In vitro induction of gonadal somatic cells

Abstract

Provided herein are methods for generating gonadal cell populations, such as ovarian somatic cells, from pluripotent stem cells, including the gonadal cell populations and intermediate cell populations generated in the methods. TIFF2023549137000020.tif65128

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Application No. 63 / 108,666, entitled “HUMAN GRANULOSA DIFFERENTIATION,” filed 2 November 2020, and U.S. Provisional Application No. 63 / 222,953, entitled “IN VITRO DERIVATION OF GONADAL SOMATIC CELLS,” filed 16 July 2021, the contents of which these applications are incorporated herein by reference in their entirety for all purposes.

[0002] Field of Invention This disclosure relates, in general terms, to a method for generating gonadal cell populations, such as ovarian somatic cells, from pluripotent stem cells. It also includes the gonadal cell populations and intermediate cell populations generated in the method. [Overview of the project]

[0003] This specification provides a method for producing a gonadal cell population, comprising: (a) culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A to produce a first intermediate cell population; (b) culturing the first intermediate cell population for a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway modulator (RAPM) to thereby produce a second intermediate cell population; and (c) culturing the second intermediate cell population for a third period in a gonadal induction medium containing BMP and FGF to produce a gonadal cell population. In some embodiments, the Specified Method for Producing a Gonadal Cell Population is provided, comprising the steps of (a) culturing mesoderm cells or mesoderm-like cells for a first period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM) to produce a second intermediate cell population, and (b) culturing the second intermediate cell population for a second period in a Gonadal induction medium containing BMP and FGF to produce a Gonadal Cell Population. In some embodiments, the Method for Producing a Gonadal Cell Population is provided, comprising the step of culturing intermediate mesoderm cells or intermediate mesoderm-like cells for a period of time in a Gonadal induction medium containing BMP and FGF to produce a Gonadal Cell Population. In some embodiments, the BMP is BMP4, BMP2, BMP7, BMP15 or any combination thereof. In some embodiments, the Gonadal induction medium comprises BMP4. In some embodiments, the Gonadal induction medium further comprises follistatin.

[0004] This specification provides a method for producing a gonadal cell population, comprising: (a) culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A to produce a first intermediate cell population; (b) culturing the first intermediate cell population for a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM) to thereby produce a second intermediate cell population; and (c) culturing the second intermediate cell population for a third period in a gonadal induction medium containing follistatin, BMP4, and FGF to produce a gonadal cell population.

[0005] This specification also provides a method for producing a gonadal cell population, comprising the steps of (a) culturing mesoderm cells or mesoderm-like cells for a first period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM) to produce a second intermediate cell population, and (b) culturing the second intermediate cell population for a second period in a gonadal induction medium containing follistatin, BMP4, and FGF to produce a gonadal cell population.

[0006] This specification also provides a method for producing a population of gonadal cells, comprising the step of culturing intermediate mesoderm cells or intermediate mesoderm-like cells for a period of time in a gonadal induction medium containing follistatin, BMP4, and FGF in order to produce a population of gonadal cells.

[0007] This specification also provides a method for producing a first intermediate cell population, comprising the step of culturing pluripotent stem cells for a period of time in a mesoderm induction medium containing activin A and a glycogen synthase kinase-3 inhibitor in order to produce a first intermediate cell population. This specification also provides a method for producing a second intermediate cell population, comprising the step of culturing mesoderm cells or mesoderm-like cells for a period of time in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM), thereby producing a second intermediate cell population. This specification also provides a method for producing a second intermediate cell population, comprising: (a) culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A and a glycogen synthase kinase-3 inhibitor to produce a first intermediate cell population; and (b) culturing the first intermediate cell population for a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway modulator (RAPM), FGF, and a glycogen synthase kinase-3 inhibitor, thereby producing a second intermediate cell population. In some part of any embodiment, the first and second intermediate cell populations are intermediate populations of differentiated pluripotent stem cells for producing a gonadal cell population.

[0008] In some part of any embodiment, at least a portion of the cells in the first intermediate cell population express Brachyury.

[0009] In some part of any embodiment, at least a portion of the cells in the second intermediate cell population express OSR1, PAX2, or LHX.

[0010] In some part of any embodiment, at least a portion of the cells in the gonadal cell population express FOXL2, NR2F2, or RUNX1.

[0011] In some of the embodiments, pluripotent stem cells are seeded at a density of approximately 10,000 to 40,000 cells per cm². In some of the embodiments, pluripotent stem cells are seeded on culture plates coated with fibronectin. In some of the embodiments, pluripotent stem cells are seeded on culture plates coated with Matrigel.

[0012] In some parts of any embodiment, the mesoderm induction medium further comprises FGF. In some parts of any embodiment, the mesoderm induction medium further comprises BMP4. In some parts of any embodiment, the mesoderm induction medium further comprises a glycogen synthase kinase-3 inhibitor. In some parts of any embodiment, the mesoderm induction medium further comprises an apoptosis inhibitor.

[0013] In some aspects of the model, the concentration of activin A in the mesoderm induction medium is approximately 30 ng / mL to approximately 70 ng / mL. In some aspects of the model, the concentration of activin A in the mesoderm induction medium is approximately 50 ng / mL.

[0014] In some aspects of the mesoderm induction medium, the FGF is FGF2. In some aspects, the concentration of FGF2 in the mesoderm induction medium is approximately 5 ng / mL to approximately 20 ng / mL. In some aspects, the concentration of FGF2 in the mesoderm induction medium is approximately 12 ng / mL.

[0015] In some embodiments, the concentration of BMP4 in the mesoderm induction medium is approximately 10 ng / mL to approximately 50 ng / mL. In some embodiments, the concentration of BMP4 in the mesoderm induction medium is approximately 30 ng / mL.

[0016] In some embodiments, the glycogen synthase kinase-3 inhibitor in the mesoderm induction medium is CHIR99021. In some embodiments, the concentration of CHIR99021 is approximately 1 μM to approximately 5 μM. In some embodiments, the concentration of CHIR99021 in the mesoderm induction medium is approximately 3 μM.

[0017] In some aspects of the mesoderm induction medium, the apoptosis inhibitor is Y-27632. In some aspects, the concentration of Y-27632 is approximately 5 μM to approximately 20 μM. In some aspects, the concentration of Y-27632 is approximately 10 μM. In some aspects of the mesoderm induction medium, the apoptosis inhibitors include Chroman 1, Emricasan, and trans-ISRIB. In some aspects, the apoptosis inhibitor is Chroman 1, and the concentration of Chroman 1 is approximately 30 nM to approximately 70 nM. In some aspects, the concentration of Chroman 1 is approximately 50 nM. In some aspects, the apoptosis inhibitor is Emricasan, and the concentration of Emricasan is approximately 2 μM to approximately 10 μM. In some aspects, the concentration of Emricasan is approximately 5 μM. In some aspects, the apoptosis inhibitor is trans-ISRIB, and the concentration of trans-ISRIB is approximately 0.2 μM to approximately 2 μM. In some embodiments, the concentration of trans-ISRIB is approximately 0.7 μM.

[0018] In some of the embodiments, the culture period in mesoderm-inducing medium is approximately 24 hours to approximately 96 hours. In some of the embodiments, the culture period in mesoderm-inducing medium is approximately 24 hours to approximately 72 hours. In some of the embodiments, the culture period in mesoderm-inducing medium is approximately 56 hours to approximately 72 hours.

[0019] In some of the embodiments, at least 90% of the cells in the first intermediate cell population express brachiuri. In some of the embodiments, at least 80% of the cells in the first intermediate cell population express one or more of MIXL1, N-cadherin, EpCam, and NCAM. In some of the embodiments, at least 90% of the cells in the first intermediate cell population express brachiuri, N-cadherin, EpCam, and NCAM. In some of the embodiments, at least 90% of the cells in the first intermediate cell population are mesoderm cells or mesoderm-like cells. In some of the embodiments, the first intermediate cell population is essentially composed of mesoderm cells or mesoderm-like cells.

[0020] In some aspects, the first intermediate cell population is replated onto a new fibronectin-coated culture plate prior to culturing in the intermediate mesoderm induction medium. In some aspects, the first intermediate cell population is enzymatically released, centrifuged, resuspended, and then replated. In some aspects of any embodiment, the first intermediate cell population is plated at a density of about 5000 to about 25000 cells / cm2.

[0021] In some aspects of any embodiment, the intermediate mesoderm induction medium further comprises FGF. In some aspects of any embodiment, the intermediate mesoderm induction medium further comprises a glycogen synthase kinase-3 inhibitor. In some aspects of any embodiment, the intermediate mesoderm induction medium further comprises activin A. In some aspects of any embodiment, the intermediate mesoderm induction medium further comprises an apoptosis inhibitor.

[0022] In some aspects of any embodiment, the method comprises culturing the first intermediate cell population (i) first in an intermediate mesoderm induction medium comprising a first concentration of an apoptosis inhibitor and (ii) then in an intermediate mesoderm induction medium comprising an apoptosis inhibitor at a second concentration or less.

[0023] In some aspects of any embodiment, the RAPM in the intermediate mesoderm induction medium is a RAR agonist. In some aspects, the RAPM comprises retinoic acid (RA) and / or TTNPB. In some aspects, the RAPM is RA. In some aspects, the concentration of RA in the intermediate mesoderm induction medium is about 0.5 μM to about 2 μM. In some aspects, the concentration of RA in the intermediate mesoderm induction medium is about 1 μM. In some aspects, the RAPM is TTNPB. In some aspects, the concentration of TTNPB in the intermediate mesoderm induction medium is about 0.2 μM to about 1 μM. In some aspects, the concentration of TTNPB in the intermediate mesoderm induction medium is about 0.5 μM.

[0024] In some aspects of the medium, FGF in the intermediate mesoderm induction medium is FGF2. In some aspects, the concentration of FGF2 is approximately 10 ng / mL to approximately 30 ng / mL. In some aspects, the concentration of FGF2 in the intermediate mesoderm induction medium is approximately 20 ng / mL.

[0025] In some aspects of the medium, the glycogen synthase kinase-3 inhibitor in the intermediate mesoderm induction medium is CHIR99021. In some aspects, the concentration of CHIR99021 is approximately 1 μM to approximately 5 μM. In some aspects, the concentration of CHIR99021 in the intermediate mesoderm induction medium is approximately 3 μM.

[0026] In some aspects of the medium, the concentration of activin A in the intermediate mesoderm induction medium is approximately 10 ng / mL to approximately 50 ng / mL. In some aspects, the concentration of activin A in the intermediate mesoderm induction medium is approximately 30 ng / mL.

[0027] In some aspects of the intermediate mesoderm induction medium, the apoptosis inhibitor is Y-27632. In some aspects, the concentration of Y-27632 is approximately 5 μM to approximately 20 μM. In some aspects, the apoptosis inhibitor in the intermediate mesoderm induction medium includes chroman-1, emricasan, and trans-ISRIB. In some aspects, the apoptosis inhibitor is chroman-1. In some aspects, the concentration of chroman-1 is approximately 30 nM to approximately 70 nM. In some aspects, the apoptosis inhibitor is emricasan. In some aspects, the concentration of emricasan is approximately 2 μM to approximately 10 μM. In some aspects, the apoptosis inhibitor is trans-ISRIB. In some aspects, the concentration of trans-ISRIB is approximately 0.2 μM to approximately 2 μM.

[0028] In some embodiments, the apoptosis inhibitor in the intermediate mesoderm induction medium is Y-27632, in which case the method includes the step of culturing the first intermediate cell population (i) first in intermediate mesoderm induction medium containing about 10 μM of Y-27632, and (ii) then in intermediate mesoderm induction medium containing about 2 μM or less of Y-27632. In some embodiments, the method includes the step of culturing the first intermediate cell population (i) first in intermediate mesoderm induction medium containing about 10 μM of Y-27632 for about 24 hours, and (ii) then in intermediate mesoderm induction medium containing about 2 μM or less of Y-27632 for about 5 to 6 days.

[0029] In some aspects of the model, the culture period in intermediate mesoderm induction medium is approximately 4 to 14 days. In some aspects of the model, the culture period in intermediate mesoderm induction medium is approximately 5 to 9 days.

[0030] In some parts of any embodiment, the apoptosis inhibitor in the intermediate mesoderm induction medium is Y-27632, and the concentration of Y-27632 during culture in the intermediate mesoderm induction medium is (a) approximately 10 μM for the first 24 hours, (b) approximately 2 μM or less from 24 to 72 hours, (c) approximately 0.5 μM or less from 72 to 120 hours, or (d) approximately 0.1 μM or less after 120 hours. In some parts of any embodiment, the first intermediate cell population is cultured for approximately 24 hours in the intermediate mesoderm induction medium containing the apoptosis inhibitor, and after approximately 24 hours, a portion of the medium is first replaced with intermediate mesoderm induction medium that does not contain the apoptosis inhibitor. In some embodiments, the said portion of the medium is approximately 80% of the medium. In some embodiments, the method further includes subsequent medium changes, which include replacing a portion of the medium approximately every 48 hours after the initial 24 hours of culture. In some embodiments, the portion of the culture medium in each subsequent culture medium exchange is approximately 80% of the culture medium.

[0031] In some parts of any embodiment, at least 90% of the cells in the second intermediate cell population express one or more of OSR1, PAX2, LHX1, and RUNX1. In some parts of any embodiment, at least 90% of the cells in the second intermediate cell population express two or more of OSR1, PAX2, LHX1, and RUNX1. In some parts of any embodiment, at least 90% of the cells in the second intermediate cell population express OSR1, PAX2, and LHX1. In some parts of any embodiment, at least 90% of the cells in the second intermediate cell population are intermediate mesoderm cells or intermediate mesoderm-like cells. In some parts of any embodiment, the second intermediate cell population is essentially composed of intermediate mesoderm cells or intermediate mesoderm-like cells.

[0032] In some aspects of the embodiment, the second intermediate cell population is replated onto a fresh fibronectin-coated culture plate before culturing in gonad induction medium. In some embodiments, the second intermediate cell population is enzymatically detached, centrifuged, resuspended, and then replated. In some embodiments, the second intermediate cell population is plated at a density of approximately 5,000 to 25,000 cells / cm².

[0033] In some aspects of the gonad induction medium, the gonad induction medium further comprises RAPM. In some aspects, the RAPM in the gonad induction medium is a RAR agonist. In some aspects, the RAPM comprises retinoic acid (RA) and / or TTNPB. In some aspects, the RAPM is RA. In some aspects, the concentration of RA in the gonad induction medium is about 0.5 μM to about 2 μM. In some aspects, the RAPM is TTNPB. In some aspects, the concentration of TTNPB in the gonad induction medium is about 0.2 μM to about 1 μM.

[0034] In some aspects of the gonad induction medium, the gonad induction medium further comprises an apoptosis inhibitor. In some aspects, the apoptosis inhibitor in the gonad induction medium is Y-27632. In some aspects, the concentration of Y-27632 is approximately 5 μM to approximately 20 μM. In some aspects, the concentration of Y-27632 is approximately 10 μM. In some aspects, the apoptosis inhibitor in the gonad induction medium comprises chroman-1, emricasan, and trans-ISRIB. In some aspects, the apoptosis inhibitor is chroman-1, and the concentration of chroman-1 is approximately 30 nM to approximately 70 nM. In some aspects, the concentration of chroman-1 is approximately 50 nM. In some aspects, the apoptosis inhibitor is emricasan, and the concentration of emricasan is approximately 2 μM to approximately 10 μM. In some aspects, the concentration of emricasan is approximately 5 μM. In some aspects, the apoptosis inhibitor is trans-ISRIB, and the concentration of trans-ISRIB is approximately 0.2 μM to approximately 2 μM. In some embodiments, the concentration of trans-ISRIB is approximately 0.7 μM.

[0035] In some part of any embodiment, the gonad induction medium further comprises follistatin.

[0036] In some embodiments, the concentration of follistatin in the gonad induction medium is approximately 10 ng / mL to approximately 50 ng / mL. In some embodiments, the concentration of follistatin in the gonad induction medium is approximately 25 ng / mL.

[0037] In some embodiments, the BMPs in the gonad induction medium include BMP4, BMP2, BMP7, BMP15, or any combination thereof. In some embodiments, the gonad induction medium includes at least BMP4 alone or in combination with another BMP. In some embodiments, BMP4 is the only BMP included in the gonad induction medium. In some embodiments, the total concentration of BMPs in the gonad induction medium is approximately 5 ng / mL to approximately 20 ng / mL or approximately 20 ng / mL to approximately 70 ng / mL. In some embodiments, the total concentration of BMPs in the gonad induction medium is approximately 10 ng / mL or approximately 50 ng / mL.

[0038] In some of the embodiments, the concentration of BMP4 in the gonad induction medium is approximately 5 ng / mL to approximately 20 ng / mL. In some of the embodiments, the concentration of BMP4 in the gonad induction medium is approximately 20 ng / mL to approximately 70 ng / mL. In some of the embodiments, the concentration of BMP4 in the gonad induction medium is approximately 10 ng / mL or approximately 50 ng / mL.

[0039] In some embodiments, the FGF in the gonad induction medium includes FGF2, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, or any combination thereof. In some embodiments, the gonad induction medium includes at least FGF2 alone or in combination with another FGF. In some embodiments, the only FGF contained in the gonad induction medium is FGF2. In some embodiments, the concentration of FGF is approximately 1 ng / mL to approximately 10 ng / mL or approximately 5 ng / mL to approximately 25 ng / mL. In some embodiments, the concentration of FGF is approximately 5 ng / mL or approximately 10 ng / mL.

[0040] In some aspects of the gonad induction medium, FGF is FGF2. In some aspects, the concentration of FGF2 is approximately 1 ng / mL to approximately 10 ng / mL. In some aspects, the concentration of FGF2 is approximately 5 ng / mL to approximately 25 ng / mL. In some aspects, the concentration of FGF2 in the gonad induction medium is approximately 5 ng / mL or approximately 10 ng / mL.

[0041] In some of the embodiments, the incubation period in gonadal induction medium is approximately 5 to 21 days. In some of the embodiments, the incubation period in gonadal induction medium is approximately 7 to 14 days.

[0042] In some aspects of any embodiment, the gonadal cell population includes ovarian somatic cells. In some aspects of any embodiment, the gonadal cell population consists of ovarian somatic cells.

[0043] In some of the embodiments, at least 20% of the cells in the gonadal population are FOXL2-positive cells. In some of the embodiments, at least 20% of the cells in the gonadal population are NR2F2-positive cells. In some of the embodiments, at least 20% of the cells in the gonadal population are KRT-19-positive cells. In some of the embodiments, at least 90% of the gonadal population consists of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. In some of the embodiments, the gonadal population essentially consists of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. In some of the embodiments, FOXL2-positive cells include granulosa cells. In some of the embodiments, NR2F2-positive cells include stromal cells and / or granulosa cells. In some of the embodiments, KRT-19-positive cells include ovarian epithelial cells.

[0044] In any part of the present invention, the gonadal cell population produced by this method is characterized by one or more of the following, compared to the corresponding gonadal cell population produced by a method that does not include RAPM in the intermediate mesoderm induction medium: (I) an increased amount of NR2F2-expressing cells in the gonadal cell population, and / or (II) an increased amount of FOXL2-expressing cells in the gonadal cell population, and / or (III) an increased amount of RUNX1-expressing cells in the gonadal cell population, and / or (IV) an increased amount of WNT6-expressing cells in the gonadal cell population, and / or (V) an increased amount of NR5A1-expressing cells in the gonadal cell population, and / or (VI) an increased amount of OSR1-expressing cells in the gonadal cell population, and / or (VII) an increased amount of LHX9-expressing cells in the gonadal cell population, and / or (VIII) an increased amount of EMX2-expressing cells in the gonadal cell population.

[0045] In any part of the present invention, the gonadal cell population produced by this method is characterized by one or more of the following, compared to the corresponding gonadal cell population produced by a method containing a lower concentration of RAPM in intermediate mesoderm induction medium: (I) an increased amount of NR2F2-expressing cells in the gonadal cell population, and / or (II) an increased amount of FOXL2-expressing cells in the gonadal cell population, and / or (III) an increased amount of RUNX1-expressing cells in the gonadal cell population, and / or (IV) an increased amount of WNT6-expressing cells in the gonadal cell population, and / or (V) an increased amount of NR5A1-expressing cells in the gonadal cell population, and / or (VI) an increased amount of OSR1-expressing cells in the gonadal cell population.

[0046] In any part of the present invention, the gonadal cell population produced by this method is characterized by one or more of the following, compared to the corresponding gonadal cell population produced by a method that does not include a glycogen synthase kinase-3 inhibitor in the intermediate mesoderm induction medium: (I) an increased amount of NR2F2-expressing cells in the gonadal cell population, and / or (II) an increased amount of FOXL2-expressing cells in the gonadal cell population, and / or (III) an increased amount of RUNX1-expressing cells in the gonadal cell population, and / or (IV) an increased amount of WNT6-expressing cells in the gonadal cell population, and / or (V) an increased amount of NR5A1-expressing cells in the gonadal cell population, and / or (VI) an increased amount of OSR1-expressing cells in the gonadal cell population, and / or (VII) an increased amount of LHX9-expressing cells in the gonadal cell population, and / or (VIII) an increased amount of EMX2-expressing cells in the gonadal cell population.

[0047] In any part of the present invention, the gonadal cell population produced by this method is characterized by one or more of the following, compared to the corresponding gonadal cell population produced by a method that includes a lower concentration of glycogen synthase kinase-3 inhibitor in the intermediate mesoderm induction medium: (I) an increased amount of NR2F2-expressing cells in the gonadal cell population, and / or (II) an increased amount of FOXL2-expressing cells in the gonadal cell population, and / or (III) an increased amount of RUNX1-expressing cells in the gonadal cell population, and / or (IV) an increased amount of WNT6-expressing cells in the gonadal cell population, and / or (V) an increased amount of NR5A1-expressing cells in the gonadal cell population, and / or (VI) an increased amount of OSR1-expressing cells in the gonadal cell population, and / or (VII) an increased amount of LHX9-expressing cells in the gonadal cell population, and / or (VIII) an increased amount of EMX2-expressing cells in the gonadal cell population.

[0048] In any part of the present invention, the second intermediate cell population produced by this method exhibits one or more of the following characteristics compared to the corresponding second intermediate cell population produced by a method that does not include RAPM in the intermediate mesoderm induction medium: (I) increased LHX1 expression in the second intermediate cell population, and / or (II) increased PAX2 expression in the second intermediate cell population, and / or (III) increased WT1 expression in the second intermediate cell population, and / or (IV) increased RUNX1 expression in the second intermediate cell population, and / or (V) increased viability of the second intermediate cell population, and / or (VI) more uniform cell morphology in the second intermediate cell population.

[0049] In any part of the present invention, the second intermediate cell population produced by the present invention exhibits one or more of the following characteristics compared to the corresponding second intermediate cell population produced by a method containing a lower concentration of RAPM in the intermediate mesoderm induction medium: (I) increased LHX1 expression in the second intermediate cell population, and / or (II) increased PAX2 expression in the second intermediate cell population, and / or (III) increased WT1 expression in the second intermediate cell population, and / or (IV) increased RUNX1 expression in the second intermediate cell population, and / or (V) increased viability of the second intermediate cell population, and / or (VI) more uniform cell morphology in the second intermediate cell population.

[0050] In any part of the present invention, the second intermediate cell population produced by this method is characterized by one or more of the following, compared to the corresponding second intermediate cell population produced by a method in which the intermediate mesoderm induction medium does not contain a glycogen synthase kinase-3 inhibitor: (I) increased LHX1 expression in the second intermediate cell population, and / or (II) increased PAX2 expression in the second intermediate cell population, and / or (III) increased WT1 expression in the second intermediate cell population, and / or (IV) increased RUNX1 expression in the second intermediate cell population, and / or (V) increased viability of the second intermediate cell population, and / or (VI) more uniform cell morphology of the second intermediate cell population.

[0051] In some part of any embodiment, the second intermediate cell population produced by this method exhibits the following characteristics compared to the corresponding second intermediate cell population produced by a method containing a lower concentration of glycogen synthase kinase-3 inhibitor in the intermediate mesoderm induction medium: (I) increased LHX1 expression in the second intermediate cell population, and / or (II) increased PAX2 expression in the second intermediate cell population, and / or (III) increased WT1 expression in the second intermediate cell population, and / or (IV) increased RUNX1 expression in the second intermediate cell population, and / or (V) increased viability of the second intermediate cell population, and / or (VI) more uniform cell morphology of the second intermediate cell population.

[0052] In any part of the present invention, the second intermediate cell population produced by this method exhibits, compared to the corresponding second intermediate cell population produced by a method that does not include RAPM, one or more of the following: (I) increased potential for differentiation into NR2F2-expressing gonadal cells, and / or (II) increased potential for differentiation into FOXL2-expressing gonadal cells, and / or (III) increased potential for differentiation into RUNX1-expressing gonadal cells, and / or (IV) increased potential for differentiation into WNT6-expressing gonadal cells, and / or (V) increased potential for differentiation into NR5A1-expressing gonadal cells, and / or (VI) increased potential for differentiation into OSR1-expressing gonadal cells.

[0053] In some part of any embodiment, the second intermediate cell population produced by this method exhibits, compared to the corresponding second intermediate cell population produced by a method containing a lower concentration of RAPM in the intermediate mesoderm induction medium, (I) an increased potential for the second intermediate cell population to differentiate into NR2F2-expressing gonadal cells, and / or (II) an increased potential for the second intermediate cell population to differentiate into FOXL2-expressing gonadal cells, and / or (III) an increased potential for the second intermediate cell population to differentiate into RUNX1-expressing gonadal cells, and / or (IV) an increased potential for the second intermediate cell population to differentiate into WNT6-expressing gonadal cells, and / or (V) an increased potential for the second intermediate cell population to differentiate into NR5A1-expressing gonadal cells, and / or (VI) an increased potential for the second intermediate cell population to differentiate into OSR1-expressing gonadal cells.

[0054] In any part of the present invention, the second intermediate cell population produced by this method is characterized by one or more of the following, compared to the corresponding second intermediate cell population produced by a method in which the intermediate mesoderm induction medium does not contain a glycogen synthase kinase-3 inhibitor: (I) increased potential for the second intermediate cell population to differentiate into NR2F2-expressing gonadal cells, and / or (II) increased potential for the second intermediate cell population to differentiate into FOXL2-expressing gonadal cells, and / or (III) increased potential for the second intermediate cell population to differentiate into RUNX1-expressing gonadal cells, and / or (IV) increased potential for the second intermediate cell population to differentiate into WNT6-expressing gonadal cells, and / or (V) increased potential for the second intermediate cell population to differentiate into NR5A1-expressing gonadal cells, and / or (VI) increased potential for the second intermediate cell population to differentiate into OSR1-expressing gonadal cells.

[0055] In any part of the present invention, the second intermediate cell population produced by this method exhibits one or more of the following characteristics compared to the corresponding second intermediate cell population produced by a method containing a lower concentration of glycogen synthase kinase-3 inhibitor in the intermediate mesoderm induction medium: (I) increased potential for differentiation into NR2F2-expressing gonadal cells, and / or (II) increased potential for differentiation into FOXL2-expressing gonadal cells, and / or (III) increased potential for differentiation into RUNX1-expressing gonadal cells, and / or (IV) increased potential for differentiation into WNT6-expressing gonadal cells, and / or (V) increased potential for differentiation into NR5A1-expressing gonadal cells, and / or (VI) increased potential for differentiation into OSR1-expressing gonadal cells.

[0056] In any part of the present invention, the gonadal cell population produced by this method is a gonadal cell population that, compared to the corresponding gonadal cell population produced by a method containing a lower concentration of RAPM in the gonadal induction medium, (I) has reduced levels of FOXL2 expression in the gonadal cell population, and / or (II) has reduced levels of NR1H4 and / or KITLG expression in the gonadal cell population, and / or (III) has increased levels of KRT-19 expression in the gonadal cell population, and / or (IV) has increased levels of cytoplasmic KRT-19 expression in the gonadal cell population, and / or (V) has increased levels of MSLN, LRRN4 and / or TMEM151A expression in the gonadal cell population.

[0057] In any part of the present invention, the gonadal cell population produced by this method is a gonadal cell population that, compared to the corresponding gonadal cell population produced by a method in which the gonadal induction medium does not contain RAPM, (I) has reduced levels of FOXL2 expression in the gonadal cell population, and / or (II) has reduced levels of NR1H4 and / or KITLG expression in the gonadal cell population, and / or (III) has increased levels of KRT-19 expression in the gonadal cell population, and / or (IV) has increased levels of cytoplasmic KRT-19 expression in the gonadal cell population, and / or (V) has increased levels of MSLN, LRRN4 and / or TMEM151A expression in the gonadal cell population.

[0058] In any part of the embodiment, the gonadal cell population is a gonadal cell population that, compared to a corresponding gonadal cell population produced by a gonadal induction process with a shorter contact period with RAPM, (I) has reduced levels of FOXL2 expression in the gonadal cell population, and / or (II) has reduced levels of NR1H4 and / or KITLG expression in the gonadal cell population, and / or (III) has increased levels of KRT-19 expression in the gonadal cell population, and / or (IV) has increased levels of cytoplasmic KRT-19 expression in the gonadal cell population, and / or (V) has increased levels of MSLN, LRRN4 and / or TMEM151A expression in the gonadal cell population.

[0059] In some aspects of any given embodiment, pluripotent stem cells are mammalian stem cells. In some aspects, pluripotent stem cells are human pluripotent stem cells. In some aspects, pluripotent stem cells are bovine stem cells. In some aspects, pluripotent stem cells are mouse pluripotent stem cells. In some aspects, pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

[0060] In some part of any embodiment, the gonadal population comprises one or more populations selected from granulosa cells, ovarian stromal cells, and epithelial cells or a combination thereof. In some part of any embodiment, the gonadal population comprises a mixture of granulosa cells, ovarian stromal cells, and epithelial cells.

[0061] This specification provides gonadal cell populations produced by any of the methods provided. In some embodiments, the gonadal population comprises one or more populations selected from granulosa cells, ovarian stromal cells, and epithelial cells or a combination thereof. In some embodiments, the gonadal population comprises a mixture of granulosa cells, ovarian stromal cells, and epithelial cells.

[0062] This specification provides a first intermediate cell population produced by any of the methods provided.

[0063] This specification provides a second intermediate cell population produced by any of the embodiments provided.

[0064] The provided embodiments include the following embodiments: 1. A step of culturing pluripotent stem cells in the presence of activin A, glycogen synthase kinase-3 inhibitor, and ROCK inhibitor in order to produce early mesoderm-like cells (iMeLCs). A step of culturing iMeLC for a first period in the presence of FGF2, glycogen synthase kinase-3 inhibitor and ROCK inhibitor. The process involves reducing the amount of ROCK inhibitor in iMeLC culture in order to produce intermediate mesoderm cells, and then culturing the iMeLC over a second period, and The process involves culturing intermediate mesoderm cells in the presence of follistatin, BMP4, FGF2, and a ROCK inhibitor to produce granulosa cells. A method for producing granulosa cells, including [the specified element]. 2. The method according to embodiment 1, wherein the glycogen synthase kinase-3 inhibitor is CHIR99021. 3. The method according to embodiment 1 or 2, wherein the ROCK inhibitor is Y-27632 or CET. 4. A method according to any of embodiments 1 to 3, wherein pluripotent stem cells are cultured for approximately 56 to 72 hours. 5. A method according to any of embodiments 1 to 4, wherein pluripotent stem cells are cultured for approximately 65 hours. 6. A method according to any one of embodiments 1 to 5, wherein pluripotent stem cells are cultured in a medium containing activin A, a glycogen synthase kinase-3 inhibitor, and a ROCK inhibitor, and the medium is replaced with fresh medium approximately every 24 hours. 7. Any method according to embodiment 1 to 6, wherein the first period is approximately 24 hours. 8. Any method according to embodiment 1 to 7, wherein the second period is approximately 5 or 6 days. 9. A method according to any one of embodiments 1 to 8, wherein iMeLC is cultured for a first period in a medium containing FGF2, a glycogen synthase kinase-3 inhibitor, and a ROCK inhibitor, and after the first period, a portion of the medium is replaced with a medium containing FGF2 and a glycogen synthase kinase-3 inhibitor but not a ROCK inhibitor. 10. The method according to embodiment 9, wherein the aforementioned portion of the culture medium constitutes approximately 80% of the culture medium. 11. The method according to embodiment 9 or 10, further comprising the step of replacing a second portion of the culture medium approximately every 48 hours during the second period. 12. The method of embodiment 11, wherein the second portion of the culture medium constitutes approximately 80% of the culture medium. 13. Any method according to embodiment 11 to 12, wherein intermediate mesoderm cells are cultured for a period of approximately 5 to 7 days. 14. Any method according to embodiment 11 to 13, wherein intermediate mesodermal cells are cultured in a medium containing follistatin, BMP4, FGF2, and a ROCK inhibitor, and a portion of the medium is replaced with fresh medium approximately every 24 to 48 hours. 15. A method in which iMeLC expresses Brachyuris, as described in any of embodiments 11 to 14. 16. A method according to any one of embodiments 11 to 13, wherein intermediate mesoderm cells express OSR1, PAX2, and LHX1. 17. A method according to any one of embodiments 11 to 16, wherein granulosa cells express FOXL2 and connexin 43. 18. Any method according to embodiment 11 to 17, wherein the pluripotent stem cells are human induced pluripotent stem cells. 19. A population comprising granulosa cells produced by any of the methods described in aspects 11 to 18.

[0065] In any of the provided embodiments, including any aspect of the provided method or the provided population, (a) one or more of the culture steps include adhesion culture, and / or (b) one or more of the culture steps include three-dimensional organoid culture. In some embodiments, one or more of the culture steps include adhesion culture. In some embodiments, one or more of the culture steps include three-dimensional organoid culture.

[0066] In any part of the embodiments provided, the method is performed in vitro. In some embodiments, the method produces an in vitro stem cell-derived gonadal cell population. In certain embodiments, the cells are an in vitro stem cell-derived gonadal cell population, such as an ovarian somatic cell population (OSC).

[0067] This specification also provides in vitro stem cell-derived gonadal cell populations comprising FOXL2-expressing cells, NR2F2-expressing cells, and / or KRT-19-expressing cells. In some embodiments, the gonadal cell population is an ovarian somatic cell population. In some embodiments, the population comprises at least a first cell type expressing FOXL2, a second cell type expressing NR2F2, and a third cell type expressing KRT-19. In some embodiments, at least 20% of the cells in the cell population are FOXL2-positive cells. In some embodiments, at least 20% of the cells in the cell population are NR2F2-positive cells. In some embodiments, at least 20% of the cells in the gonadal cell population are KRT19-positive cells. In some embodiments, (a) at least 20% of the cells in the cell population are FOXL2-positive cells, and / or (b) at least 20% of the cells in the cell population are NR2F2-positive cells, and / or (c) at least 20% of the cells in the gonadal cell population are KRT19-positive cells. In some embodiments, at least 90% of the gonadal cell population consists of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells, and optionally, the gonadal somatic cell population consists essentially of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. In some embodiments, the FOXL2-positive cells include granulosa cells, the NR2F2-positive cells include ovarian stromal cells and / or granulosa cells, and the KRT-19-positive cells include ovarian epithelial cells.

[0068] In part of any aspect of the provided gonadal cell population, the cell population differentiates from pluripotent stem cells. In part of any aspect, the gonadal cell population is induced in a process comprising: (a) culturing pluripotent stem cells for a period of one phase in a mesoderm induction medium containing activin A to produce a first intermediate cell population; (b) culturing the first intermediate cell population for a period of two phases in an intermediate mesoderm induction medium containing a retinoic acid pathway modulator (RAPM) to thereby produce a second intermediate cell population; and (c) culturing the second intermediate cell population for a period of three phases in a gonadal induction medium containing BMP and FGF and optionally follistatin to produce a gonadal cell population.

[0069] In some aspects of the invention, the gonadal cell population is cryopreserved or cryopreserved. In some aspects, a composition comprising the gonadal cell population is provided. In some aspects, the composition further comprises a cryoprotectant. [Brief explanation of the drawing]

[0070] Representative embodiments of the present invention are disclosed with reference to the following drawings. It should be understood that the embodiments described are not limited to the details shown.

[0071] [Figure 1] Figures 1A and 1B illustrate the characteristics of mesoderm induction, showing the morphology of iMeLCs (Figure 1A) and the staining of iMeLCs and non-inducible cells with a fluorescently labeled antibody against brachiuri (Figure 1B). [Figure 2] Figures 2A-2D illustrate the characteristics of intermediate mesoderm induction, showing IM cells on day 5 of culture (Figure 2A), the expression pattern of OSR1 across differentiated iPSCs, iMeLCs, and IMs (Figure 2B), the expression pattern of PAX2 across differentiated iPSCs, iMeLCs, and IMs (Figure 2C), and the expression pattern of LHX1 across differentiated iPSCs, iMeLCs, and IMs (Figure 2D). [Figure 3A]Figures 3A-3C illustrate the characteristics of gonadal cell differentiation, showing passaged gonadal cells on day 7 of culture (Figure 3A), staining for transcription factors highly expressed in FOXL2-granulosa cells (Figure 3B), and representative immunofluorescence images of IM, kidney, and granulosa cells stained for FOXL2 (second row of Figure 3C) and connexin 43 (third row of Figure 3C). [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 4] Figure 4 shows the CD24 expression pattern during in vitro gonadal cell differentiation from iPSCs. [Figure 5A] Figures 5A-5C illustrate the effects of retinoic acid (RA) on IM induction. RA / TTNPB treatment resulted in increased cell viability and uniform cell morphology (Figure 5A). The expression of IM markers LHX1 and PAX2 increased with increasing RA and CHIR concentrations (Figure 5B). Further IM lineage markers WT1 and RUNX1 also showed similar increases in expression with increasing RA or TTNPB concentrations (Figure 5C). [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 6A] Figures 6A-6E show that IM cells treated with retinoic acid (RA) exhibited improved viability and induction of gonadal cell differentiation. Figure 6A illustrates that in in vitro gonadal cells, bulk RNA-seq experiments without RA treatment showed increased expression of granulosa cell markers FOXL2, RUNX1, NR2F2, WNT6, and KRT19 at all stages. Figure 6B shows that in long-term culture of granulosa cells, RA-treated cells exhibited improved viability and increased expression of FOXL2 and RUNX1. Figure 6C shows immunofluorescence staining of granulosa cells for FOXL2 and NR2F2. Figure 6D shows that continuous treatment with RA during differentiation of the second intermediate cell population increased KRT19 expression. Figure 6E shows immunofluorescence staining for KRT19 on day 28 of gonadal cell culture. [Figure 6B]See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 6D] See the explanation in Figure 6A. [Figure 6E] See the explanation in Figure 6A. [Figure 7] Figure 7 shows qRT-PCR results for the granulosa cell marker Foxl2, ovarian stromal cell Nr2f2, and ovarian epithelial cell KRT19. Expression levels at day 13 of the mouse differentiation protocol were compared to basic granulosa basal medium supplemented with retinoic acid (RA). Positive and negative controls were included as e13 mouse ovaries and mPSCs, respectively. [Figure 8] Figure 8 shows immunofluorescence staining on day 13 of the mouse differentiation protocol after 7 days of granulosa induction, illustrating the cell count and expression levels of Foxl2, NR2F2, and KRT19 with and without the addition of 1 μM retinoic acid in addition to granulosa basal medium growth factor. [Figure 9] Figure 9A shows the relative expression levels of markers for bi-potent ovarian somatic cell precursors after OSC induction by various FGF family members. Figure 9B shows the relative expression levels of markers for mature ovarian granulosa cells after OSC induction by various FGF family members. [Figure 10] Figure 10A shows the relative expression levels of markers for bidifferentiating ovarian somatic cell precursors after OSC induction by various BMP isoforms. Figure 10B shows the relative expression levels of markers for mature ovarian granulosa cells after OSC induction by various BMP isoforms. [Figure 11]Figure 11A shows the relative expression of the steroid-producing enzyme CYP19A1 at various stages of OSC differentiation (iPSC stage; IM D5 - intermediate mesoderm day 5; OSC D7 basic - day 7 of OSC induction in granulosa basic medium; OSC D7 double - day 7 of OSC induction in granulosa double medium). Figure 11B shows secreted estradiol when OSCs were treated with the indicated amount of dhT for 24 hours (four bars on the left) or 48 hours (two bars on the right). [Figure 12A] Figures 12A, 12C, and 12E show the relative expression levels of GATA4, a marker of bidifferentiating ovarian somatic cell precursors, after OSC induction with the indicated amounts of follistatin (Foll), FGF2 (FGF), and BMP4 (BMP). Figures 12B, 12D, and 12F show the relative expression levels of FOXL2, a marker of mature ovarian granulosa cells, after OSC induction with the indicated amounts of follistatin (Foll), FGF2 (FGF), and BMP4 (BMP). [Figure 12B] See the explanation in Figure 12A. [Figure 12C] See the explanation in Figure 12A. [Figure 12D] See the explanation in Figure 12A. [Figure 12E] See the explanation in Figure 12A. [Figure 12F] See the explanation in Figure 12A. [Figure 13A]Figure 13A is a panel of immunofluorescence images showing the expression of granulosa marker FOXL2 and ovarian epithelial marker KRT19 when OSCs are induced in the absence of retinoic acid (RA). Figure 13B is a panel of immunofluorescence images showing the expression of granulosa marker FOXL2 and ovarian epithelial marker KRT19 when OSCs are induced in the presence of 500 nM retinoic acid (RA). Figures 13C and 13D show the relative expression levels of FOXL2, a marker for granulosa cells, and KRT19, a marker for ovarian epithelial cells, respectively, when OSCs are induced in the indicated concentrations of RA. Figures 13E and 13F show the relative expression levels of FOXL2, a marker for granulosa cells, and KRT19, a marker for ovarian epithelial cells, respectively, when OSCs are induced in the indicated concentrations of RA over the indicated duration. [Figure 13B] See the explanation in Figure 13A. [Figure 13C] See the explanation in Figure 13A. [Figure 13D] See the explanation in Figure 13A. [Figure 13E] See the explanation in Figure 13A. [Figure 13F] See the explanation in Figure 13A. [Figure 14A] Figures 14A and 14B represent heatmaps of the top 200 highly variable genes and principal component analysis of normalized and processed data, respectively, showing changes in gene expression profiles during the transition from the first intermediate stage to the second intermediate stage and from the second intermediate stage to OSC. Figure 14C represents distinct clusters of increased gene expression observed among the more than 10,000 cell populations analyzed in this experiment. Figures 14D, 14E, 14F, and 14G show the expression of bipolarization potential gonadal cell markers, ovarian stromal markers, granulosa markers, and ovarian epithelial markers in cells at days 7 and 14 of gonadal induction (OSC induction), with or without retinoic acid treatment (RA), using either basic granulosa medium (basic) or double granulosa medium (double), respectively. [Figure 14B] See the explanation in Figure 14A. [Figure 14C] See the explanation in Figure 14A. [Figure 14D] See the explanation in Figure 14A. [Figure 14E] See the explanation in Figure 14A. [Figure 14F] See the explanation in Figure 14A. [Figure 14G] See the explanation in Figure 14A. [Figure 15] Figure 15 shows immunofluorescence images indicating the presence of ovarian somatic cells (GATA4 and NR5A1 expressing cells) and ovarian epithelial / mesothelial cells (KRT19 expressing cells). [Modes for carrying out the invention]

[0072] Detailed description of the invention In some respects, this disclosure relates to a method for generating gonadal cell populations, such as ovarian somatic cells, from pluripotent stem cells. It also includes the gonadal cell populations and intermediate cell populations generated in the method.

[0073] Any publications referenced herein, including patent documents, scientific articles, and databases, are incorporated herein in whole for all purposes to the same extent as if each individual publication were incorporated herein by reference individually. If any definition provided herein conflicts with or otherwise contradicts any definition provided herein in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition provided herein shall prevail over the definition incorporated herein by reference.

[0074] Section headings used in this specification serve only structural purposes and should not be construed as limiting the subjects described.

[0075] general technique The techniques and procedures described or referred to herein are generally well understood and can be used by those skilled in the art using conventional methodologies, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., 4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012), Current Protocols in Molecular Biology (FMAusubel, et al. eds., 2003), Methods in Enzymology series (Academic Press, Inc.), PCR 2:A Practical Approach (MJMacPherson, BD Hames and GRTaylor eds., 1995), Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988), Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RIFreshney, 6 thed.,J.Wiley and Sons,2010)、Oligonucleotide Synthesis(M.J.Gait,ed.,1984)、Methods in Molecular Biology,Humana Press、Cell Biology:A Laboratory Notebook(J.E.Cellis,ed.,Academic Press,1998)、Introduction to Cell and Tissue Culture(J.P.Mather and P.E.Roberts,Plenum Press,1998)、Cell and Tissue Culture:Laboratory Procedures(A.Doyle,J.B.Griffiths,and D.G.Newell,eds.,J.Wiley and Sons,1993-8)、Handbook of Experimental Immunology(D.M.Weir and C.C.Blackwell,eds.,1996)、Gene Transfer Vectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987)、PCR:The Polymerase Chain Reaction(Mullis et al.,eds.,1994)、Current Protocols in Immunology(J.E.Coligan et al.,eds.,1991)、Short Protocols in Molecular Biology(Ausubel et al.,eds.,J.Wiley and Sons,2002)、Immunobiology(C.A.Janeway et al.,2004)、Antibodies(P.Finch,1997)、Antibodies:A Practical Approach(D.Catty.,ed.,IRL Press,1988-1989)、Monoclonal Antibodies:A Practical Approach(P.Shepherd and C.Dean,eds.It is commonly used with widely adopted methodologies, such as those described in *Using Antibodies: A Laboratory Manual* (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), *The Antibodies* (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995), and *Cancer: Principles and Practice of Oncology* (VT DeVita et al., eds., JBLippincott Company, 2011).

[0076] definition In interpreting this specification, the following definitions apply, and wherever appropriate, a word used in the singular form also includes the plural form, and vice versa. In the event of any conflict between any of the following definitions and any document incorporated herein by reference, the following definition shall prevail.

[0077] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise indicated.

[0078] The aspects and embodiments of the present invention described herein are understood to include the terms “including,” “consisting of,” and “essentially consisting of.”

[0079] As used herein, the term “about” refers to the normal range of error for each value, as readily apparent to those skilled in the art. Where “about” is used herein with respect to a value or parameter, it encompasses (and describes) aspects directed toward that value or parameter itself.

[0080] As used herein, the term “homogeneous” refers to something whose structure or composition is consistently or uniform throughout. In some instances, the term refers to cells having a consistent state of maturity, marker expression, or phenotype within a given population.

[0081] As used herein, the term “inhibit” refers to an action that blocks, reduces, eliminates, or otherwise antagonizes the presence or activity of a particular target. For example, inhibiting tau protein phosphorylation may refer to any action that leads to a decrease, reduction, antagonization, elimination, blockade, or otherwise diminishing of tau protein phosphorylation. Inhibition may refer to partial or complete inhibition. In another example, inhibition of nucleic acid expression may include, but is not limited to, a reduction in nucleic acid transcription, a reduction in mRNA abundance (e.g., silencing mRNA transcription), mRNA degradation, or inhibition of mRNA translation.

[0082] As used herein, the term “suppress” may mean an action that reduces, diminishes, prohibits, restricts, reduces, or otherwise diminishes the presence or activity of a particular target. Suppression may mean partial or complete suppression. For example, suppressing tau protein phosphorylation may mean any action that reduces, diminishes, prohibits, restricts, reduces, or otherwise diminishes tau protein phosphorylation. In another example, suppression of nucleic acid expression may include, but is not limited to, a reduction in nucleic acid transcription, a reduction in mRNA abundance (e.g., silencing mRNA transcription), mRNA degradation, or inhibition of mRNA translation.

[0083] As used herein, the term “enhance” may refer to an action that improves, increases, enhances, or otherwise increases the presence or activity of a particular target. For example, enhancing steroid production may refer to any action that improves, increases, enhances, or otherwise increases steroid production.

[0084] As used herein, the term “modulate” may refer to an action that alters, modulates, fluctuates, or otherwise alters the presence or activity of a particular target. For example, modulating a signaling pathway may include, but are not limited to, any action that alters, modulates, fluctuates, or otherwise alters the activity of a signaling pathway. In some examples, “modulate” means to enhance the presence or activity of a particular target. In some examples, “modulate” means to suppress the presence or activity of a particular target. For example, modulating the amount of retinoic acid signaling may include, but are not limited to, suppressing or enhancing the amount of retinoic acid signaling.

[0085] As used herein, the term “induce” may mean to initiate, promote, stimulate, establish, or otherwise bring about a certain outcome. For example, inducing the expression of a mutant gene may mean any action that initiates, promotes, stimulates, establishes, or otherwise brings about the desired expression of a mutant gene. In another example, inducing the expression of a nucleic acid may include, but is not limited to, the initiation of nucleic acid transcription or the initiation of mRNA translation. In some examples, inducing a germ layer may mean any action that leads to, or is designed to lead to, initiating, promoting, stimulating, establishing, or otherwise bringing about the desired induction of a germ layer.

[0086] As used herein, “stem cells” refers to any non-somatic cell unless otherwise defined. Any cell that is neither a terminally differentiated cell nor a terminally committed cell may be called a stem cell. This includes embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, progenitor cells, and partially differentiated progenitor cells. Stem cells may be totipotent, pluripotent, or multipotent. Any cell that has the potential to differentiate into two different types of cells is considered a stem cell in this application.

[0087] As used herein, “pharmaceutically acceptable” or “pharmacologically compatible” means a material that is not biologically or otherwise undesirable, for example, a material that can be incorporated into a pharmaceutical composition administered to a patient without causing a serious undesirable biological effect or interacting in an adverse manner with any of the other components of the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the requirements of toxicity and manufacturing testing and are listed in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0088] The methods for determining any of the structural and functional features described herein are known in the art.

[0089] Induction, differentiation, and maturation of gonadal progeny cells Human iPSCs have become a powerful tool for modeling human diseases and hold great potential for translational research in target discovery and drug development.

[0090] Recent advances have shown that oocytes can be generated from pluripotent stem cells in vitro; however, these induced cells still require a suitable somatic environment to fully develop as germ cells. Early primordial germ cells or in vitro induced primordial germ cell-like cells mature into gametes only when combined with correctly constructed ovarian somatic cells. This specification provides a method for producing gonadal cell populations, particularly ovarian somatic cells, from pluripotent stem cells. In some embodiments, the provided cell populations can be used, for example, in a method for supporting in vitro oocyte development in relation to in vitro gamete formation.

[0091] Stem cell differentiation proceeds through various stages that can be identified by changes in gene expression. Methods for generating gonadal cells (such as ovarian somatic cells) by progressively converting stem cells through a series of steady-state stable conditions are described. In some embodiments, conditions are optimized to maximize purity and efficiency in order to obtain a pure, homogeneous ovarian somatic cell culture. In other embodiments, conditions are optimized to obtain a certain heterogeneous ovarian somatic cell culture containing granulosa cells, ovarian stromal cells, and ovarian epithelial cells. In some embodiments, two key intermediate steps for granulosa cell differentiation are provided herein. In some embodiments, two key intermediate steps for ovarian somatic cell differentiation are provided herein. In some embodiments, the two key intermediate steps include first differentiating pluripotent stem cells into mesoderm intermediates, then into intermediate mesoderm populations, and then inducing the cells into gonadal cell populations (ovarian somatic cells, OSCs, etc.).

[0092] In some embodiments, the protocols described herein can be used for different stem cell lines from different mammals (e.g., humans, mice, and cattle, but not limited to these). For robust differentiation, the expression of certain markers can be optimized at each step by varying the concentration and duration of the inducer (e.g., cytokines or small molecules). More importantly, a precise balance and organization of different ovarian somatic cell types (e.g., granulosa cells, epithelial cells, and stromal cells, but not limited to these) may be required to form a functional "mini-ovary" to facilitate gamete production and maturation; therefore, the final composition of ovarian somatic cells can be optimized by varying the concentration and duration of the inducer. The findings herein support the demonstration that, during the induction of intermediate mesoderm and / or gonadal cell populations, the use of specific compounds (e.g., retinoic acid and glycogen synthase kinase 3, but not limited to these) and incubation at different timings can produce gonadal cell populations containing various aggregates of ovarian somatic cells, including granulosa cells, epithelial cells, and stromal cells. Such optimizations are incorporated into robust, universal methods that will also work with cell lines of different genetic backgrounds, as demonstrated herein. Such methods and cell populations could also be used to modulate and facilitate the development and maturation of pluripotent stem cell-derived oocytes generated by different protocols.

[0093] Induction of gonadal cell population Method for generating gonadal cell populations and intermediate cell populations In some aspects, methods for generating gonadal cell populations from pluripotent stem cells are provided. In some embodiments, the Specified provides a method for producing gonadal cell populations, comprising: (a) culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A to produce a first intermediate cell population; (b) culturing the first intermediate cell population for a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM) to thereby produce a second intermediate cell population; and (c) culturing the second intermediate cell population for a third period in a gonadal induction medium containing BMP and FGF to produce a gonadal cell population.

[0094] In some embodiments, the gonad induction medium further comprises follistatin.

[0095] In some aspects, methods for generating gonadal cell populations from pluripotent stem cells are provided. In some embodiments, the Specified provides a method for producing gonadal cell populations, comprising: (a) culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A to produce a first intermediate cell population; (b) culturing the first intermediate cell population for a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM) to thereby produce a second intermediate cell population; and (c) culturing the second intermediate cell population for a third period in a gonadal induction medium containing follistatin, BMP4, and FGF to produce a gonadal cell population.

[0096] In some embodiments, a method for producing a gonadal cell population is provided, comprising the steps of (a) culturing mesoderm cells or mesoderm-like cells for a first period in an intermediate mesoderm induction medium containing RAPM to produce a second intermediate cell population, and (b) culturing the second intermediate cell population for a second period in a gonadal induction medium containing BMP and FGF to produce a gonadal cell population. In some embodiments, the gonadal induction medium further comprises follistatin. In some embodiments, a method for producing a gonadal cell population is provided, comprising the steps of (a) culturing mesoderm cells or mesoderm-like cells for a first period in an intermediate mesoderm induction medium containing RAPM to produce a second intermediate cell population, and (b) culturing the second intermediate cell population for a second period in a gonadal induction medium containing follistatin, BMP4 and FGF to produce a gonadal cell population.

[0097] In some embodiments, a method for producing a gonadal cell population is provided, comprising the step of culturing intermediate mesoderm cells or intermediate mesoderm-like cells for a period of time in a gonadal induction medium comprising BMP and FGF in order to produce a gonadal cell population. In some embodiments, the gonadal induction medium further comprises follistatin. In some embodiments, a method for producing a gonadal cell population is provided, comprising the step of culturing intermediate mesoderm cells or intermediate mesoderm-like cells for a period of time in a gonadal induction medium comprising follistatin, BMP4 and FGF in order to produce a gonadal cell population.

[0098] In some embodiments, a method is provided for producing a first intermediate cell population, comprising the step of culturing pluripotent stem cells for a period of time in a mesoderm-inducing medium containing activin A and a glycogen synthase kinase-3 inhibitor in order to produce a first intermediate cell population.

[0099] In some embodiments, a method is provided for producing a second intermediate cell population, comprising the step of culturing mesoderm cells or mesoderm-like cells in a medium containing RAPM for a period of time, thereby producing a second intermediate cell population.

[0100] In some embodiments, a method for producing a second intermediate cell population is provided, comprising the steps of (a) culturing pluripotent stem cells for a first period in a medium containing activin A and a glycogen synthase kinase-3 inhibitor to produce a first intermediate cell population, and (b) culturing the first intermediate cell population for a second period in a medium containing RAPM, FGF, and a glycogen synthase kinase-3 inhibitor, thereby producing a second intermediate cell population.

[0101] In some embodiments of any one of the methods described herein,

[0102] Gonadal cell population and intermediate cell population In some aspects, an in vitro stem cell-derived gonadal cell population is provided, comprising FOXL2-expressing cells, NR2F2-expressing cells, and / or KRT-19-expressing cells, and optionally, this gonadal cell population is an ovarian somatic cell population. In some aspects, the gonadal cell population is produced by a process comprising: (a) culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A to produce a first intermediate cell population; (b) culturing the first intermediate cell population for a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway modulator (RAPM) to thereby produce a second intermediate cell population; and (c) culturing the second intermediate cell population for a third period in a gonadal induction medium containing BMP and FGF to thereby generate a gonadal cell population. In some aspects, the gonadal induction medium further comprises follistatin.

[0103] Gonadal cell populations and intermediate cell populations produced by the methods described herein are also provided. In some embodiments, gonadal cell populations produced by a method comprising the steps of (a) culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A to produce a first intermediate cell population, (b) culturing the first intermediate cell population for a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM) to thereby produce a second intermediate cell population, and (c) culturing the second intermediate cell population for a third period in a gonadal induction medium containing follistatin, BMP4, and FGF to thereby produce a gonadal cell population. In some embodiments, the Specified provides a gonadal cell population produced by a method comprising: (a) culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A to produce a first intermediate cell population; (b) culturing the first intermediate cell population for a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway modulator (RAPM) to thereby produce a second intermediate cell population; and (c) culturing the second intermediate cell population for a third period in a gonadal induction medium containing BMP and FGF to thereby produce a gonadal cell population. In some embodiments, the gonadal induction medium further comprises follistatin.

[0104] In some embodiments, the Specified provides a gonadal cell population produced by a method comprising (a) culturing mesoderm cells or mesoderm-like cells in an intermediate mesoderm induction medium containing RAPM for a first period to produce a second intermediate cell population, and (b) culturing the second intermediate cell population in a gonadal induction medium containing follistatin, BMP4, and FGF for a second period to produce a gonadal cell population. In some embodiments, the Specified provides a gonadal cell population produced by a method comprising (a) culturing mesoderm cells or mesoderm-like cells in an intermediate mesoderm induction medium containing RAPM for a first period to produce a second intermediate cell population, and (b) culturing the second intermediate cell population in a gonadal induction medium containing BMP4 and FGF for a second period to produce a gonadal cell population. In some embodiments, the gonadal induction medium further comprises follistatin.

[0105] In some embodiments, a gonadal cell population is provided, which is produced by a method comprising culturing intermediate mesoderm cells or intermediate mesoderm-like cells for a period of time in a gonadal induction medium comprising follistatin, BMP4, and FGF. In some embodiments, a gonadal cell population is provided, which is produced by a method comprising culturing intermediate mesoderm cells or intermediate mesoderm-like cells for a period of time in a gonadal induction medium comprising BMP and FGF. In some embodiments, the gonadal induction medium further comprises follistatin.

[0106] In some embodiments, a first intermediate cell population is provided, which is produced by a method comprising the step of culturing pluripotent stem cells for a period of time in a mesoderm induction medium containing activin A and a glycogen synthase kinase-3 inhibitor, thereby generating a first intermediate cell population.

[0107] In some embodiments, a second intermediate cell population is provided, which is produced by a method comprising the step of culturing mesoderm cells or mesoderm-like cells in a medium containing RAPM for a period of time, thereby producing a second intermediate cell population.

[0108] In some embodiments, a second intermediate cell population is provided, produced by a method comprising the steps of (a) culturing pluripotent stem cells for a first period in a medium containing activin A and a glycogen synthase kinase-3 inhibitor to produce a first intermediate cell population; and (b) culturing the first intermediate cell population for a second period in a medium containing RAPM, FGF, and a glycogen synthase kinase-3 inhibitor, thereby producing a second intermediate cell population.

[0109] Induction medium, differentiation protocol, and precursor cells In some embodiments of the methods or cell populations described herein, the pluripotent stem cells are human pluripotent stem cells or mouse pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are mammalian pluripotent stem cells. In some embodiments, the pluripotent stem cells are mouse or human pluripotent stem cells. In some embodiments, the pluripotent stem cells are mouse pluripotent stem cells. In some embodiments, the pluripotent stem cells are human pluripotent stem cells. In some embodiments, the pluripotent stem cells are bovine pluripotent stem cells.

[0110] In some embodiments, pluripotent stem cells are 1 cm 2 They are seeded at a density of approximately 10,000 to 40,000 cells per area. In some embodiments,

[0111] In some embodiments of the methods or cell populations described herein, the concentration of activin A in the mesoderm induction medium is approximately 30 ng / mL to approximately 70 ng / mL. In some embodiments, the concentration of activin A in the mesoderm induction medium is approximately 50 ng / mL. In some embodiments, the concentration of activin A in the mesoderm induction medium is approximately one of the following concentrations: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 300, 400, or 500 ng / mL, or any concentration in between. In some embodiments, the concentration of activin A in mesoderm induction medium is approximately one of the following concentrations: about 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 ng / mL, or any concentration in between.

[0112] In some embodiments of the methods or cell populations described herein, the mesoderm induction medium further comprises FGF. In some embodiments, FGF is one or more of FGF2, FGF4, or FGF9. In some embodiments, FGF is FGF2. In some embodiments, the mesoderm induction medium comprises FGF2, and the concentration of FGF2 is approximately one of any of the following concentrations: 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / mL, or any concentration in between. In some embodiments, the concentration of FGF2 in the mesoderm induction medium is approximately 5 ng / mL to approximately 20 ng / mL. In some embodiments, the concentration of FGF2 in mesoderm induction medium is approximately 12 ng / mL.

[0113] In some embodiments of the methods or cell populations described herein, the mesoderm induction medium further comprises BMP4. In some embodiments, the concentration of BMP4 in the mesoderm induction medium is approximately one of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 300, 400 or 500 ng / mL, or any concentration in between. In some embodiments, the concentration of BMP4 in the mesoderm induction medium is approximately one of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 ng / mL, or any concentration in between. In some embodiments, the concentration of BMP4 in the mesoderm induction medium is approximately 10 ng / mL to approximately 50 ng / mL. In some embodiments, the concentration of BMP4 in the mesoderm induction medium is approximately 30 ng / mL.

[0114] In some embodiments of the method or cell population described herein, the mesoderm induction medium further comprises a glycogen synthase kinase-3 inhibitor. In some embodiments, the glycogen synthase kinase-3 inhibitor is CHIR99021. In some embodiments, the concentration of CHIR99021 in mesoderm induction medium is approximately one of the following concentrations: 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 20.0, 30.0, 40.0, 50.0 μM, or any concentration between them. In some embodiments, the concentration of CHIR99021 is approximately 1 μM to approximately 5 μM. In some embodiments, the concentration of CHIR99021 is approximately 3 μM.

[0115] In some embodiments of the methods or cell populations described herein, the mesoderm induction medium further comprises an apoptosis inhibitor. In some embodiments, the apoptosis inhibitor in the mesoderm induction medium is Y-27632. In some embodiments, the concentration of Y-27632 is approximately one of the following concentrations: 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / mL, or any concentration in between. In some embodiments, the concentration of Y-27632 is approximately 5 μM to approximately 20 μM. In some embodiments, the concentration of Y-27632 is approximately 10 μM. In some embodiments, the apoptosis inhibitor in the mesoderm induction medium comprises chroman-1, emricasan, and trans-ISRIB. In some embodiments, the concentration of chroman-1 is approximately 30 nM to approximately 70 nM, the concentration of emricasan is approximately 2 μM to approximately 10 μM, and / or the concentration of trans-ISRIB is approximately 0.2 μM to approximately 2 μM. In some embodiments, the concentration of chroman-1 is approximately 50 nM, the concentration of emricasan is approximately 5 μM, and / or the concentration of trans-ISRIB is approximately 0.7 μM.

[0116] In some embodiments of the methods or cell populations described herein, the period of culture in mesoderm-inducing medium is approximately one of 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 120, 144, 168, 192 hours, or any length between them. In some embodiments, the period of culture in mesoderm-inducing medium is at least approximately one of 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 120, 144, 168, 192 hours. In some embodiments, the incubation period in mesoderm-inducing medium is approximately 24 hours to approximately 96 hours. In some embodiments, the incubation period in mesoderm-inducing medium is approximately 24 hours to approximately 72 hours. In some embodiments, the incubation period in mesoderm-inducing medium is approximately 56 hours to approximately 72 hours.

[0117] In some embodiments, the first intermediate cell population is replated onto a fresh fibronectin-coated culture plate before culturing in intermediate mesoderm induction medium, and optionally, the first intermediate cell population is enzymatically detached, centrifuged, resuspended, and then replated. In some embodiments, the first intermediate cell population is approximately 5000 to 25000 cells / cm². 2 They are plated at a density of approximately 25,000 to 75,000 cells / cm³. In some embodiments, the first intermediate cell population is approximately 25,000 to 75,000 cells / cm³. 2 They are plated at a density of approximately 75,000 to 150,000 cells / cm³. In some embodiments, the first intermediate cell population is approximately 75,000 to 150,000 cells / cm³. 2 They are plated at a density of approximately 5,000 to 25,000 cells / cm³. In some embodiments, mesoderm cells or mesoderm-like cells are plated at a density of approximately 5,000 to 25,000 cells / cm³. 2 They are plated at a density of approximately 25,000 to 75,000 cells / cm³. In some embodiments, mesoderm cells or mesoderm-like cells are plated at a density of approximately 25,000 to 75,000 cells / cm³. 2is plated at a density of. In some embodiments, the mesoderm cells or mesoderm-like cells are at a density of about 75,000 to about 150,000 cells / cm 2 is plated.

[0118] In some embodiments by any one of the methods or cell populations described herein, the RAPM in the intermediate mesoderm induction medium is a RAR agonist. In some embodiments, the RAPM comprises retinoic acid (RA) and / or TTNPB. In some embodiments, the RAPM is RA. In some embodiments, the RAPM is TTNPB. In some embodiments, the concentration of RA in the intermediate mesoderm induction medium is approximately any one of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 20.0, 30.0, 40.0, 50.0 μM, or one of any concentration between them. In some embodiments, the concentration of TTNPB is approximately any one of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 20.0 μM, or one of any concentration between them. In some embodiments, the concentration of RA in the intermediate mesoderm induction medium is from about 0.5 μM to about 2 μM, and / or the concentration of TTNPB in the intermediate mesoderm induction medium is from about 0.2 μM to about 1 μM. In some embodiments, the concentration of RA in the intermediate mesoderm induction medium is about 1 μM, and / or the concentration of TTNPB in the intermediate mesoderm induction medium is about 0.5 μM.

[0119] In some embodiments of the methods or cell populations described herein, the intermediate mesoderm induction medium further comprises FGF. In some embodiments, FGF is one or more of FGF2, FGF4, or FGF9. In some embodiments, FGF is FGF2. In some embodiments, the intermediate mesoderm induction medium comprises FGF2, and the concentration of FGF2 is approximately one of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / mL, or any concentration in between. In some embodiments, the concentration of FGF2 in the intermediate mesoderm induction medium is approximately 10 ng / mL to approximately 30 ng / mL. In some embodiments, the concentration of FGF2 in the intermediate mesoderm induction medium is approximately 20 ng / mL.

[0120] In some embodiments of the methods or cell populations described herein, the intermediate mesoderm induction medium further comprises a glycogen synthase kinase-3 inhibitor. In some embodiments, the glycogen synthase kinase-3 inhibitor is CHIR99021. In some embodiments, the concentration of CHIR99021 in intermediate mesoderm induction medium is approximately one of the following concentrations: 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 20.0, 30.0, 40.0, 50.0 μM, or any concentration between them. In some embodiments, the concentration of CHIR99021 is approximately 1 μM to approximately 5 μM. In some embodiments, the concentration of CHIR99021 is approximately 3 μM.

[0121] In some embodiments of the methods or cell populations described herein, different concentrations of the inducer (e.g., growth factors or small molecules) and exposure durations may result in the acquisition of final ovarian somatic cell populations with different compositions.

[0122] In some embodiments, RAPM and / or glycogen synthase kinase-3 inhibitors with different concentrations and exposure durations can be used in the induction of the second intermediate cell population. In some embodiments, the use of RAPM and / or glycogen synthase kinase-3 inhibitors with different concentrations and exposure durations during induction results in different developmental potential of the resulting second intermediate cell population. In some embodiments, RAPM comprises retinoic acid (RA) and / or TTNPB. In some embodiments, RAPM is RA. In some embodiments, RAPM is TTNPB.

[0123] In some embodiments of the methods or cell populations described herein, the intermediate mesoderm induction medium further comprises an apoptosis inhibitor. In some embodiments, the apoptosis inhibitor in the intermediate mesoderm induction medium is Y-27632. In some embodiments, the concentration of Y-27632 is approximately one of the following concentrations: 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / mL, or any concentration in between. In some embodiments, the concentration of Y-27632 is approximately 5 μM to approximately 20 μM. In some embodiments, the concentration of Y-27632 is approximately 10 μM. In some embodiments, the apoptosis inhibitor in the intermediate mesoderm induction medium comprises chroman-1, emricasan, and trans-ISRIB. In some embodiments, the concentration of chroman-1 is approximately 30 nM to approximately 70 nM, the concentration of emricasan is approximately 2 μM to approximately 10 μM, and / or the concentration of trans-ISRIB is approximately 0.2 μM to approximately 2 μM. In some embodiments, the concentration of chroman-1 is approximately 50 nM, the concentration of emricasan is approximately 5 μM, and / or the concentration of trans-ISRIB is approximately 0.7 μM. In some embodiments, the method comprises the step of culturing a first intermediate cell population (i) first in intermediate mesoderm induction medium containing approximately 10 μM of Y-27632, and (ii) second in intermediate mesoderm induction medium containing approximately 2 μM or less of Y-27632. In some embodiments, the method includes the steps of (i) first culturing a first intermediate cell population in an intermediate mesoderm induction medium containing approximately 10 μM of Y-27632 for approximately 24 hours, and (ii) then culturing it in an intermediate mesoderm induction medium containing approximately 2 μM or less of Y-27632 for approximately 5 to 6 days.

[0124] In some embodiments of the methods or cell populations described herein, the period of culture in intermediate mesoderm induction medium is approximately one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 30, 35, 42, 49 days, or any length between them. In some embodiments, the period of culture in intermediate mesoderm induction medium is at least approximately one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 30, 35, 42, 49 days. In some embodiments, the incubation period in intermediate mesoderm induction medium is approximately 4 to 14 days. In some embodiments, the incubation period in intermediate mesoderm induction medium is approximately 5 to 9 days.

[0125] In some embodiments, the second intermediate cell population is replated onto a fresh fibronectin-coated culture plate before culturing in gonad induction medium, and optionally, the second intermediate cell population is enzymatically detached, centrifuged, resuspended, and then replated. In some embodiments, the second intermediate cell population is approximately 5000 to 25000 cells / cm². 2 They are plated at a density of approximately 25,000 to 75,000 cells / cm³. In some embodiments, the second intermediate cell population is approximately 25,000 to 75,000 cells / cm³. 2 They are plated at a density of approximately 75,000 to 150,000 cells / cm³. In some embodiments, the second intermediate cell population is approximately 75,000 to 150,000 cells / cm³. 2 They are plated at a density of approximately 5,000 to 25,000 cells / cm³. In some embodiments, mesoderm cells or mesoderm-like cells are plated at a density of approximately 5,000 to 25,000 cells / cm³. 2 They are plated at a density of approximately 5,000 to 25,000 cells / cm³. In some embodiments, intermediate mesoderm cells or intermediate mesoderm-like cells are plated at a density of approximately 5,000 to 25,000 cells / cm³. 2 They are plated at a density of approximately 25,000 to 75,000 cells / cm³. In some embodiments, intermediate mesoderm cells or intermediate mesoderm-like cells are plated at a density of approximately 25,000 to 75,000 cells / cm³. 2They are plated at a density of approximately 75,000 to 150,000 cells / cm³. In some embodiments, intermediate mesoderm cells or intermediate mesoderm-like cells are plated at a density of approximately 75,000 to 150,000 cells / cm³. 2 It is plated at this density.

[0126] In some embodiments of the methods or cell populations described herein, the concentration of follistatin in the gonad induction medium is approximately one of the following concentrations: 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 200, 500 ng / mL, or any concentration in between. In some embodiments, the concentration of follistatin in the gonad induction medium is approximately 10 ng / mL to approximately 50 ng / mL. In some embodiments, the concentration of follistatin in the gonad induction medium is approximately 25 ng / mL.

[0127] In some embodiments of the methods or cell populations described herein, the concentration of BMP4 in the gonad induction medium is approximately one of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 300, 400, or 500 ng / mL, or any concentration in between. In some embodiments, the concentration of BMP4 in the gonad induction medium is approximately one of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any concentration in between. In some embodiments, the concentration of BMP4 in the gonad induction medium is approximately one of the following concentrations: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 ng / mL, or any concentration in between. In some embodiments, the concentration of BMP4 in the gonad induction medium is approximately 5 ng / mL to approximately 20 ng / mL. In some embodiments, the concentration of BMP4 in the gonad induction medium is approximately 30 ng / mL to approximately 70 ng / mL. In some embodiments, the concentration of BMP4 in the gonad induction medium is approximately 10 ng / mL. In some embodiments, the concentration of BMP4 in the gonad induction medium is approximately 50 ng / mL.

[0128] In some embodiments of the methods or cell populations described herein, the BMPs in the gonad induction medium include BMP4, BMP2, BMP7, BMP15, or any combination thereof. In some embodiments, the concentration of BMPs in the gonad induction medium is approximately one of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 300, 400, or 500 ng / mL, or any concentration in between. In some embodiments, the concentration of BMPs in the gonad induction medium is approximately one of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any concentration in between. In some embodiments, the concentration of BMP in the gonad induction medium is approximately one of the following concentrations: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 ng / mL, or any concentration between them. In some embodiments, the concentration of BMP in the gonad induction medium is approximately 5 ng / mL to approximately 20 ng / mL. In some embodiments, the concentration of BMP in the gonad induction medium is approximately 30 ng / mL to approximately 70 ng / mL. In some embodiments, the concentration of BMP in the gonad induction medium is approximately 10 ng / mL. In some embodiments, the concentration of BMP in the gonad induction medium is approximately 20 ng / mL. In some embodiments, the concentration of BMP in the gonad induction medium is approximately 50 ng / mL.

[0129] In some embodiments of the methods or cell populations described herein, the FGF in the gonad induction medium is one or more of FGF2, FGF4, or FGF9. In some embodiments, the FGF in the gonad induction medium is FGF2. In some embodiments, the gonad induction medium contains FGF2, and the concentration of FGF2 is approximately one of any of the following concentrations: 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / mL, or any concentration in between. In some embodiments, the concentration of FGF2 in the gonad induction medium is approximately 1 ng / mL to approximately 10 ng / mL. In some embodiments, the concentration of FGF2 in the gonad induction medium is approximately 5 ng / mL to approximately 25 ng / mL. In some embodiments, the concentration of FGF2 in the gonad induction medium is approximately 1 ng / mL to approximately 10 ng / mL. In some embodiments, the concentration of FGF2 in the gonad induction medium is approximately 5 ng / mL to approximately 25 ng / mL. In some embodiments, the concentration of FGF2 in the gonad induction medium is approximately 5 ng / mL. In some embodiments, the concentration of FGF2 in the gonad induction medium is approximately 10 ng / mL.

[0130] In some embodiments, the FGF in the gonad induction medium includes FGF2, FGF4, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, or any combination thereof. In some embodiments, the concentration of FGF is approximately one of the following concentrations: 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / mL, or any concentration between them. In some embodiments, the concentration of FGF in the gonad induction medium is approximately 1 ng / mL to approximately 10 ng / mL. In some embodiments, the concentration of FGF in the gonad induction medium is approximately 5 ng / mL to approximately 25 ng / mL. In some embodiments, the concentration of FGF in the gonad induction medium is approximately 1 ng / mL to approximately 10 ng / mL. In some embodiments, the concentration of FGF in the gonad induction medium is approximately 5 ng / mL to approximately 25 ng / mL. In some embodiments, the concentration of FGF in the gonad induction medium is approximately 5 ng / mL. In some embodiments, the concentration of FGF in the gonad induction medium is approximately 10 ng / mL.

[0131] In some embodiments of the methods or cell populations described herein, the gonadal medium further comprises RAPM. In some embodiments, the RAPM in the gonadal induction medium is a RAR agonist. In some embodiments, the RAPM comprises retinoic acid (RA) and / or TTNPB. In some embodiments, the RAPM is RA. In some embodiments, the RAPM is TTNPB. In some aspects, the concentration of RA in gonad induction medium is approximately one of the following concentrations, or any concentration between them: 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 20.0, 30.0, 40.0, 50.0 μM. In some embodiments, the concentration of TTNPB in gonad induction medium is approximately one of the following concentrations: 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 20.0 μM, or any concentration in between. In some embodiments, the concentration of RA in gonad induction medium is approximately 0.5 μM to approximately 2 μM, and / or the concentration of TTNPB in gonad induction medium is approximately 0.2 μM to approximately 1 μM. In some embodiments, the concentration of RA in the gonad induction medium is approximately 1 μM, and / or the concentration of TTNPB in the gonad induction medium is approximately 0.5 μM.

[0132] In some embodiments of the methods or cell populations described herein, the gonad induction medium further comprises an apoptosis inhibitor. In some embodiments, the apoptosis inhibitor in the gonad induction medium is Y-27632. In some embodiments, the concentration of Y-27632 is approximately one of the following concentrations: 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / mL, or any concentration in between. In some embodiments, the concentration of Y-27632 is approximately 5 μM to approximately 20 μM. In some embodiments, the concentration of Y-27632 is approximately 10 μM. In some embodiments, the apoptosis inhibitor in the gonad induction medium comprises chroman-1, emricasan, and trans-ISRIB. In some embodiments, the concentration of chroman-1 is approximately 30 nM to approximately 70 nM, the concentration of emricasan is approximately 2 μM to approximately 10 μM, and / or the concentration of trans-ISRIB is approximately 0.2 μM to approximately 2 μM. In some embodiments, the concentration of chroman-1 is approximately 50 nM, the concentration of emricasan is approximately 5 μM, and / or the concentration of trans-ISRIB is approximately 0.7 μM.

[0133] In some embodiments of the methods or cell populations described herein, the period of culture in gonad induction medium is approximately one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 30, 35, 42, 49, 56, 63, 70 days, or any length between them. In some embodiments, the period of culture in gonad induction medium is at least approximately one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 30, 35, 42, 49, 56, 63, 70 days. In some embodiments, the incubation period in gonadal induction medium is approximately 5 to 21 days. In some embodiments, the incubation period in gonadal induction medium is approximately 7 to 14 days.

[0134] In some embodiments, this specification provides a method for producing a gonadal cell population, comprising: (a) culturing pluripotent stem cells in mesoderm induction medium containing about 50 ng / mL of activin A and about 3 μM of CHIR99021 for about 56 to about 72 hours, thereby producing a first intermediate cell population in which at least about 80% of the cells in the population express brachiuri; and (b) culturing in intermediate mesoderm induction medium containing about 1 μM of RA, about 3 μM of CHIR99021 and about 20 ng / mL of FGF2 for about 5 to 9 days. A method is provided comprising the steps of (c) culturing a first intermediate cell population to produce a second intermediate cell population in which at least about 80% of the cells in the population express PAX2, and (c) culturing the second intermediate cell population in a gonad induction medium containing about 25 ng / mL follistatin, about 10 ng / mL BMP4, and about 5 ng / mL FGF for about 7 to about 14 days to produce a gonadal cell population in which at least about 20% of the cells in the population express FOXL2 and / or NR2F2.

[0135] Characterization of gonadal cell populations and intermediate cell populations In some embodiments of either the method or cell population described herein, a gonadal cell population can be characterized by one or more expression markers. In some embodiments, at least a portion of the cells in the gonadal cell population express one or more of FOXL2, NR2F2, WNT6, KITLG, and NR1H4. In some embodiments, at least a portion of the cells in the gonadal cell population express one or more of NR2F2, GPC3, and COL1A1. In some embodiments, at least a portion of the cells in the gonadal cell population express one or more of NR2F2, GPC3, and COL1A1. In some embodiments, at least a portion of the cells in the gonadal cell population express one or more of KRT-19 and UPK3B. In some embodiments, at least a portion of the cells in the gonadal cell population express one or more of FOXL2, NR2F2, and KRT-19.

[0136] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express FOXL2. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express FOXL2. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express FOXL2.

[0137] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express NR2F2. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express NR2F2. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express NR2F2.

[0138] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express RUNX1. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express RUNX1. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express RUNX1.

[0139] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express LGR5. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express LGR5. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express LGR5.

[0140] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express WNT6. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express WNT6. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express WNT6.

[0141] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express KITLG. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express KITLG. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express KITLG.

[0142] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express NR1H4. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express NR1H4. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express NR1H4.

[0143] Granulosa cells mature into steroid-producing cells, and these cells can convert androgens such as testosterone into estrogens such as estradiol. One of the key enzymes responsible for steroid production is aromatase-CYP19A1.

[0144] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express CYP19A1. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express CYP19A1. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express CYP19A1.

[0145] In some embodiments, gonadal cell populations secrete estradiol when treated with dihydroxytestosterone (dhT). In some embodiments, the estradiol secretion level of mature gonadal cell populations after dhT treatment is at least approximately 10%, 20%, 50%, 80%, 100%, 10 times, 20 times, 50 times, 100 times, 1000 times, 10000 times, 100000 times, or 1,000000 times higher than that of untreated cells. In some embodiments, the estradiol secretion levels of mature glandular cell populations after treatment with dhT are at least approximately 10%, 20%, 50%, 80%, 100%, 10 times, 20 times, 50 times, 100 times, 1000 times, 10000 times, 100000 times, or 1,000000 times higher than those of bipolar glandular cell populations. In some embodiments, the estradiol secretion levels of mature glandular cell populations after treatment with dhT are at least approximately 10%, 20%, 50%, 80%, 100%, 10 times, 20 times, 50 times, 100 times, 1000 times, 100000 times, 1000000 times, or 1,000000 times higher than those of intermediate cell populations expressing mesodermal markers. In some embodiments, estradiol secretion levels in mature glandular cell populations after treatment with dhT are at least approximately 10%, 20%, 50%, 80%, 100%, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1,000,000-fold higher than in intermediate cell populations expressing intermediate mesoderm markers. In some embodiments, estradiol secretion levels in mature glandular cell populations after treatment with dhT are at least approximately 10%, 20%, 50%, 80%, 100%, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 1000-fold, 10000-fold, 100000-fold, or 1,000,000-fold higher than in pluripotent stem cells.

[0146] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express GPC3. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express GPC3. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express GPC3.

[0147] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express COL1A1. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express COL1A1. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express COL1A1.

[0148] In some embodiments, at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first gonadal cell population express KRT19. In some embodiments, at least approximately 20% of the cells in the gonadal cell population express KRT19. In some embodiments, at least approximately 50% of the cells in the gonadal cell population express KRT19.

[0149] In some embodiments, at least 90% of the cells in the gonadal cell population are FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. In some embodiments, the gonadal cell population consists essentially of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. In some embodiments, FOXL2-positive cells include granulosa cells. In some embodiments, NR2F2-positive cells include stromal cells and / or granulosa cells. In some embodiments, KRT-19-positive cells include ovarian epithelial cells.

[0150] In some embodiments, the gonadal population includes ovarian somatic cells (OSCs). In some embodiments, the gonadal population is an ovarian somatic cell population. In some embodiments, the gonadal population includes bipolar somatic cells (e.g., bipolar somatic ovarian cells). In some embodiments, bipolar somatic cells express one or more of GATA4, WT1, LHX9, and ZFPM2. In some embodiments, the gonadal population includes mature ovarian somatic cells. In some embodiments, the gonadal population includes one or more of granulosa cells, ovarian stromal cells, and / or ovarian epithelial cells. In some embodiments, granulosa cells express one or more of FOXL2, KITLG, and / or NR1H4. In some embodiments, ovarian stromal cells express NR2F2. In some embodiments, ovarian epithelium expresses one or more of KRT19, MSLN, TMEM151A, and / or LRRN4.

[0151] In some embodiments of either the method or cell population described herein, the first intermediate cell population can be characterized by one or more expression markers. In some embodiments, at least a portion of the cells in the first intermediate cell population express one or more of brachiuri, MIXL1, N-cadherin, ECPAM, and NCAM.

[0152] In some embodiments, at least a portion of the cells in the first intermediate cell population express brachiuri. In some embodiments, at least a portion of the cells in the first intermediate cell population express MIXL1. In some embodiments, at least a portion of the cells in the first intermediate cell population express N-cadherin. In some embodiments, at least a portion of the cells in the first intermediate cell population express EpCam. In some embodiments, at least a portion of the cells in the first intermediate cell population express NCAM. In some embodiments, at least a portion of the cells in the first intermediate cell population express GSC. In some embodiments, at least a portion of the cells in the first intermediate cell population express two or more of brachiuri, MIXL1, N-cadherin, EpCam, and NCAM. In some embodiments, at least a portion of the cells in the first intermediate cell population express three or more of brachiuri, MIXL1, N-cadherin, EpCam, and NCAM.

[0153] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first intermediate cell population express brachiuri. In some embodiments, at least approximately 80% of the cells in the first intermediate cell population express brachiuri. In some embodiments, at least approximately 90% of the cells in the first intermediate cell population express brachiuri. In some embodiments, at least approximately 95% of the cells in the first intermediate cell population express brachiuri.

[0154] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first intermediate cell population express MIXL1. In some embodiments, at least approximately 80% of the cells in the first intermediate cell population express MIXL1. In some embodiments, at least approximately 90% of the cells in the first intermediate cell population express MIXL1.

[0155] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first intermediate cell population express N-cadherin. In some embodiments, at least approximately 80% of the cells in the first intermediate cell population express N-cadherin. In some embodiments, at least approximately 90% of the cells in the first intermediate cell population express N-cadherin.

[0156] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first intermediate cell population express EPCAM. In some embodiments, at least approximately 80% of the cells in the first intermediate cell population express EPCAM. In some embodiments, at least approximately 90% of the cells in the first intermediate cell population express EPCAM.

[0157] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first intermediate cell population express N-cadherin. In some embodiments, at least approximately 80% of the cells in the first intermediate cell population express N-cadherin. In some embodiments, at least approximately 90% of the cells in the first intermediate cell population express N-cadherin.

[0158] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first intermediate cell population express NCAM. In some embodiments, at least approximately 80% of the cells in the first intermediate cell population express NCAM. In some embodiments, at least approximately 90% of the cells in the first intermediate cell population express NCAM.

[0159] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first intermediate cell population express GSCs. In some embodiments, at least approximately 80% of the cells in the first intermediate cell population express GSCs. In some embodiments, at least approximately 90% of the cells in the first intermediate cell population express GSCs.

[0160] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first intermediate cell population are mesoderm cells. In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the first intermediate cell population are mesoderm-like cells. In some embodiments, mesoderm-like cells may refer to cells that display one or more markers of the corresponding mesoderm in vivo. In some embodiments, mesoderm-like cells may refer to cells that have some or all of the developmental potential of the corresponding mesoderm in vivo. In some embodiments, at least about 90% of the cells in the first intermediate cell population are mesoderm cells. In some embodiments, at least about 90% of the cells in the first intermediate cell population are mesoderm-like cells. In some embodiments, at least about 90% of the cells in the first intermediate cell population are mesoderm cells or mesoderm-like cells.

[0161] In some embodiments of either the method or cell population described herein, the second intermediate cell population can be characterized by one or more expression markers. In some embodiments, at least a portion of the cells in the second intermediate cell population express one or more of OSR1, PAX2, LHX1, and RUNX1.

[0162] In some embodiments, at least a portion of the cells in the second intermediate cell population expresses OSR1. In some embodiments, at least a portion of the cells in the second intermediate cell population expresses PAX2. In some embodiments, at least a portion of the cells in the second intermediate cell population expresses LHX1. In some embodiments, at least a portion of the cells in the second intermediate cell population expresses WT1. In some embodiments, at least a portion of the cells in the second intermediate cell population expresses SALL1. In some embodiments, at least a portion of the cells in the second intermediate cell population expresses GSC. In some embodiments, at least a portion of the cells in the second intermediate cell population expresses one or more of OSR1, PAX2, LHX1, RUNX1, WT1, and SALL1. In some embodiments, at least a portion of the cells in the second intermediate cell population expresses two or more of OSR1, PAX2, LHX1, RUNX1, WT1, and SALL1. In some embodiments, at least a portion of the cells in the second intermediate cell population express three or more of the following: OSR1, PAX2, LHX1, RUNX1, WT1, and SALL1.

[0163] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the second intermediate cell population express LHX1. In some embodiments, at least approximately 90% of the cells in the second intermediate cell population express LHX1. In some embodiments, at least approximately 95% of the cells in the second intermediate cell population express LHX1.

[0164] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the second intermediate cell population express PAX2. In some embodiments, at least approximately 90% of the cells in the second intermediate cell population express PAX2. In some embodiments, at least approximately 95% of the cells in the second intermediate cell population express PAX2.

[0165] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the second intermediate cell population express OSR1. In some embodiments, at least approximately 80% of the cells in the second intermediate cell population express OSR1. In some embodiments, at least approximately 90% of the cells in the second intermediate cell population express OSR1.

[0166] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the second intermediate cell population express RUNX1. In some embodiments, at least approximately 80% of the cells in the second intermediate cell population express RUNX1. In some embodiments, at least approximately 90% of the cells in the second intermediate cell population express RUNX1.

[0167] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the second intermediate cell population express WT1. In some embodiments, at least approximately 80% of the cells in the second intermediate cell population express WT1. In some embodiments, at least approximately 90% of the cells in the second intermediate cell population express WT1.

[0168] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the second intermediate cell population express SALL1. In some embodiments, at least approximately 80% of the cells in the second intermediate cell population express SALL1. In some embodiments, at least approximately 90% of the cells in the second intermediate cell population express SALL1.

[0169] In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the second intermediate cell population are intermediate mesoderm cells. In some embodiments, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells in the second intermediate cell population are intermediate mesoderm-like cells. In some embodiments, intermediate mesoderm-like cells may refer to cells that display one or more markers of the corresponding intermediate mesoderm in vivo. In some embodiments, mesoderm-like cells may refer to cells that have some or all of the developmental potential of the corresponding intermediate mesoderm in vivo. In some embodiments, at least about 80% of the cells in the second intermediate cell population are intermediate mesoderm cells. In some embodiments, at least about 90% of the cells in the second intermediate cell population are intermediate mesoderm-like cells. In some embodiments, at least 90% of the cells in the second intermediate cell population are intermediate mesoderm cells and / or intermediate mesoderm-like cells.

[0170] In some embodiments, the intermediate mesoderm induction medium shows an increased potential for the second intermediate cell population to differentiate into (I) NR2F2-expressing gonadal cells, and / or (II) FOXL2-expressing gonadal cells, and / or (III) RUNX1-expressing gonadal cells, and / or (IV) WNT6-expressing gonadal cells, and / or, compared to the corresponding second intermediate cell population produced by a method that does not include RAPM. Alternatively, the potential of the second intermediate cell population to differentiate into (V)NR5A1-expressing gonadal cells is increased, and / or the potential of the second intermediate cell population to differentiate into (VI)OSR1-expressing gonadal cells is increased, and / or the potential of the second intermediate cell population to differentiate into (VII)DLK1- and / or GPC3-expressing gonadal cells is increased, and / or the potential of the second intermediate cell population to differentiate into (VIII)LHX9-expressing gonadal cells is increased, and / or the potential of the second intermediate cell population to differentiate into (IX)KRT19-expressing gonadal cells is increased.

[0171] In some embodiments, the potential of the second intermediate cell population to differentiate into (I) NR2F2-expressing gonadal cells is increased, and / or (II) the potential of the second intermediate cell population to differentiate into FOXL2-expressing gonadal cells is increased, and / or (III) the potential of the second intermediate cell population to differentiate into RUNX1-expressing gonadal cells is increased, and / or (IV) the potential of the second intermediate cell population to differentiate into WNT6-expressing gonadal cells is increased, and The potential of the second intermediate cell population to differentiate into / or (V)NR5A1-expressing gonads is increased, as is the potential of the second intermediate cell population to differentiate into / or (VI)OSR1-expressing gonads, as is the potential of the second intermediate cell population to differentiate into / or (VII)DLK1- and / or GPC3-expressing gonads, as is the potential of the second intermediate cell population to differentiate into / or (VIII)LHX9-expressing gonads, as is the potential of the second intermediate cell population to differentiate into / or (IX)KRT19-expressing gonads.

[0172] In some embodiments, the intermediate mesoderm induction medium shows an increased potential for (I) the second intermediate cell population to differentiate into NR2F2-expressing gonadal cells, and / or (II) the second intermediate cell population to differentiate into FOXL2-expressing gonadal cells, and / or (III) the second intermediate cell population to differentiate into RUNX1-expressing gonadal cells, and / or (IV) the second intermediate cell population to differentiate into WNT6-expressing gonadal cells, compared to the corresponding second intermediate cell population produced by a method that does not include glycogen synthase kinase-3 inhibitors. Furthermore, the potential of the second intermediate cell population to differentiate into (V)NR5A1-expressing gonads is increased, as well as the potential of the second intermediate cell population to differentiate into (VI)OSR1-expressing gonads, as well as the potential of the second intermediate cell population to differentiate into (VII)DLK1- and / or GPC3-expressing gonads, as well as the potential of the second intermediate cell population to differentiate into (VIII)LHX9-expressing gonads, as well as the potential of the second intermediate cell population to differentiate into (IX)KRT19-expressing gonads.

[0173] In some embodiments, the intermediate mesoderm induction medium shows an increased potential for (I) the second intermediate cell population to differentiate into NR2F2-expressing gonadal cells, and / or (II) the second intermediate cell population to differentiate into FOXL2-expressing gonadal cells, and / or (III) the second intermediate cell population to differentiate into RUNX1-expressing gonadal cells, and / or (IV) the second intermediate cell population to differentiate into WNT6-expressing gonadal cells, compared to the corresponding second intermediate cell population produced by a method containing a lower concentration of glycogen synthase kinase-3 inhibitor. In addition, the potential of the second intermediate cell population to differentiate into (V)NR5A1-expressing gonads is increased, as well as the potential of the second intermediate cell population to differentiate into (VI)OSR1-expressing gonads, as well as the potential of the second intermediate cell population to differentiate into (VII)DLK1- and / or GPC3-expressing gonads, as well as the potential of the second intermediate cell population to differentiate into (VIII)LHX9-expressing gonads, as well as the potential of the second intermediate cell population to differentiate into (IX)KRT19-expressing gonads.

[0174] In some embodiments, the concentration of RAPM and the duration of exposure to RAPM can be adjusted during induction of the gonadal somatic cell population. In some embodiments, adjusting the concentration of RAPM and the duration of exposure to RAPM during induction results in different cell fate and composition of the resulting gonadal somatic cell population. In some embodiments, RAPM comprises retinoic acid (RA) and / or TTNPB. In some embodiments, RAPM is RA. In some embodiments, RAPM is TTNPB.

[0175] In some aspects, RAPM is RA. In some aspects by either the method or cell population described herein, the gonad induction medium contains RA at a concentration that is at least approximately one of the following: 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 20.0, 30.0, 40.0, 50.0 μM, or any concentration in between. In some embodiments, gonad induction is performed in the presence of RA (at any one of the concentrations mentioned above, for example, but not limited to 0.1, 0.5, or 1.0 μM) for at least approximately 2, 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 72, 96, 120, 144, 168, 192, or 240 hours.

[0176] In some embodiments of either the method or cell population described herein, compared to the corresponding gonadal cell population produced by a method containing a lower concentration of RAPM in the gonadal induction medium, (I) the amount of FOXL2 expression in the gonadal cell population is reduced, and / or (II) the amount of NR1H4 and / or KITLG expression in the gonadal cell population is reduced, and / or (III) the amount of KRT-19 expression in the gonadal cell population is increased, and / or (IV) the amount of cytoplasmic KRT-19 expression in the gonadal cell population is increased, and / or (V) the amount of MSLN, LRRN4 and / or TMEM151A expression in the gonadal cell population is increased.

[0177] In some embodiments of either the method or cell population described herein, the gonadal induction medium exhibits the following compared to the corresponding gonadal cell population produced by a method that does not include RAPM: (I) reduced FOXL2 expression in the gonadal cell population, and / or (II) reduced NR1H4 and / or KITLG expression in the gonadal cell population, and / or (III) increased KRT-19 expression in the gonadal cell population, and / or (IV) increased cytoplasmic KRT-19 expression in the gonadal cell population, and / or (V) increased expression of MSLN, LRRN4 and / or TMEM151A in the gonadal cell population.

[0178] In some embodiments of either the method or cell population described herein, compared to the corresponding gonadal cell population produced by a gonadal induction step with a shorter contact period with RAPM, (I) the amount of FOXL2 expression in the gonadal cell population is reduced, and / or (II) the amount of NR1H4 and / or KITLG expression in the gonadal cell population is reduced, and / or (III) the amount of KRT-19 expression in the gonadal cell population is increased, and / or (IV) the amount of cytoplasmic KRT-19 expression in the gonadal cell population is increased, and / or (V) the amount of MSLN, LRRN4 and / or TMEM151A expression in the gonadal cell population is increased.

[0179] A population of gonadal cells (e.g., ovarian somatic cells) produced by one of the methods provided is also provided.

[0180] This specification also provides in vitro stem cell-derived gonadal cell populations comprising FOXL2-expressing cells, NR2F2-expressing cells, and / or KRT-19-expressing cells. In some embodiments, the gonadal cell population is an ovarian somatic cell population. In some embodiments, the population comprises at least a first cell type expressing FOXL2, a second cell type expressing NR2F2, and a third cell type expressing KRT-19. In some embodiments, at least 20% of the cells in the cell population are FOXL2-positive cells, for example, at least 25%, at least 30%, at least 40%, or at least 50% of the cells in the population are FOXL2-positive cells. In some embodiments, at least 20% of the cells in the cell population are NR2F2-positive cells, for example, at least 25%, at least 30%, at least 40%, or at least 50% of the cells in the population are NR2F2-positive cells. In some embodiments, at least 20% of the cells in a gonadal cell population are KRT19-positive cells, and for example, at least 25%, at least 30%, at least 40%, or at least 50% of the cells in the population are KRT192-positive cells. In some embodiments, (a) at least 20% of the cells in a cell population are FOXL2-positive cells, and / or (b) at least 20% of the cells in a cell population are NR2F2-positive cells, and / or (c) at least 20% of the cells in a gonadal cell population are KRT19-positive cells. In some embodiments, (a) about 20% to 40% of the cells in a cell population are FOXL2-positive cells, and / or (b) about 20% to 40% of the cells in a cell population are NR2F2-positive cells, and / or (c) about 20% to 40% of the cells in a gonadal cell population are KRT19-positive cells. In some embodiments, at least 85% of the gonadal cell population are FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. In some embodiments, at least 90% of the gonadal cell population consists of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. In some embodiments, at least 95% of the gonadal cell population consists of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells.In some embodiments, the gonadal cell population consists essentially of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. In some embodiments, FOXL2-positive cells include granulosa cells, NR2F2-positive cells include ovarian stromal cells and / or granulosa cells, and KRT-19-positive cells include ovarian epithelial cells.

[0181] In some aspects of the provided gonadal somatic cell population, the cell population differentiates from pluripotent stem cells. In some aspects, the gonadal somatic cell population (e.g., ovarian somatic cells) can be produced by any of the methods provided herein. In some aspects of any aspect, the gonadal somatic cell population is induced in a process comprising: (a) culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A to produce a first intermediate cell population; (b) culturing the first intermediate cell population for a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM) to thereby produce a second intermediate cell population; and (c) culturing the second intermediate cell population for a third period in a gonadal induction medium containing BMP and FGF and optionally follistatin to produce a gonadal cell population.

[0182] In some aspects of the provided embodiment, a population of gonadal cells is cryopreserved or is cryopreserved. In some aspects, a composition comprising a population of gonadal cells is provided. In some aspects, the composition further comprises a cryoprotective substance. In some aspects of the provided embodiment, the method further comprises the step of formulating a population of gonadal cells, e.g., a population of cells produced or harvested by the provided method, together with a cryoprotective substance (also called a cryopreservative). In some aspects, the cryoprotective substance is selected from glycerol, propylene glycol, dimethyl sulfoxide (DMSO), or a combination thereof. In some aspects, the cryoprotective substance comprises DMSO. In some aspects, the cryoprotective substance is DMSO.

[0183] In some embodiments, the formulation buffer contains a cryopreservative. In some embodiments, cells are formulated with a cryopreservative solution containing a 1.0% to 30% DMSO solution, for example, a 5% to 20% DMSO solution or a 5% to 10% DMSO solution. In some embodiments, the cryopreservative solution is or contains, for example, PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. In some embodiments, the cryopreservative solution is or contains, for example, at least 7.5% or about 7.5% DMSO. In some embodiments, the method may include one or more processing steps, which may involve washing differentiated cells to replace them with a cryopreservative solution. In some embodiments, cells are frozen, e.g., cryopreserved or cryopreserved, in culture medium and / or solution at final concentrations of 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or approximately 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or 1%-15%, 6%-12%, 5%-10%, or 6%-8% DMSO. In certain embodiments, cells are frozen, e.g., cryopreserved or cryoprotected, in culture medium and / or solution at final concentrations of 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% HSA, or approximately 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% HSA, or 0.1% to -5%, 0.25% to 4%, 0.5% to 2%, or 1% to 2% HSA. In some embodiments, the gonadal cell population produced by this method (e.g., ovarian somatic cells) is formulated with approximately 10% DMSO. In some embodiments, one or more compositions are stored in advance by freezing and are thawed before further use.

[0184] Exemplary examples The provided embodiments include the following embodiments: 1. (a) A step of culturing pluripotent stem cells in a mesoderm induction medium containing activin A for a first period in order to produce a first intermediate cell population. (b) A step of culturing a first intermediate cell population over a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM), thereby producing a second intermediate cell population, and (c) A step of culturing the second intermediate cell population for a third period in a gonadal induction medium containing BMP and FGF in order to produce a gonadal cell population. A method for producing a population of gonadal cells, including [the specified term]. 2. (a) A step of culturing pluripotent stem cells in a mesoderm induction medium containing activin A for a first period in order to produce a first intermediate cell population. (b) A step of culturing a first intermediate cell population over a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM), thereby producing a second intermediate cell population, and (c) A step of culturing the second intermediate cell population for a third period in a gonadal induction medium containing follistatin, BMP4, and FGF in order to produce a gonadal cell population. A method for producing a population of gonadal cells, including [the specified term]. 3. (a) A step of culturing mesoderm cells or mesoderm-like cells for a first period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM), thereby producing a second intermediate cell population, and (b) A step of culturing a second intermediate cell population for a second period in a gonadal induction medium containing BMP and FGF in order to produce a gonadal cell population. A method for producing a population of gonadal cells, including [the specified term]. 4. (a) A step of culturing mesoderm cells or mesoderm-like cells for a first period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM), thereby producing a second intermediate cell population, and (b) A step of culturing a second intermediate cell population for a second period in a gonadal induction medium containing follistatin, BMP4, and FGF in order to produce a gonadal cell population. A method for producing a population of gonadal cells, including [the specified term]. 5. A step of culturing intermediate mesoderm cells or intermediate mesoderm-like cells for a period of time in a gonadal induction medium containing BMP and FGF in order to produce a population of gonadal cells. A method for producing a population of gonadal cells, including [the specified term]. 6. A step of culturing intermediate mesoderm cells or intermediate mesoderm-like cells for a period of time in a gonadal induction medium containing follistatin, BMP4, and FGF in order to produce a population of gonadal cells. A method for producing a population of gonadal cells, including [the specified term]. 7. A step of culturing pluripotent stem cells for a certain period of time in a mesoderm induction medium containing activin A and a glycogen synthase kinase-3 inhibitor in order to produce a first intermediate cell population. A method for producing a first intermediate cell population, including [a specific cell type]. 8. A step of culturing mesoderm cells or mesoderm-like cells for a certain period of time in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM), thereby producing a second intermediate cell population. A method for producing a second intermediate cell population, including [a specific cell type]. 9. (a) A step of culturing pluripotent stem cells for a first period in a mesoderm induction medium containing activin A and a glycogen synthase kinase-3 inhibitor in order to produce a first intermediate cell population, and (b) A step of culturing the first intermediate cell population over a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM), FGF, and a glycogen synthase kinase-3 inhibitor, thereby producing a second intermediate cell population. A method for producing a second intermediate cell population, including [a specific cell type]. 10. A method according to any one of embodiments 1 to 4 and 9, wherein at least a portion of the cells in the first intermediate cell population express brachiuri. 11. A method according to any one of embodiments 1 to 4, 8 and 9, wherein at least a portion of the cells in the second intermediate cell population express OSR1, PAX2, or LHX. 12. A method according to any one of embodiments 1 to 6, wherein at least a portion of the cells in a population of gonadal cells express FOXL2, NR2F2, or RUNX1. 13. Pluripotent stem cells, 1 cm 2 A method according to any of embodiments 1, 2, 7 and 9-11, wherein the cells are seeded at a density of approximately 10,000 to 40,000 cells per cell. 14. A method according to any of embodiments 1, 2, 7 and 9-13, wherein pluripotent stem cells are seeded on a culture plate coated with fibronectin. 15. A method according to any of embodiments 1, 2, 7 and 9-13, wherein pluripotent stem cells are seeded on a culture plate coated with Matrigel. 16. Any method according to embodiment 1 to 15, wherein the mesoderm induction medium further contains FGF. 17. Any method according to embodiment 1 to 16, wherein the mesoderm induction medium further comprises BMP4. 18. Any method according to embodiment 1 to 17, wherein the mesoderm induction medium further comprises a glycogen synthase kinase-3 inhibitor. 19. Any method according to embodiment 1 to 18, wherein the mesoderm induction medium further comprises an apoptosis inhibitor. 20. Any method according to embodiment 1 to 19, wherein the concentration of activin A in the mesoderm induction medium is approximately 30 ng / mL to approximately 70 ng / mL. 21. The method according to embodiment 20, wherein the concentration of activin A in the mesoderm induction medium is approximately 50 ng / mL. 22. Any method according to embodiment 16 to 21, wherein the FGF in the mesoderm induction medium is FGF2. 23. The method of embodiment 22, wherein the concentration of FGF2 in the mesoderm induction medium is approximately 5 ng / mL to approximately 20 ng / mL, and optionally, the concentration of FGF2 in the mesoderm induction medium is approximately 12 ng / mL. 24. Any of the methods in embodiments 17 to 23, wherein the concentration of BMP4 in the mesoderm induction medium is approximately 10 ng / mL to approximately 50 ng / mL, and optionally, the concentration of BMP4 in the mesoderm induction medium is approximately 30 ng / mL. 25. Any method according to embodiment 18 to 24, wherein the glycogen synthase kinase-3 inhibitor in the mesoderm induction medium is CHIR99021. 26. The method according to embodiment 25, wherein the concentration of CHIR99021 is approximately 1 μM to approximately 5 μM. 27. The method according to embodiment 26, wherein the concentration of CHIR99021 in the mesoderm induction medium is approximately 3 μM. 28. In mesoderm induction medium, (A) The apoptosis inhibitor is Y-27632, and optionally the concentration of Y-27632 is approximately 5 μM to approximately 20 μM, or (B) The apoptosis inhibitor comprises chroman-1, emricasan, and trans-ISRIB, optionally with a concentration of chroman-1 of approximately 30 nM to 70 nM, a concentration of emricasan of approximately 2 μM to 10 μM, and a concentration of trans-ISRIB of approximately 0.2 μM to 2 μM. Any of the methods described in embodiments 19 to 27. 29. In mesoderm induction medium, (A) The concentration of Y-27632 is approximately 10 μM, or (B) The concentration of Chroman-1 is approximately 50 nM, the concentration of Emricasane is approximately 5 μM, and the concentration of trans-ISRIB is approximately 0.7 μM. The method of embodiment 28. 30. Any method according to embodiment 1 to 29, wherein the culture period in mesoderm induction medium is approximately 24 hours to approximately 96 hours. 31. The method of embodiment 30, wherein the culture period in mesoderm induction medium is approximately 24 hours to approximately 72 hours. 32. The method of embodiment 31, wherein the culture period in mesoderm induction medium is approximately 56 hours to approximately 72 hours. 33. Any method according to embodiment 1 to 32, wherein at least 90% of the cells in the first intermediate cell population express brachiuri. 34. Any method according to embodiment 1 to 33, wherein at least 80% of the cells in the first intermediate cell population express one or more of MIXL1, N-cadherin, EpCam, and NCAM. 35. A method according to any one of embodiments 1 to 34, wherein at least 90% of the cells in the first intermediate cell population express brachiuri, N-cadherin, EpCam, and NCAM. 36. Any method according to embodiment 1 to 35, wherein at least 90% of the cells in the first intermediate cell population are mesoderm cells or mesoderm-like cells. 37. The method according to aspect 36, wherein the first intermediate cell population essentially consists of mesoderm cells or mesoderm-like cells. 38. Any method according to embodiment 1 to 37, wherein the first intermediate cell population is replated onto a new fibronectin-coated culture plate before culturing in intermediate mesoderm induction medium, and optionally, the first intermediate cell population is enzymatically detached, centrifuged, resuspended, and then replated. 39. The first intermediate cell population is approximately 5,000 to 25,000 cells / cm². 2 The method of embodiment 38, wherein plating is performed at a density of . 40. Any method according to embodiment 1 to 39, wherein the intermediate mesoderm induction medium further contains FGF. 41. Any method according to embodiment 1 to 40, wherein the intermediate mesoderm induction medium further comprises a glycogen synthase kinase-3 inhibitor. 42. Any method according to embodiment 1 to 41, wherein the intermediate mesoderm induction medium further comprises activin A. 43. Any method according to embodiment 1 to 42, wherein the intermediate mesoderm induction medium further comprises an apoptosis inhibitor. 44. The first intermediate cell population, (i) First, in an intermediate mesoderm induction medium containing a first concentration of an apoptosis inhibitor, (ii) Next, in an intermediate mesoderm induction medium containing an apoptosis inhibitor at a concentration of 2 or less, The method according to embodiment 43, which includes a step of culturing. 45. Any method according to embodiment 1 to 44, wherein RAPM in the intermediate mesoderm induction medium is a RAR agonist, and optionally RAPM comprises retinoic acid (RA) and / or TTNPB. 46. ​​(c) RAPM is RA, and the concentration of RA in the intermediate mesoderm induction medium is approximately 0.5 μM to approximately 2 μM, and / or (d) RAPM is TTNPB, and the concentration of TTNPB in the intermediate mesoderm induction medium is approximately 0.2 μM to approximately 1 μM. Any method of embodiment 1 to 45. 47. (c) The concentration of RA in the intermediate mesoderm induction medium is approximately 1 μM, and / or (d) The concentration of TTNPB in the intermediate mesoderm induction medium is approximately 0.5 μM. The method according to aspect 46. 48. Any method according to embodiment 40 to 47, wherein the FGF in the intermediate mesoderm induction medium is FGF2, and the concentration of FGF2 is approximately 10 ng / mL to approximately 30 ng / mL. 49. The method according to embodiment 48, wherein the concentration of FGF2 in the intermediate mesoderm induction medium is approximately 20 ng / mL. 50. Any method according to embodiment 41 to 49, wherein the glycogen synthase kinase-3 inhibitor in the intermediate mesoderm induction medium is CHIR99021, and the concentration of CHIR99021 is approximately 1 μM to approximately 5 μM. 51. The method according to embodiment 50, wherein the concentration of CHIR99021 in the intermediate mesoderm induction medium is approximately 2 μM or approximately 3 μM. 52. Any method according to embodiment 42 to 51, wherein the concentration of activin A in the intermediate mesoderm induction medium is approximately 10 ng / mL to approximately 50 ng / mL. 53. The method according to embodiment 52, wherein the concentration of activin A in the intermediate mesoderm induction medium is approximately 30 ng / mL. 54. In mesoderm induction medium, (A) The apoptosis inhibitor is Y-27632, and the concentration of Y-27632 is approximately 5 μM to approximately 20 μM, or (B) The apoptosis inhibitor contains chroman-1, emricasan, and trans-ISRIB, with a concentration of chroman-1 of approximately 30 nM to 70 nM, a concentration of emricasan of approximately 2 μM to 10 μM, and a concentration of trans-ISRIB of approximately 0.2 μM to 2 μM. The method described in any of embodiments 43 and 46-53. 55. The apoptosis inhibitor in the intermediate mesoderm induction medium is Y-27632. The above method provides the first intermediate cell population, (i) First, in an intermediate mesoderm induction medium containing approximately 10 μM of Y-27632, (ii) Next, in an intermediate mesoderm induction medium containing Y-27632 at a concentration of approximately 2 μM or less, Including the process of culturing, Any method of aspects 45 to 54. 56. The first intermediate cell population, (i) First, in an intermediate mesoderm induction medium containing approximately 10 μM Y-27632, for approximately 24 hours, (ii) Next, in an intermediate mesoderm induction medium containing Y-27632 at a concentration of approximately 2 μM or less, for approximately 5 to 6 days, A method according to any of embodiments 45 to 55, which includes a step of culturing. 57. Any method according to embodiment 1 to 56, wherein the culture period in intermediate mesoderm induction medium is approximately 4 to 14 days. 58. Any method according to embodiment 1 to 57, wherein the culture period in intermediate mesoderm induction medium is approximately 5 to 9 days. 59. The apoptosis inhibitor in the intermediate mesoderm induction medium is Y-27632, and the concentration of Y-27632 during culture in the intermediate mesoderm induction medium is (a) The concentration is approximately 10 μM for the first 24 hours. (b) The concentration is approximately 2 μM or less for 24 hours to 72 hours. (c) Between 72 and 120 hours, the concentration is approximately 0.5 μM or less, or (d) After 120 hours, the concentration is approximately 0.1 μM or less. Any method described in aspects 1 to 58. 60. A method according to any of embodiments 44 to 59, wherein a first intermediate cell population is cultured for approximately 24 hours in an intermediate mesoderm induction medium containing an apoptosis inhibitor, and after approximately 24 hours, a portion of the medium is first replaced with an intermediate mesoderm induction medium that does not contain an apoptosis inhibitor. 61. The method according to embodiment 60, wherein the aforementioned portion of the culture medium constitutes approximately 80% of the culture medium. 62. The method of embodiment 60 or 61, further comprising a subsequent medium exchange, wherein the medium exchange includes replacing a portion of the medium approximately every 48 hours after the first 24 hours of incubation. 63. The method of embodiment 62, wherein the portion of the culture medium in each subsequent culture medium exchange is approximately 80% of the culture medium. 64. Any method according to embodiment 1 to 63, wherein at least 90% of the cells in the second intermediate cell population express one or more of OSR1, PAX2, LHX1, and RUNX1. 65. Any method according to embodiment 1 to 64, wherein at least 90% of the cells in the second intermediate cell population express two or more of OSR1, PAX2, LHX1, and RUNX1. 66. A method according to any one of embodiments 1 to 65, wherein at least 90% of the cells in the second intermediate cell population express OSR1, PAX2, and LHX1. 67. Any method according to embodiment 1 to 66, wherein at least 90% of the cells in the second intermediate cell population are intermediate mesoderm cells or intermediate mesoderm-like cells. 68. The method of aspect 67, wherein the second intermediate cell population essentially consists of intermediate mesoderm cells or intermediate mesoderm-like cells. 69. Any method according to embodiment 1 to 68, wherein the second intermediate cell population is replated onto a new fibronectin-coated culture plate before culturing in gonad induction medium, and optionally, the second intermediate cell population is enzymatically detached, centrifuged, resuspended, and then replated. 70. The second intermediate cell population is approximately 5,000 to 25,000 cells / cm². 2 A method according to embodiment 69, wherein plating is performed at a density of . 71. Any method according to embodiment 1 to 70, wherein the gonad induction medium further comprises RAPM. 72. Any method according to embodiment 1 to 71, wherein the gonad induction medium further comprises an apoptosis inhibitor. 73. Any method according to embodiments 1, 3, 5 and 10-72, wherein the gonad induction medium further comprises follistatin. 74. Any method according to embodiment 1 to 73, wherein the concentration of follistatin in the gonad induction medium is approximately 10 ng / mL to approximately 50 ng / mL. 75. The method according to embodiment 74, wherein the concentration of follistatin in the gonad induction medium is approximately 25 ng / mL. 76. Any method according to embodiments 1, 3, 5 and 10-75, wherein the BMP in the gonad induction medium includes BMP4, BMP2, BMP7, BMP15 or any combination thereof. 77. Any method according to embodiments 1, 3, 5 and 10-76, wherein the total concentration of BMP in the gonad induction medium is approximately 5 ng / mL to approximately 20 ng / mL or approximately 20 ng / mL to approximately 70 ng / mL. 78. The method of embodiment 77, wherein the total concentration of BMP in the gonad induction medium is approximately 10 ng / mL or approximately 50 ng / mL. 79. Any method according to embodiment 1 to 78, wherein the concentration of BMP4 in the gonad induction medium is approximately 5 ng / mL to approximately 20 ng / mL or approximately 20 ng / mL to approximately 70 ng / mL. 80. The method according to embodiment 79, wherein the concentration of BMP4 in the gonad induction medium is approximately 10 ng / mL or approximately 50 ng / mL. 81. Any method according to embodiment 1 to 80, wherein the FGF in the gonad induction medium includes FGF2, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, or any combination thereof. 82. Any method according to embodiment 1 to 81, wherein the concentration of FGF is approximately 1 ng / mL to approximately 10 ng / mL or approximately 5 ng / mL to approximately 25 ng / mL. 83. The method of embodiment 82, wherein the concentration of FGF is approximately 5 ng / mL or approximately 10 ng / mL. 84. Any method according to embodiment 1 to 83, wherein the FGF in the gonad induction medium is FGF2, and the concentration of FGF2 is approximately 1 ng / mL to approximately 10 ng / mL or approximately 5 ng / mL to approximately 25 ng / mL. 85. The method of embodiment 84, wherein the concentration of FGF2 in the gonad induction medium is approximately 5 ng / mL or approximately 10 ng / mL. 86. Any method according to embodiment 71 to 85, wherein RAPM in the gonad induction medium is a RAR agonist, and optionally RAPM comprises retinoic acid (RA) and / or TTNPB. 87. (c) RAPM is RA, and the concentration of RA in the gonad induction medium is approximately 0.5 μM to approximately 2 μM, and / or (d) RAPM is TTNPB, and the concentration of TTNPB in the gonad induction medium is approximately 0.2 μM to approximately 1 μM. The method of embodiment 86. 88. In gonad induction medium, (A) The apoptosis inhibitor is Y-27632, and optionally the concentration of Y-27632 is approximately 5 μM to approximately 20 μM, or (B) The apoptosis inhibitor comprises chroman-1, emricasan, and trans-ISRIB, optionally with a concentration of chroman-1 of approximately 30 nM to 70 nM, a concentration of emricasan of approximately 2 μM to 10 μM, and a concentration of trans-ISRIB of approximately 0.2 μM to 2 μM. Any method described in aspects 72 to 87. 89. In gonad induction medium, (A) The concentration of Y-27632 is approximately 10 μM, or (B) The concentration of Chroman-1 is approximately 50 nM, the concentration of Emricasane is approximately 5 μM, and the concentration of trans-ISRIB is approximately 0.7 μM. The method of aspect 88. 90. Any method according to embodiment 1 to 89, wherein the incubation period in gonad induction medium is approximately 5 to 21 days. 91. The method of embodiment 90, wherein the incubation period in gonad induction medium is approximately 7 to 14 days. 92. Any method according to embodiment 1 to 91, wherein the gonadal cell population includes ovarian somatic cells. 93. Any method according to embodiment 1 to 92, wherein the gonadal cell population consists of ovarian somatic cells. 94. Any method according to aspect 1 to 93, wherein at least 20% of the cells in the gonadal cell population are FOXL2-positive cells. 95. Any method according to embodiment 1 to 94, wherein at least 20% of the cells in the gonadal cell population are NR2F2-positive cells. 96. Any method according to aspect 1 to 95, wherein at least 20% of the cells in the gonadal cell population are KRT19-positive cells. 97. Any method according to aspect 1 to 96, wherein at least 90% of the gonadal cell population consists of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. 98. The method of aspect 97, wherein the gonadal cell population essentially consists of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. 99. The method of aspect 97 or 98, wherein FOXL2-positive cells include granulosa cells, NR2F2-positive cells include stromal cells and / or granulosa cells, and KRT-19-positive cells include ovarian epithelial cells. 100. Corresponding gonadal cell populations produced by a method that does not include RAPM in the intermediate mesoderm induction medium. In comparison, (I) The amount of NR2F2-expressing cells in the gonadal cell population is increased, and / or (II) The amount of FOXL2-expressing cells in the gonadal cell population is increased, and / or (III) The amount of RUNX1-expressing cells in the gonadal cell population is increased, and / or (IV) The amount of WNT6-expressing cells in the gonadal cell population is increased, and / or (V) The amount of NR5A1-expressing cells in the gonadal cell population is increased, and / or (VI) The amount of OSR1-expressing cells in the gonadal cell population is increased, and / or (VII) The amount of LHX9-expressing cells in the gonadal cell population is increased, and / or (VIII) The amount of EMX2-expressing cells in the gonadal cell population is increasing. Any method from Embodiments 1 to 99. 101. Corresponding gonadal cell populations generated by a method using intermediate mesoderm induction medium containing lower concentrations of RAPM. In comparison, (I) The amount of NR2F2-expressing cells in the gonadal cell population is increased, and / or (II) The amount of FOXL2-expressing cells in the gonadal cell population is increased, and / or (III) The amount of RUNX1-expressing cells in the gonadal cell population is increased, and / or (IV) The amount of WNT6-expressing cells in the gonadal cell population is increased, and / or (V) The amount of NR5A1-expressing cells in the gonadal cell population is increased, and / or (VI) The amount of OSR1-expressing cells in the gonadal cell population is increasing. Any method from Embodiments 1 to 99. 102. Corresponding gonadal cell populations produced in intermediate mesoderm induction medium by a method that does not include glycogen synthase kinase-3 inhibitors. In comparison, (I) The amount of NR2F2-expressing cells in the gonadal cell population is increased, and / or (II) The amount of FOXL2-expressing cells in the gonadal cell population is increased, and / or (III) The amount of RUNX1-expressing cells in the gonadal cell population is increased, and / or (IV) The amount of WNT6-expressing cells in the gonadal cell population is increased, and / or (V) The amount of NR5A1-expressing cells in the gonadal cell population is increased, and / or (VI) The amount of OSR1-expressing cells in the gonadal cell population is increased, and / or (VII) The amount of LHX9-expressing cells in the gonadal cell population is increased, and / or (VIII) The amount of EMX2-expressing cells in the gonadal cell population is increasing. Any method from Embodiments 1 to 99. 103. Corresponding gonadal cell populations produced by a method involving intermediate mesoderm induction medium containing lower concentrations of glycogen synthase kinase-3 inhibitor. In comparison, (I) The amount of NR2F2-expressing cells in the gonadal cell population is increased, and / or (II) The amount of FOXL2-expressing cells in the gonadal cell population is increased, and / or (III) The amount of RUNX1-expressing cells in the gonadal cell population is increased, and / or (IV) The amount of WNT6-expressing cells in the gonadal cell population is increased, and / or (V) The amount of NR5A1-expressing cells in the gonadal cell population is increased, and / or (VI) The amount of OSR1-expressing cells in the gonadal cell population is increased, and / or (VII) The amount of LHX9-expressing cells in the gonadal cell population is increased, and / or (VIII) The amount of EMX2-expressing cells in the gonadal cell population is increased, The method according to any one of aspects 1 to 99. 104. A corresponding second intermediate cell population generated by a method in which the intermediate mesoderm induction medium does not contain RAPM compared to (I) The amount of LHX1 expression in the second intermediate cell population is increased, and / or (II) The amount of PAX2 expression in the second intermediate cell population is increased, and / or (III) The amount of WT1 expression in the second intermediate population is increased, and / or (IV) The amount of RUNX1 expression in the second intermediate cell population is increased, and / or (V) The viability of the second intermediate cell population is increased, and / or [[ID=2​​​​​​​​​​​​​​​​​​​​​​106. The corresponding second intermediate cell population is produced in an intermediate mesoderm induction medium by a method that does not include glycogen synthase kinase-3 inhibitors. In comparison, (I) Increased LHX1 expression in the second intermediate cell population, and / or (II) Increased PAX2 expression in the second intermediate cell population, and / or (III) Increased levels of WT1 expression in the second intermediate population, and / or (IV) Increased RUNX1 expression in the second intermediate cell population, and / or (V) The survival rate of the second intermediate cell population is increasing, and / or (VI) The cell morphology of the second intermediate cell population is more uniform. Any method from Embodiments 1 to 99. 107. The corresponding second intermediate cell population produced by a method involving intermediate mesoderm induction medium containing a lower concentration of glycogen synthase kinase-3 inhibitor. In comparison, (I) Increased LHX1 expression in the second intermediate cell population, and / or (II) Increased PAX2 expression in the second intermediate cell population, and / or (III) Increased levels of WT1 expression in the second intermediate population, and / or (IV) Increased RUNX1 expression in the second intermediate cell population, and / or (V) The survival rate of the second intermediate cell population is increasing, and / or (VI) The cell morphology of the second intermediate cell population is more uniform. Any method from Embodiments 1 to 99. 108. The corresponding second intermediate cell population is produced by an intermediate mesoderm induction medium using a method that does not include RAPM. In comparison, (I) The potential of the second intermediate cell population to differentiate into NR2F2-expressing gonadal cells is increased, and / or (II) The potential of the second intermediate cell population to differentiate into FOXL2-expressing gonadal cells is increased, and / or (III) The potential of the second intermediate cell population to differentiate into RUNX1-expressing gonadal cells is increased, and / or (IV) The potential of the second intermediate cell population to differentiate into WNT6-expressing gonadal cells is increased, and / or (V) The potential of the second intermediate cell population to differentiate into NR5A1-expressing gonadal cells is increased, and / or (VI) The potential of the second intermediate cell population to differentiate into OSR1-expressing gonadal cells is increasing. Any method from Embodiments 1 to 99. 109. The corresponding second intermediate cell population produced by a method using intermediate mesoderm induction medium containing a lower concentration of RAPM. In comparison, (I) The potential of the second intermediate cell population to differentiate into NR2F2-expressing gonadal cells is increased, and / or (II) The potential of the second intermediate cell population to differentiate into FOXL2-expressing gonadal cells is increased, and / or (III) The potential of the second intermediate cell population to differentiate into RUNX1-expressing gonadal cells is increased, and / or (IV) The potential of the second intermediate cell population to differentiate into WNT6-expressing gonadal cells is increased, and / or (V) The potential of the second intermediate cell population to differentiate into NR5A1-expressing gonadal cells is increased, and / or (VI) The potential of the second intermediate cell population to differentiate into OSR1-expressing gonadal cells is increasing. Any method from Embodiments 1 to 99. 110. The corresponding second intermediate cell population is produced in an intermediate mesoderm induction medium by a method that does not include glycogen synthase kinase-3 inhibitors. In comparison, (I) The potential of the second intermediate cell population to differentiate into NR2F2-expressing gonadal cells is increased, and / or (II) The potential of the second intermediate cell population to differentiate into FOXL2-expressing gonadal cells is increased, and / or (III) The potential of the second intermediate cell population to differentiate into RUNX1-expressing gonadal cells is increased, and / or (IV) The potential of the second intermediate cell population to differentiate into WNT6-expressing gonadal cells is increased, and / or (V) The potential of the second intermediate cell population to differentiate into NR5A1-expressing gonadal cells is increased, and / or (VI) The potential of the second intermediate cell population to differentiate into OSR1-expressing gonadal cells is increasing. Any method from Embodiments 1 to 99. 111. The corresponding second intermediate cell population is generated by a method that includes a lower concentration of glycogen synthase kinase-3 inhibitor in the intermediate mesoderm induction medium. Compared (I) The potential of the second intermediate cell population to differentiate into NR2F2-expressing gonadal cells is increased, and / or (II) The potential of the second intermediate cell population to differentiate into FOXL2-expressing gonadal cells is increased, and / or (III) The potential of the second intermediate cell population to differentiate into RUNX1-expressing gonadal cells is increased, and / or (IV) The potential of the second intermediate cell population to differentiate into WNT6-expressing gonadal cells is increased, and / or (V) The potential of the second intermediate cell population to differentiate into NR5A1-expressing gonadal cells is increased, and / or (VI) The potential of the second intermediate cell population to differentiate into OSR1-expressing gonadal cells is increasing. Any method from Embodiments 1 to 99. 112. Corresponding gonadal cell populations generated by a method using gonadal induction medium containing lower concentrations of RAPM. In comparison, (I) Decreased levels of FOXL2 expression in the gonadal cell population, and / or (II) Decreased expression levels of NR1H4 and / or KITLG in the gonadal cell population, and / or (III) Increased KRT-19 expression in the gonadal cell population, and / or (IV) an increased amount of cytoplasmic KRT-19 expression in the gonadal cell population, and / or (V) an increased amount of expression of MSLN, LRRN4 and / or TMEM151A in the gonadal cell population, The method according to any one of aspects 1 to 111. 113. A corresponding gonadal cell population generated by a method in which the gonadal induction medium does not contain RAPM compared to (I) a decreased amount of FOXL2 expression in the gonadal cell population, and / or (II) a decreased amount of expression of NR1H4 and / or KITLG in the gonadal cell population, and / or (III) an increased amount of KRT-19 expression in the gonadal cell population, and / or (IV) an increased amount of cytoplasmic KRT-19 expression in the gonadal cell population, and / or (V) an increased amount of expression of MSLN, LRRN4 and / or TMEM151A in the gonadal cell population, The method according to any one of aspects 1 to 111. 114. A corresponding gonadal cell population generated by a gonadal induction process with a shorter period of contact with RAPM [[ID=²5]]compared to (I) a decreased amount of FOXL2 expression in the gonadal cell population, and / or (II) a decreased amount of expression of NR1H4 and / or KITLG in the gonadal cell population, and / or (III) an increased amount of KRT-19 expression in the gonadal cell population, and / or (IV) an increased amount of cytoplasmic KRT-19 expression in the gonadal cell population, and / or (V) an increased amount of expression of MSLN, LRRN4 and / or TMEM151A in the gonadal cell population, The method according to any one of aspects 1 to 111. 115. The method according to any one of aspects 1 to 114, wherein the pluripotent stem cells are mammalian stem cells. 116. Any method according to embodiment 1 to 115, wherein the pluripotent stem cells are human pluripotent stem cells. 117. Any method according to embodiment 1 to 115, wherein the pluripotent stem cells are bovine stem cells. 118. Any method according to embodiment 1 to 115, wherein the pluripotent stem cells are mouse pluripotent stem cells. 119. Any method according to embodiment 115 to 118, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. 120. Any method according to embodiments 1-6 and 10-119, wherein the gonadal population comprises one or more populations selected from granulosa cells, ovarian stromal cells, and epithelial cells or a combination thereof. 121. Any method according to embodiments 1-6 and 10-120, wherein the gonadal population comprises a mixture of granulosa cells, ovarian stromal cells, and epithelial cells. 122. A population of gonadal cells produced by any of the methods described in embodiments 1-6 and 10-121. 123. A population of gonadal cells according to embodiment 122, comprising one or more populations selected from granulosa cells, ovarian stromal cells, and epithelial cells or a combination thereof. 124. A population of gonadal cells according to embodiment 122 or embodiment 123, comprising a mixture of granulosa cells, ovarian stromal cells, and epithelial cells. 125. A first intermediate cell population produced by any of the methods described in Embodiment 7 and 13-119. 126. A second intermediate cell population produced by any of the methods described in aspects 6-11 and 13-119. 127. A step of culturing pluripotent stem cells in the presence of activin A, glycogen synthase kinase-3 inhibitor and ROCK inhibitor in order to produce early mesoderm-like cells (iMeLCs). A step of culturing iMeLC for a first period in the presence of FGF2, glycogen synthase kinase-3 inhibitor and ROCK inhibitor. The process involves reducing the amount of ROCK inhibitor in iMeLC culture in order to produce intermediate mesoderm cells, and then culturing the iMeLC over a second period, and The process involves culturing intermediate mesoderm cells in the presence of follistatin, BMP4, FGF2, and a ROCK inhibitor to produce granulosa cells. A method for producing granulosa cells, including [the specified element]. 128. The method according to embodiment 127, wherein the glycogen synthase kinase-3 inhibitor is CHIR99021. 129. The method according to embodiment 127 or 128, wherein the ROCK inhibitor is Y-27632 or CET. 130. Any method according to embodiments 127 to 129, wherein pluripotent stem cells are cultured for approximately 56 to 72 hours. 131. Any method according to embodiment 127 to 130, wherein pluripotent stem cells are cultured for approximately 65 hours. 132. Any method according to embodiments 127 to 131, wherein pluripotent stem cells are cultured in a medium containing activin A, a glycogen synthase kinase-3 inhibitor, and a ROCK inhibitor, and the medium is replaced with fresh medium approximately every 24 hours. 133. Any method according to embodiments 127 to 132, wherein the first period is approximately 24 hours. 134. Any method of embodiments 127 to 133, wherein the second period is approximately 5 or 6 days. 135. Any method according to embodiments 127 to 134, wherein iMeLC is cultured for a first period in a medium containing FGF2, a glycogen synthase kinase-3 inhibitor, and a ROCK inhibitor, and after the first period, a portion of the medium is replaced with a medium containing FGF2 and a glycogen synthase kinase-3 inhibitor but not a ROCK inhibitor. 136. The method of embodiment 135, wherein the aforementioned portion of the culture medium constitutes approximately 80% of the culture medium. 137. The method of embodiment 135 or 136, further comprising the step of replacing a second portion of the culture medium approximately every 48 hours during the second period. 138. The method of embodiment 137, wherein the second portion of the culture medium constitutes approximately 80% of the culture medium. 139. Any method according to embodiments 127 to 138, wherein intermediate mesoderm cells are cultured for a period of approximately 5 to 7 days. 140. Any method according to embodiments 127 to 139, wherein intermediate mesodermal cells are cultured in a medium containing follistatin, BMP4, FGF2, and a ROCK inhibitor, and a portion of the medium is replaced with fresh medium approximately every 24 to 48 hours. 141. Any method of embodiment 127 to 140, wherein iMeLC expresses brachiuri. 142. A method according to any one of embodiments 127 to 139, wherein intermediate mesoderm cells express OSR1, PAX2, and LHX1. 143. Any method according to embodiments 127 to 142, wherein granulosa cells express FOXL2 and connexin 43. 144. Any method according to embodiment 127 to 143, wherein the pluripotent stem cells are human induced pluripotent stem cells. 145. A population containing granulosa cells produced by any of the methods described in aspects 127 to 144. 146. (a) One or more of the culture steps include adherent culture, and / or (b) One or more of the culture steps include three-dimensional organoid culture, Any method or group described in aspects 1 to 145. 147. A method or group according to any of embodiments 1 to 146, wherein one or more of the culture steps include adhesion culture. 148. A method or group according to any of embodiments 1 to 147, wherein one or more of the culture steps include three-dimensional organoid culture. 149. An in vitro stem cell-derived gonadal somatic cell population, comprising FOXL2-expressing cells, NR2F2-expressing cells, and / or KRT-19-expressing cells, and optionally being an ovarian somatic cell population. 150. A population of gonadal somatic cells according to embodiment 149, comprising at least a first cell type expressing FOXL2, a second cell type expressing NR2F2, and a third cell type expressing KRT-19. 151. (a) At least 20% of the cells in the cell population are FOXL2-positive cells, and / or (b) At least 20% of the cells in the cell population are NR2F2-positive cells, and / or (c) At least 20% of the cells in the gonadal cell population are KRT19-positive cells. A population of gonadal cells according to embodiment 149 or 150. 152. At least 90% of the gonadal cell population consists of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells, and optionally, The gonadal somatic cell population essentially consists of FOXL2-positive cells, NR2F2-positive cells, and / or KRT-19-positive cells. A population of gonadal somatic cells according to any of the embodiments 149 to 151. 153. A population of gonadal cells of embodiment 151 or 152, wherein FOXL2-positive cells include granulosa cells, NR2F2-positive cells include ovarian stromal cells and / or granulosa cells, and KRT-19-positive cells include ovarian epithelial cells. 154. (a) A step of culturing pluripotent stem cells in a mesoderm induction medium containing activin A for a first period in order to produce a first intermediate cell population. (b) A step of culturing a first intermediate cell population over a second period in an intermediate mesoderm induction medium containing a retinoic acid pathway regulator (RAPM), thereby producing a second intermediate cell population, and (c) A step of culturing the second intermediate cell population for a third period in a gonadal induction medium containing BMP and FGF and optionally follistatin in order to produce a gonadal cell population. A population of gonadal cells induced in any of embodiments 149 to 153, which includes a process. [Examples]

[0185] This application may be better understood by referring to the following non-limiting embodiments provided herein as exemplary embodiments. The following embodiments are presented to illustrate the embodiments in more detail, but should not be construed as limiting the broad scope of this application. While certain embodiments of the invention have been shown and described herein, it will be obvious that such embodiments are provided for illustrative purposes only. Those skilled in the art will be able to conceive of numerous variations, alterations, and substitutions without departing from the spirit and scope of the invention. It should be understood that various alternative embodiments of the embodiments described herein may be used in carrying out the methods described herein.

[0186] Example 1: Culture medium preparation In some embodiments, the following culture media were prepared for use in one or more of the following examples, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 81%, and 82% of each of the following culture media.

[0187] (Table 1) GK2 medium preparations TIFF2023549137000002.tif64158

[0188] (Table 2) IM medium preparations TIFF2023549137000003.tif55170

[0189] (Table 3) Granulomem Basic Medium Preparations TIFF2023549137000004.tif71169

[0190] (Table 4) Granule membrane double culture medium preparation TIFF2023549137000005.tif70168

[0191] (Table 5) Granulomea BMP7 medium preparation TIFF2023549137000006.tif71169

[0192] (Table 6) Alternative 50ng differentiation preparation TIFF2023549137000007.tif72152

[0193] (Table 7) CHIR differentiation medium preparations TIFF2023549137000008.tif68151

[0194] (Table 8) MEF medium preparations TIFF2023549137000009.tif63168

[0195] (Table 9) iMeLC medium preparations TIFF2023549137000010.tif55170

[0196] Example 2: Production of iMeLC cells Stem cell differentiation proceeds through various stages that can be identified by changes in gene expression. We describe a method for generating ovarian somatic cells by progressively converting stem cells through a series of steady-state stable conditions that mimic fetal ovarian embryonic development. For each step, conditions were optimized to maximize purity and efficiency to obtain a pure, homogeneous ovarian somatic cell culture. Two key intermediate steps in granulosa cell differentiation and marker genes for each of these steps were identified. The following protocol was tested in two different stem cell lines. For robust differentiation, the expression of certain markers was optimized at each step by varying the concentration and duration of inducing substances (e.g., cytokines or small molecules). Such optimizations are incorporated into a robust, universal method that should work even in cell lines with different genetic backgrounds.

[0197] Before handling the cells, each well of a 12-well plate was coated with 610 μL of fibronectin solution prepared using 600 μL of PBS (room temperature) + 10 μL of fibronectin (1 mg / ml; Millipore, FC010, kept on ice), and incubated at 37°C for 1 hour. Then, the medium was removed from the human induced pluripotent stem cells (hiPSCs), and the cells were gently washed with PBS (room temperature). Next, the PBS was removed, and 500 μL of 1:1 TrypLE Select + 500 μL of 0.5 mM EDTA solution (37°C) was added to each well of the iPSCs, followed by incubation at 37°C for 2-3 minutes. Next, the cells were removed by spraying 1 mL of MEF medium into the wells, and resuspended into a single-cell suspension by pipetting 3-5 times. After counting the cells, they were centrifuged at 1200 rpm for 5 minutes. The centrifuged cells were resuspended in GK2 medium, resulting in approximately 10 × 10⁶ cells. 6 The cells were then counted again, and 70K cells (number of wells × 70K) were transferred to 1.5 mL (15 mL if collecting from many wells) tubes. These hiPSCs were centrifuged and resuspended in iMeLC medium to obtain approximately 70k cells / mL.

[0198] The fibronectin-coated solution was removed from the coated wells, and the wells were washed once with PBS. 1 mL of iMeLC medium containing the above 70,000 iPSCs was added to each well. The plated cells were cultured in an incubator at 37°C and 5% CO2 for 56–72 hours (preferably 65 hours). The iMeLC medium was changed every 24 hours. Approximately 2–4 million iMeLCs were produced in each well from one 12-well plate.

[0199] iMeLCs were characterized by the expression of transcription factors such as brachiuri. Protein expression in iMeLCs was detected using fluorescently labeled antibodies. Figure 1A shows the morphology of iMeLCs in phase-contrast imaging, and Figure 1B shows high brachiuri expression 48 hours after stem cell induction (bottom panel). Cells not treated with CHIR and activin (middle panel) show no brachiuri expression or low brachiuri expression.

[0200] Example 3: Production of IM cells By extending iMeLC culture to 65 hours, a homogeneous population of iMeLCs was secured, which were then induced into IM cells. IM cells are characterized by the expression of PAX2, OSR1, and LHX1. Culture conditions for extending IM cell culture over several passages and multiple weeks were identified. This allows for large-scale culture of IM cells and also provides a pure, homogeneous culture that can be differentiated into downstream lineages. Furthermore, bulk IM cells can be frozen to provide an excellent batch control for reproducible differentiation of granulosa cells in subsequent steps.

[0201] The iMeLC medium was removed from the wells of Example 2 and washed with 1 mL of PBS. 500 μL of TrypLE was added to each well, and the plate was incubated at 37°C for 2 minutes. 500 μL of MEF medium was added to each well, and a single-cell suspension was obtained by pipetting up and down three times. The suspension was transferred to a 1.5 mL (15 mL if collecting many wells) tube, and the remaining cells were collected in another 500 μL of MEF medium.

[0202] The cell suspension was centrifuged at 1200 RPM for 5 minutes, and the supernatant was discarded. The centrifuged cells were resuspended in 1 ml of GK2 medium, and the cells were counted. Approximately 720,000 cells (from a 24-well plate: 30,000 cells per well) were added to 12 mL of IM medium (prepared in Example 1, but without the ROCK inhibitor). 12 μL of the ROCK inhibitor Y-27632 (1000×) or 36 μL of CET (3:1000) (final concentration of CET in the medium: 50 nM chroman 1, 5 μM emricasane, 0.7 μM trans-ISRIB) was added to bring the total to 60,000 cells / mL. 500 μL of this solution was placed in each well of a fibronectin-coated 24-well plate (approximately 30,000 cells / well). The plates were cultured for 24 hours, and then 80% of the medium was replaced with fresh IM medium that did not contain ROCK inhibitors. 80% of the medium was replaced again every other day until the cells reached confluence (approximately 5-6 days). Once the cells reached confluence, at least 50,000 cells (25kJ / cm³) were passed through each well. 2 The cells were then passaged into fresh fibronectin-coated 24-well plates. Approximately 4 to 5 million IM cells / 24-well plate were produced.

[0203] Figure 2 shows the morphology of cultured IM cells on day 5, as seen in phase-contrast imaging. Cells were stained for OSR1, PAX2, and LHX1. IM cells (bottom panels in Figures 2B-D) show high expression for all tested markers compared to iPSCs and iMeLCs. The unimodal distribution of signals indicates the purity and homogeneity of cultured IM cells.

[0204] Example 4: Differentiation into granulosa and other lineages The depleted media was removed from the IM cells, and the cells were washed with 500 μL of PBS at room temperature. 200 μL of TrypLE (37°C) was added, and the cells were incubated at 37°C for 2 minutes. 300 μL of MEF medium (room temperature) was added to each well, and a single-cell suspension was obtained by pipetting up and down three times. This suspension was transferred to a 15 mL tube. The remaining cells were collected in another 500 μL of MEF medium. The transferred cells were centrifuged at 1200 RPM for 5 minutes, and the supernatant was discarded. The cells were resuspended in 1 ml of GK2 medium, and the cells were counted.

[0205] 500 μL (approximately 30,000 cells) of differentiation medium containing a ROCK inhibitor (either Y-27 or CET, as previously mentioned) was added to each well of a fibronectin-coated 24-well plate. Separate experiments were performed using each of the following differentiation media (37°C): granulosa basic, granulosa double, granulosa BMP7, alternative differentiation medium (50 ng FGF9), CHIR differentiation medium, and IM (control).

[0206] In all cases, the plates were cultured for 24 hours, and then 80% of the differentiation medium was replaced with fresh medium. 80% of the medium was replaced again every other day until the cells reached confluence (approximately 5-6 days). Once the cells reached confluence, this process was repeated until at least 30,000 cells (15k cells / cm³) were obtained per well after each passage. 2 IM cells were passaged into fresh fibronectin-coated 24-well plates to achieve the following result. After approximately 5-7 days of granulosa cell differentiation, 2-4 million granulosa cells were produced per 24-well plate.

[0207] On day 7 of differentiation, cells were collected to stain for FOXL2, a transcription factor that characterizes granulosa cells. Figure 3A shows the morphology of granulosa cells (left). Figure 3B shows the distribution of FOXL2+ cells under various culture conditions, with the percentage of FOXL2+ cells being highest in granulosa differentiation. Immunofluorescence staining for FOXL2 and connexin 43 shows FOXL2 expression in granulosa cells compared to IM and kidney cells (Figure 3C).

[0208] Example 5: Identification of intermediate stages in granulosa differentiation IM cells are multipotent progenitor cells that can differentiate into multiple lineages, including ovarian somatic cells. Ovarian somatic cells have multiple types, and progenitor cells committed to the ovarian somatic cell lineage and capable of differentiating into these cell types have been identified. CD24 was identified as a highly expressed cell surface marker in early differentiation, but its expression decreases as the cells commit to the ovarian somatic cell fate. This provides us with a guiding trajectory for granulosa cell differentiation for downstream applications.

[0209] Example 6: Generation of intermediate mesoderm and fetal ovarian somatic cells, including Foxl2+ granulosa cells, from human pluripotent stem cells. Culture medium preparations (Table 10) IM induction medium preparations TIFF2023549137000011.tif70160

[0210] A. Generation of a first intermediate cell population expressing markers representative of the mesoderm. A 12-well plate was coated with 610 μL of fibronectin solution prepared using 600 μL of PBS (room temperature) + 10 μL of fibronectin (1 mg / ml; Millipore, FC010, kept on ice), and incubated at 37°C for 1 hour. The medium was then removed from the human induced pluripotent stem cells (hiPSCs), and the cells were gently washed with PBS (room temperature). Next, the PBS was removed, and 500 μL of 1:1 TrypLE Select was added to each well of the iPSCs, followed by incubation at 37°C for 2-3 minutes. The cells were then removed by spraying 1 mL of MEF medium into the wells and resuspended into a single-cell suspension by pipetting 3-5 times. After counting the cells, they were centrifuged at 1200 rpm for 5 minutes. The centrifuged cells were resuspended in GK2 medium, resulting in approximately 10 × 10⁶ cells. 6The cells were then counted again, and 60K cells (number of wells × 60K) were transferred to 1.5 mL (15 mL if collecting many wells) tubes. These hiPSCs were centrifuged and resuspended in iMeLC medium (see Table 9) (mesoderm induction medium) to approximately 60k cells / mL.

[0211] The fibronectin-coated solution was removed from the coated wells, and 1 mL of iMeLC medium containing the above 60,000 iPSCs was added to each well. The plated cells were cultured in an incubator at 37°C and 5% CO2 for 56–72 hours (preferably 65 hours). The iMeLC medium was changed every 24 hours. Approximately 2–4 million iMeLCs were produced in each well from one 12-well plate.

[0212] iMeLCs were characterized by the expression of transcription factors such as brachiuri. Protein expression in iMeLCs was detected using fluorescently labeled antibodies. Figure 1A shows the morphology of iMeLCs in phase-contrast imaging, and Figure 1B shows high brachiuri expression 48 hours after stem cell induction (bottom panel). Cells not treated with CHIR and activin (middle panel) show no brachiuri expression or low brachiuri expression.

[0213] B. Generation of a second intermediate cell population expressing markers representative of the intermediate mesoderm. The iMeLC medium was removed from the wells of the first intermediate cell population (those expressing a marker representative of the mesoderm), and the wells were washed with 1 mL of PBS. 500 μL of TrypLE was added to each well, and the plate was incubated at 37°C for 2 minutes. 500 μL of MEF medium was added to each well, and a single-cell suspension was obtained by pipetting up and down three times. The suspension was transferred to a 1.5 mL (15 mL if collecting many wells) tube, and the remaining cells were collected in another 500 μL of MEF medium.

[0214] The cell suspension was centrifuged at 1200 RPM for 5 minutes, and the supernatant was discarded. The centrifuged cells were resuspended in 1 ml of GK2 medium, and the cells were counted. Approximately 720,000 cells (from a 24-well plate: 30,000 cells per well) were added to 12 mL of IM induction medium (prepared according to Table 10, but without the ROCK inhibitor). 12 μL of the ROCK inhibitor Y-27632 (1000×) or 36 μL of CET (3:1000) (final concentration of CET in the medium: 50 nM chroman 1, 5 μM emricasane, 0.7 μM trans-ISRIB) was added to bring the total to 60,000 cells / mL. 500 μL of this solution was placed in each well of a fibronectin-coated 24-well plate (approximately 30,000 cells / well). The plates were cultured for 24 hours, and then 80% of the medium was replaced with fresh IM induction medium that did not contain ROCK inhibitors. 80% of the medium was replaced again every other day until the cells reached confluence (approximately 5-6 days). Once the cells were confluenced, they were subcultured into fresh fibronectin-coated 24-well plates to a concentration of at least 50,000 cells (25 k / cm²) per well after subculturing. Approximately 4-5 million second intermediate cells were produced per 24-well plate.

[0215] To further determine the effects of GSK3 inhibitors and retinoic acid pathway modulators (RAPMs) on the efficacy of inducing the second intermediate cell population, cells were incubated with IM induction medium containing increased levels of RAPMs and GSK inhibitors, following the replating of the first intermediate cell population described above. IM cells treated with retinoic acid (RA) showed improved viability and induction of gonadal cell differentiation (data omitted).

[0216] Figure 5A shows the morphology of the resulting second intermediate cell population, as observed by phase-contrast imaging, 5 days after incubation of the first intermediate cell population in IM induction medium containing GK2 medium, FGF2, CHIR, and either 0.5 μM, 1 μM, or 2 μM of RAPM TTNPB, according to Table 10. As shown in Figure 5A, increasing RA concentration resulted in increased cell viability and uniform cell morphology.

[0217] Figure 5B shows the expression of intermediate mesoderm markers LHX1 and PAX2 in the resulting second intermediate cell population after incubation of the first intermediate cell population in IM induction medium containing GK2 medium and FGF2; 1 μM, 2 μM, or 3 μM of the GSK3 inhibitor CHIR; and 50 nM, 100 nM, 500 nM, or 1 μM of RA, as described in Table 10. As shown in Figure 5B, increasing the concentration of RA and / or CHIR resulted in increased expression of LHX1 (upper panel) and PAX2 (lower panel).

[0218] Figure 5C shows the expression of intermediate mesoderm markers WT1 and RUNX1 in the resulting second intermediate cell population after incubation of the first intermediate cell population in IM induction medium containing GK2 medium and FGF2; 0.5 μM, 1 μM, or 2 μM of the GSK3 inhibitor CHIR; and 0 μM, 0.1 μM, 0.5 μM, or 1 μM of RA or TTNPB, as described in Table 10. As shown in Figure 5C, increasing the concentration of RA and / or CHIR resulted in increased expression of WT1 (left panel) and RUNX1 (right panel).

[0219] C. Generation of gonadal cells A second intermediate cell population expressing markers representative of the intermediate mesoderm was generated according to the protocol described above, incubating the first intermediate cell population in GK2 medium and IM induction medium containing FGF2; 0.5 μM, 1 μM, or 2 μM of any GSK3 inhibitor CHIR; and 0 μM, 0.1 μM, 0.5 μM, or 1 μM of any RA or TTNPB.

[0220] The depleted culture medium was removed from the second intermediate cell population expressing a marker representative of the intermediate mesoderm, and the cells were washed with 500 μL of PBS at room temperature. 200 μL of TrypLE was added, and the cells were incubated at 37°C for 2 minutes. 300 μL of MEF medium (room temperature) was added to each well, and a single-cell suspension was obtained by pipetting up and down three times. This suspension was transferred to a 15 mL tube. The remaining cells were collected in another 500 μL of MEF medium. The transferred cells were centrifuged at 1200 RPM for 5 minutes, and the supernatant was discarded. The cells were resuspended in 1 ml of GK2 medium, and the cells were counted.

[0221] Approximately 60,000 second intermediate population cells generated under each of the above conditions were reconstituted in 500 μL of differentiation medium containing a ROCK inhibitor (either Y-27632 or CET, as previously mentioned) and replated into each well of a fibronectin-coated 24-well plate. Cells replated from each of the second intermediate populations (generated under various concentrations of CHIR and RAPM) were incubated in the granulosa basal medium shown in Table 3, with or without additional supplementation of 500 nM RA.

[0222] Cells were longitudinally collected for RNA-seq analysis, immunofluorescence staining, and flow cytometry. When assayed with bulk RNA-seq experiments, Figure 6A shows that when no RA treatment was performed at any stage, the resulting gonadal cells showed increased expression of gonadal cell markers FOXL2, RUNX1, NR2F2, WNT6, and KRT19, with FOXL2 being a transcription factor that characterizes granulosa cells. Figure 6B shows that in long-term culture, incubation in differentiation medium containing RA led to increased viability (cells without RA treatment did not survive until differentiation D21) and increased expression of granulosa cell markers FOXL2 and RUNX1.

[0223] As shown by immunofluorescence staining in Figure 6C, high concentrations of RA in the IM induction medium during the generation of the second intermediate cell population resulted in high levels of FOXL2-positive and NR2F2-positive cells in the gonadal cell population. We also observed a simultaneous increase in KRT19 expression following RA treatment, as shown in Figure 6D. This was confirmed by immunostaining for KRT19 in cells continuously treated with RA for 28 days. We observed that a delicate balance between the duration and concentration of RA treatment during the generation of the second intermediate cell population and gonadal cells led to a fluctuating percentage of FOXL2 and KRT19-positive cells (data omitted). This could be leveraged to achieve desirable heterogeneity for downstream applications.

[0224] Example 7: Generation of intermediate mesoderm and fetal ovarian somatic cells containing Foxl2+ granulosa cells from mouse pluripotent stem cells When a granulosa differentiation protocol designed using human pluripotent stem cells was adapted to a mouse model system, it resulted in the induction of Foxl2+ granulosa-like cells from mouse pluripotent stem cells. To replicate early ovarian development, mouse pluripotent stem cells were treated with growth factors in a two-dimensional tissue culture environment. First, the cells were oriented to the first intermediate cell population, then to the second intermediate cell population, and finally to the fetal ovarian somatic cell fate. At each stage, the gene and protein expression of stage-specific markers were evaluated by analyzing the cells using qRT-PCR and protein staining.

[0225] A. Generation of the first mouse intermediate cell population in vitro Mouse pluripotent stem cells were cultured, maintained, and dissociated using a previously published technique (Chow et al. npg Regenerative Medicine 2020, PMID:32351711). Before initiating induction toward the first intermediate cell population, 24-well plates were coated with 0.5 mL of 100 μg / mL Matrigel diluted in DMEM-F12 at room temperature for 1 hour or overnight at 4°C. Next, 50,000 mouse pluripotent stem cells were seeded into priming medium in each well of the 24-well plate. The cells were grown in a 5% CO2 incubator at 37°C for 48 hours. The cells were then treated with mouse mesoderm induction medium (Table 14) for 48 hours. The resulting cells expressed high levels of the mesoderm marker, brachiuri, by qRT-PCR and immunostaining (data omitted).

[0226] B. Generation of a second mouse intermediate cell population in vitro The first intermediate cell population was washed once with sterile PBS at room temperature, and then treated with mouse intermediate mesoderm induction medium (Table 15) for 48–72 hours. The resulting cells expressed the intermediate mesoderm markers Pax2 and Lhx1 by qRT-PCR (data omitted).

[0227] C. Generation of mouse fetal ovarian somatic cells in vitro The second mouse intermediate cell population was washed once with sterile PBS at room temperature and then treated with mouse gonad induction medium (basal medium or basal medium supplemented with 1 μM RA; Table 16) for 7 days. The resulting cells were harvested for RT-PCR analysis and immunofluorescence staining. Immunofluorescence staining for Foxl2 on cells treated with or without RA revealed that the addition of 1 μM retinoic acid led to an increase in the total number of cells expressing the Foxl2 protein (Figure 8, leftmost panel). When assayed by qRT-PCR, Figure 7 shows that the resulting induced gonad cells expressed markers for granulosa cells (Foxl2), stromal cells (Nr2f2), and ovarian epithelial cells (KRT19) at levels comparable to or higher than those of fetal ovarian cells. As shown by immunohistochemistry in Figure 8, the resulting induced glandular cells expressed protein markers for granulosa cells (Foxl2), stromal cells (Nr2f2), and ovarian epithelial cells (Krt19).

[0228] Culture medium preparations: (Table 11) TIFF2023549137000012.tif65166

[0229] (Table 12) TIFF2023549137000013.tif67166

[0230] (Table 13) TIFF2023549137000014.tif38128

[0231] (Table 14) TIFF2023549137000015.tif45128

[0232] (Table 15) TIFF2023549137000016.tif64166

[0233] (Table 16) TIFF2023549137000017.tif54166

[0234] Example 8: Induction of OSCs by various BMP isoforms This example illustrates the effect of various BMP isoforms on the ability to induce gonadal somatic cells (e.g., ovarian somatic cells -- OSCs) from a second intermediate population. Gonadal somatic cells were induced in granulosa diplied medium containing different BMP isoforms, and the expression of bipolarization potential gonadal markers and granulosa markers in the resulting cells was measured.

[0235] Following the protocol described in Example 6, a first intermediate cell population expressing a marker representative of the mesoderm and a second intermediate cell population expressing a marker representative of the intermediate mesoderm were generated.

[0236] Cells replated from the second intermediate population were incubated in granulosa diplex medium (also known as gonad induction medium or OSC induction medium) as shown in Table 17 with 20 ng / mL of either BMP4, BMP2, BMP7, or BMP15. In the resulting cells, the expression of bidifferentiation potential gonad markers (WT1, LHX9, GADD45G, and GATA4) and granulosa markers (FOXL2, NR1H4, and KITLG) was measured by qPCR.

[0237] As shown in Figure 9A, OSC induction using media containing BMP2, BMP7, or BMP15 resulted in expression of bipolarization potential gonad markers that was equivalent to or higher than that of OSC induction using media containing BMP4.

[0238] As shown in Figure 9B, OSC induction using media containing BMP2, BMP7, or BMP15 resulted in granulosa marker expression that was equivalent to or higher than that using the corresponding media containing BMP4.

[0239] These results indicate that OSCs can be induced using granular membrane dip medium containing BMP4, BMP2, BMP7, BMP15, or combinations thereof.

[0240] (Table 17) Granulomea dip medium with different BMP isoforms TIFF2023549137000018.tif56145

[0241] Example 9: Induction of OSCs by various fibroblast growth factor (FGF) family members Fibroblast growth factor (FGF) is a large protein family that is important for signaling events in a wide variety of processes. This example illustrates the effect of various FGF family members on their ability to induce gonadal somatic cells (e.g., ovarian somatic cells -- OSCs) from a second intermediate population. Briefly, gonadal cells were induced in granulosa diplied medium containing different FGFs, and the expression of bidifferentiation potential gonadal markers and granulosa markers in the resulting cells was measured.

[0242] Following the protocol described in Example 6, a first intermediate cell population expressing a marker representative of the mesoderm and a second intermediate cell population expressing a marker representative of the intermediate mesoderm were generated.

[0243] Cells replated from the second intermediate population were incubated in the granulosa diplex medium (also known as gonad induction medium or OSC induction medium) shown in Table 18 with the same concentration of FGF2, 9, 10, 16, 17, 18, or 19. In the resulting cells, the expression of bipolarization potential gonad markers (WT1, LHX9, GADD45G, and GATA4) and granulosa markers (FOXL2, NR1H4, and KITLG) was measured.

[0244] As shown in Figure 10A, OSC induction with media containing FGF9, 10, 16, 17, 18, and 19 resulted in expression of bipolarization potential gland markers that was equivalent to or higher than that with the corresponding media containing FGF2.

[0245] As shown in Figure 9B, OSC induction using media containing FGF9, 10, 16, 17, 18, and 19 resulted in comparable expression of granulosa markers compared to OSC induction using the corresponding media containing BMP4.

[0246] These results indicate that OSCs can be induced using granular membrane dip medium containing FGF2, 9, 10, 16, 17, 18, or 19, or combinations thereof.

[0247] (Table 18) Granulomea dip medium with different BMP isoforms TIFF2023549137000019.tif56128

[0248] Example 10: OSCs derived from pluripotent stem cells are functional. Granulosa cells mature into steroid-producing cells, and these cells can convert androgens such as testosterone into estrogens such as estradiol. One of the key enzymes responsible for steroid production is aromatase-CYP19A1.

[0249] To assay whether gonadal somatic cells (e.g., ovarian somatic cells -- OSCs) generated from pluripotent stem cells are functional, CYP19A1 expression was assayed by qPCR, and steroid production was measured by ELISA for estradiol.

[0250] Following the protocol described in Example 6, a first intermediate cell population expressing a mesoderm-representative marker, a second intermediate cell population expressing an intermediate mesoderm-representative marker, and OSCs were generated.

[0251] After 7 days of induction, CYP19A1 expression was measured in OSCs by qPCR. As shown in Figure 11A, CYP19A1 expression increased in OSCs generated using basic induction medium, and further increased in OSCs generated using induction medium containing twice the amount of growth factor as basic induction medium.

[0252] To further demonstrate this functionality of granulosa cells, estradiol secreted into the culture medium after treatment of OSCs with dihydroxytestosterone (dhT) was measured by ELISA. As shown in Figure 11B, after 24 hours of incubation, estradiol levels observed in the culture medium increased with increasing concentrations of dHT treatment. In addition, when OSCs were treated with 50 ng / ml dHT, a further increase in estradiol levels was observed with longer incubation times (48 hours) compared to 24 hours of incubation.

[0253] These results demonstrate that mature granulosa cells can be generated using the OSC induction protocol described in the above-mentioned examples.

[0254] Example 11: Concentration titration of BMP4, follistatin, and FGF for OSC induction In some initial experiments, the differentiation potential of the second intermediate population was investigated by using a medium containing 10 ng / ml BMP4, 25 ng / ml follistatin, and 5 ng / ml FGF2 to induce gonadal somatic cells (e.g., ovarian somatic cells - OSCs). During OSC induction, various concentrations of BMP4, follistatin, and FGF were titrated, and the resulting cells were examined for the expression of bidifferentiation potential gonadal markers and granulosa markers.

[0255] A first intermediate cell population expressing a mesoderm-representative marker, and a second intermediate cell population expressing an intermediate mesoderm-representative marker, were obtained using the protocol described in Example 6. OSCs were induced based on the protocol described in Example 6, except that various concentrations of BMP4, follistatin, and FGF were tested.

[0256] B MP4 First, we investigated OSC induction conditions by varying BMP4 concentrations. Induction in the complete absence of BMP4 led to low survival rates, but all other BMP4 concentrations tested resulted in similar, equivalent morphologies. Gene expression in the resulting cells was tested by qPCR for the bipolar glandular cell marker (GATA4) and the granulosa cell marker (FOXL2) (Figures 12A and 12B, respectively). GATA4 expression increased with increasing BMP4 concentration, and FOXL2 expression appeared to peak at a BMP4 concentration of 10 ng / ml under the tested conditions.

[0257] In subsequent experiments, based on the expression results for precursor and mature OSC populations, 20 ng / ml of BMP4 was used in OSC induction tests.

[0258] Follistatin Next, as previously mentioned, the BMP4 concentration was kept constant at 20 ng / ml, and four different concentrations of follistatin were tested for OSC induction. Gene expression in the resulting cells was then examined by qPCR for the bipolar glandular cell marker (GATA4) and the granulosa cell marker (FOXL2) (Figures 12C and 12D, respectively). GATA4 expression in Figure 12C demonstrated that OSCs could be generated from precursors in the absence of follistatin. The generation of mature OSCs by FOXL2 expression was also investigated. As shown in Figure 12D, a slight increase in FOXL2 expression was observed as the follistatin concentration increased. Subsequent experiments further explored OSC induction in the absence of follistatin or at low concentrations of follistatin.

[0259] FGF2 Next, the BMP4 concentration was kept constant at 20 ng / mL, and follistatin was removed (0 ng / mL). Five different concentrations of FGF2 were then tested in OSC-induced conditions. Gene expression in the resulting cells was examined by qPCR for potentially bidifferentiating glandular cells (GATA4) and granulosa cells (FOXL2) (Figures 12E and 12F, respectively). As shown in Figures 12E and 12F, both GATA4 and FOXL2 expression reached saturation at 20 ng / mL of FGF2.

[0260] result As shown by the comprehensive concentration titration of BMP4, follistatin, and FGF2 described above, BMP4 promotes the precursor population (bidifferentiated potential gonadal cells), but these results indicate that, under the tested conditions, its effect on OSCs appears to saturate at 20 ng / ml. Similarly, we found that the effect of FGF2 on the generation of both precursor and mature populations also saturates at 20 ng / ml under the tested conditions. On the other hand, while GATA4 expression appeared to increase when follistatin was completely removed, GATA4-expressing precursors can potentially arise from other lineages in addition to OSCs. Therefore, the effect of follistatin concentration was also examined based on the results of FOXL2 expression. A slight increase in FOXL2 expression was observed with 25 ng / ml of follistatin, but no increase in FOXL2 expression was observed at higher follistatin concentrations. These results indicate that OSC induction can be performed both without follistatin and with the various concentrations of follistatin tested. Further experiments will be needed to understand the role of follistatin in OSC induction.

[0261] Example 12: Effects of RA time gradient and concentration gradient on cell fate in pregranulosa-versus-epithelial cells Previous experiments by the inventors have shown that if RA is present for more than two days during the induction of gonadal somatic cells (e.g., ovarian somatic cells - OSCs), more cells will express higher levels of KRT19 and cytoplasmic KRT19. Both of these indicate a higher ratio of epithelial / mesothelial cells. On the other hand, if RA is not present during OSC induction, the proportion of cells expressing FOXL2 will be higher. This indicates a higher proportion of granulosa cells.

[0262] To further understand the effect of RA during OSC induction on the delicate balance between epithelial / mesothelial cells and granulosa cells, the inventors created a 10-step concentration gradient of RA from 0 μm to 10 μM. The inventors observed a decrease in nuclear positivity for FOXL2 staining and a corresponding increase in KRT19 staining, peaking at RA concentrations around 500 nM, as RA concentration increased (Figures 13A, 13B, 13C, 13D). Next, the inventors investigated whether the duration of RA exposure affected this balance between FOXL2-expressing and KRT19-expressing cells. This test was conducted at three different RA concentrations: 100 nM, 500 nM, and 1 μM. One quarter of the wells received no RA, the next quarter received RA for the first 20 hours, another quarter received RA for 48 hours, and the remaining quarter continued RA exposure for the entire experiment (7 days). All cells were fixed on day 7 and stained for FOXL2 and KRT-19. FOXL2 expression was quantified by the percentage of FOXL2-positive nuclei, while KRT-19 expression was measured by the mean intensity of KRT-19 staining normalized to the number of nuclei based on DAPI staining.

[0263] Contrary to previously observed trends, even short exposure to RA led to an increase in KRT19-positive cells and a decrease in FOXL2-positive cells (Figures 13E, 13F). These results confirm that even short pulses or low concentrations of RA are sufficient to induce and increase epithelial / mesothelial cells during OSC induction.

[0264] Example 13 Characterization of OSCs based on gene expression profiles from qPCR, single-cell RNA sequencing, and bulk RNA sequencing for marker genes. To characterize OSCs at the gene expression level, we collected longitudinal samples for bulk RNA-seq experiments. Cells were flash-frozen as iPSCs in the first and second intermediate stages (i.e., the first and second intermediate cell populations), and as OSCs (with and without retinoic acid treatment, at OSC induction D7 and D14). Heatmaps of the top 200 highly variable genes and principal component analysis of normalized and processed data showed dramatic changes in gene expression profiles during the transition from the first to the second intermediate stage and from the second intermediate stage to OSCs (Figures 14A and 14B). We observed that retinoic acid-treated OSCs exhibited increased expression in clusters of genes enriched with respect to hormone levels and cell proliferation regulation (Figure 14A). To further characterize the heterogeneity of differentiating OSCs, we collected OSCs on days 2 and 7 with and without retinoic acid treatment and subjected them to single-cell analysis using a 10X genomics platform according to the manufacturer's protocol. Five distinctly different clusters of increased gene expression were observed among the more than 10,000 cells analyzed in this experiment. We observed that these clusters corresponded to pregranulosa, stromal cells, proliferative precursors, and epithelial cells, as characterized by marker gene expression (Figure 14C). Furthermore, to validate gene expression readouts based on these sequencing studies, the inventors performed qPCR assays for biopotential gonadal markers—GATA4, WT1, LHX9, and ZFPM2; stromal cell marker—NR2F2; granulosa cell markers—FOXL2, KITLG, and NR1H4; and epithelial cell markers—KRT19, TMEM151A, and LRRN4 (Figures 14D, 14E, 14F, and 14G, respectively). For most of these genes, the inventors observed increased expression on days 7 and 14 after OSC induction under basic or doubled conditions with or without retinoic acid treatment.

[0265] Example 14: qPCR and immunofluorescence assays in induced ovarian somatic cells A first intermediate cell population expressing a mesoderm-representative marker, and a second intermediate cell population expressing an intermediate mesoderm-representative marker, according to the protocol described in Example 6. Gonadal somatic cells (e.g., ovarian somatic cells -- OSCs) were induced according to the protocol described in Example 6.

[0266] To further validate the identification of OSCs, gene expression was analyzed for the granulosa marker gene FOXL2 and the bipolar gonad genes LHX9 and WT1. The expression of all three genes was upregulated as the culture duration increased. The inventors also observed an increase in the expression of the mature granulosa cell markers KITLG and NR1H4 as the culture duration increased (data omitted).

[0267] While qPCR assays are effective for evaluating gene expression in bulk, they cannot adequately demonstrate the heterogeneity of cell types present in culture. To assess heterogeneity in OSC differentiation and the localization of different cell types, cell aggregates were sectioned and stained for immunofluorescence imaging. As shown in Figure 15, ovarian somatic cells were induced (indicated by GATA4 and NR5A1-expressing cells) as well as ovarian epithelial / mesothelial cells (indicated by KRT-19-expressing cells).

[0268] These results demonstrate that this differentiation protocol can be used to induce the described somatic cell types from pluripotent stem cells.

[0269] The specific embodiments disclosed are provided for purposes such as illustrating various aspects of the invention, and the scope of the invention is not limited to those specific embodiments. Various modifications of the compositions and methods described will become apparent from the description and teaching herein. Such modifications can be carried out without departing from the true scope and spirit of this disclosure and are included within the scope of the invention.

Claims

1. (a) culturing pluripotent stem cells in a mesoderm induction medium comprising activin A for a first period of time to produce a population comprising cells, at least a portion of said cells expressing Brachyury; (b) culturing the cells, including the cells expressing Brachyury, in an intermediate mesoderm induction medium that includes a retinoic acid pathway regulator (RAPM), for a second period of time, thereby producing a population comprising cells, at least a portion of which express OSR1 or LHX1; and (c) culturing the cells, including the cells expressing OSR1 or LHX1, in a gonadal induction medium comprising BMP and FGF for a third period of time to produce a gonadal cell population.

2. A method for producing a gonadal cell population comprising:

2. (a) culturing cells, including cells that express Brachyury, in an intermediate mesoderm induction medium that includes a retinoic acid pathway modulator (RAPM), for a first period of time, thereby producing a population comprising cells, at least a portion of the cells expressing OSR1 or LHX1; (b) culturing the cells, including cells expressing OSR1 or LHX1, in a gonadal induction medium comprising BMP and FGF for a second period of time to produce a gonadal cell population.

2. A method for producing a gonadal cell population comprising:

3. Culturing the cells, including cells expressing OSR1 or LHX1, in a gonadal induction medium comprising BMP and FGF for a period of time to produce a gonadal cell population.

2. A method for producing a gonadal cell population comprising:

4. 4. The method of any one of claims 1 to 3, wherein the gonadal induction medium further comprises follistatin.

5. Culturing pluripotent stem cells in a mesoderm induction medium comprising activin A and a glycogen synthase kinase-3 inhibitor for a period of time to produce a population comprising cells, at least a portion of which express Brachyury. A method for producing a cell that expresses Brachyury, comprising:

6. Culturing the cells expressing Brachyury in intermediate mesoderm induction medium containing a retinoic acid pathway modulator (RAPM) for a period of time, thereby producing a population comprising cells, at least a portion of which express OSR1 or LHX1. A method for producing a cell expressing OSR1 and / or LHX1, comprising:

7. (a) culturing pluripotent stem cells in a mesoderm induction medium comprising activin A and a glycogen synthase kinase-3 inhibitor for a first period of time to produce a population comprising cells, at least a portion of the cells expressing Brachyury; (b) culturing the cells, including the cells expressing Brachyury, in an intermediate mesoderm induction medium comprising a retinoic acid pathway regulator (RAPM), FGF and a glycogen synthase kinase-3 inhibitor, for a second period of time, thereby producing a population comprising cells, at least a portion of which express OSR1 or LHX1. A method for producing a cell expressing OSR1 and / or LHX1, comprising:

8. The method of any one of claims 1 to 4, wherein at least a portion of the cells in the gonadal cell population express FOXL2, NR2F2 or RUNX1.

9. Pluripotent stem cells are 1 cm 2 and / or are seeded at a density of about 10,000 to about 40,000 cells per well; Pluripotent stem cells are seeded onto fibronectin-coated culture plates, or Pluripotent stem cells are seeded onto culture plates coated with Matrigel; The method of any one of claims 1, 4, and 6-8.

10. and / or the mesoderm induction medium further comprises FGF; The mesoderm induction medium further comprises BMP4, a glycogen synthase kinase-3 inhibitor, and / or an apoptosis inhibitor; The method of any one of claims 1, 4, 5, and 7 to 9.

11. The method of any one of claims 1, 4, 5, and 7-10, wherein the concentration of Activin A in the mesoderm induction medium is about 30 ng / mL to about 70 ng / mL.

12. The method of claim 10 or 11, wherein the FGF in the mesoderm induction medium is FGF2.

13. the concentration of FGF in the mesoderm induction medium is between about 5 ng / mL and about 20 ng / mL; and / or the concentration of BMP4 in the mesoderm induction medium is between about 10 ng / mL and about 50 ng / mL; and / or the glycogen synthase kinase-3 inhibitor in the mesoderm induction medium is CHIR99021, and the concentration of CHIR99021 is about 1 μM to about 5 μM; The method according to any one of claims 10 to 12.

14. The method of any one of claims 1, 4, 5, and 7-13, wherein the period of culturing in the mesoderm induction medium is from about 24 hours to about 96 hours.

15. A method described in any one of claims 1, 4, 5, and 7-14, wherein at least 80% of the cells expressing Brachyury express one or more of MIXL1, N-cadherin, EpCam, and / or NCAM.

16. A method described in any one of claims 1, 4, 5, and 7-15, wherein at least 90% of cells expressing Brachyury express one or more of MIXL1, N-cadherin, EpCam, and / or NCAM.

17. The method of any one of claims 1, 2, 4, and 6-16, wherein the intermediate mesoderm induction medium further comprises FGF, a glycogen synthase kinase-3 inhibitor, activin A, and / or an apoptosis inhibitor.

18. the FGF in the intermediate mesoderm induction medium is FGF2, and the concentration of FGF2 is about 10 ng / mL to about 30 ng / mL; and / or the glycogen synthase kinase-3 inhibitor in the intermediate mesoderm induction medium is CHIR99021, and the concentration of CHIR99021 is about 1 μM to about 5 μM; and / or the concentration of activin A in the intermediate mesoderm induction medium is about 10 ng / mL to about 50 ng / mL; 18. The method of claim 17.

19. A method described in any one of claims 1, 2, 4, and 6-18, wherein at least 70% of cells expressing OSR1 or LHX1 further express one or more of PAX2 and / or RUNX1.

20. A method described in any one of claims 1, 2, 4, and 6-19, wherein at least 90% of cells expressing OSR1 or LHX1 also express one or more of PAX2 and / or RUNX1.

21. The method of any one of claims 1, 4, and 8 to 21, wherein the cells expressing Brachyury or the cells expressing OSR1 or LHX1 are replated onto new fibronectin-coated culture plates prior to culture in gonadal induction medium.

22. The method of any one of claims 1 to 4 and 8 to 21, wherein the gonad induction medium further comprises RAPM and / or an apoptosis inhibitor.

23. 23. The method of any one of claims 4 and 8-22, wherein the concentration of follistatin in the gonad induction medium is from about 10 ng / mL to about 50 ng / mL.

24. the BMP in the gonad induction medium comprises BMP4, BMP2, BMP7, BMP15 or any combination thereof; and / or The total concentration of BMP in the gonad induction medium is about 5 ng / mL to about 20 ng / mL or about 20 ng / mL to about 70 ng / mL; The method of any one of claims 1 to 4 and 8 to 23.

25. The method of any one of claims 23 to 24, wherein the RAPM in the intermediate mesoderm induction medium and / or in the gonad induction medium is a RAR agonist.

26. (a) the RAPM is RA, and the concentration of RA in the intermediate mesoderm induction medium and / or in the gonad induction medium is about 0.5 μM to about 2 μM; and / or (b) the RAPM is TTNPB, and the concentration of TTNPB in the intermediate mesoderm induction medium and / or in the gonad induction medium is about 0.2 μM to about 1 μM; 26. The method of claim 25.

27. in mesoderm induction medium, in intermediate mesoderm induction medium, and / or in gonad induction medium, (A) the apoptosis inhibitor is Y-27632, or (B) Apoptosis inhibitors include chroman 1, emricasan and trans-ISRIB; 27. The method of any one of claims 23 to 26.

28. in mesoderm induction medium and / or in gonad induction medium, (A) The concentration of Y-27632 is about 10 μM, or (B) The concentration of chroman 1 is about 50 nM, the concentration of emricasan is about 5 μM, and the concentration of trans-ISRIB is about 0.7 μM; 28. The method of claim 27.

29. A cell comprising a cell expressing Brachyury. (i) First, in an intermediate mesoderm induction medium containing about 10 μM Y-27632, for about 24 hours, (ii) Next, the cells were incubated in an intermediate mesoderm induction medium containing about 2 μM or less of Y-27632 for about 5 to 6 days. Culturing the culture medium The method of any one of claims 1, 4, 5, and 8 to 28.

30. The method of any one of claims 1, 2, 4, and 6-29, wherein the period of culturing in the intermediate mesoderm induction medium is about 5-9 days.

31. The method of any one of claims 1 to 4 and 8 to 30, wherein the period of culturing in the gonad induction medium is from about 5 days to about 21 days.

32. The method of any one of claims 1-4 and 8-31, wherein the gonadal cell population comprises ovarian somatic cells.

33. The method of any one of claims 1-4 and 8-32, wherein at least 20% of the cells in the gonadal cell population express one or more of FOXL2, NR2F2, and KRT19.

34. The method of any one of claims 1 to 4 and 8 to 33, wherein at least 90% of the gonadal cell population are FOXL2 positive cells, NR2F2 positive cells and / or KRT-19 positive cells.

35. The method of any one of claims 1, 2, 4, and 7-34, wherein the pluripotent stem cells are mammalian stem cells.

36. The method of any one of claims 1, 2, 4, and 7-35, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

37. The method of any one of claims 1-4 and 8-36, wherein the gonadal population comprises one or more populations selected from granulosa cells, ovarian stromal cells and epithelial cells or a combination thereof.

38. A gonadal cell population produced by the method of any one of claims 1 to 4 and 8 to 37.

39. 38. A population comprising cells produced by the method of any one of claims 5 and 10-37, wherein at least a portion of said cells express Brachyury.

40. 38. A population comprising cells produced by the method of any one of claims 6 and 10-37, wherein at least a portion of the cells express OSR1 or LHX1.

41. Culturing pluripotent stem cells in the presence of activin A, a glycogen synthase kinase-3 inhibitor, and a ROCK inhibitor to produce early mesoderm-like cells (iMeLCs); culturing the iMeLCs in the presence of FGF2, a glycogen synthase kinase-3 inhibitor, and a ROCK inhibitor for a first period of time; reducing the amount of ROCK inhibitor in the iMeLC culture and then culturing the iMeLCs for a second period of time to produce intermediate mesoderm cells; Culturing intermediate mesoderm cells in the presence of follistatin, BMP4, FGF2 and a ROCK inhibitor to produce granulosa cells.

2. A method for producing granulosa cells comprising:

42. 42. A population comprising granulosa cells produced by the method of claim 41.

43. (a) one or more of the culturing steps comprises adherent culture; and / or (b) one or more of the culturing steps comprises three-dimensional organoid culture; 43. The method or population of any one of claims 1 to 42.

44. An in vitro stem cell derived gonadal somatic cell population comprising FOXL2-expressing cells, NR2F2-expressing cells and / or KRT-19-expressing cells, and optionally an ovarian somatic cell population.

Images