al., 2002) and the hair follicle (Amoh et al., 2012) stands out. In the ovary of various species of mammals, it has been found the existence of pluripotent cells of epithelial and / or stromal origin from which, a part, can differentiate in culture cells that express some characteristic markers of nerve cells (Bukovsky et al. ., 2008; Parte et al., 2011; Gong et al., 2010; Stimpfel et al., 2014), a process promoted by the addition of some steroid hormones to the culture medium.
     40
    The conditions of the culture systems for obtaining CTN / CPN from the specification, expansion and differentiation of embryonic stem cells and induced pluripotent cells are currently well known (Yan et al., 2013, Naka et al., 2008). There are commercial culture media formulated to advance at each stage of the in vitro bypass process. In general, the defined means that are used in the specification and expansion stage, contain, in addition to essential additives, the EGF and FGF-2 mitogens, fundamental in the process of specifying pluripotent cells to the neuroepithelial lineage, in the maintenance of the undifferentiated status of these cells, in their proliferative activity and in their capacity for self-renewal (Eisellova et al., 2009, Nieto-Estévez et al., 2013). The terminal differentiation of 50 cells already committed to the neural lineage is achieved by removing the factors
    proliferation inducers, and incorporating differentiation factors to the defined environment, such as BDNF (brainderivedneurotrophicfactor). GDNF (glial cell dedved neurotrophic factor), PDGF (platelet-dedved growth factor), or fetal bovine serum (1%), in addition to essential additives.
     5
    Despite all the progress made, research is still underway to determine alternative methods that are simpler, more efficient and faster.

    References
     10
    Achilli T.M., Meyer J., Morgan J.R. (2012). Expert Opin. Biol. Ther. 12 (10): 1347-1360.

    Amoh Y., Mii S., Aki R., Hamada Y., Kawahara K., Hoffman R.M., Katsuoka K. (2012). Cell Cycle 11 (18): 3513-3517.
     fifteen
    Beck H.C., Petersen J., Felthaus O., Schmalz G., Morsczeck (2011). Neurochemical Research 36 (11): 2002-2007.

    Biswas D., and Jiang P (2016). lnt. J. Mol. Sci. 17: 226, Art. ID 17020226.
     twenty
    Bukovsky A. Claude M.R., Svetlikova M. (2008). Cell Cycle 7: 3577-3583.

    Chambers S.M., Fasano C.A., Papapetrou E.P., Tomishima M., Sadelain M., Studer L., (2009). Nat. Biotechnol. 27: 275-280.
     25
    Cheng L., Hu W. et al. (2014). Cell Research 24: 665-679.

    Eisellova L., Matulka K., Kriz V., Kunova M., Schmidtova Z. et al., (2009). Stem Cells 27: 1847-1857.
     30
    Gage F.H., Temple, S. (2013). Neuron vol. 80 nº 3: 588-601.

    Gil-Perotín S., Durán-Moreno M., Cebrián-Silla A., Ramírez M., García-Belda P., García-Verdugo J.M. (2013). The Anatomical Record 296: 1435-1452.
     35
    Gong SP, Lee ST, JH, Lee, EJ, Kim, DY, Lee, G., Chi, SJ, Ryu, 8-K., Lee, CH, Yum, KE, Lee, HJ, Han JY, Tilly, JL , Lim, JM (2010). Fertility and Sterility 93 (8) .Doi: 10.1016 / j.fertnstert.2009.12.053.

    Koch P., Breuer P., Peitz M., Jungverdorben J., Kesavan, J., Poppe D., et al. (2011). 40 Nature 480: 543-546.

    Lee H., Al Shamy G., Elkabetz Y., Schofield C.M., Harrsion N.L., Panagiotakos G., Socci N.O., Tabar V., Studer L. (2007). Stem Cells 25: 1931-1939.
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    Naka H., Nakamura S., Shimazaki T., Okano H. (2008). Nature Protocol Exchange, No. 479, doi: 10.1038 / nprot. 2008.168.

    Nieto-Estévez V., Pignatelli J., Aráuzo-Bravo M.J., Hurtado-Chong A., Vicario-Abejón C. (2013). PLoS ONE 8 (1): e53594. fifty
    Part S., Bhartiya D., Telang J., Daithankar V., Salvi V., Zaveri K., Hinduja 1 (2011). Stem Cells and Development 20 (8): 1451-1464.

    Reynolds B.A., Weiss S. (1992). Science 255: 1707-1710.
     5
    Romero-Ramos M., Vourc'h P., Young H.E., Lucas P.A., Wu Y., Chivatacarn O., Laman R., Dunkelman N., EI-Kalay A., Chesselet M-F. (2002). J. Neurosci. Res. 69: 894-907.

    Stimpfel M., Cerkovnik P., Novakovic S., Maver A., Virant-Kiun 1 (2014). J. Assist Play Genet 31: 959-974. 10

    Takahashi K., Yamanaka S. (2006). Cell126: 663-676.

    Thier M., Worsdorfer P., Lakes Y.B., Gorris R., Herms S., Opitz T., Seiferling D., Quandel T., Hoffman P., Nothen M.N., Brüstle O., Edenhofer F. (2012). Cell Stem Cell10: 473-479. fifteen

    Tropepe V., Sibilia M., Ciruna B.G., Rossant J., Wagner E. F., van der Kooy D. (1999). Dev. Biol. 208: 166-188.

    Wang Z, Oron E, Nelson 8, Razis, S, lvanova N (2012). Cell Stem Cell 10: 440-454. twenty

    Yan Y., Shin S., Jha B.S., Liu Q., Sheng J., Li F., Zhang M., Davis J., Bharti K., Zeng X., Rhao M., Malik N., Vemuri M.C. (2013). Stem Cells Translational Medicine 2: 862-870.

    Zhang J. and Jiao J. (2015). Biomed Research International 2015, Art.ID 727542: 1-14. 25

    Detailed description of the invention

    Method to obtain neural stem cells and neural progenitor cells from non-embryonic ovarian cortical tissue. 30

    One aspect of the present invention relates to a method for obtaining neural stem cells and neural progenitor cells (CTN / CPN) from postnatal-prepubertal ovarian cortical tissue.It is an alternative method with advantages over the methodologies in use. current in various species of mammals, from preimplantation embryos (embryonic stem cells), from genetically reprogrammed somatic cells (induced pluripotent cells), or from the fetal or postnatal brain for purely experimental purposes, since samples that have been used be taken beforehand and preserved. Such samples may come from mammalian animals, including the human species. This method includes the following 40 steps:

    a) fragment a sample of female ovarian cortical tissue in postnatal-prepubertal age until fragments of dimensions equal to or less than 1.0 x 1.0 mm are obtained;
     Four. Five
    b) enzymatically digest fragments of ovarian cortical tissue;

    c) mechanically disintegrate the digested fragments from step b);

    d) remove the preantral ovarian follicles and oocytes from the fragments broken down in step e), by filtration;
    e) incubate the cells obtained by steps a) - d) for 6-21 days in fibronectin-upholstered plates and in complete culture medium, which includes essential or serum-free maintenance medium, supplemented with albumin, insulin, transferrin, selenium and cholesterol, in which induction factors and / or neural expansion are not included.
     5
    Neural induction factors are understood as those factors that exert their action on pluripotent cells and cause the process of cellular specification towards the neuroepithelial lineage. Due to neural expansion factors. those that stimulate the proliferation of neural stem cells and neural progenitors are understood. Examples of neural induction factors are: FGF2 (fibroblast growth factor type 2), Nogina, Cordina, Folistatina, Cerberus, XNr3 and Sonic hedgehog (SHH), with the epidermal growth factor (EGF) being the main expansion factor used, as well as FGF2, HGF (hepatocyte growth factor), and IGF-I (insulin growth factor type 1).
     fifteen
    The culture medium may include antibiotics and / or antifungals to prevent contamination by bacteria and fungi.

    In addition, the complete medium may contain follicle-stimulating hormone (FSH) as an additive, which increases the yield in obtaining CTN / CPN. Preferably, 20 FSH is added to the medium at a concentration between 20-100 ng / ml.

    The method of the invention can be applied to samples of postnatal-prepubertal ovarian cortical tissue from mammals, including human mammals.
     25
    In this specification, the terms "ovarian cortical tissue" and "cortex" are used interchangeably.

    Using this method, cells from the postnatal-prepubertal cortical tissue of the ovary are isolated and cultured and cells that express genes 30 and characteristic CTN / CPN proteins are obtained in a homogeneous and consistent manner. After 6-21 days of culture, abundant neurospheres are obtained, spheroids whose cells express characteristic genes of CTN / CPN, such as: nestin, vimentin, SOX2, PAX6, p75 and transcripts characteristic of neural differentiation, such as: DCX (neurons) , GFAP (astrocytes) and Olig4 (oligodendrocytes). 35

    The cells obtained by the method of the invention can be used in the development of experimental models for basic research in neurogenesis and in autotransplant models in regenerative medicine of the nervous system.
     40
    In this invention, the cells of origin can be obtained using samples that have been taken from ovarian cortical tissue, which represents a remarkable qualitative advance compared to conventional systems for their isolation from preimplantation embryos, allowing to overcome the ethical and legal barriers that they affect, in this sense, the human species. On the other hand, this method provides greater simplicity in the procedures for obtaining these cells when using experimental animals. The definition of the specific method and the conditions of the culture, which allow to obtain a uniform cell population, with molecular identity corresponding to neural stem cells and neural progenitors, brings significant advances with respect to the usual procedures of isolation of these cells, 50 found in neurogenic niches of the brain in postnatal and adult age,
    such as the dentate gyrus of the hippocampus or the subependymal zone. The high performance of the process of the invention, in the production of cells with CTN / CPN markers, allows to propose autologous autologous transplantation of cells committed to the neural lineage, in neurological pathology cell therapy programs. In addition, the CTN / CPN obtained can be used in experimental models of neurogenesis in vitro, in directed differentiation of CTN / CPN to generate various types of nerve cells of interest in regenerative medicine of the nervous system. as well as for the evaluation of neurotoxicity of various drugs, contaminants and chemical compounds.
     10
    Brief description of the figures

    FIGURE 1. Representative graphs of the characterization of the initial cell suspension, by flow cytometry, demonstrating the expression and localization of the specific surface antigens of embryonic stem cells. TRA-1-60 and SSEA4, whose marking was evaluated individually (B, C) and combined (D), with respect to the negative control (A), represented by - / -.

    FIGURE 2. Representative microphotographs of the process of formation of spheroids similar to embryoid bodies, during the first (A, B) and second (C-F) week in 20 culture.

    FIGURE 3. Photomicrographs showing the exit of large cells from the spheroids, which actively migrate (A, B) to specific areas of the culture support, in which they appear to regroup (C). The cells that leave the spheroids have a high volumetric nucleus ratio: cytoplasm and frequent mitosis (D, E).

    FIGURE 4. Representative microphotographs of the differentiation processes of the cells that emerge from the spheroids, which acquire morphological appearance of differentiated nerve cells (A, B, E), and in differentiation (C, D, F). 30

    FIGURE 5. Graphical representation of the temporal evolution (D, time in days) of the diameters of the spheroids (Ø μm), in complete medium (C) and in complete medium with FSH as additive (FSH). The numbers (1, 2, 3, 4) and the letters (a, b) indicate statistically significant differences (p <0.01), due to the time within each treatment (C 35 or FSH) and between treatments at each time point, respectively.

    FIGURE 6. Graphical representation of the relative quantification (RQ) of the transcripts characteristic of pluripotentiality, SOX2 (A), OCT4 (B) and Nanog (C) determined by RT-PCRQ, during the three weeks of the culture, on the days ( D) corresponding sampling, with respect to their expression levels in the starting cell suspension (0, horizontal axis), in complete culture medium (C) and complete culture medium with FSH (FSH). The numbers (1, 2, 3) and the letters (a, b) indicate statistically significant differences (p <0.01) in the temporal expression of each transcript and due to the addition of FSH to the culture medium, respectively. Four. Five

    FIGURE 7. Graphical representation of the relative quantification (RQ) of the characteristic markers of CTN / CPN. nestin (A), vimentin (B), PAX6 (C) and p75 (D), determined by RT-PCRQ, during the three weeks of the culture on the days (D) of sampling (10, 15, 21), with respect to the expression levels of each transcript in the starting cell suspension (0, horizontal axis). The numbers (1, 2, 3) and the letters (a, b) indicate
    Statistically significant differences (p <0.01), in the temporal expression of each transcript and due to the addition of FSH to the culture medium at each time point, respectively.

    FIGURE 8. Graphical representation of the relative quantification (RQ) of the characteristic markers of neural differentiation, doublecortin (DCX; A). glia acid fibrillar protein (GFAP; B), and oligodendrocyte transcription factor 4 (OLIG4; C), determined by RT-PCRQ, during the three weeks of culture, on the corresponding sampling days (D), with respect to at the expression levels of each transcript in the starting cell suspension (0, horizontal axis). The numbers (1, 2), and the letters (a, b) 10 indicate statistically significant differences (p <0.01), in the temporal expression of each transcript and due to the addition of FSH to the culture medium at each time point, respectively.

    FIGURE 9. Representative microphotographs of the immunolocalization of nestin (A), vimentin (B), PAX6 (C) and p75 (0), in the spheroids obtained by the culture method of the invention.

    FIGURE 10. Representative images of the characterization analysis of the cell population by flow cytometry, carried out on the starting cell suspension 20 prior to culture (A), and on day 6 of culture, in the M1 groups (8 ), M2 (C) and M3 (D). Nestine (NEST), SOX2 (SOX), PAX6 (PAX) and p75 (P75) are analyzed.

    FIGURE 11. A-B: Semi-thin sections stained with methylene blue. A: Spheroid in which two parts are distinguished: the central or "core" region and the peripheral, structurally similar to an epithelium. B: Detail of the image A, at higher magnifications, which shows the interaction between the cells of the central and peripheral region of the spheroid. C-G: Ultramicrophotographs of the spheroids. C: Cell that has left the spheroid. D: Clear cells are located in the peripheral region of the spheroid. E: The central region of the spheroid contains dark cells and compacted by apposition of its membranes and abundant cell degeneration. F, G Image D, at higher magnifications, allows cells with indented nuclei (arrowheads) and cytoplasms to be observed with abundant coughs of rough endoplasmic reticulum (arrows), mitochondria (m) and filaments (f). Calibration bar: 100 μm (A); 50 μm (B); 1 μm (C, G); 5 μm (D); 10 μm (E); 2 μm (F). 35

    FIGURE 12. A-C: Ultramicrophotographs of spheroids showing details of the cytoplasm of clear cells: A: Clustered filaments (arrow). B: Abundant structures similar to lysosomes and phagosomes (arrow). C: Apical surface of clear cells with abundant well developed microvilli; interdigitation in the apical area 40 of the membranes (arrowhead). The arrows indicate the presence of adherent intercellular junctions. D: Dark cells in the inner region of the spheroid showing reticulated nucleus (arrow) and apposition of membranes. E: Intercellular junctions and contacts in clear cells: adherent junctions (small arrows), strong interdigitation (arrowhead) and cytoplasmic processes with labile interdigitations 45 with adjacent cells (large arrow). F: Images of cell degeneration (probably apoptosis) in the central region of the spheroid. Calibration bar: 0.2 μm (A); 1 μm (B, C, D); 2 μm (E); 5 μm (F).

     fifty

    Embodiment of the invention

    1.- Method of obtaining CTN / CPN

    1.1.- Biological starting material: for this example, ovarian cortical tissue of 5 prepubber sheep (Ovis aries) was used. The biological material was obtained in the slaughterhouses of Alpedrete (Carnes Alpedrete, SAT), Colmenar Viejo (Cárnica Colmenar, SC) and Leganés (Transformación Ganadera de Leganés, SA), in Madrid. The ovaries were dissected from the canal, immediately after slaughter, and transported to the laboratory in sterile containers containing culture medium (M199, Sigma, ref. M7528), 10 with antibiotic and antifungal (antibiotic, antifungal 100X Gibco. Life Technologies, ref. 15240), in a refrigerator, at 2-8ºC. Upon arrival at the laboratory, they were transferred to containers with renewed medium with antibiotic and antifungal, prior to processing.

    1.2.- Obtaining fragments of the ovarian cortical tissue: the ovaries were placed 15 on Petri dishes, on ice, and repeated cuts were made, in areas devoid of macroscopically visible antral follicles and under the stereoscopic microscope, until obtaining fragments of smaller dimensions at 1.0 X 1.0 mm. On average. About 15-20 ovaries were used in each experimental session and about 40 sessions were carried out for the development of the method. twenty

    1.3. Media preparation:

    1.3.1.- Preparation of the enzyme digestion medium: 0.08 g of bovine serum albumin (BSA. Sigma, ref. A9418) were weighed. 0.12 g of collagenase type IA (Sigma, 25 ref. C2674). 20 ml of Hanks solution with Ca2 + and Mg2 + (Sigma, ref. H9269), previously tempered at 37 ° C, 2000 Uf of DNAse (Sigma, ref. 04513) were added. The mixture was sterilized by passing through a 0.22 μm pore filter (Millex GP, ref. SLGP033RS, Millipore) and 200 ~ 1 of antibiotic and antifungal were added.
     30
    1.3.2.- Preparation of the resuspension medium: 0.04 g of BSA were weighed and 10 ml of Hanks solution without Ca2 + or Mg2 +, previously tempered at 37 ° C, and 1000 Ul of DNAse were added; The mixture was sterilized by filtration and 100 μl of antibiotic and antifungal were added.
     35
    1.3.3.- Preparation of the complete medium and control medium: 0.20g of BSA were weighed and 50 ml of M199 medium were added with 25mMHepes (Sigma ref. M7528), previously tempered at 37 ° C, 500 μl of STI (Insulin, transferrin and selenium, Sigma, ref. I3146-5ML), 100 μl of Synthecol (cholesterol, Sigma ref. S5442) and FSH (follicle stimulating hormone) sheep (0 or 50 ng / ml, in control groups (M2) and in treated groups with FSH (M3), 40 respectively; National Institute of Diabetes & Digestive & Kidney Diseases, Ref. NIDDK-oFSH-18 AFP58620). The mixture was sterilized by filtration and 500 ~ 1 of antibiotic and antifungal were added. This culture medium was used in the cultures of one month of duration, which served to establish the method and perform the molecular, structural and ultrastructural characterization of the spheroids. The two treatment groups (control and FSH) were maintained throughout three weeks of culture, in all experimental repetitions.

    1.3.4.- Preparation of the positive control medium: its composition is identical to that of the complete control medium (without FSH), supplemented with EGF (20 ng / ml; Sigma Ref.E9644) and 50 FGF-2 (20 ng / ml ; Sigma Ref. F0291). This culture medium (M1) was used in the group
    positive control of neural induction, in the experiments performed to compare comparatively the efficiency of the culture method presented. in which the cultivation period was one week.

    1.4.- Enzymatic digestion of cortex fragments: In the laminar flow cabinet, the cortex fragments, obtained as described in example 1.2, were transferred to a vial with enzymatic digestion medium. The mixture was kept under gentle and permanent stirring at 37 ° C for 30 minutes; centrifuged (500 g, 10 min, at laboratory temperature); the supernatant was removed and 10 ml of resuspension medium was added.
     10
    1.5.- Resuspension and mechanical disintegration of fragments: in the laminar flow cabinet and with Pasteur pipettes of decreasing tip diameter, the suspension was subjected to repeated aspiration, in order to achieve mechanical disruption of the fragments not broken down by the enzyme ; centrifuged (500 g, 12 min, at laboratory temperature); the supernatant medium was removed, and the pellet was resuspended with complete medium.

    1.6.- Preparation of the cell suspension by filtration: to remove the developing preantral ovarian follicles and oocytes from the cell suspension, this was subjected to a successive filtration process through 100 μm, 70 μm mesh and finally 20 , 40 μm of plot (Cell Strainer, Beckton Dickinson, BD Falcon).

    1.7.- Analysis of cell viability: an aliquot was removed from the suspension (20 μI), the same volume of trypan blue solution (Sigma, ref. T8154) was added and a count of viable and non-viable cells was performed under a microscope optical, in the Neubauer chamber. 25 The average viability percentages were between 70 and 90%.

    1.8.- Cultivation of ovarian cortical cells.

    1.8.1.- Preparation of culture plates: 24-well plates (Nunclon Delta, 30 ref. 142475) were upholstered with a solution of fibronectin (Sigma, ref. F4759; 10 μg / ml in M199 medium), and they were kept in the oven at 37 ° C, 5% CO2 and saturation humidity, for 24 hours prior to cultivation.

    1.8.2.- Cell culture: the fibronectin solution was removed, a volume of cell suspension containing 500,000 living cells was dispensed, in each well and tempered complete culture medium was added, to a final volume of 500 μl.

    • In the experiments that allowed establishing the culture method and performing the molecular, structural and ultrastructural characterization of the spheroids, 40 cells were cultured for 3 weeks at 37 ° C, with 5% CO2 and moisture saturation.

    • Once the culture method was established, in the experiments whose objective was to define the time course of the process of specification and expansion, to obtain CTN / CPN, the cells were grown for 6 days at 37 ° C, with 5% CO2 and 45 humidity to saturation.

    • The culture medium was replaced by a renewed medium of the same composition, every 48 hours and stored at -20º C in identified individual vials, for subsequent analysis of hormones and local regulatory factors. fifty

    2.- Method validation tests and results

    2.1.- The ovarian cortical tissue contains pluripotent cells, antigenically similar to embryonic stem cells. The analysis of the cell population of the starting suspension was performed, by flow cytometry, using antibodies 5 for TRA-1-60 and SSEA4, (Milli-MarkTM, Merck-Millipore, Anti-SSEA-4, Clone MC-813 -70 PE conjugate, ref. FCMAB 116P and Anti-TRA-1-60, clone TRA-1-60 FITC conjugate, ref. FCMAB 115F), which specifically recognize embryonic stem cell membrane glycoproteins of the human species. The cell suspension was prepared as described in examples 1.4-1.6. 500,000 live cells were dispensed into each of 4 10 eppendorf vials, washed with 500 IJI of tempered phosphate buffer solution (PBS) and resuspended in incubation medium (PBS, 2mM EDTA. Sigma ref. E-6758), 1% of BSA, without antibodies (negative control) or with antibodies (5 μI of each antibody individually and in combination), to a final volume of 50 μI, keeping the incubation for 30 minutes at 2-8ºC, protected from light. Then, 15,500 μI of PBS at 2-8 ° C was added to each vial, the samples were centrifuged at 500 g for 10 minutes, the supernatant was removed and each pellet was resuspended with paraformaldehyde solution (Sigma, ref. P6148) at 0 , 5% in PBS. The samples were protected from light, at 2-8 ° C, until analysis by flow cytometry, 24 hours later, in samples of the cell population comprised between 25,000 and 50,000 cells. twenty

    Results: 14.99% of the cells presented immunoreactivity to TRA-1-60 and between 1.50% and 4% to SSEA4 (Figure 1). Almost all of the cells that expressed this last marker, colocalized TRA-1-60. 12.96% of the cells located TRA-1-60 exclusively. This indicates that approximately 15% of the cells of the ovarian cortical tissue are adult stem cells, antigenically similar to embryonic stem cells.

    2.2.- With this method of culture, the cells of the ovarian cortical tissue form spheroids similar to embryoid bodies. During the 3 weeks of culture, in each one of the experimental replicas, two weekly points of evaluation of the dynamics of cellular organization in the culture were carried out, and the morphometric evaluation of the development of spheroids generated in vitro, in the that the diameter of an approximate number of 200 was determined and its evolution during the cultivation period. The measurements were made on photographic images of the cultures under an inverted microscope (Nikon Eclipse Ti S) equipped with a digital camera (Nikon DS-Fi1) and image analysis software (NIS-D-Elements, Nikon). The influence of the addition of FSH to the complete culture medium on the development of spheroids in vitro was evaluated. The findings related to cell proliferation, migration and differentiation processes towards specialized cell types were reported. 40

    Results: from the beginning of the culture period, in particular from day 3, it was observed that a significant proportion of cells initiated the formation of aggregates that underwent a progressive increase in their diameters, until forming compact spheroids at the end of the first week of crop (Figure 2). On average, between 65 and 75 spheroids were counted in each culture well. As notable findings, the presence of frequent mitosis during the entire culture period (Figure 3D, E), the appearance of patterns of orderly migration of large diameter cells with a high nucleus-cytoplasm ratio from the spheroid to the support of surrounding culture (Figure 3A-C), from the end of the first week, and the presence of 50 abundant cells with differentiated neural morphology (Figure 4), during the second
    cultivation week The spheroids reached maximum development in the second week, with diameters between 120 and 200 μm. who experienced a decrease (p <0.01) from the end of the second week onwards (Figure 5).

    The addition of FSH hormone to the complete culture medium, caused significant increases in the diameters of the spheroids (p <0.01), in the second and third week of culture, with respect to cultures not exposed to the hormone, as shown in the graph corresponding (Figure 5).

    2.3.- The spheroids have an intense and consistent alkaline phosphatase activity throughout the culture. The detection of alkaline phosphatase activity is used as an initial qualitative analysis in the characterization of pluripotent cells.

    Each week, starting from day 5 of culture, the alkaline phosphatase activity analysis was performed in a representative number of wells. A commercial kit 15 (Alkaline Phosphatase Detection Kit. Millipore ref. SCR004, Merck-Millipore) was used according to the manufacturer's instructions.

    Results: intense alkaline phosphatase activity was detected in all spheroids during the culture period. which turned out to be maximum in the first two weeks of it. In the third week, in spheroids of larger diameter, this activity was restricted to peripheral cells.

    2.4.- The molecular characterization of the spheroids throughout the culture demonstrates the predominant expression of pluripotentiality transcripts and the neuroepithelial lineage.

    2.4.1.- Expression of specific genes of pluripotentiality.

    The expression of the transcription factors OCT4, Nanog and SOX2, was analyzed by quantitative RT-30 PCR, in the cell suspension prepared before culture (day 0) and in three time sampling points during the three weeks of culture (days 10 , 15 and 21), in total RNA extracts obtained from lysates of 300 spheroids per treatment and sampling point in each experiment, with lysis buffer (Cell lysis buffer solution; Ambion, ref. 8540G2), on ice. The RNA was extracted in purification columns (Kit 35 RNeasymini kit, Quiagen, ref. 74104) and after treatment with DNAse (Turbo DNA Free Kit, Ambion ref. AM1907) it was subjected to analysis by quantitative RT-PCR, in the Unit of Genomics Antonia Martín Gallardo of the Madrid Science Park on the Cantoblanco Campus, according to usual procedures. The design of the primers used in the analysis was based on the sequences of messenger RNAs 40 corresponding to OCT4, Nanog, and SOX2 in the sheep species, published by the NCBI (National Center of Biotechnology Information), whose access numbers are detailed in table 1.

    The expression levels of each transcript, determined in the cell suspension before culture (OD), were adopted as reference values for the relative quantification of each of them over time in culture, in each group (C vs FSH).

    Results: As shown in Figure 6, the expression of transcripts associated with pluripotentiality. OCT4, Nanog and SOX2, remained in the spheroids throughout the three weeks of culture, with temporary variations. The most remarkable result of
    These experiments were the increase in the expression of SOX2 recorded on day 10 of culture, with respect to the levels of expression of this transcript in the starting cell suspension (Figure 6A). The SOX2 transcript was maintained above baseline levels (D0) throughout the culture, being higher in the FSH-treated group (FSH) at 21 days (D21), compared to the control group (C). Against the increase in the levels of SOX2 transcription, OCT4 (Figure 6B) and Nanog (Figure 6C), they simultaneously experienced a decrease in expression with respect to their reference values (D0) which, in the case of OCT 4 (Figure 6B), traced back to them on day 21 in culture (D21), in the presence of FSH (Figure 6B).
     10
    When cells are pluripotent, both in adult and embryonic trunks, the balanced expression of these three transcripts (OCT4, NANOG and SOX2) keeps the cell in an undifferentiated status and capable of permanent self-renewal. In the first step of choosing the cellular destination, the specification produces the predominance in the expression of one of the three transcripts. The predominance in the expression of SOX2, over that of OCT4 and NANOG, directs the cellular specification towards the neuroepithelial lineage, that is to say: the cell begins the process of derivation towards that destination. The first cell type that arises is the neural stem cell, with high expression of SOX2, in addition to nestin, vimentin and others. The neural stem cell is particular because it is still multipotent and, if it is exposed to the action of certain factors, it can be differentiated to cells of 20 different tissues. When the neural stem cell advances in its differentiation process, the expression of SOX2 decreases.

    2.4.2.- Expression of characteristic transcripts of neural differentiation and specification. 25

    The expression of nestin, vimentin, PAX6 and p75 as marker transcripts of neural specification and of DCX, GFAP and Olig4, as transcripts associated with neural differentiation (neurons, astrocytes and oligodendrocytes, respectively) were analyzed. The procedure described in section 2.4.1 was followed. The sequences of the primers 30 used are detailed in Table 1.

    The expression levels of each transcript, determined in the cell suspension before culture (D0), were adopted as reference values for the relative quantification of each of them over time in culture in each group (C vs FSH) , 35 as in 2.4.1.

    Results: The transcription of nestin, vimentin, the neurotrophin receptor p75 and PAX6, characteristic markers of CTN / CPN, was predominant at all time points of analysis throughout the culture. The expression of the four markers 40 increased (p <0.01) with respect to their transcription levels in the starting cell suspension (Figure 7 A-D). In the case of nestin (figure 7A), vimentin (figure 7B) and neurotrophin receptor p75 (figure 7D), the increase in the level of expression with respect to the reference levels (D0) was observed in the sampling of day 10 (D10) of culture, and in the case of PAX6 (Figure 7C), its transcription experienced sustained increases in time, quantified from day 10 onwards (D10-D15-D21), being significantly higher in the presence of FSH than in his absence (C). The expression of p75 (Figure 7D) decreased from day 10 of culture, this decrease being significant on day 15 in the presence of FSH (FSH) and on day 21 in complete medium without FSH (C). fifty
    Also predominant was the expression of the transcripts associated with neural differentiation (Figure 8), doublecortin (OCX; figure 8A), glia acid fibrillar protein (AP GF; figure 8B) and oligodendrocyte transcription factor 4 (Oiig4; figure 8C ). The relative expression analysis of the three markers at each time point demonstrated the predominance of GFAP and DCX expression over the transcription of Olig4 (GFAP> DCX> Olig4). 5 The increase in the expression of DCX and AP GF in the spheroids was remarkable, in the interval from the beginning of the culture (D0) to day 10 (D10), with subsequent decrease (D15, D21) to the reference values ( D0), both in the presence (FSH) and in the absence of FSH (C). In relation to the expression variations of Olig4, the addition of FSH to the complete culture medium caused significant increases in its transcription 10 (p <0.01), on days 10 and 15 of the culture.

    The expression levels of each transcript, determined in the cell suspension before culture (D0), were adopted as reference values for the relative quantification of each of them over time in culture in each group (C vs FSH) . fifteen

    2.4.3.- Relative expression of transcripts from the three germinative strata (ectoderm, endoderm and mesoderm), pluripotentiality and neural induction and specification.
     twenty
    A relative quantification analysis of characteristic transcripts of ectoderm (nestin), endoderm (alpha-fetoprotein) and mesoderm (Brachyury) was performed. The procedure described in section 2.4.1 was followed. The sequences of the oligonucleotides used are detailed in Table 1.
     25
    Results: The expression of pluripotential transcripts, OCT 4, SOX2 and Nanog, was maintained throughout the culture, but at a significantly lower level (p <0.01) with respect to the expression of nestin, p75, vimentin, DCX, GFAP and Olig4, in the first two weeks, and with respect to PAX6 in the second and third week. The expression of pluripotentiality transcripts remained at levels similar or even somewhat higher than the expression of the characteristic genes of endoderm specification (AFP) and mesoderm (Brachyury). The expression of Brachyury, decreased in the first week of culture with respect to the starting levels (p <0.01) and remained with levels in the detection limit of the technique in the second and third week. The expression of AFP, residual as well as that of Brachyury, experienced an increase in the first and second week of culture, with respect to the quantified values in the starting cell suspension. The predominant expression was therefore that of transcripts of specification and neural differentiation.




    2.5.- The cells that make up the spheroids express mostly CTN / CPN markers: immunolocation experiments. 5

    In representative samples of spheroids obtained on days 10, 15 and 21 of culture, the immunolocalization of nestin, vimentin, PAX6 and the p75 neurotrophin receptor was carried out. This analysis was carried out on serial cuts of the spheroids included in paraffin in groups (30-40 spheroids per treatment group and time in 10 each block), using primary antibodies and commercial immunohistochemical reagents (Novolink Polymer Detection System, Novocastra ref. RE7150 -K), following the manufacturer's instructions.

    Incubation with primary antibodies was carried out in a humid chamber, under the following conditions: the anti-PAX6 antibody (ref. 030765, Sigma-Aldrich, lnc, Saint Louise, USA) was used at a 1: 400 dilution, in incubation overnight at 2-8 ° C; anti-p75 antibody (NGFR p75; ref. N3908, Sigma Aldrich, lnc, Saint Louise, USA) was used at a 1: 1500 dilution, overnight incubation at 2-8 ° C; anti-5 vimentin antibody (clone V9 Dako Glostrup, Denmark) was used at a 1: 500 dilution, with incubation for 1 hour at laboratory temperature; the anti-nestin antibody (ref. N5413 Sigma Aldrich, lnc, Saint Louise, USA) was used at a 1: 200 dilution with 1 hour incubation at laboratory temperature. Immunolocation was revealed with diaminobenzidine and the cuts were contrasted with hematoxylin. After assembly, the image and count analysis of positive and negative cells was performed for each marker.

    Results: The percentages of cells with immunolocalization of the CTN / CPN, nestin, vimentin, p75 and PAX6 markers, during the three weeks of culture, are detailed in Table 2. Neither the time in culture nor the addition of FSH in the medium they influenced the percentages of cells with immunolocalization of nestin and vimentin. The addition of FSH to the medium, reduced the percentage of cells with immunolocalization of PAX6 only on day 10 of culture, and progressively increased the percentages of immunolocalization of this marker, which were significant on day 21 of culture, compared to day 10. The 20 adding FSH to the medium, reduced the percentages of cells with immunolocation of p75 on days 10 and 15 (p <0.05), without affecting them over time in culture. In figure 9, representative images of the immunolocalization experiments of nestin (figure 9A), vimentin (figure 9B), PAX6 (figure 9C) and p75 (figure 9D) are presented. 25

    Table 2. Results of the immunolocation of the markers associated with the neuroepithelial lineage, nestin, vimentin, p75 and PAX6.

     30

    Different numbers indicate significant differences (p <0.05; ANOVA) between different time points for each antigen, in each group (e vs FSH). The different letters indicate significant differences (p <0.05; ANOVA) in the immunolocation of each marker, associated with the addition of FSH to the complete culture medium (e vs FSH), 35 within each time point of analysis.
    2.6.- The process of specification takes place from day 6 of culture, when the cells mostly place four characteristic markers of CTN / CPN and remain in a proliferative state.

    A cell characterization analysis was performed by flow cytometry during the first 5 days of culture, with the objective of studying the temporal evolution in the expression and immunolocation of characteristic markers of neural stem cells and neural progenitors and a cell proliferation marker. An experimental group exposed to the M1 medium, described in example 1.3.3., Was included as a positive control medium. For it. The cells were cultured according to the procedure indicated in section 1.8. In these experiments, media and solutions without phenol red were used in order to avoid autofluorescence phenomena in cell preparations, whereby equivalent products, free of this pH indicator, were used. For cell labels prior to flow cytometry, primary anti-SOX2 antibodies (ref. S9072, Sigma Aldrich, lnc, Saint Louise, USA) were used. nestina (ref. N5413 Sigma 15 Aldrich, lnc, Saint Louise, USA), p75 (ref.N3908, Sigma Aldrich, lnc, Saint Louise, USA), PAX6 (ref. 030765, Sigma-Aldrich, lnc, Saint Louise, USA ) and K67 (ref. PA0118, Leica Biosystems, Nussloch, Germany), marked with PERCP fluorochromes (Abcam Per CP Conjugation Kit ref. ab102907; Abcam, Cambridge, UK), Alexa488 (Apex Antibody Labeling Kits, ref. A 10468: lnvitrogen, Thermo Fisher Scientific, Massachusetts USA), PE 20 (Zenon Rabbit lgG Labelling Kits, ref. Z25355, lnvitrogen Thermo Fisher Scientific, Massachusetts, USA) and Alexa 647 (Zenon Rabbit lgG Labelling Kits, ref. Z25308, Invitrogen Thermo Fisher Scientific , Massachusetts, USA), in order to reveal the colocalization of the corresponding antigens and their temporal evolution in culture. The analyzes were performed on days 0, 3 and 6 in culture. On day 0, after preparing the cell suspension prior to culture, the cells were subjected to fixation in 2% paraformaldehyde solution in PBS at pH 7.4, for 15 minutes at 2-8 ° C, followed by permeabilization with PBS pH7.4 with Triton X100 (1/1000) for 5 minutes at 2-8 ° C and subsequent blocking in PBS with 5% BSA for 5 minutes at 2-8 ° C. On days 3 and 6, the cultures were treated with Accutase solution (Stem Pro Accutase Gibco lnvitrogen. 30 Ref. A 11105-01), following the manufacturer's recommendations. In all cases, the resulting cell suspensions were distributed in eppendorf vials (200,000 to 500,000 cells per vial) and incubated for 60 minutes at 2-80C in the dark, with antibodies previously labeled with fluorochromes, in single and in combination. Then, they were washed by the addition of refrigerated PBS and resuspended in 0.5% paraformaldehyde solution in PBS at pH 7.4, until flow cytometric analysis was carried out 24 hours later at the Cytometry Center and Fluorescence Microscopy of the Complutense University of Madrid.

    Results: The results showed that in the cell suspension prior to culture there are 40.32% of cells with the Nestin + / SOX2 + / Pax6 + / Ki67 + phenotype, characteristic of CTN / CPN. In the positive control group (M1), on day 3, the percentage was 45%, compared to 4.45% in M2 and 1.21% in M3. On day 6 of cultivation, these percentages were 98.45% in the M1 group, 83.16% in the M2 and 88.77% in the M3 group. The results show that, on day 6 of culture, neural induction is achieved in a majority of the three treatment groups. The spheroids of the M2 and M3 groups are composed of cells that express mostly antigen phenotype corresponding to CTN / CPN. Part of the most representative results are presented in Figure 10.
     fifty
    Against an approximate percentage of 10% of cells with immunoreactivity for SOX2 before culture, of which more than 99% placed nestin and Ki67 (A), on day 6 of culture, SOX2 was located in 98% (M1, B ), 92.3% (M2, C) and 94% (M3, D) of the cells that, in addition, showed majority colocalization with nestin (NEST), PAX6 (PAX) and p75 (P75), characteristic markers of CTN / CPN 5

    2.7.- The characterization of spheroids by ultrastructural analysis corroborates that these are neurospheres.

    The ultrastructural analysis by transmission electron microscopy was carried out in 10 sampled spheroids during the second week of culture. The spheroids were subjected to fixation in 4% paraformaldehyde solution and 0.5% glutaraldehyde, in PBS for 2 hours at room temperature. After washing with PBS, the spheroids were transferred to a solution of 1% osmium tetroxide in PBS, for 1 hour, dehydrated by immersion in increasing concentrations of ethanol-15 acetone and were finally included in Epon 812 resin. Semi-thin (0.5 μm thick) and ultra-thin (50-70 nm thick) sections with an ultramicrotome (Leica-Reichert Ultracut E), which were stained respectively with methylene blue and uranyl lead acetate citrate. The sections were examined under the transmission electron microscope (Jeol Jem 1010), at the Center for Electron Microscopy of the Universidad 20 Complutense de Madrid.

    Results: The ultrastructural analysis of the spheroids (Figures 11 and 12), allowed to highlight numerous similarities between these and the neurospheres, spheroids integrated by CTN / CPN, described by other authors (Gil-Perotin et al., 2013). The observation of 25 semi-thin sections (Figure 11A-B), allowed to distinguish two areas of cellular distribution in the structure of the spheroid: the central or "core" region and the peripheral area, composed of cells arranged as epithelium, among which Some large cells that protrude and leave the spheroid (Figure 11C) stand out. Among the similarities of the spheroids of the invention with the neurospheres, the following stand out: two main cell types are distinguished, according to their electronic density, the light cells (figure 11D) and dark ones (figure 11E), distributed in the sphere so organized. Both types of cells have a great abundance of free ribosomes, dispersed chromatin patterns and nucleoli with cross-linked configuration, indicating that the cells are very active from the point of view of protein synthesis (Figure 11F, G). The cells 35 located on the periphery of the sphere are mostly clear and are organized in one or several layers. In the central area of the sphere, dark cells predominate, which show a more disordered spatial organization. Stresses, in light cells, the abundant presence of filament bundles (Figure 12A), which is rarely observed in dark cells, and the abundance of lysosomes and phagosomes 40 (Figure 12B). In the spheroids of the present invention, as well as in the neurospheres characterized by the authors referred to above, the clear cells of the periphery have abundant intercellular junctions of adherent type, which play an important role in the processes of cellular aggregation and migration that characterize to the formation and development of neurospheres (figure 12C, E). The image analysis of the 45 clear cells of the periphery of the spheroids of the present invention revealed an intense cellular metabolism, given the characteristics of its cytoplasm and the abundance of organelles, associated with the active exchange of nutrients and, possibly, regulatory factors. . In the stratum or cellular strata of the periphery, numerous interdigitations are also distinguished as a type of intercellular junction (Figure 12C, E). In addition, 50 highlights the high incidence of clear cell migration activity plus
    superficial, which first appear flattened and progressively protrude, until detached from the spheroid and migrate (Figure 11C). The cells of the spheroid are morphologically heterogeneous, often with dark nuclei, and with a high incidence of phagocytosis, apoptosis and cell necrosis phenomena, which predominate in the central cell mass of the spheroid (Figure 12F), while the periphery is 5 composed of more active cells with intense biosynthetic activity and intercellular junctions as described in this example.

    With the results obtained in the examples set forth herein, we can conclude that the spheroids of the invention are neurospheres. 10
    权利要求:
    Claims (8)
    [1]

    1. Method for obtaining neural stem cells and neural progenitor cells (CTN / CPN) from samples of postnatal / prepubertal ovarian cortical tissue that includes the following steps:
    a) fragment a sample of postnatal / prepubertal ovarian cortical tissue to obtain fragments of dimensions equal to or less than 1.0 x 1.0 mm;
    b) enzymatically digest the fragments of ovarian cortical tissue from step a); 10
    c) mechanically disintegrate the digested fragments from step b);
    d) remove the preantral ovarian follicles and oocytes from the fragments disaggregated from step 15
    e), by filtration; e) incubate the cells obtained by steps a) - d) for 6-21 days in plates covered with fibronectin and in complete culture medium, which includes essential or maintenance-free serum, supplemented with bovine serum albumin, insulin, transferrin, selenium and cholesterol, 20
    where induction factors and / or expansion of the neural lineage are not included.

    [2]
    2. Method according to claim 1 wherein the culture medium of step e) includes follicle-stimulating hormone (FSH). 25

    [3]
    3. Method according to claim 2 wherein the concentration of FSH is 50 ng / ml of culture medium.

    [4]
    4. Method according to any of the preceding claims wherein the culture medium of step e) includes HEPES buffer, antibiotics and / or antifungal.

    [5]
    5. Method according to any of the preceding claims wherein the incubation of step e) is maintained for 6 days.
     35
    [6]
    6. Method according to any of the preceding claims wherein the samples of ovarian cortical tissue are derived from a mammal.

    [7]
    7. Method according to claim 6 wherein the mammal is of the sheep species.
     40
    [8]
    8. Method according to claim 6 wherein the mammal is of the human species.

     Four. Five
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    CN111719003A|2020-07-14|2020-09-29|安徽省天长市周氏羊业有限公司|RBP1 gene and application thereof, sheep ovary in-vitro development quality evaluation method and amplification primer|GB0025088D0|2000-10-13|2000-11-29|Univ Edinburgh|Stem cells|
    WO2008020657A1|2006-08-14|2008-02-21|Seoul National University Industry Foundation|Acquired pluripotency through ovarian niche|
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