WO2020205464A1 - Cellules de follicules ovariens et constructions pour le traitement de la fertilité et une hormonothérapie substitutive - Google Patents

Cellules de follicules ovariens et constructions pour le traitement de la fertilité et une hormonothérapie substitutive Download PDF

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WO2020205464A1
WO2020205464A1 PCT/US2020/025111 US2020025111W WO2020205464A1 WO 2020205464 A1 WO2020205464 A1 WO 2020205464A1 US 2020025111 W US2020025111 W US 2020025111W WO 2020205464 A1 WO2020205464 A1 WO 2020205464A1
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cells
granulosa
oogonia
ovarian
theca
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PCT/US2020/025111
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English (en)
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Sittadjody SIVANANDANE
Russel C. SEQUEIRA
John D. Jackson
Anthony Atala
James J. Yoo
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Wake Forest University Health Sciences
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Priority to US17/449,015 priority Critical patent/US20220010270A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells

Definitions

  • Loss of ovarian function leading to infertility and hormonal imbalance can be from natural causes (e.g., premature ovarian failure) or due to surgical removal of ovaries as a part of cancer treatment or other gynecological -related issues.
  • donor eggs vitrified oocytes from a donor
  • assisted reproductive techniques It was previously believed that eggs (oocytes) are formed from their mother cells (oogonia) during the embryonic period, and all female babies are born with a definite set of pre-formed oocytes. This definite pool of pre-formed oocytes declines with age in addition to the monthly procurement of a few oocytes for maturation and ovulation.
  • OSCs oogonia stem cells
  • HRT hormone replacement therapy
  • a method of providing a culture of oogonia stem cells comprising: (a) providing a mixed population of ovarian cells isolated from an ovary tissue (e.g., adult human ovary tissue); (b) separating and collecting ovarian cells that are positive for: i) DEAD box helicase peptide 4 (DDX4, also known as Vasa homologue), and/or ii) interferon-induced transmembrane protein 3 (IFITM3, also known as fragilis), from said mixed population, to provide a double positive population of cells; and (c) culturing the double positive population of cells, wherein the double positive population of cells comprises oogonia stem cells.
  • ovary tissue e.g., adult human ovary tissue
  • IFITM3 interferon-induced transmembrane protein 3
  • the separating is carried out with fluorescent-activated cell sorting (FASC), immunomagnetic bead sorting, or magnetic activated cell sorting (MASC).
  • FSC fluorescent-activated cell sorting
  • MSC magnetic activated cell sorting
  • the providing step comprises: isolating ovarian cells from an ovary tissue; and culture expanding the ovarian cells in a germ-line stem cell media.
  • the method further includes a step of collecting ovarian cells that are not positive for either DDX4 or IFITM3, to provide a second population comprising cells that can differentiate into granulosa and/or theca cells. In some embodiments, the method further comprises differentiating the cells of the second population into granulosa and/or theca cells.
  • the method further includes culturing the oogonia stem cells with granulosa and theca cells (optionally produced as taught herein from cells not positive for either DDX4 or IFITM3), to differentiate the oogonia stem cells into oocytes.
  • the culturing comprises including the oogonia stem cells in an in vitro follicle construct or a microcapsule comprising said granulosa and theca cells.
  • the in vitro follicle construct or a microcapsule comprises an outer layer comprising the theca cells and an inner layer and/or core comprising the granulosa cells and the double positive population comprising oogonia stem cells.
  • the culturing includes contacting the oogonia stem cells with a combination of retinoic acid, follicle-stimulating hormone, and/or estradiol.
  • an in vitro follicle construct comprising: live mammalian oogonia stem cells, or an oocyte differentiated from an oogonia stem cell (optionally wherein the oocyte is positive for DDX4 and DAZL); live mammalian granulosa cells; and live mammalian theca cells, optionally where the cells are provided in a hydrogel carrier (e.g., collagen hydrogel).
  • the cells are provided in a cell culture apparatus such as a dish or plate.
  • one, two, or all three of the live mammalian ovarian granulosa cells, live mammalian ovarian theca cells, and live mammalian oogonia stem cells are provided according to a method as described herein above and below.
  • the construct is multilayered with the oogonia-stem cells in the center, the granulosa cells around them as a second layer, and the theca cells around the second layer as a third layer.
  • the oogonia stem cells are included in the construct in an amount of from 500 cells to 5000 cells; the granulosa cells are included in the construct in an amount of from 1,000 cells to 1 x 10 9 cells; and/or the theca cells are included in the construct in an amount of from 1,000 cells to 1 x 10 9 cells.
  • Also provided is a method for performing in vitro fertilization comprising: providing an in vitro follicle construct as taught herein, wherein the construct comprises the oocyte differentiated from the oogonia stem cells (optionally wherein the oocyte is positive for DDX4 and DAZL); collecting the oocyte; and fertilizing the oocyte, to thereby perform in vitro fertilization.
  • a method of providing a culture of granulosa and/or theca cells comprising one or more of the steps of: (a) providing a mixed population of ovarian cells isolated from an ovary tissue (e.g ., adult human ovary tissue); (b) separating and collecting ovarian cells that are not positive for either: i) DEAD box helicase peptide 4 (DDX4, also known as Vasa homologue), or ii) interferon-induced transmembrane protein 3 (IFITM3, also known as fragilis), from said mixed population, to provide a population of sorted cells; (c) culturing the population of sorted cells; and (d) differentiating the sorted cells into granulosa and/or theca cells, wherein the granulosa cells are positive for FSH receptor and aromatase enzyme, and wherein the theca cells are positive for LH receptor and CYP17A1.
  • DDX4 DEAD box helicase peptide 4
  • the separating is carried out with fluorescent-activated cell sorting (FASC), immunomagnetic bead sorting, or magnetic activated cell sorting (MASC).
  • FSC fluorescent-activated cell sorting
  • MSC magnetic activated cell sorting
  • the providing step comprises: isolating ovarian cells from an ovary tissue; and culture expanding the ovarian cells in a germ-line stem cell media.
  • the method further includes forming an in vitro follicle construct or a microcapsule with the granulosa and/or theca cells, said microcapsule comprising: a core comprising live mammalian granulosa cells; and an auxiliary layer surrounding said core and comprising live mammalian theca cells.
  • FIG. 1 Schematic of bio-engineered ovarian follicle formation. Briefly, egg progenitor cells or oogonia stem cells (OSCs) positive for DDX4 and/or IFTIM3 are isolated from the ovary. Isolated OSCs are expanded and characterized. Cells negative for these OSC markers are differentiated into somatic cells namely granulosa (GC) and theca (TC). During the bio-engineering of follicle process, first the OSCs are made into organoids, followed by the addition of GC and then finally TC.
  • OSCs oogonia stem cells
  • Bio-engineered follicles are exposed to follicle- stimulating hormone (FSH) and luteinizing hormone (LH) in a cyclic fashion followed by exposure to a LH surge.
  • FSH follicle- stimulating hormone
  • LH luteinizing hormone
  • the production of secondary oocyte from the LH-surge exposed bio engineered follicles is evaluated and characterized.
  • FIG. 2 Primary culture of ovary cells. Cells isolated from ovaries using enzymatic digestion were cultured in germ-line stem cell media (A-D). Some cells in the culture form distinct clusters on top of other cells (E-H). The scale bar in micrograph is 100 pm.
  • FIG. 3 Flow cytometry analysis. 79.4% of cluster forming cells are DDX4 positive.
  • FIG. 4 Cluster forming cells stain for DDX4. Overall majority of the cells in the clusters are DDX4 positive.
  • FIG. 5 Flow cytometry analysis. 97.7% of MACS-sorted cells are DDX4 positive, indicating that these cells maintain their stem cell properties in culture.
  • FIG. 6 Immunofluorescent staining of MACS-sorted cells. MACS sorted cells that are positive for DDX4 (a surface protein) maintain their stem cells properties in culture as shown by the immunofluorescent staining for DDX4 and Fragilis. The scale bar in the micrograph is 50 pm.
  • DDX4 a surface protein
  • FIG. 7 Flow cytometry analysis. 94.5% of expanded and MACS-sorted cells are DDX4 positive.
  • FIG. 8 Immunofluorescent staining of MACS-sorted cells from culture expanded ovarian cells.
  • MACS sorted cells that are culture expanded before the magnetic sorting are positive for DDX4 (a surface protein), maintaining their stem cells properties in culture as shown by the immunofluorescent staining for DDX4 and Fragilis.
  • the scale bar in the micrograph is 50 pm.
  • FIG. 9 Immunofluorescent staining of MACS-sorted ovarian cells in culture.
  • FIG. 10 Representative images of MACS-sorted ovarian cells in cultured stained for specific markers. Top row: DDX4 and IFITM3, the oogonia stem cell marker; Middle row: Nanog and SSEA1, pluripotent stem cell markers; and bottom row: OCT4 and SOX2, pluripotent stem cell markers. The scale bar in the micrograph is 50 pm.
  • FIG. 10. Immunofluorescent staining of differentiated granulosa and theca cells.
  • FIG. 11 Engineered organoids from Oogonia stem cells (OSCs).
  • A Formation of ovarian cell organoids in AggreWellTM system;
  • B Ovarian organoid harvested from collagen gel after one week of culture;
  • FIG. 12 Engineered organoids with three different cell types of ovarian follicles.
  • the schematic of native follicle and expected organization of engineered follicle are provided on the left.
  • Epi-fluorescent image of engineered follicle was provided on the right.
  • OSCs were positioned in the core (indicated by arrow), granulosa cells layered around the OSC core and theca cells in the periphery.
  • GC granulosa cell
  • OSC oogonia-stem cells
  • TC theca cell.
  • RA retinoic acid
  • FSH follicle-stimulating hormones
  • E2 17 b-estradiol
  • Subjects as used herein are, in general, mammalian subjects. While human subjects are preferred, the subjects may in some embodiments be other animals, such as dogs and cats for veterinary purposes. Subjects are generally female. While the subjects may be of any suitable age, the subjects are typically adults.
  • Treat refers to any type of treatment that imparts a benefit to a subject, including but not limited to delaying the onset or reducing the severity of at least one symptom associated with hormonal imbalance or dysregulation in the subject, and/or promoting or enhancing fertility of the subject.
  • treating may be for infertility due to iatrogenic causes (especially patients treated with chemo/radiation) as well as naturally declining fertility with advancing age/menopause, to increase or enhance the reproductive lifespan of women who desire to have children later on in life with improved conception rates/viable pregnancies.
  • “Pharmaceutically acceptable” as used herein means that the microcapsule or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
  • Cells used to carry out the present invention are, in general, live mammalian cells collected from a suitable donor.
  • Donors are, in general, mammalian (e.g ., human, dog, cat, rabbit, rat, mouse, monkey, chimpanzee, horse, pig, goat, sheep).
  • the donor may be of the same species as the subject being treated, or of a different species.
  • the donor may be the same subject undergoing treatment (i.e., autogenic), where suitable cells were harvested from the subject and provided as fresh tissue or stored for subsequent use (e.g., frozen).
  • Ovarian cells may be isolated from donors and cultured as taught herein or in accordance with techniques known in the art. See, e.g, Sanjay K. Agarwal et ah, Leptin Antagonizes the Insulin-Like Growth Factor-I Augmentation of Steroidogenesis in Granulosa and Theca Cells of the Human Ovary, J. Clin Endocrinol Metab 84: 1072-1076 (1999); Jon C. Havelock et ak, Ovarian granulosa cell lines, Molecular and Cellular Endocrinology 228, 67-78 (2004); Jessica K.
  • the isolated ovarian cells are culture expanded.
  • the cells are culture expanded in a germ line stem cell media supplemented with stem cell promoting growth factors.
  • Such media may include ingredients such as, but not limited to, minimum essential medium alpha (aMEM), antibiotic/antimycotic (e.g., 1000 U/ml penicillin, 1000 pg/ml strep, 25 pg/ml amphotericin B), ITS (e.g, 5 mg/ml insulin, 5 mg/ml transferrin and 5 pg/ml selenium), glutamine (e.g, 2 mM), sodium pyruvate (e.g, 1 mM), nonessential amino-acids (e.g, 1 mM), fetal bovine serum (e.g, 10%), b- mercaptoethanol (e.g, 0.1 mM), petrescine (e.g, 60 pM), epidermal growth factor (e.g, 10 ng/ml
  • Sorting can be performed based upon any unique properties that distinguish one cell type from another, e.g., density, size, unique markers, unique metabolic pathways, nutritional requirements, protein expression, protein excretion, etc.
  • cells may be selected based on density and size with the use of centrifugal gradients.
  • Unique markers may be selected with fluorescent-activated cell sorting (FASC), immunomagnetic bead sorting, magnetic activated cell sorting (MASC), panning, etc.
  • Unique metabolic pathways and nutritional requirements may be exploited by varying the makeup and/or quantity of nutritional ingredients of the medium on which cells are grown, particularly in a serum-free environment. Protein expression and/or excretion may be detected with various assays, e.g., ELISA.
  • ovarian cells as taught herein may be selected/separated based on being positive for both of: i) DEAD box helicase peptide 4 (DDX4, also known as Vasa homologue), and ii) interferon-induced transmembrane protein 3 (IFITM3, also known as fragilis) (sometimes referred to herein as "double positive" population of cells in that the population is positive for both markers - i.e., contains cells positive for either or both of the markers).
  • DDX4 DEAD box helicase peptide 4
  • IFITM3 interferon-induced transmembrane protein 3
  • Such double positive cells may be differentiated into oogonia cells, and therefore are referred to as "oogonia stem cells.”
  • Ovarian cells that are not double positive may also be separated and collected for use, e.g., to provide granulosa and/or theca cells.
  • markers may be used for separation with methods known in the art, such as with the use of marker- specific antibodies in fluorescent-activated cell sorting (FASC), immunomagnetic bead sorting, and/or magnetic activated cell sorting (MASC).
  • ovarian stem cells may be stimulated into forming granulosa cells with a combination of different hormones and/or growth factors in a sequential fashion in accordance with procedures known in the art.
  • the reagents, hormones and growth factors may include one or more of: all-trans retinoic acid, growth hormone, follicle-stimulating hormone, anti-mullerian hormone, b-estradiol, inhibin-a and -b, basic fibroblast growth factor, epidermal growth factor, and transforming growth factor-b. See Liu et al., 2016; Molecular Medicine reports 13:5053-5058.
  • ovarian stem cells e.g., ovarian cells that are not double positive for DDX4 and IFITM3 may be differentiated into theca cells with a combination of various hormones and/or growth factors in accordance with procedures known in the art.
  • the reagents, hormones and growth factors used for theca cell differentiation may include one or more of: Putrescine, D-(+)-glucose, Puyruvic acid, DL-Lactic acid, Ascorbic acid, 2- mercaptoethanol, D-biotin, epidermal growth factor, basic fibroblast growth factor, Glial cell- derived neurotrophic factor, insulin-like growth factor-I, stem cell factor, leukemia inhibitor factor, luteinizing hormone, b-estradiol and progesterone. See Honda et al., 2007; PNAS 104(30): 12389-12394.
  • the oogonia stem cells may be stimulated to form oocytes with a combination of retinoic acid, follicle-stimulating hormone, and/or estradiol.
  • the ovarian cells may be formed into an in vitro follicle construct including live mammalian oogonia stem cells, or an oocyte differentiated from an oogonia stem cell; live mammalian granulosa cells; and live mammalian theca cells.
  • the cells may be provided in a hydrogel carrier.
  • the cells may be provided in a suitable cell culture apparatus such as a dish or plate (e.g., a 3D spheroid plate such as AggreWellTM).
  • the construct may be multilayered with the oogonia stem cells in the center, the granulosa cells around the oogonia stem cells as a second layer, and the theca cells around the second layer as a third layer.
  • the construct/layers may or may not have cells encapsulated in one or more semipermeable membrane(s).
  • the in vitro follicle construct may be of any suitable size, such as from 10, 20 or 30 microns in diameter, up to 1000, 2000, or 5000 microns in diameter.
  • the in vitro follicle construct may contain any suitable amount of cells.
  • oogonia stem cells are included in the construct in an amount of from 500 cells to 5000 cells; granulosa cells are included in an amount of from 1,000 or 2,000 cells to 1 x 10 6 , 1 x 10 8 , or 1 x 10 9 cells; and theca cells are included in an amount of from 1,000 or 2,000 cells to 1 x 10 6 , 1 x 10 8 , or 1 x 10 9 cells.
  • Mesenchymal stem cells if present, may be included in the construct in any amount from 500 or 1000 cells to 0.5 x 10 6 , to 0.5 x 10 8 , 0.5 x 10 9 , or 1 x 10 9 cells.
  • the in vitro follicle construct may be refrigerated and/or cryopreserved for subsequent use, and/or cultured for subsequent use, as desired.
  • the oocyte is positive for DDX4 and DAZL (deleted in azoospermia like). See Varras, "Marker of stem cells in human ovarian granulosa cells: is there a clinical significance in ART?” Journal of Ovarian Research 5:36 (2012).
  • the formed oocyte may be collected from an in vitro follicle construct and fertilized by in vitro fertilization procedures.
  • Methods for in vitro fertilization are known, and generally involve combination of an egg and sperm in vitro to form an embryo, which may then be implanted into a uterus of a subject. See, e.g., U.S. Patent Nos. 4,589,402, 4,725,579, WO 1989/004366, WO 1992/020359.
  • the cells are encapsulated to form a microcapsule. Encapsulation of live cells can be carried out in accordance with known techniques or variations thereof that will be apparent to those skilled in the art. See, e.g., U.S. Patent No. 9,283,251 to Opara et ak, the disclosures of which are incorporated by reference herein in their entirety.
  • the microcapsules contain theca and/or granulosa cells differentiated from ovarian stem cells as provided herein, and/or contain oogonia stem cells (or "double positive" cells) or oocytes differentiated therefrom.
  • Microcapsules useful in the present invention may optionally have at least one semipermeable membrane surrounding a cell-containing core and/or layer(s).
  • the semipermeable membrane may permit the diffusion of nutrients, biologically active molecules and/or other selected products through the surface membrane and into the microcapsule core.
  • the surface membrane contains pores of a size that determines the molecular weight cut-off of the membrane.
  • the membrane pore size may be chosen to allow the passage of estrogen, and in some embodiments progesterone, from within the capsule to the external environment, but to exclude the entry of host immune response factors (where the administered microcapsules contain cells that are not autologous).
  • Such a semipermeable membrane is typically formed from a poly cation such as a polyamine (e.g ., polylysine and/or polyornithine).
  • U.S. Patent No. 4,391,909 to Lim et al. describes a method in which cells are suspended in sodium alginate in saline, and droplets containing cells are produced. Droplets of cell-containing alginate flow into calcium chloride in saline. The negatively charged alginate droplets bind calcium and form a calcium alginate gel.
  • microcapsules are washed in saline and incubated with poly-L-lysine or poly-L-ornithine (or combinations thereof); the positively charged poly-l-lysine and/or poly-L-ornithine displaces calcium ions and binds (ionic) negatively charged alginate, producing an outer poly-electrolyte semipermeable membrane.
  • An exterior coating of sodium alginate may be added by washing the microcapsules with a solution of sodium alginate, which ionically bonds to the poly-L-lysine and/or poly-L- ornithine layer (this serves to reduce any inflammatory response that may be provoked in the subject by contact of the poly cationic membrane to tissue).
  • a “double-wall” microcapsule can be produced by following the same procedure as for single-wall microcapsules, but prior to incubation with sodium citrate, the microcapsules are again incubated with poly-l-lysine and sodium alginate.
  • Chang et al. U.S. Patent No. 5,084,350 teaches microcapsules enclosed in a larger matrix, where the microcapsules are liquefied once the microcapsules are within the larger matrix.
  • Tsang et al. U.S. Patent No. 4,663,286 teaches encapsulation using an alginate polymer, where the gel layer is cross-linked with a polycationic polymer such as polylysine, and a second layer formed using a second polycationic polymer (such as polyornithine); the second layer can then be coated by alginate.
  • U.S. Patents No. 5,801,033 and 5,573,934 to Hubbell et al. describe alginate/polylysine microspheres having a final polymeric coating (e.g., polyethylene glycol (PEG)); Sawhney et al., Biomaterials 13:863 (1991) describe alginate/polylysine microcapsules incorporating a graft copolymer of poly-l- lysine and polyethylene oxide on the microcapsule surface, to improve biocompatibility;
  • U.S. Patent No. 5,227,298 to Weber et al. describes a method for providing a second alginate gel coating to cells already coated with polylysine alginate; both alginate coatings are stabilized with polylysine.
  • U.S. Patent No. 5,578,314 to Weber et al. provides a method for microencapsulation using multiple coatings of purified alginate.
  • the alginate-polylysine microcapsules can be incubated in sodium citrate to solubilize any calcium alginate that has not reacted with poly-l-lysine, /. e. , to solubilize the internal core of sodium alginate containing the cells, thus producing a microcapsule with a liquefied cell-containing core portion.
  • Such microcapsules are referred to herein as having "chelated", “hollow” or "liquid” cores.
  • the microcapsules may be treated or incubated with a physiologically acceptable salt such as sodium sulfate or like agents, in order to increase the durability of the microcapsule, while retaining or not unduly damaging the physiological responsiveness of the cells contained in the microcapsules. See, e.g ., U.S. Patent No. 6,783,964 to Opara.
  • a physiologically acceptable salt such as sodium sulfate or like agents
  • Microcapsules may be of any suitable size, such as from 10, 20 or 30 microns in diameter, up to 1000, 2000, or 5000 microns in diameter. Microcapsules may contain any suitable amount of cells. For example, in some embodiments, granulosa cells are included in the microcapsules in an amount of from 1,000 or 2,000 cells per microcapsule up to 1 x 10 6 , 1 x 10 8 , or 1 x 10 9 cells per microcapsule; and the theca cells are included in the microcapsules in an amount of from 1,000 or 2,000 cells per microcapsule up to 1 x 10 6 , 1 x 10 8 , or 1 x 10 9 cells per microcapsule.
  • Mesenchymal stem cells may be included in said microcapsules in any amount from 500 or 1000 cells per microcapsule up to 0.5 x 10 6 , to 0.5 x 10 8 , 0.5 x 10 9 , or 1 x 10 9 cells per microcapsule. See US 2016/0166620 to Opara et al.
  • Microcapsules of the present invention may be administered after production, refrigerated and/or cryopreserved for subsequent use, and/or cultured for subsequent use, as desired.
  • Microcapsules of the invention may be washed (e.g, in sterile physiological saline solution) prior to formulation and/or administration, as needed depending upon their manner of production.
  • Microcapsules of the present invention may be administered for hormone therapy per se or formulated for administration by any suitable technique, such as by mixing with sterile physiological saline solution.
  • the microcapsules may be administered by any suitable technique, including but not limited to surgical implantation or injection (either of which may be carried out subcutaneously, intraperitoneally, intramuscularly, or into any other suitable compartment, particularly into the omentum, such as by deposition into a surgically created omental pouch. Dosage of cells administered can be determined in accordance with known techniques or variations thereof that will be apparent to those skilled in the art.
  • Subjects or patients to be treated with hormone replacement therapy may include subjects afflicted with, or at increased risk of, one or more of osteoporosis, hot flashes, irregular period, vaginal atrophy, vaginal and/or bladder infection, incontinence (e.g., urge incontinence, stress incontinence), fatigue, sleep disturbances, irritability, mood swings, depression, loss of muscle mass, increased fat tissue, thinning and loss of skin elasticity, loss of bone tissue, impaired cognition etc., which may be associated with menopause, hysterectomy, ovariectomy, or other conditions for which estrogen or hormone replacement therapy is employed.
  • incontinence e.g., urge incontinence, stress incontinence
  • fatigue e.g., sleep disturbances, irritability
  • mood swings depression
  • loss of muscle mass e.g., increased fat tissue, thinning and loss of skin elasticity, loss of bone tissue, impaired cognition etc.
  • impaired cognition etc. which may be associated
  • Oogonia stem cells are purified by the presence of two markers: a) DEAD box helicase peptide 4 (DDX4 also referred as Vasa homologue) and b) interferon-induced transmembrane protein 3 (IFITM3 also referred as fragilis).
  • the double positive cells were characterized to be the OSCs, and stem cells that are negative for OSC markers were characterized to be stem cells from granulosa and theca cells.
  • a bio-fabrication process was developed to construct and mimic an ovarian follicular structure using three functional cell types of ovaries, namely: a) oocytes or oogonia; b) granulosa cells; and c) theca cells, which is schematically illustrated in FIG. 1.
  • a maturation system was developed to differentiate oocytes from their stem cells by incorporating them in ovarian follicle-like organoids, where granulosa and theca cells form the other components of the organoids. These somatic cells (granulosa and theca cells) induce the differentiation and maturation of oocytes from the OSCs.
  • This technology may be used for the treatment for infertility due to iatrogenic causes (especially patients treated with chemo/radiation) as well as naturally declining fertility with advancing age/menopause. This could increase or enhance the reproductive lifespan of women who desire to have children later on in life with improved conception rates/viable pregnancies.
  • this approach may serve as a source of HRT with correction of hormonal imbalance that responds to patient’s natural hormone axis/pathway and delivers more controlled, steady physiologic doses compared to current methods of HRT.
  • Ovaries Whole ovary or partial ovary or biopsy samples of ovary (fresh or frozen tissue) can be used for isolating cells.
  • the cells that form the integral part of ovarian follicle include: a) oocyte, b) granulosa and c) theca cells.
  • oocytes are the germ cells and the other two types are somatic cells that act as supporting cells. From our initial study with the procured ovaries, we have observed that the aforementioned ovarian cells do not exist in a differentiated form or as functional cells. Instead, they exist as stem cells in ovaries as reported by others and by us in our prior rat studies.
  • Oogonia stem cells serve as the mother cell for the production of an oocyte (clinically referred to as egg cell), whereas, other stem cells in the ovary differentiate into granulosa and theca cells. These stem cells upon proper stimulation are capable of differentiating into these functional cells.
  • Oogonia stem cells may be isolated by one of the following methods: a) culturing the primary ovarian cells until they form distinct stem cell clusters; b) by magnetic activated cell sorting using oogonia stem cell markers; c) by fluorescent activated cell sorting using oogonia stem cell markers; or d) by culturing the cells scraped off from the surface of ovaries as ovarian surface epithelium cells.
  • Granulosa and theca stem cells may be isolated by magnetic activated cell sorting or fluorescent activated cell sorting using pluripotent stem cell markers (OCT-4, Nanog, SSEA-4, SOX-2).
  • Approach- 1 Isolating OSCs from the cluster forming primary ovarian cells. Even though the initial population of cells that are isolated from the ovaries yields a mixture of all kinds of ovarian cells, certain cell types aggregate and form clusters in the culture condition as shown in FIG. 2 (E-H).
  • the primary cells isolated from ovaries were cultured in germ-line stem cell media (minimum essential medium alpha (aMEM) containing antibiotic/antimycotic (1000 El/ml penicillin, 1000 pg/ml strep, 25 pg/ml amphotericin B), ITS (5 mg/ml insulin, 5 mg/ml transferrin and 5 pg/ml selenium), 2 mM glutamine, 1 mM sodium pyruvate, 1 mM nonessential amino-acid, 10% fetal bovine serum, 0.1 mM b- mercaptoethanol, 60 pM petrescine, 10 ng/ml epidermal growth factor (Invitrogen), 1 ng/ml basic fibroblast growth factor, 40 ng/ml glial-derived neurotrophic factor, 10 ng/ml leukemia- inhibitory factor, 100 ng/ml insulin-like growth factor, 100 ng/ml Stem cell factor).
  • aMEM minimum
  • OSCs are known to express a specific protein referred to as DEAD box polypeptide4 (DDX4), and this was used as a marker in our study to screen for OSCs in the cluster-forming cells. Briefly, the cells were incubated with antibody for DDX4 for 1 hr. Then, the cells were incubated with fluorescent-conjugated secondary antibody after washing of any unbound primary antibody. The cells were then analyzed by flow cytometry for the presence of fluorescent-labelled antibody complex. About 79.4% of cells were observed to be positive for the OSC marker (FIG. 3).
  • DDX4 DEAD box polypeptide4
  • the cluster forming cells in culture were also stained for DDX4 marker. As shown in the FIG. 4, a significant number of cells in the cluster stain positive for DDX4 validating flow cytometry data that a majority of the cluster forming cells are OSCs.
  • Approach-2 Enriching the oogonia stem cells by a magnetic sorting method
  • the MACS-sorted ovarian cells were expanded in culture and characterized. Immunofluorescent staining confirms the presence of OSCs in culture. With the help of flow cytometry, the percentage of OSCs among the cultured cells was assessed to determine how many cultured cells still carry their stem cell character after expansion. It was observed that about 98% of the MACS-sorted cells maintain their stem cell properties in culture (FIG. 5). Immunofluorescent staining for DDX4 and fragilis supported the flow cytometry data that the MACS-sorted cells in culture are OSCs (FIG. 6).
  • Approach-3 Expanding the primary ovarian cells first and then enriching the OSCs by magnetic sorting method
  • aMEM germ-line stem cell media
  • antibiotic/antimycotic 1000 U/ml penicillin, 1000 pg/ml strep, 25 pg/ml amphotericin B
  • ITS 5 mg/ml insulin, 5 mg/ml transferrin and 5 pg/ml selenium
  • 2 mM glutamine 1 mM sodium pyruvate, 1 mM nonessential amino-acid, 10% fetal bovine serum, 0.1 mM b-mercaptoethanol, 60 pM petrescine, 10 ng/ml epidermal growth factor (Invitrogen), 1 ng/ml basic fibroblast growth factor, 40 ng
  • the stem cells isolated from the human ovary samples are heterogeneous.
  • the three different cells of ovary that constitutes ovarian follicles (oocytes, granulosa and theca cells) are known to be derived from different stem cells.
  • Cells isolated were expanded, characterized and used to fabricate into ovarian follicle-like structures.
  • Ovarian stem cells are cultured and expanded by using appropriate stem cell media until they reach sufficient number (1 x 10 6 to 1 x 10 7 cells) to differentiate them into respective functional ovarian cells.
  • Oogonia stem cells are cultured using germline stem cell media to maintain their stem-cell properties and to induce differentiation into oocytes.
  • Granulosa cells (GC) and theca cells (TC) play a pivotal role in the maturation of oocytes.
  • GC Granulosa cells
  • TC theca cells
  • these two cell types were differentiated from the pluripotent stem cells of the ovary.
  • Pluripotent stem cells are stimulated into granulosa cells with a combination of different hormones and growth factors in a sequential fashion.
  • the reagents, hormones and growth factors include: all-trans retinoic acid, growth hormone, follicle-stimulating hormone, anti-mullerian hormone, b- estradiol, inhibin-a and -b, basic fibroblast growth factor, epidermal growth factor, and transforming growth factor-b. See Liu et ah, 2016; Molecular Medicine reports 13:5053- 5058. Similarly, pluripotent stem cells differentiate into theca cells with a combination of various hormones and growth factors.
  • the reagents, hormones and growth factors used for theca cell differentiation include: Putrescine, D-(+)-glucose, Puyruvic acid, DL-Lactic acid, Ascorbic acid, 2-mercaptoethanol, D-biotin, epidermal growth factor, basic fibroblast growth factor, Glial cell-derived neurotrophic factor, insulin-like growth factor-I, stem cell factor, leukemia inhibitor factor, luteinizing hormone, b-estradiol and Progesterone. See Honda et ah, 2007; PNAS 104(30): 12389-12394. During the long-term culture of OSCs, their stem cell characteristics were assessed to investigate how these cells maintain their stem cell properties in culture.
  • Pluripotent stem cells that were treated with hormones and growth factors in their culture media differentiated into granulosa and theca cells. The differentiation into granulosa and theca cells was confirmed and characterized by the presence of cell-specific markers. Granulosa cells showed the expression of FSH receptor and aromatase enzyme, whereas theca cells exhibited the presence of LH receptor and CYP17A1, as shown in FIG. 10.
  • OSCs Oogonia-stem cells obtained from either cluster-forming cells in the primary culture of ovarian cells or from MACS/FACS sorting method was adopted to obtain OSCs using two specific markers which validate the reports from literature. In contrast to prior reports, however, we used both antibodies to select the cells to form a double positive population of cells, whereas other investigators have used only one antibody for selection. These isolated OSCs were then used for the construction of follicle-like organoids using an AggreWellTM system (STEMCELL Technologies Inc., Cambridge, Massachusetts). Once the cells formed organoids in AggreWellTM system, they were then transferred to 3 mg/ml collagen gel. After one week in collagen, the organoids were assessed histologically for the maturation of oocyte or oocyte-like cells.
  • the organoids that were bio-engineered using the ovarian cells when assessed for oocyte-like cell, showed no such visible oocyte-like cells. Even though the ovarian cells formed a follicle-like structure, they failed to produce any oocyte-like cells in culture (FIG. 11). Without wishing to be bound to any particular theory, this may have been due to a need for additional cues to be pushed into meiosis.
  • Example 2 Bio-engineering human follicle-like ovarian constructs using OSCs, granulosa (GCs) and theca cells (TCs) for in vitro oocyte maturation
  • the OSCs were used along with the granulosa and theca cells. Each cell type is pre-stained with different fluorescent cell tracker dye in order to localize them in the engineered constructs.
  • the pre-stained OSCs were used to make the core of the follicle-like construct. Once the formation of the core was confirmed, the second cell type granulosa were added to the construct and allowed to form second layer around the OSC core. Finally, the third cell type, theca cells, were added onto the periphery of the construct.
  • the epi-fluorescent image of an engineered human follicle made with this method is presented in FIG. 12 along with the schematic of native follicle and expected organization of engineered follicle.
  • the engineered follicle formed a multilayered ovary organoid, with the oogonia-stem cells (OSCs) in the center, granulosa around them and theca as a final outer layer. It was also noted that OSCs in some of the organoids moved to periphery of the construct or were found off-centered (as indicated by the arrow).
  • OSCs oogonia-stem cells
  • Example 3 In vitro oogenesis from laboratory-engineered ovary organoids
  • the oogonia stem cells are mitotically diving cells, which need to enter meiosis to produce oocytes. Thus they may need special cues to push the mitotically dividing OSCs into meiosis. It has been reported in embryonic development where the oogonia cells are converted into primary oocytes, that one of the response factors of retinoic acid (RA), STRA8 increase during the transition of oogonia into oocyte (Le Bouffant et ah, 2010, Human Reproduction 25(10): 2579-2590; Feng et al., 2014, Molecular and Cellular Endocrinology 382: 488-497), which may indicate a role of retinoic acid in this transition.
  • RA retinoic acid
  • RA follicle-stimulating hormone
  • E 2 estradiol
  • the laboratory-bioengineered ovary organoids produced oocyte-like structures that stained positive for early markers of oocytes, namely DDX4 and DAZL (FIG. 13). Further maturation of these oocyte-like structures will be carried out and characterized for late markers of oocytes.
  • Example 4 Stem cell selection for somatic cells and egg progenitors
  • First step is to isolate OSCs, the egg-progenitor cells and the stem cells for somatic cells from the donor human ovaries.
  • OSCs the egg-progenitor cells and the stem cells for somatic cells from the donor human ovaries.
  • surface marker-based cell sorting method Since there are quite a few methods reported to isolate OSCs (including the use of surface markers), we chose to use surface marker-based cell sorting method. Since there are some concerns and controversies in the use of DDX4, we used an additional marker called Fragilis along with DDX4 thereby maximizing the yield of OSCs. In other words, the cells we isolate in our lab via magnetic sorting method could be positive for either DDX4 or Fragilis or both, to maximize our chance of obtaining more OSCs.
  • the isolated OSCs are expanded and sub-cultured, as well as assessed for the maintenance of their stem cell characteristics.
  • Example 5 Somatic cell differentiation from their stem cells and bio-engineering ovarian follicle
  • Somatic cells like GCs and TCs are part of functional ovarian follicles (Follicles beyond the primary follicle stage, not primordial follicle stage); however, in human ovaries, the number of maturing follicles is very low. Therefore, these cells (GCs and TCs) are not available in the ovarian tissue from which OSCs are isolated.
  • OSCs ovarian tissue from which OSCs are isolated.
  • We used the remaining ovary cells after OSC isolation which contain somatic stem cells to differentiate into GCs and TCs. The differentiated cells were confirmed by immunofluorescence staining for respective markers of GCs and TCs.
  • LH-receptor and CYP17 are used as markers for TCs and the expression of FSH-receptor and CYP19 (aromatase enzyme) are used as markers for GCs.
  • the OSCs were made into an organoid by culturing them in an ultra-low attachment round-bottom culture plates.
  • the OSC organoids were exposed to 1 mM retinoic acid (RA) in order to induce meiosis.
  • RA retinoic acid
  • the entry of meiosis was confirmed by immunofluorescence staining for an early marker of meiosis DAZL (deleted in azoospermia-like protein). This confirms the egg is at the primary oocyte stage.
  • the laboratory-differentiated GC were added to the RA-primed OSC organoid as mentioned earlier, followed by the final addition of TC.
  • This three cell type organoid mimics the structural architecture of an ovarian follicle.
  • the follicle-like organoids are treated with exogenous pituitary gonadotropins in similar fashion that exists during the follicular phase in a reproductively active ovary.
  • the gonadotropins-treated bio-engineered ovarian follicles produced sex steroids proving that a functional ovarian unit was produced.
  • EGF EGF (10 ng/ml); FGF (10 ng/ml); GDNF (40 ng/ml); IGF-I (100 ng/ml); SCF or kit-ligand (100 ng/ml); BMP-4 (20 ng/ml); SDF (20 ng/ml); Forskolin (5 pM); N- Acetyl cysteine (1 mg/ml).
  • EGF 10 ng/ml
  • FGF 10 ng/ml
  • GDNF 40 ng/ml
  • IGF-I 100 ng/ml
  • SCF or kit-ligand 100 ng/ml
  • BMP-4 20 ng/ml
  • SDF 20 ng/ml
  • Forskolin 5 pM
  • N- Acetyl cysteine N- Acetyl cysteine (1 mg/ml).
  • Example 6 In vitro oogenesis and fertilizable eggs
  • the final stage in the production of fertilizable eggs is the completion of meiosis-I.
  • the somatic cells help in the maturation of primary oocytes; however, they hinder the progression of meiosis-I and completion by introducing a check point.
  • the only way to relieve this check point is to luteinize the somatic cells by using high levels of LH, which is termed as a LH surge.
  • LH surge Low levels of LH

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Abstract

L'invention concerne un procédé de fourniture d'une culture de cellules souches d'ovogonies comprenant des cellules souches d'ovogonies. Le procédé peut en outre comprendre la culture des cellules souches d'ovogonies avec des cellules de la granulosa et des cellules thécales pour différencier les cellules souches d'ovogonies en ovocytes. Dans certains modes de réalisation, la culture comprend l'inclusion des cellules souches d'ovogonies dans une construction de follicules in vitro ou une microcapsule comprenant lesdites cellules de la granulosa et lesdits cellules thécales.
PCT/US2020/025111 2019-03-29 2020-03-27 Cellules de follicules ovariens et constructions pour le traitement de la fertilité et une hormonothérapie substitutive WO2020205464A1 (fr)

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CN113913370A (zh) * 2021-12-06 2022-01-11 天津市农业科学院 N-乙酰半胱氨酸在绵羊卵巢颗粒细胞体外培养中的应用

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US20160237402A1 (en) * 2013-10-07 2016-08-18 Northeastern University Methods and Compositions for Ex Vivo Generation of Developmentally Competent Eggs from Germ Line Cells Using Autologous Cell Systems

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CN113913370A (zh) * 2021-12-06 2022-01-11 天津市农业科学院 N-乙酰半胱氨酸在绵羊卵巢颗粒细胞体外培养中的应用

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