A method of preparing human ovarian somatic-like cells

Abstract

The present application provides a method for preparing human ovarian somatic cell-like cells. The present application establishes a method for preparing human ovarian somatic cell-like cells in vitro, which comprises stage-based culture based on human stem cells (including human pluripotent stem cells and human totipotent stem cells). The method of the present application can efficiently obtain functional human ovarian somatic cell-like cells, promote the differentiation ability of female germ stem cells (FGSC) cultured in vitro, and provide a new approach for assisted reproduction and treatment of infertility.

Description

Technical Field

[0001] This invention belongs to the field of cell biology, and more specifically, this invention relates to a method for differentiating human pluripotent stem cells into human ovarian somatic cell-like cells. Background Technology

[0002] Infertility affects the quality of life and marital stability of countless families, as well as the mental health of patients and their family members, and has become one of the major health and social problems. Many factors contribute to infertility, including malnutrition, stress, organic problems such as tumors of the pituitary gland, ovaries, or hypothalamus, and endocrine disorders.

[0003] FGSCs have various clinical applications, including treating female infertility, preserving female fertility, and delaying menopause. In previous work, the inventors were the first in the world to discover the presence of FGSCs in the ovaries of newborn mice. Using C-terminal DDX4 antibody, germ cells were sorted from the ovaries of newborn mice with magnetic beads, and FGSC cell lines were established after in vitro culture. Transplanting FGSCs into the ovaries of infertile mice can produce functional oocytes and offspring (Zou, K., Z. Yuan, Z. Yang, et al., Production of offspring from a germline stem cell line derived from neonatal ovaries. Nat Cell Biol, 2009.11(5):p.631-636). Subsequently, White et al. used the same antibody to flow cytometry isolate human FGSCs from the ovaries of reproductive-age women (White, YAR, DC Woods, Y. Takai, et al., Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. Nat Med, 2012, 18(3): p. 413-421). Increasingly, studies have found FGSCs in the ovaries of mice, rats, and humans. Clinical studies have shown that injecting human FGSC mitochondria into a patient's own oocytes can improve oocyte quality and increase the success rate of in vitro fertilization (IVF) (Fakih, MH, MEShmoury, J. Szeptycki, et al., The AUGMENT). SMTreatment: Physician Reported Outcomes of the Initial GlobalPatient Experience. JFIV Reprod Med Genet 2015.3(3):p.154).

[0004] Human FGSC has opened up new avenues for delaying female menopause, treating female infertility, and maintaining fertility.

[0005] In vitro differentiation of human FGSCs requires the support and induction of human ovarian somatic cell-like cells. However, the source of human ovarian somatic cell-like cells suitable for in vitro differentiation of human FGSCs is extremely limited or nonexistent.

[0006] Stem cells are a general term for a group of cells with the potential for self-renewal and differentiation. Based on their differentiation potential, stem cells can be classified into totipotent stem cells, pluripotent stem cells, and unipotent stem cells. Although stem cells have been extensively studied and successfully used to induce differentiation into various tissue cells, such as liver cells, technical challenges remain in inducing human ovarian somatic cell-like cells using stem cells as the starting cell. Summary of the Invention

[0007] The purpose of this invention is to provide a method for differentiating human pluripotent stem cells into human ovarian somatic cell-like cells and its application.

[0008] In a first aspect of the invention, a method for preparing human ovarian somatic cell-like cells is provided, the method being based on a staged culture of human stem cells, comprising:

[0009] (1) Cell pre-culture;

[0010] (2) Treat (1) cells with GSK3 inhibitor;

[0011] (3) Treat (2) cells with B / A / W, wherein the B / A / W is BMP4, ActivinA and Wnt3a;

[0012] (4) The cells of (3) were treated with Follistain, BMP4 and serum;

[0013] (5) The cells in (4) were treated with Follistain and BMP4 to obtain a culture containing human ovarian somatic cell-like cells.

[0014] In one or more embodiments, the “process” includes “induction” or “induced processing”.

[0015] In one or more embodiments, the method is an in vitro / ex vivo culture method.

[0016] In one or more embodiments, after step (3), the cells obtained express mesoendodermal markers of brachyury, mixl1, and pax2.

[0017] In one or more embodiments, the human stem cells include human pluripotent stem cells (such as human induced pluripotent stem cells) and human pluripotent stem cells (such as human embryonic stem cells).

[0018] In one or more embodiments, the "ovarian somatic cell-like cell" refers to a cell expressing Foxl2, Cyp19a1, and Ptch1.

[0019] In one or more embodiments, in steps (1) to (3), suspension culture is performed, and in steps (4) to (5), adherent culture is performed.

[0020] In one or more embodiments, in step (1), cells are cultured in a basal medium; in steps (2) to (5), GSK3 inhibitor, B / A / W, Follistain, BMP4, and serum are added to the basal medium; preferably, the basal medium is a stem cell culture medium (a culture medium suitable for culturing stem cells); more preferably, the basal medium is E8, mTesr, or DMEM / F12 basal culture medium; more preferably, the basal medium is a basal medium supplemented with 20±10% KSR, 1±0.5% NEAA, 1±0.5% Glutamax, 0.7±0.2 β-mercaptoethanol, and 4-10 ng / ml bFGF; or

[0021] In one or more embodiments, in step (1), the cell pre-culture time is 1 to 4 days; preferably 1.5 to 3 days; more preferably 1.5 to 2.5 days (e.g., 2 days).

[0022] In one or more embodiments, the culture medium used for suspension culture is a liquid culture medium (culture solution). The culture medium used for adherent culture is a corresponding liquid culture medium (culture solution).

[0023] In one or more embodiments, suspension culture is performed using low-adhesion culture dishes.

[0024] In one or more embodiments, during adherent culture, cells are adhered to a culture dish treated with gelatin; preferably, the gelatin treatment is 0.2%.

[0025] In one or more embodiments, E8 is GIBCO's Essential 8. TM The culture medium, mTeSR1, is from Stemcell. Both of these media can be used for culturing pluripotent stem cells.

[0026] In one or more embodiments, in step (2), the GSK3 inhibitor includes the following: CHIR99021, LiCl, BIO or Ly2090314; preferably CHIR99021; preferably the concentration of CHIR99021 is 3 to 8 μM (e.g. 4, 4.5, 5, 5.5, 6 or 7 μM), more preferably 4 to 6 μM.

[0027] In one or more embodiments, in step (2), the culture time is 30 to 48 hours (e.g., 32, 34, 36, 38, 40, 42 or 44 hours).

[0028] In one or more embodiments, in step (3), the BMP4 concentration is 4 to 15 ng / ml (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng / ml), preferably 9 to 12 ng / ml.

[0029] In one or more embodiments, the concentration of Activin A is 5 to 15 ng / ml (e.g., 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng / ml), preferably 9 to 12 ng / ml.

[0030] In one or more embodiments, the Wnt3a concentration is 5–15 ng / ml (e.g., 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng / ml), preferably 9–12 ng / ml.

[0031] In one or more embodiments, in step (3), the culture time is 2 to 9 days (e.g., 2.5, 3, 4, 5, 6, 7 or 8 days), preferably 2.5 to 7 days; more preferably 3 to 6 days.

[0032] In one or more embodiments, in step (4), the concentration of Folistain is 15 to 40 ng / ml (e.g., 18, 20, 22, 25, 28, 30, 32 or 35 ng / ml), preferably 20 to 30 ng / ml, and more preferably 22 to 28 ng / ml.

[0033] In one or more embodiments, in step (4), the BMP4 concentration is 4 to 15 ng / ml (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng / ml), preferably 9 to 12 ng / ml.

[0034] In one or more embodiments, in step (4), the serum concentration is 3 to 10% (v / v) (preferably 4 to 8%, such as 5%, 6%, 7% (v / v)).

[0035] In one or more embodiments, in step (4), the culture time is 3 to 12 hours (e.g., 4, 5, 6, 7, 8, 9, 10 or 11 hours), preferably 4 to 10 hours; more preferably 5 to 8 hours.

[0036] In one or more embodiments, in step (5), the concentration of follistain is 15 to 40 ng / ml (e.g., 18, 20, 22, 25, 28, 30, 32 or 35 ng / ml), preferably 20 to 30 ng / ml, and more preferably 22 to 28 ng / ml.

[0037] In one or more embodiments, in step (5), the BMP4 concentration is 4–15 ng / ml (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ng / ml), preferably 9–12 ng / ml; or

[0038] In one or more embodiments, in step (5), the culture time is 4 to 12 days (e.g., 4.5, 5, 6, 7, 8, 9, 10 or 11 days), preferably 5 to 10 days; more preferably 6 to 8 days.

[0039] In another aspect of the invention, an application of any of the methods described above is provided for the preparation of human ovarian somatic cell-like cells through staged culture based on human stem cells.

[0040] In another aspect of the present invention, a human ovarian somatic cell-like cell (including cell culture) obtained by induction of human stem cells is provided, which is prepared by any of the methods described above; preferably, it has the characteristics selected from the group consisting of: the human ovarian somatic cell-like cell is predominantly epithelial-like in morphology, with fibroblast-like cells, and is short spindle-shaped or triangular; the human ovarian somatic cell-like cell is positive for Foxl2, cyp19a1, and Ptch1 markers (including gene / transcriptional and protein levels); and / or, the human ovarian somatic cell-like cell can promote the differentiation of human female reproductive stem cells.

[0041] In another aspect of the invention, the application of the aforementioned human ovarian somatic cell-like cells (including cell cultures) is provided for promoting the differentiation of human female reproductive stem cells into human oocytes.

[0042] In another aspect of the present invention, a method for promoting the differentiation of human female reproductive stem cells is provided, comprising: using the human ovarian somatic cell-like cells as supporting cells, culturing human female reproductive stem cells on a cell layer of the human ovarian somatic cell-like cells or co-culturing human female reproductive stem cells with the human ovarian somatic cell-like cells.

[0043] In another aspect of the present invention, a kit for in vitro preparation of human ovarian somatic cell-like cells is provided, comprising:

[0044] Culture medium 1: includes basal culture medium and GSK3 inhibitor; preferably, the GSK3 inhibitor includes CHIR99021, LiCl, BIO or Ly2090314, more preferably CHIR99021; preferably, the amount of CHIR99021 is such that when used to prepare functional human ovarian somatic cell-like cells in vitro, it can form a concentration of 3-8 μM, more preferably 4-6 μM in the culture system;

[0045] Culture medium 2: includes basal culture medium and B / A / W, wherein B / A / W is BMP4, ActivinA and Wnt3a or a mixture thereof; preferably, when used for the in vitro preparation of functional human ovarian somatic cell-like cells, the BMP4 is able to form a concentration of 4-15 ng / ml, more preferably 9-12 ng / ml in the culture system, the ActivinA is able to form a concentration of 5-15 ng / ml, more preferably 9-12 ng / ml in the culture system, and the Wnt3a is able to form a concentration of 5-15 ng / ml, more preferably 9-12 ng / ml in the culture system;

[0046] Culture medium 3 includes basal culture medium, as well as follistain, BMP4, and serum; preferably, when used for the in vitro preparation of functional human ovarian somatic cell-like cells, the follistain is able to form a concentration of 15–40 ng / ml, more preferably 20–30 ng / ml, and more preferably 22–28 ng / ml in the culture system; the BMP4 is able to form a concentration of 4–15 ng / ml, more preferably 9–12 ng / ml in the culture system; and the serum is at a concentration of 3–10% (v / v) (preferably 4–8%, such as 5%, 6%, or 7% (v / v)) in the culture system.

[0047] Culture medium 4 includes basal culture medium as well as Folistain and BMP4; preferably, when used for the in vitro preparation of functional human ovarian somatic cell-like cells, the Folistain is able to form a concentration of 15-40 ng / ml, more preferably 20-30 ng / ml, and more preferably 22-28 ng / ml in the culture system, and the BMP4 is able to form a concentration of 4-15 ng / ml, more preferably 9-12 ng / ml in the culture system.

[0048] In another aspect of the invention, the application of the kit is provided for preparing human ovarian somatic cell-like cells based on staged culture of human stem cells.

[0049] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0050] Figure 1 Bright field image of human pluripotent stem cells differentiating into human ovarian somatic cell-like cells.

[0051] Figure 2 RT-PCR detection of cell markers at various stages of cell differentiation, with GAPDH used as an internal control.

[0052] Figure 3 Comparison of Brachyury gene expression in different schemes (5C: 5uM Chir99021; B: BMP4; A: Activin A; W: Wnt3a).

[0053] Figure 4 Quantitative analysis of PCR products (group 5C) showed that the human ovarian somatic cell-like cells (OSLC) obtained by this differentiation protocol expressed Foxl2, Cyp19a1, and Ptch1 at high levels.

[0054] Figure 5 Immunocytochemistry was used to identify human ovarian somatic cell-like cells derived from hPSCs.

[0055] Figure 6 Determination of estrogen secretion from human ovarian somatic cell-like cells derived from hPSCs. Human ovarian somatic cell-like cells derived from hPSCs were treated with 50 μg / L testosterone for 48 hours, and the supernatant was collected for ELISA to determine estrogen levels. D14.5 represents the culture time to the fifth stage of induction of human ovarian somatic cell-like cells, i.e., day 14.5 of the entire induction differentiation process; D0 represents cells before the start of induction.

[0056] Figure 7 Human female reproductive stem cells were induced to differentiate by human ovarian somatic cell-like cells; the expression of GDF9 and ZP3 in oocytes formed after female reproductive stem cells were induced by human ovarian somatic cell-like cells was detected by immunofluorescence.

[0057] Figure 8 RT-qPCR was used to detect the expression of oocyte marker genes c-KIT, FIGLA, GJA4, GDF9, and ZP3 in oocytes induced from female germ cells by human ovarian somatic cell-like cells; +OSLC, female germ cells induced by human ovarian somatic cell-like cells; Control, female germ cells induced without human ovarian somatic cell-like cells; *, P<0.05, **, P<0.01, ***, P<0.001. Detailed Implementation

[0058] Based on in-depth research, the inventors have developed a method for preparing human ovarian somatic cell-like cells in vitro, including cell culture in stages based on human stem cells or human pluripotent stem cells. This method can efficiently obtain functional human ovarian somatic cell-like cells and effectively promote the differentiation capacity of female reproductive stem cells cultured in vitro, providing a new approach for assisted reproduction and infertility treatment.

[0059] the term

[0060] As used herein, the term "cell culture medium" refers to a "basal culture medium" which is a generally suitable culture medium for cell (stem cell) culture that provides sufficient nutrients to meet the needs of cell (stem cell) growth.

[0061] As used herein, the terms "female reproductive stem cells" or "ovarian reproductive stem cells" are used interchangeably, and these may be cells of natural origin or cells derived from the expansion / passage of natural cells. Additionally, they may be cells that have undergone genetic modification or genetic engineering.

[0062] As used herein, the terms “ovarian somatic cell-like cells” and “ovarian somatic cells” are used interchangeably. These cells may be naturally derived cells or cells derived from the expansion / passage of natural cells, or cells obtained through induction; in addition, they may be cells that have been genetically modified or genetically engineered.

[0063] As used in this article, human ovarian somatic cell-like cells refer to "ovarian somatic cell-like cells" that are positive for Foxl2, Cyp19a1, and Ptch1, also known as "ovarian somatic cells".

[0064] As used in this article, “functional” means that human ovarian somatic cell-like cells obtained according to the described methods have the same or similar functions as expected.

[0065] As used in this article, “differentiation” refers to the developmental process of lineage commitment. “Lineage” refers to the pathway of cell development.

[0066] Culture and induction methods

[0067] This invention focuses on the large-scale in vitro preparation of functional human ovarian somatic cell-like cells, revealing a method for differentiating stem cells (including human pluripotent stem cells or human embryonic stem cells, preferably human pluripotent stem cells) into human ovarian somatic cell-like cells. This method utilizes a human pluripotent stem cell culture system, adds key differentiation-inducing factors, and induces in vitro differentiation of pluripotent stem cells to obtain functional human ovarian somatic cell-like cells.

[0068] Therefore, the present invention provides a method for preparing human ovarian somatic cell-like cells in vitro, comprising: (1) pre-culturing cells; (2) treating the cells of (1) with a GSK3 inhibitor; (3) treating the cell product of (2) with B / A / W, wherein the B / A / W is BMP4, Activin A, and Wnt3a; (4) treating the cells of (3) with Follistain, BMP4, and serum; and (5) treating the cells of (4) with Follistain and BMP4 to obtain a culture containing human ovarian somatic cell-like cells. During the culture period, fresh culture medium may be used as needed to adjust the culture conditions.

[0069] The human embryonic stem cells or human stem cells involved in this invention are commercially available and do not necessarily require the destruction of embryos to obtain. Human embryonic stem cells and pluripotent stem cells were successfully lined up as early as 1998. For example, in 1998, Thomson's group established five human embryonic stem cell lines from 14 blastocysts: H1, H13, H14, H7, and H9; Gearhart's group isolated primitive stem cells from the gonadal crest and mesentery of aborted fetuses aged 5-9 weeks, aiming to avoid the ethical problems caused by directly using embryos. See Chao Lan et al., "Research Progress of Human Embryonic Stem Cells," *Advances in Modern Obstetrics and Gynecology*, July 2003, Vol. 12, No. 4. Based on the above work, in February 2000, the Wisconsin Alumni Research Foundation (WARF) established WiCell, a non-governmental, non-profit affiliate that distributes human embryonic stem cells to qualified scientists at a low cost. There are many institutions that provide these ready-made human embryonic stem cells.

[0070] In 2006, Takahashi et al. screened four effective transcription factors (Oct4, Sox2, c-Myc, and Klf4) from 24 transcription factors and used retroviruses to transfer them into mouse fetal or adult MEF cells, thereby reversing the "biological clock" and successfully converting them into induced pluripotent stem cells (iPSCs). These iPSCs can be derived from the body's own cells or from pluripotent stem cells that have already been expanded, cultured, passaged, or established in the field.

[0071] In this invention, the female reproductive stem cells can be derived from the body or from female reproductive stem cells that have already been expanded, cultured, passaged, or established in the field. For example, the inventors have established female reproductive stem cell lines in previous studies. For instance, a method for culturing (expanding) female reproductive stem cells includes: placing the female reproductive stem cells in an embryonic fibroblast (STO cell) feeder layer and culturing them in a cell culture medium containing sodium pyruvate, L-glutamine, mercaptoethanol, non-essential amino acids, epidermal growth factor, human basic fibroblast growth factor, glial cell growth factor, and leukemia inhibitory factor. This method can culture female reproductive stem cells but will not induce them to further differentiate into oocytes.

[0072] In the method of this invention, cell induction is performed using suspension culture in the early stage and adherent culture in the later stage. Unless otherwise stated, the culture medium used for culture or induction is a liquid culture medium (culture solution).

[0073] In a specific embodiment of the present invention, a specific scheme for inducing differentiation from human stem cells is provided, which includes the following steps:

[0074] First, human pluripotent stem cells were suspended and cultured in a low-adhesion culture dish for 2 days to obtain embryoid bodies.

[0075] Based on the previous step, mesoendodermal differentiation is performed, including treatment with an agonist of the WNT signaling pathway (GSK3 inhibitor, such as CHIR99021) for 30 to 48 hours.

[0076] Based on the previous step, add Wnt3a, Activin A, and BMP4 for 3-6 days;

[0077] Building on the previous step, the cells were then placed in a culture dish treated with 0.2% gelatin.

[0078] Based on the previous step, add Follistain, BMP4, and serum for 5-8 hours.

[0079] Based on the previous step, add Follistain and BMP4 processing for 6-8 days.

[0080] In the specific implementation schemes above, the concentration of CHIR99021 is 5 μM, the concentration of BMP is 5-10 ng / ml, the concentration of Activin A is 6-10 ng / ml, the concentration of Follistain is 25 ng / ml, and the concentration of WNT3A is 6-10 ng / ml.

[0081] The inventors' test results show that the obtained mesoendodermal cells can express brachyury, mixl1, and pax2; and the obtained human ovarian somatic cell-like cells can express foxl2, cyp19a1, and ptch1.

[0082] Human ovarian somatic cell-like cells obtained by reverse transcription polymerase chain reaction and immunocytochemistry were identified as being able to efficiently promote the differentiation of human female reproductive stem cells into oocytes.

[0083] The method of this invention utilizes human stem cells to mimic the developmental pattern of human ovarian somatic cells, first differentiating them in vitro into early mesodermal cells, and then further inducing differentiation into human ovarian somatic cell-like cells. Several key features and advantages of this method are as follows: First, the entire culture system uses cell growth factors and chemical molecules, without introducing exogenous genes involved in reprogramming or transdifferentiation (without altering the genome structure), thus avoiding interference with the genomic stability of the original stem cells by exogenous genes and the transplantation safety risks associated with exogenous cells. Second, it can efficiently obtain human ovarian somatic cell-like cells with the function of secreting estrogen and progesterone. Third, the efficiently obtained human ovarian somatic cell-like cells are in ideal cell state and can well support the differentiation of female germline stem cells into oocytes.

[0084] The human ovarian somatic cell-like cells obtained by this invention can preferably be used as supporting cells for female reproductive stem cells, promoting the efficient differentiation of female reproductive stem cells into human oocytes.

[0085] The human ovarian somatic cell-like cells obtained by this invention can be introduced into the ovarian tissue of an organism for necessary treatment or adjuvant therapy. Administration within the body may include, but is not limited to, local injection (e.g., catheter administration or direct intraovarian injection), systemic injection, intravenous injection, intrauterine injection, and parenteral administration. Recipients may include, but are not limited to, subjects with difficulty conceiving, undergoing infertility treatment, those who have been treated for cancer, and those who have received cytotoxic therapy (e.g., chemotherapy or radiation therapy) or a combination thereof.

[0086] The method and products of this invention not only provide an excellent model for studying the molecular mechanism of human ovarian somatic cell development, but also provide a new approach for the clinical application of human ovarian somatic cells, which can be applied to assisted reproduction and infertility treatment.

[0087] Culture medium / kit

[0088] The present invention also provides culture media for each stage of induction culture, including the aforementioned culture media 1 to culture media 4.

[0089] In addition to the specific cytokines or chemical components listed in the embodiments of this invention, cytokines or chemical components known in the art that have the same or similar functions may also be used in this invention. Analogs, homofunctional proteins (such as homofunctional proteins of growth factors), or compounds of the specifically listed components, equivalent compounds inducing the same target, analogs, derivatives, and / or their salts, hydrates, or precursors may also be used to replace the specifically listed components to achieve the same technical effect. These analogs, homofunctional proteins, or compounds should also be included in this invention. Analogs of compounds include, but are not limited to, isomers and racemates of compounds. Compounds have one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, cis, or trans isomers. The term "precursor of a compound" refers to a compound, when applied or treated by appropriate methods, that can be converted in a culture medium into any of the above-mentioned compounds, or a salt or solution of any of the above-mentioned compounds.

[0090] As a preferred embodiment of the present invention, the culture medium may also contain components for preventing bacterial contamination of cell culture, such as Gram-positive and Gram-negative bacterial contamination, such as some antibiotics.

[0091] The cell culture medium (basal culture medium) may be, for example, but not limited to, DMEM / F12, MEM, DMEM, RPMI 1640, Neuronal basal, etc. It should be understood that those skilled in the art are familiar with the preparation or purchase methods of the aforementioned basic cell culture medium. Preferred cell culture media are provided in the embodiments of the present invention.

[0092] The present invention also provides a kit containing culture media 1 to 4 as described in the present invention; preferably, it further contains pluripotent stem cells. Preferably, if necessary, the kit further contains culture media / reagents for isolating and maintaining cells. Preferably, the kit also includes instructions for use, thereby facilitating application by those skilled in the art in research or clinical practice.

[0093] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0094] Example 1: Differentiation of human pluripotent stem cells into human ovarian somatic cell-like cells

[0095] In this embodiment, human pluripotent stem cells were used as the base cells to prepare human ovarian somatic cell-like cells.

[0096] Phase 1

[0097] Human induced pluripotent stem cells (iPSCs, obtained from the National Stem Cell Transformation Resource Bank of Shanghai East Hospital (Tongji University Affiliated East Hospital)) were cultured in suspension in low-adhesion culture flasks at an inoculum size of 5 x 10⁻⁶. 5 Cells / flask. The basal medium was DMEM, 20% KSR, 1% NEAA, 1% Glutamax, and 0.7% β-mercaptoethanol (Sigma). The culture conditions were: suspension culture at 37°C and 5% CO2 constant temperature; after 2 days of culture, embryoid bodies were formed.

[0098] Let stand for 5 minutes to allow the embryoids to settle naturally. These cell spheres can be used for subsequent culture.

[0099] In addition, during this stage, human embryonic stem cells (H9-ES, purchased from Shanghai Chuanqiu Biotechnology Co., Ltd.) can be used to replace human induced pluripotent stem cells.

[0100] Phase Two

[0101] Replace with fresh culture medium and proceed with the second stage of culture.

[0102] During this stage, the inventors adopted two different cultivation methods.

[0103] (1) Option 1 (B / A / W)

[0104] Add B / A / W to the basal medium DMEM / F12 (containing 20% ​​KSR, 1% NEAA, 1% Glutamax, and 0.7% β-mercaptoethanol), that is, add:

[0105] BMP4: Final concentration 5 ng / mL;

[0106] Activin A: final concentration 6 ng / mL; and

[0107] Wnt3a: Final concentration 6 ng / mL;

[0108] Incubate for 24 hours.

[0109] (2) Option 2 (5C)

[0110] Add 5 μM of CHIR99021, an agonist of the WNT signaling pathway, to the basal medium DMEM / F12 (containing 20% ​​KSR, 1% NEAA, 1% Glutamax, and 0.7% β-mercaptoethanol). Incubate for 36 hours.

[0111] Other culture conditions for Scheme 1 and Scheme 2 above: suspension culture at 37℃ and 5% CO2 constant temperature.

[0112] After standing, the cell spheres will naturally settle, and these cell spheres can be used for subsequent culture.

[0113] Phase Three

[0114] Replace with fresh culture medium and proceed with the third stage of culture.

[0115] The basal medium DMEM / F12 (containing 20% ​​KSR, 1% NEAA, 1% Glutamax, and 0.7% β-mercaptoethanol) was treated for 3 days with Wnt3a (final concentration 6 ng / ml), Activin A (final concentration 6 ng / ml), and BMP4 (final concentration 10 ng / ml).

[0116] After standing, the cell spheres will naturally settle, and these cell spheres can be used for subsequent culture.

[0117] Phase 4

[0118] Replace with fresh culture medium and proceed with the fourth stage of culture.

[0119] Building upon the previous step, cells were attached to culture dishes treated with 0.2% gelatin. Follicle-like protein 1 (Follistain or Fstl1) (final concentration 25 ng / ml), BMP4 (final concentration 10 ng / ml), and FBS (final concentration 5%) were added to the basal medium DMEM / F12 (containing 20% ​​KSR, 1% NEAA, 1% Glutamax, and 0.7% β-mercaptoethanol) for 5-8 hours.

[0120] Phase 5

[0121] Replace with fresh culture medium and proceed with the fifth stage of culture.

[0122] The basal medium DMEM / F12 (containing 20% ​​KSR, 1% NEAA, 1% Glutamax, and 0.7% β-mercaptoethanol) was treated for 8 days with the addition of Folistain (25 ng / ml) and BMP4 (10 ng / ml); no serum was added.

[0123] Bright-field plot of human pluripotent stem cells differentiated into human ovarian somatic cell-like cells obtained by the above method is shown below. Figure 1As shown in the figure, hPSCs are human pluripotent stem cells, EB is embryoid body, ME is mesoendothelial cell, and OSLC is human ovarian somatic cell-like cells. As can be seen from the figure, this invention has obtained human ovarian somatic cell-like cells in ideal condition and in large quantities.

[0124] Example 2: Detection of cell markers at different stages of cell differentiation

[0125] In this embodiment, reverse transcription polymerase chain reaction (RT-PCR) was used to detect cell markers at various stages of cell differentiation (induced by group 5C).

[0126] The RT-PCR detection method is as follows:

[0127] (1) Total RNA extraction

[0128] (a) Transfer the collected cells (cells at various stages of cell differentiation) into 1 ml of Trizol, pipette repeatedly 3-5 times, and let stand at room temperature for 5 min. After standing, add 200 μl of chloroform, mix vigorously up and down and left and right for 20 s, and let stand at room temperature for 5 min.

[0129] (b) Centrifuge at 12,000 rpm for 15 min at 4℃;

[0130] (c) Collect the supernatant into another new enzyme-free centrifuge tube, add an equal amount of isopropanol to the supernatant, mix well from top to bottom and left to right, and let stand at room temperature for 10 minutes.

[0131] (d) Centrifuge at 12000 rpm for 10 min at 4℃;

[0132] (e) Discard the supernatant, add 1 ml of 70% anhydrous ethanol, shake to mix, and centrifuge at 7600 rpm for 5 min at 4°C.

[0133] (f) Discard the supernatant, air dry at room temperature for 2-5 minutes, add an appropriate amount of enzyme-free water, mix well, and then use a nucleic acid quantification instrument to measure the RNA concentration and purity.

[0134] (2) Preparation of cDNA by reverse transcription

[0135] Based on the RNA concentration measured above and Hifair III 1 st According to the requirements of the strand Synthesis SuperMix for qPCR (catalog number: 11141ES10) kit, take 500ng of total RNA for reverse transcription.

[0136] (3) PCR

[0137] Based on the primer sequences in Table 1 and The PCR Master Mix (With Dye) (Catalog No.: 10102ES03) kit requires PCR to be performed.

[0138] Table 1

[0139]

[0140]

[0141] The electrophoresis results of the PCR products are as follows: Figure 2 The mesoendothelial cells (ME) obtained by this differentiation protocol can express Brachyury (Bra), Mixl1, and Pax2 ( Figure 2 The human ovarian somatic cell-like cells (OSLCs) obtained by this differentiation protocol can express Foxl2 and Cyp19a1. Figure 2 These are typical markers of human ovarian somatic cell-like cells.

[0142] Quantitative analysis of PCR products showed that ( Figure 4 The human ovarian somatic cell-like cells (OSLCs) obtained by this differentiation protocol expressed high levels of Foxl2, Cyp19a1, and Ptch1.

[0143] For "Scheme 1" (B / A / W) and "Scheme 2" (5C) in the second stage of Example 1, the inventors used PCR to analyze the Brachyury markers in the cell cultures after culturing under both schemes. The results are as follows: Figure 3 .according to Figure 3 It is evident that 5C induction yields mesoendodermal cells more efficiently, resulting in significantly more mesoendodermal cells in ideal cell condition.

[0144] Example 3: Identification of human ovarian somatic cell-like cells derived from hPSCs

[0145] In this embodiment, immunocytochemistry was used to identify human ovarian somatic cell-like cells (induced by group 5C) derived from hPSCs.

[0146] The immunocytochemical detection methods are as follows:

[0147] (a) Cells were fixed with 4% paraformaldehyde (PFA) fixative and incubated at room temperature for 30 min;

[0148] (b) Discard the fixative and wash three times with calcium- and magnesium-free PBS for 5 minutes each time;

[0149] (c) Block cells with blocking solution (PBS solution containing 1% BSA + 0.2% Triton-X 100) for 1 h;

[0150] (d) The antibodies (Foxl2, diluted 1:100; Cyp19A1, diluted 1:100; Ptch1, diluted 1:100) were placed in antibody dilution buffer (PBS solution containing 5% BSA + 0.2% Triton-X 100);

[0151] (e) Discard the blocking solution; add the diluted primary antibody to the sample and incubate overnight at 4°C;

[0152] (f) The next day, the primary antibody was discarded, and the cells were washed three times with PBS for 5 minutes each time;

[0153] (g) Dilute the secondary antibody (anti-goat IgG) in antibody dilution buffer (PBS solution containing 5% BSA);

[0154] (h) Remove the PBS, add the diluted secondary antibody (1:200) to the sample, and incubate at room temperature for 1 h;

[0155] (i) Discard the secondary antibody and wash with PBS 3 times, 5 min each time;

[0156] (j) Add DAPI solution (10ug / ml) and let stand at room temperature for 2-3 minutes;

[0157] (k) Discard the DAPI solution and wash three times with PBS, 5 min each time;

[0158] (l) Discard PBS, mount with anti-fluorescence quenching agent, and observe and photograph under an inverted fluorescence microscope.

[0159] according to Figure 5 It is evident that human ovarian somatic cell-like cells derived from hPSCs express Cyp19a1, Foxl2, and Ptch1 proteins.

[0160] Regarding the suspension culture and adherent culture schemes, the inventors have found that suspension culture is preferred in stages 1 to 3, followed by adherent culture.

[0161] Simultaneously, the inventors measured hormone secretion from human ovarian somatic cell-like cells (induced by the 5C group) derived from hPSCs. After treating hPSC-derived human ovarian somatic cell-like cells with 50 μg / L testosterone (added starting on day 6 of phase 5) for 48 hours, the supernatant was collected for ELISA to measure estrogen levels. The results showed that the human ovarian somatic cell-like cells of this invention had high levels of progesterone (prog) and estradiol (E2). Figure 6 ).

[0162] Example 4: The ability of human ovarian somatic cell-like cells to promote the differentiation of female reproductive stem cells cultured in vitro.

[0163] In this embodiment, the inventors investigated the effect of the aforementioned induced human ovarian somatic cell-like cells on the differentiation capacity of female reproductive stem cells cultured in vitro.

[0164] First, the inventors seeded human female germline stem cells into induced human ovarian somatic cell-like cells (induced by group 5C) and cultured them. A differentiation system without the addition of human ovarian somatic cell-like cells served as a control.

[0165] Next, the inventors seeded human female reproductive stem cells onto a human ovarian somatic cell-like cell layer and cultured them for 5 days in a culture medium containing bFGF, EGF, insulin, transferrin, and RA. Subsequently, the inventors used immunofluorescence chemical analysis to detect the expression of marker proteins in the oocytes. The results showed that the human oocytes produced by the above method expressed oocyte-specific proteins GDF9 and ZP3, such as... Figure 7 .

[0166] Meanwhile, the inventors used RT-qPCR to detect the expression of oocyte marker genes c-KIT, FIGLA, GJA4, GDF9, and ZP3 in human oocytes generated by the above method. The results showed that these marker genes were all significantly upregulated, such as... Figure 8 .

[0167] These results indicate that induced human ovarian somatic cell-like cells have the ability to promote the differentiation of female reproductive stem cells, with excellent results and ideal state of the induced products.

[0168] The hormones secreted by human ovarian somatic cell-like cells produced by the above method were identified as being able to secrete estrogen and progesterone, and to efficiently promote the differentiation of human female reproductive stem cells.

[0169] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for preparing human ovarian somatic cell-like cells, characterized in that, The method is based on the phased culture of human stem cells, including: (1) Pre-culture cells for 1.5 to 3 days, then culture the cells in basal medium; the cells are human pluripotent stem cells; (2) The cells of (1) were treated with a GSK3 inhibitor; the GSK3 inhibitor was CHIR99021, with a concentration of 3-8 μM; the culture time was 30-48 hours. (3) Treat the cells of (2) with B / A / W, wherein B / A / W is BMP4, Activin A and Wnt3a; the concentration of BMP4 is 4-15 ng / ml, the concentration of Activin A is 5-15 ng / ml, and the concentration of Wnt3a is 5-15 ng / ml; the culture time is 2-7 days; (4) Treat (3) cells with Follistain, BMP4 and serum; Follistain concentration is 15-40 ng / ml, BMP4 concentration is 4-15 ng / ml, serum concentration is 3-10% (v / v); culture time is 3-12 hours; (5) The cells in (4) were treated with Follistain and BMP4 to obtain a culture containing human ovarian somatic cell-like cells; In steps (2) to (5), each component is added to the basic culture medium, which is a stem cell culture medium.

2. The method as described in claim 1, characterized in that, In steps (1) to (3), suspension culture is performed, and in steps (4) to (5), adherent culture is performed.

3. The method as described in claim 1, characterized in that, The basal culture medium was DMEM / F12 basal culture medium supplemented with 20±10% KSR, 1±0.5% NEAA, 1±0.5% Glutamax, 0.7±0.2 β-mercaptoethanol, and 4–10 ng / ml bFGF.

4. The method as described in claim 1, characterized in that, In step (1), the cell pre-culture time is 1.5 to 2.5 days.

5. The method as described in claim 1, characterized in that, In step (2), the concentration of the GSK3 inhibitor CHIR99021 is 4–6 μM.

6. The method as described in claim 1, characterized in that, In step (3), The concentration of BMP4 was 9–12 ng / ml; The concentration of Activin A was 9–12 ng / ml; The concentration of Wnt3a was 9–12 ng / ml; The incubation period is 2.5 to 7 days.

7. The method as described in claim 1, characterized in that, In step (4), The concentration of follistain is 20–30 ng / ml; The concentration of BMP4 was 9–12 ng / ml; Serum concentrations ranged from 3% to 10% (v / v); The incubation time is 4 to 10 hours.

8. The method as described in claim 1, characterized in that, In step (5), Follistain concentration is 15–40 ng / ml; The concentration of BMP4 is 4–15 ng / ml; The incubation period is 4 to 12 days.

9. The method as described in claim 8, characterized in that, In step (5), The concentration of follistain is 20–30 ng / ml; The concentration of BMP4 was 9–12 ng / ml; The incubation period is 5 to 10 days.

10. A human ovarian somatic cell-like cell obtained by induction of human pluripotent stem cells, which is prepared by the method described in any one of claims 1 to 9.

11. The human ovarian somatic cell-like cells obtained by induction of human pluripotent stem cells as described in claim 10, characterized in that, It has the characteristics selected from the following group: The human ovarian somatic cell-like cells described are predominantly epithelial in morphology, exhibiting fibroblast-like cells that are short spindle-shaped or triangular. The human ovarian somatic cell-like cells showed positive results for Foxl2, cyp19a1, and Ptch1 markers; and / or The human ovarian somatic cell-like cells described herein can promote the differentiation of human female reproductive stem cells.

12. The application of the human ovarian somatic cell-like cells according to claim 10, used to promote the differentiation of human female reproductive stem cells into human oocytes; the application is an in vitro, non-therapeutic application.

13. A method for promoting the differentiation of human female reproductive stem cells, characterized in that, The method includes: using the human ovarian somatic cell-like cells as described in claim 10 as supporting cells, culturing human female reproductive stem cells on the cell layer of the human ovarian somatic cell-like cells or co-culturing human female reproductive stem cells with the human ovarian somatic cell-like cells.

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