Method for producing ovarian somatic cell-like cells and method for inducing differentiation of primate pluripotent stem cells into ovarian somatic cell-like cells

Abstract

This method for producing ovarian somatic cell-like cells comprises: step 1 for culturing primate pluripotent stem cells in a medium containing a GSK3 inhibitor and a ROCK inhibitor; step 2 for culturing the cells from step 1 in a medium containing BMP4, retinoic acid and a MEK inhibitor; and step 3 for culturing the cells from step 2 in a basal medium to give ovarian somatic cell-like cells. This method for inducing the differentiation of primate pluripotent stem cells into ovarian somatic cell-like cells comprises inducing the differentiation of primate pluripotent stem cells into ovarian somatic cell-like cells with the use of the aforesaid method for producing ovarian somatic cell-like cells.

Description

[Technical Field]

[0001] The present invention relates to a method for producing ovarian somatic cell-like cells and a method for inducing differentiation of primate pluripotent stem cells into ovarian somatic cell-like cells. This application claims priority to U.S. Patent No. 63 / 168,263, filed provisionally in the United States on March 31, 2021, the contents of which are incorporated herein by reference. [Background technology]

[0002] Germ cells originate from primordial germ cells, differentiate into sperm or eggs, and then fuse to form new individuals. Meanwhile, the ovary, a reproductive organ responsible for the production, maturation, and ovulation of eggs, originates from the intermediate mesoderm formed after gastrulation. It differentiates into a single layer of coelomic epithelium, a portion of which then proliferates and thickens to form the reproductive ridge. After a certain period of time has passed since fertilization, the cells are stimulated by more sex-specific stimuli and differentiate into the granulosa and interstitial cells that make up the ovary.

[0003] It has been reported that mouse primordial germ cells (PGCs) and PGC-like cells (PGCLCs) induced from pluripotent stem cells differentiate into functional sperm and eggs that can contribute to offspring when aggregated and cultured in vitro with mouse fetal gonad somatic cells (see, for example, Patent Document 1). However, although this method matures eggs in vitro, it requires the collection of ovarian somatic cells (follicles) that nourish the eggs from the living body. This makes it difficult to apply this method to various animals, including humans, from which ovarian tissue is difficult to collect.

[0004] Meanwhile, the present inventors have reported a method for inducing differentiation of mouse pluripotent stem cells into ovarian somatic cell-like cells (see, for example, Non-Patent Document 1).

[0005] When the method described in Patent Document 1 is used, human or cynomolgus monkey PGCLCs do not initiate meiosis like germ cells in vivo, even when aggregated with mouse fetal ovarian somatic cells for a long period of several months. The main reasons for this are thought to be a mismatch between the differentiation and maturation of germ cells, which takes several months, and the maturation of mouse gonad somatic cells, which takes only a few days, and differences in secreted factors between species.

[0006] Furthermore, even if attempts are made to obtain ovarian somatic cell-like cells using pluripotent stem cells of primates such as humans using the method described in Non-Patent Document 1, it is not possible to induce differentiation into ovarian somatic cell-like cells because the time and factors required for the maturation of gonadal somatic cells differ between mice and primates such as humans. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2017 / 047799 [Non-patent literature]

[0008] [Non-Patent Document 1] Yoshino T et al., “Generation of ovarian follicles from mouse pluripotent stem cells.”, Science, Vol. 373, No. 6552, p. eabe0237, 2021. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in consideration of the above circumstances, and provides a method for producing ovarian somatic cell-like cells, which can efficiently obtain ovarian somatic cell-like cells at any developmental stage from primate pluripotent stem cells, and a method for inducing differentiation of primate pluripotent stem cells into ovarian somatic cell-like cells. [Means for solving the problem]

[0010] As a result of extensive research to achieve the above-mentioned object, the inventors discovered that primate pluripotent stem cells can be induced to differentiate into mesoderm by culturing them in the presence of a GSK3 inhibitor and a ROCK inhibitor, and then induced to differentiate into intermediate mesoderm that expresses OSR1 and WT1 by culturing them in the presence of BMP4, retinoic acid, and a MEK inhibitor, and that further differentiation into FOXL2-positive ovarian somatic cell-like cells can be achieved by continuing to culture them in a basal medium, thereby completing the present invention.

[0011] That is, the present invention includes the following aspects. (1) A method for producing ovarian somatic cell-like cells, comprising: Step 1: culturing primate pluripotent stem cells in a medium containing a GSK3 inhibitor and a ROCK inhibitor; Step 2: culturing the cells obtained after step 1 in a medium containing BMP4, retinoic acid, and a MEK inhibitor; Step 3: culturing the cells obtained after step 2 in a basal medium to obtain ovarian somatic cell-like cells; A manufacturing method comprising: (2) The method according to (1), wherein the culture in step 1 is carried out for 3 days or more. (3) The method of (1) or (2), wherein the culturing in step 2 is carried out for 3 days or more. (4) The method according to any one of (1) to (3), wherein the culture in step 3 is carried out for one week or more. (5) The method of any one of (1) to (4), wherein the medium in step 1 further contains BMP4. (6) Step 1 is Step 1-1: culturing primate pluripotent stem cells in a medium containing a GSK3 inhibitor and a ROCK inhibitor; Step 1-2 of culturing the cells after step 1-1 in a medium containing a GSK3 inhibitor and Activin A; The method for producing a semiconductor device according to any one of (1) to (5), comprising: (7) The method according to any one of (1) to (6), wherein the culture in step 1 is plate culture. (8) The method according to (7), wherein a container coated with a cell scaffold is used in the flat culture. (9) The method according to (7) or (8), wherein the medium in step 2 further contains a ROCK inhibitor. (10) The method according to any one of (1) to (9), wherein the medium in step 1 further contains bFGF. (11) The method for production according to any one of (1) to (10), wherein the medium in the step 2 further contains a hedgehog signal activator. (12) Step 3 Step 3-1: culturing the cells obtained after step 2 in a medium containing a MEK inhibitor; Step 3-2 of culturing the cells obtained after step 3-1 in a basal medium to obtain ovarian somatic cell-like cells; The method for producing a semiconductor device according to any one of (1) to (11), comprising: (13) The method according to any one of (1) to (12), wherein the ovarian somatic cell-like cells are fetal ovarian somatic cell-like cells. (14) The method according to any one of (1) to (13), wherein the primate pluripotent stem cells are derived from a cynomolgus monkey or a human. (15) A method for inducing differentiation of primate pluripotent stem cells into ovarian somatic cell-like cells, comprising: A method comprising inducing differentiation from primate pluripotent stem cells into ovarian somatic cell-like cells using the production method according to any one of (1) to (14). [Effects of the Invention]

[0012] According to the production method and differentiation induction method of this embodiment, ovarian somatic cell-like cells at any developmental stage can be efficiently obtained from primate pluripotent stem cells. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the configuration of the constructs introduced into each gene locus of the human iPS cell line in Example 1. [Figure 2] FIG. 1 shows a protocol for inducing differentiation of mouse ES cells into ovarian somatic cell-like cells. [Figure 3A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in "1." of Example 1. [Figure 3B] 1 shows a bright-field image (left side) and fluorescent images (center and right side) of an embryoid body in "1." of Example 1. [Figure 3C] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP in "1." of Example 1. [Figure 4A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in "2." of Example 1. [Figure 4B] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP in "2." of Example 1. [Figure 5A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in "3." of Example 1. [Figure 5B] 1 shows a bright-field image (left side) and fluorescent images (center and right side) of an embryoid body in "3." of Example 1. [Figure 5C] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP in "3." of Example 1. [Figure 5D] This figure shows the results of examining the time course of OSR1 and NR5A1 expression when the period of step 1 in "3." in Example 1 was set to 4 days. The upper row shows the number of days of induction, the middle row shows bright-field images, and the lower row shows FACS plots of OSR1-tdTomato and NR5A1-EGFP. [Figure 6A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in "4." of Example 1. [Figure 6B] 1 shows a bright-field image (top row) and fluorescent images (middle and bottom rows) of embryoid bodies in "4." of Example 1. [Figure 6C] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP in "4." of Example 1. [Figure 7A]FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in "5." of Example 1. [Figure 7B] 1 shows a bright-field image (left side) and fluorescent images (center and right side) of an embryoid body in "5." of Example 1. [Figure 7C] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP in "5." of Example 1. [Figure 8A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in "6." of Example 1. [Figure 8B] 1 shows a bright-field image (left side) and fluorescent images (center and right side) of an embryoid body in "6." of Example 1. [Figure 8C] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP in "6." of Example 1. [Figure 9A] 1 shows a bright-field image (left) and fluorescent images (center and right) of embryoid bodies in the presence of PD0325901 (1.0 μM) in "7." of Example 1. [Figure 9B] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP in the presence of PD0325901 (1.0 μM) in "7." of Example 1. [Figure 9C] 1 shows a bright-field image (left side) and fluorescent images (center and right side) of embryoid bodies in the absence of PD0325901 in "7." of Example 1. [Figure 9D] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP in the absence of PD0325901 in "7." of Example 1. [Figure 10A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in "8." of Example 1. [Figure 10B] 1 shows bright-field images (left columns for each condition) and fluorescent images (center and right columns for each condition) of embryoid bodies in "8." of Example 1. [Figure 10C]1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP in "8." of Example 1. [Figure 11A] 1 shows a bright-field image (left) and fluorescent images (center and right) of embryoid bodies at a KSR concentration of 5% in "9." of Example 1. [Figure 11B] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP under the condition of a KSR concentration of 5% in "9." of Example 1. [Figure 11C] 1 shows a bright-field image (left) and fluorescent images (center and right) of embryoid bodies at a KSR concentration of 7.5% in "9." of Example 1. [Figure 11D] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP under the condition of a KSR concentration of 7.5% in "9." of Example 1. [Figure 12A] 1 shows a bright-field image (left) and fluorescent images (center and right) of an embryoid body on day 15 of induction in "10." of Example 1. [Figure 12B] 10 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP on day 15 of induction in "10." of Example 1. [Figure 12C] 1 shows a bright-field image (left) and fluorescent images (center and right) of an embryoid body on day 23 of induction in "10." of Example 1. [Figure 12D] 1 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP on day 23 of induction in "10." of Example 1. [Figure 13A] 1 shows a bright-field image (left) and fluorescent images (center and right) of an embryoid body on day 15 of induction in "11." of Example 1. [Figure 13B] 11 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP on day 15 of induction in "11." of Example 1. [Figure 13C] 1 shows a bright-field image (left) and fluorescent images (center and right) of an embryoid body on day 23 of induction in "11." of Example 1. [Figure 13D]11 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP on day 23 of induction in "11." of Example 1. [Figure 14A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in “12.” of Example 1. [Figure 14B] 1 shows a bright-field image (left) and fluorescent images (center and right) of embryoid bodies grown in the absence of sonic hedgehog (SHH) in "12." of Example 1. [Figure 14C] 12 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP under conditions in which SHH was not added in "12." of Example 1. [Figure 14D] 1 shows a bright-field image (left) and fluorescent images (center and right) of embryoid bodies under SHH-added conditions in "12." of Example 1. [Figure 14E] 12 shows FACS plots of OSR1-tdTomato and NR5A1-EGFP under SHH-addition conditions in "12." of Example 1. [Figure 15A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in “13.” of Example 1. [Figure 15B] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "13." of Example 1. [Figure 16A] 1 shows FACS plots of OSR1-tdTomato and NR5A1(SF1)-EGFP in "14." of Example 1. [Figure 16B] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "14." of Example 1. [Figure 17] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "15." of Example 1. [Figure 18A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in "16." of Example 1. [Figure 18B] 16 shows images of the changes over time of cells in "16." of Example 1. [Figure 18C] 1 shows FACS plots of OSR1-tdTomato and NR5A1(SF1)-EGFP in "16." of Example 1. [Figure 18D] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "16." of Example 1. [Figure 18E] 1 shows a fluorescent immunostained image of the embryoid body in "16." of Example 1. [Figure 18F] 1 shows a fluorescent immunostained image of the embryoid body in "16." of Example 1. [Figure 18G] 1 shows FACS plots of OSR1-tdTomato and NR5A1(SF1)-EGFP in "16." of Example 1. [Figure 18H] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "16." of Example 1. [Figure 19A] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "17." of Example 1. [Figure 19B] 1 shows FACS plots of OSR1-tdTomato and NR5A1(SF1)-EGFP in "17." of Example 1. [Figure 20A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells in "18." of Example 1. [Figure 20B] 1 is a bright-field image of the embryoid body in "18." of Example 1. [Figure 20C] 1 is a bright-field image of the embryoid body in "18." of Example 1. [Figure 20D] 18 shows FACS plots of OSR1-tdTomato and NR5A1(SF1)-EGFP in "18." of Example 1. [Figure 20E] 1 shows a fluorescent immunostained image of the embryoid body in "18." of Example 1. [Figure 20F] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "18." of Example 1. [Figure 20G]FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using human iPS cells established based on the results up to "18." in Example 1. [Figure 21] 1 shows FACS plots of PDGFRα and SF1 in "19." of Example 1. [Figure 22A] 1 shows a bright-field image (left) of an embryoid body in "20." of Example 1 and FACS plots of OSR1-tdTomato and NR5A1(SF1)-EGFP. [Figure 22B] 1 shows a fluorescent immunostained image of an embryoid body on day 37 of induction in "20." of Example 1. [Figure 22C] 1 shows a fluorescent immunostained image of an embryoid body on day 57 of induction in "20." of Example 1. [Figure 22D] 1 shows a fluorescent immunostained image of an embryoid body on day 86 of induction in "20." of Example 1. [Figure 22E] 1 shows a fluorescent immunostained image of an embryoid body on day 86 of induction in "20." of Example 1. [Figure 22F] 1 shows a fluorescent immunostained image of an embryoid body on day 86 of induction in "20." of Example 1. [Figure 23A] 1 shows FACS plots from days 7 to 10 of induction in "21." of Example 1. [Figure 23B] 1 shows FACS plots from days 11 to 15 of induction in "21." of Example 1. [Figure 24A] FIG. 1 shows the structure of the construct introduced into the TBXT locus of cynomolgus monkey ES cells in Example 2 and changes in the structure of the construct after introduction. [Figure 24B] FIG. 1 shows the structure of the construct introduced into the NR5A1 locus of cynomolgus monkey ES cells in Example 2 and changes in the structure of the construct after introduction. [Figure 24C] FIG. 1 shows the structure of the construct introduced into the OSR1 locus of cynomolgus monkey ES cells in Example 2 and changes in the structure of the construct after introduction. [Figure 24D]FIG. 1 shows the structure of the construct introduced into the FOXL2 locus of cynomolgus monkey ES cells in Example 2 and the change in the structure of the construct after introduction. [Figure 24E] FIG. 1 shows the structure of the construct introduced into the WT1 locus of cynomolgus monkey ES cells in Example 2 and changes in the structure of the construct after introduction. [Figure 24F] FIG. 1 shows the structure of the construct introduced into the GATA4 locus of cynomolgus monkey ES cells in Example 2 and the change in the structure of the construct after introduction. [Figure 25A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "1." of Example 2. [Figure 25B] 1 shows bright-field images (left side, center, and upper left on the right side) and fluorescent images (upper right side, lower left, and lower right) of embryoid bodies in "1." of Example 2. [Figure 25C] 1 shows FACS plots of OSR1-tdTomato and TBXT-EGFP in "1." of Example 2. [Figure 26A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "2." of Example 2. [Figure 26B] 1 shows FACS plots of OSR1-tdTomato and TBXT-EGFP in "2." of Example 2. [Figure 27A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "3." of Example 2. [Figure 27B] 10 shows FACS plots of OSR1-tdTomato and TBXT-EGFP on day 4 of induction in "3." of Example 2. [Figure 27C] 10 shows FACS plots of OSR1-tdTomato and TBXT-EGFP on day 6 of induction in "3." of Example 2. [Figure 28A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "4." of Example 2. [Figure 28B] 1 shows FACS plots of OSR1-tdTomato and TBXT-EGFP in "4." of Example 2. [Figure 29A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "5." of Example 2. [Figure 29B] 1 shows FACS plots of OSR1-tdTomato and TBXT-EGFP in "5." of Example 2. [Figure 30A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "6." of Example 2. [Figure 30B] 10 shows FACS plots of OSR1-tdTomato and TBXT-EGFP under conditions in which Y-27632 was added for one day in "6." of Example 2. [Figure 30C] 10 shows FACS plots of OSR1-tdTomato and TBXT-EGFP under conditions in which Y-27632 was added for two days in "6." of Example 2. [Figure 31A] This is a diagram showing the protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "7." of Example 2. In the diagram, "MC" means medium change. The same meaning is used hereinafter. [Figure 31B] 1 shows FACS plots of OSR1-tdTomato and TBXT-EGFP in "7." of Example 2. [Figure 31C] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "7." of Example 2. [Figure 32A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "8." of Example 2. [Figure 32B] 1 shows FACS plots from days 2 to 14 of induction in "8." of Example 2. [Figure 33A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "9." of Example 2. [Figure 33B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "9." of Example 2. [Figure 34A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "10." of Example 2. [Figure 34B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "10." of Example 2. [Figure 34C] 1 is a bright-field image of the embryoid body in "10." of Example 2. [Figure 35A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "11." of Example 2. [Figure 35B] 11 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "11." of Example 2. [Figure 36A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "12." of Example 2. [Figure 36B] 12 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "12." of Example 2. [Figure 37A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "13." of Example 2. [Figure 37B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "13." of Example 2. [Figure 37C] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "13." of Example 2. [Figure 38A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "14." of Example 2. [Figure 38B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "14." of Example 2. [Figure 39A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "15." of Example 2. [Figure 39B] 15 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "15." of Example 2. [Figure 39C] 15 is a bright-field image of the embryoid body in "15." of Example 2. [Figure 40A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "16." of Example 2. [Figure 40B] 16 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under the condition that the durations of step 1, step 2, and step 3 in "16." of Example 2 were 2-2-12 (days). [Figure 40C] 16 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under the condition that the durations of step 1, step 2, and step 3 in "16." of Example 2 were 3-2-11 (days). [Figure 40D] 16 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under the conditions of 4-2-10 (days) periods for step 1, step 2, and step 3 in "16." of Example 2. [Figure 40E] 16 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under conditions in which the durations of step 1, step 2, and step 3 in "16." of Example 2 were 2-3-11 (days). [Figure 40F] 16 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under the condition where the periods of step 1, step 2, and step 3 in "16." of Example 2 were 3-3-10 (days). [Figure 40G]16 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under conditions in which the periods of step 1, step 2, and step 3 in "16." of Example 2 were 4-3-9 (days). [Figure 40H] 16 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP on day 17 of induction, where culture was continued for an additional day under the conditions of 3-3-10 (days) periods for steps 1, 2, and 3 in "16." of Example 2. [Figure 40I] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "16." of Example 2. [Figure 40J] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "16." of Example 2. [Figure 40K] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "16." of Example 2. [Figure 40L] 16 shows a fluorescent immunostained image of an embryoid body on day 17 of induction in "16." of Example 2. [Figure 41A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "17." of Example 2. [Figure 41B] 17 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP on days 16 and 28 of induction in "17." of Example 2. [Figure 41C] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "17." of Example 2. [Figure 42A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "18." of Example 2. [Figure 42B] 18 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "18." of Example 2. [Figure 43A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "19." of Example 2. [Figure 43B] 1 is a bright-field image of the embryoid body in "19." of Example 2. [Figure 43C] 19 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "19." of Example 2. [Figure 44A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "20." of Example 2. [Figure 44B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "20." of Example 2. [Figure 45A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "21." of Example 2. [Figure 45B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "21." of Example 2. [Figure 46A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "22." of Example 2. [Figure 46B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "22." of Example 2. [Figure 47A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "23." of Example 2. [Figure 47B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "23." of Example 2. [Figure 48A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "24." of Example 2. [Figure 48B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "24." of Example 2. [Figure 49A]FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "25." of Example 2. [Figure 49B] 10 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under conditions in which SHH was not added in "25." of Example 2. [Figure 49C] 10 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under SHH-addition conditions in "25." of Example 2. [Figure 50A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "26." of Example 2. [Figure 50B] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in "26." of Example 2. [Figure 51A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "27." of Example 2. [Figure 51B] 10 shows FACS plots of the controls FOXL2-tdTomato and NR5A1-EGFP in "27." of Example 2. [Figure 51C] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under control (CK-) and cytokine-added conditions in "27." of Example 2. [Figure 51D] 10 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under conditions of no BMP4 addition and with BMP4 addition in "27." of Example 2. [Figure 51E] 10 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under conditions of no addition of SHH and with addition of SHH in "27." of Example 2. [Figure 51F] 1 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP in the absence of PD0325901 and with the addition of PD0325901 in "27." of Example 2. [Figure 51G] 10 shows FACS plots of FOXL2-tdTomato and NR5A1-EGFP under conditions of no retinoic acid (RA) addition and RA addition in "27." of Example 2. [Figure 52A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "28." of Example 2. [Figure 52B] 10 is a FACS plot of FOXL2-tdTomato and NR5A1(SF1)-EGFP in "28." of Example 2. [Figure 52C] This is a diagram showing the results of RNAseq in "28." of Example 2. [Figure 53A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "29." of Example 2. [Figure 53B] 10 is a FACS plot of PE-TexasRed and NR5A1(SF1)-EGFP in "29." of Example 2. [Figure 54A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "30." of Example 2. [Figure 54B] 1 shows a FACS plot under conditions in which PD0325901 was not added in "30." of Example 2. [Figure 54C] 1 shows a FACS plot under the condition of adding PD0325901 in "30." of Example 2. [Figure 54D] 10 is a fluorescent image of the cell mass at "30." in Example 2. [Figure 55A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "31." of Example 2. [Figure 55B] 1 shows FACS plots of WT1-tdTomato and GATA4-mTagBFP2 in "31." of Example 2. [Figure 55C] 10 is a FACS plot of APC and NR5A1(SF1)-EGFP in "31." of Example 2. [Figure 56A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "32." of Example 2. [Figure 56B] 10 shows FACS plots of WT1-tdTomato and NR5A1(SF1)-EGFP in "32." of Example 2. [Figure 57A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "33." of Example 2. [Figure 57B] 10 shows FACS plots of WT1-tdTomato and NR5A1(SF1)-EGFP in "33." of Example 2. [Figure 57C] 1 is a graph showing the results of quantitative PCR measurement of each marker gene in "33." of Example 2. [Figure 58A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "34." of Example 2. [Figure 58B] 1 shows FACS plots from days 12 to 22 of induction in "34." of Example 2. [Figure 59A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells in "35." of Example 2. [Figure 59B] 10 is a FACS plot of FOXL2-tdTomato and NR5A1(SF1)-EGFP in "35." of Example 2. [Figure 60A] FIG. 1 shows a protocol for inducing differentiation into ovarian somatic cell-like cells using cynomolgus monkey ES cells established based on the results up to "35." in Example 2 (top row), and FACS plots on days 3 to 14 of induction (bottom row). [Figure 60B] FIG. 1 shows a diagram (upper panel) illustrating a protocol for inducing differentiation of mouse ES cells into ovarian somatic cell-like cells, and FACS plots (lower panel) on days 2 to 6 after induction. DETAILED DESCRIPTION OF THE INVENTION

[0014] <Method of producing ovarian somatic cell-like cells> The method for producing ovarian somatic cell-like cells of this embodiment (hereinafter sometimes simply referred to as "the production method of this embodiment") includes the following steps. Step 1: culturing primate pluripotent stem cells in a medium containing a GSK3 inhibitor and a ROCK inhibitor; Step 2: culturing the cells obtained after step 1 in a medium containing BMP4, retinoic acid, and a MEK inhibitor; and Step 3: culturing the cells obtained after step 2 in a basal medium to obtain ovarian somatic cell-like cells.

[0015] According to the production method of this embodiment, ovarian somatic cell-like cells at any developmental stage can be efficiently obtained from primate pluripotent stem cells. That is, the production method of this embodiment can also be said to be a method of inducing differentiation of primate pluripotent stem cells into ovarian somatic cell-like cells.

[0016] Each step constituting the manufacturing method of this embodiment will be described in detail below.

[0017] [Process 1] In step 1, primate pluripotent stem cells are cultured in a medium containing a GSK3 inhibitor and a ROCK inhibitor.

[0018] As used herein, "pluripotent stem cells" refer to undifferentiated cells that possess both the "self-renewal ability" of allowing proliferation while maintaining an undifferentiated state and the "pluripotency" of allowing differentiation into all three germ layer lineages. Examples of pluripotent stem cells include, but are not limited to, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic germ cells (EG cells) derived from primordial germ cells, multipotent germline STEM cells (mGS cells) isolated during the establishment and culture of GS cells from testicular tissue, and Muse cells isolated from bone marrow mesenchymal cells. The pluripotent stem cells listed above can be obtained by known methods.

[0019] The pluripotent stem cells used in step 1 are derived from primates. "Primates" refers to mammals belonging to the order Primates, and examples of primates include the suborder Prosimian, such as lemurs, lorises, and tree shrews, and the suborder Anthropoid, such as monkeys (including cynomolgus monkeys), apes, and humans. Of these, cynomolgus monkeys or humans are preferred.

[0020] As used herein, "iPS cells" refers to differentiated somatic cells that can be reprogrammed into cells of various tissues or organs by introducing several genes into them. In the method of this embodiment, the iPS cells used to induce differentiation of primordial germ cells may be derived from primary cultures of somatic cells collected from an appropriate donor, or from established cell lines. Since iPS cells can be induced to differentiate into any germ layer, somatic cells used to prepare iPS cells may, in principle, be derived from either ectodermal or endodermal germ layers. Cells from sources such as skin, hair, gums, and blood, which are easy to collect with minimal invasiveness, are suitable as somatic cells for use in preparing iPS cells. Methods known in the art can be used to prepare iPS cells. Specifically, preparation methods can be used that are described in known publications such as "Okita K. et al., "Generation of germline-competent induced pluripotent stem cells." Nature, Vol. 448, p313-317, 2007." (Reference 1) and "Hamanaka S. et al., "Generation of germline-competent rat induced pluripotent stem cells." PLoS One, Vol. 6, Issue 7, e22008, 2011." (Reference 2).

[0021] ES cells can be obtained by known methods. For example, they can be established by collecting an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on feeder cells derived from fibroblasts. ES cells can also be established by culturing early embryos produced by nuclear transfer of the nucleus of a somatic cell.

[0022] The medium used in step 1 contains a GSK3 inhibitor and a ROCK inhibitor as essential additives.

[0023] GSK3 inhibitors are compounds that have inhibitory activity against glycogen synthase 3 (GSK3). Examples of such compounds include CHIR99021 (CAS No.: 252917-06-9), SB216763 (CAS No.: 280744-09-4), SB415286 (CAS No.: 264218-23-7), CHIR98014 (CAS No.: 252935-94-7), AZD1080 (CAS No.: 612487-72-6), and LY2090314 (CAS No.: 603288-22-8), with CHIR99021 being preferred.

[0024] The concentration of the GSK3 inhibitor in the culture medium is preferably 5 μM or higher, more preferably 10 μM or higher, even more preferably 12 μM or higher, and particularly preferably 14 μM or higher. When the concentration of the GSK3 inhibitor is equal to or higher than the above-mentioned lower limit, WNT signaling can be activated, and differentiation into mesoderm can be more efficiently achieved. The upper limit of the concentration of the GSK3 inhibitor is not particularly limited, but can be, for example, 20 μM. The unit "μM" indicates a concentration that is 1 / 1,000,000 of the molecular weight (mol / L) in 1 liter of growth medium, and the same applies hereinafter.

[0025] ROCK inhibitors are compounds that have inhibitory activity against Rho-associated kinase (ROCK). Examples of such compounds include Y-27632 (CAS number: 146986-50-7), Fasudil (CAS number: 105628-07-7), Y39983 (CAS number: 203911-26-6), Wf-536 (CAS number: 539857-64-2), SLx-2119 (CAS number: 911417-87-3), azabenzimidazole-aminofurazans (CAS number: 850664-21-0), DE-104, H-1152P (CAS number: 872543-07-6), and Rho kinase α inhibitors (ROKα). inhibitor), XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostain, BA-210, BA-207, Ki-23095, VAS-012, etc. Among these, Y-27632 is preferred.

[0026] The concentration of the ROCK inhibitor in the medium can usually be 1 μM to 20 μM, preferably 5 μM to 15 μM, and more preferably 8 μM to 12 μM. When the concentration of the ROCK inhibitor is within the above range, cell death in step 1 can be more effectively suppressed.

[0027] When human pluripotent stem cells are used, it is preferable that the medium used in step 1 further contains BMP4 (bone morphogenetic protein 4), which promotes the expression of GATA4 and allows the cells to differentiate into mesoderm more efficiently. On the other hand, when using cynomolgus monkey pluripotent stem cells, it is preferable that the medium used in step 1 does not contain BMP4. This can further improve the efficiency of inducing NR5A1-positive cells.

[0028] When the medium used in step 1 contains BMP4, the concentration of BMP4 in the medium can be 0.1 ng / mL or higher, preferably 0.3 ng / mL or higher, more preferably 0.5 ng / mL or higher, and even more preferably 0.7 ng / mL or higher. On the other hand, the concentration of BMP4 in the medium can be 10 ng / mL or lower, preferably 5 ng / mL or lower, more preferably 3 ng / mL or lower, and even more preferably 1.3 ng / mL or lower. By maintaining the BMP4 concentration within the above range, GATA4 expression can be promoted, allowing for more efficient differentiation into mesoderm.

[0029] When cynomolgus monkey pluripotent stem cells are used, it is preferable that the medium used in step 1 further contains bFGF (basic fibroblast growth factor; also known as FGF2), which allows for more efficient differentiation into mesoderm. On the other hand, when human pluripotent stem cells are used, it is preferable that the medium used in step 1 does not contain bFGF, which can further improve the efficiency of inducing NR5A1-positive cells.

[0030] When the medium used in step 1 contains bFGF, the concentration of bFGF in the medium can be 0.1 ng / mL or higher, preferably 0.5 ng / mL or higher, more preferably 1 ng / mL or higher, and even more preferably 3 ng / mL or higher. On the other hand, the concentration of bFGF in the medium can be 10 ng / mL or lower, preferably 9 ng / mL or lower, more preferably 8 ng / mL or lower, and even more preferably 7 ng / mL or lower. By keeping the bFGF concentration within the above range, differentiation into mesoderm can be more efficiently achieved.

[0031] The medium used in step 1 may or may not contain EGF (epidermal growth factor).

[0032] Examples of the basal medium to which the above-mentioned additives are added in step 1 include, but are not limited to, αMEM medium, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Minimum Essential Medium (MEM), Eagle MEM, Dulbecco's Modified Eagle Medium (DMEM), Glasgow MEM (GMEM), Improved MEM Zinc Option, IMDM, Medium 199 medium, DMEM / F12 medium, StemPro-34SFM medium, Ham's medium, RPMI 1640 medium, HTF medium, Fischer's medium, Advanced DMEM, Advanced DMEM / F12, Advanced MEM, Advanced RPMI medium, and mixed media thereof. Among these, when pluripotent stem cells derived from cynomolgus monkeys are used, GMEM is preferred, and when pluripotent stem cells derived from humans are used, Advanced DMEM or Advanced DMEM / F12 is preferred, with Advanced DMEM / F12 being more preferred.

[0033] The medium may be a serum-containing medium or a serum-free medium. A serum-free medium is preferably used. Serum-free medium (SFM) refers to a medium that does not contain either untreated or unpurified serum, and includes a medium containing purified blood-derived components or animal tissue-derived components (growth factors, etc.). The concentration of serum (e.g., fetal bovine serum (FBS), human serum, etc.) can be 0 v / v% or more and 20 v / v% or less, preferably 0 v / v% or more and 5 v / v% or less, more preferably 0 v / v% or more and 2 v / v% or less, and even more preferably 0 v / v% (i.e., serum-free). SFM may contain any serum substitute. Examples of serum replacements include albumin (e.g., albumin substitutes such as lipid-rich albumin and recombinant albumin; plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, and equivalents thereof. Other examples include KnockOut (registered trademark) Serum Replacement (KSR), GlutaMax (registered trademark), and the like. These may be used alone or in combination of two or more types.

[0034] The medium may contain other known additives. The additives are not particularly limited, but examples include growth factors other than those mentioned above, polyamines, minerals, sugars (e.g., glucose, etc.), organic acids (e.g., pyruvic acid, lactic acid, etc.) and their salts (sodium salts, potassium salts, etc.), amino acids (e.g., non-essential amino acids (NEAA), L-glutamine, etc.), reducing agents (e.g., 2-mercaptoethanol, etc.), vitamins (e.g., ascorbic acid, d-biotin, etc.), steroids, antibiotics (e.g., streptomycin, penicillin, etc.), buffers (e.g., HEPES, etc.), and nutritional additives (e.g., B27 supplement, N2 supplement, StemPro-Nutrient Supplement, etc.). These can be used alone or in combination of two or more. Each additive can be contained within a known concentration range.

[0035] Examples of basal media that are preferably used for human pluripotent stem cells include Advanced DMEM or Advanced DMEM / F12 containing KSR (preferable concentration: 6.0 v / v% to 9.0 v / v%), 2-mercaptoethanol (preferable concentration: 0.01 mM to 0.5 mM), penicillin and streptomycin (preferable concentration: 50 U / mL to 150 U / mL), and GlutaMax (registered trademark) (preferable concentration: 0.5 mM to 5.0 mM).

[0036] Examples of basal media that are more preferably used for human pluripotent stem cells include Advanced DMEM / F12 containing KSR (preferable concentration: 7.0 v / v% to 8.0 v / v%), 2-mercaptoethanol (preferable concentration: 0.05 mM to 0.15 mM), penicillin and streptomycin (preferable concentration: 80 U / mL to 120 U / mL), and GlutaMax (registered trademark) (preferable concentration: 1.5 mM to 2.5 mM).

[0037] Examples of basal media that are preferably used for cynomolgus monkey pluripotent stem cells include GMEM containing KSR (preferable concentration: 7.0 v / v% to 8.0 v / v%), 2-mercaptoethanol (preferable concentration: 0.05 mM to 0.15 mM), NEAA (preferable concentration: 0.05 mM to 0.15 mM), pyruvate (preferable concentration: 0.5 mM to 1.5 mM), penicillin and streptomycin (preferable concentration: 80 U / mL to 120 U / mL), and L-glutamine (preferable concentration: 1.5 mM to 2.5 mM).

[0038] The culture in step 1 can be carried out, for example, by seeding primate pluripotent stem cells in a known cell non-adhesive or low-adhesive culture vessel and culturing them. The culture conditions are not limited to the following, but can be, for example, in an atmosphere of 1 v / v% to 10 v / v% carbon dioxide and 90 v / v% to 99 v / v% air. The culture temperature is about 30°C or higher and 40°C or lower, preferably about 37°C. The culture period is preferably 2 days or more, more preferably 3 days or more and 5 days or less.

[0039] When using human pluripotent stem cells, in order to promote more appropriate differentiation into ovarian somatic cells, the culture in step 1 is preferably carried out by seeding primate pluripotent stem cells in a known cell adhesive culture vessel and culturing them on a plate.

[0040] It is preferable to use a cell-adhesive culture vessel coated with a cell scaffold for the planar culture in step 1. As the cell scaffold, a known extracellular matrix (ECM) can be used, and specific examples include, but are not limited to, Matrigel (registered trademark) (manufactured by BD Biosciences), which contains fibronectin, vitronectin, laminin, entactin, and collagen IV.

[0041] When human pluripotent stem cells are used, step 1 preferably includes the following steps 1-1 and 1-2. Step 1-1: culturing primate pluripotent stem cells in a medium containing a GSK3 inhibitor and a ROCK inhibitor; Step 1-2: culturing the cells obtained after step 1-1 in a medium containing a GSK3 inhibitor and Activin A.

[0042] The GSK3 inhibitor and ROCK inhibitor used in steps 1-1 and 1-2 can be those exemplified above. In steps 1-1 and 1-2, the additives can be added to the basal medium exemplified above.

[0043] Activin A, used in step 1-2, is a member of the TGF-β family that is produced by many cell types during development. Addition of Activin A enables differentiation into ovarian somatic cells through posteriorization.

[0044] The concentration of Activin A in the medium can be 1 ng / mL to 30 ng / mL, preferably 3 ng / mL to 20 ng / mL, more preferably 5 ng / mL to 15 ng / mL, and even more preferably 8 ng / mL to 12 ng / mL. Having an Activin A concentration within this range enables differentiation into ovarian somatic cells by posteriorization.

[0045] The culture conditions for steps 1-1 and 1-2 are not limited to the following, but can be, for example, performed in an atmosphere of 1 v / v% to 10 v / v% carbon dioxide and 90 v / v% to 99 v / v% air. The culture temperature is about 30°C or higher and 40°C or lower, preferably about 37°C. The culture period may be any number of days such that the total culture period of steps 1-1 and 1-2 is the culture period of step 1 described above, and each of steps 1-1 and 1-2 is preferably 1 day or more, more preferably 2 days or more and 3 days or less.

[0046] Whether the cells obtained in step 1 have differentiated into mesoderm can be confirmed by analyzing the expression levels of marker genes such as OSR1 and TBXT using known methods such as immunostaining, FACS analysis, quantitative PCR, and RNAseq.

[0047] [Process 2] In step 2, the cells obtained after step 1 are cultured in a medium containing BMP4, retinoic acid, and a MEK inhibitor.

[0048] The BMP4 used in step 2 can be the same as that described in step 1 above.

[0049] In step 2, the concentration of BMP4 in the culture medium can be 0.1 ng / mL or higher, preferably 0.3 ng / mL or higher, more preferably 0.5 ng / mL or higher, and even more preferably 0.7 ng / mL or higher. On the other hand, the concentration of BMP4 in the culture medium can be 10 ng / mL or lower, preferably 5 ng / mL or lower, more preferably 3 ng / mL or lower, and even more preferably 1.3 ng / mL or lower. By maintaining the BMP4 concentration within the above range, GATA4 expression can be promoted, allowing for more efficient differentiation into intermediate mesoderm.

[0050] In step 2, the concentration of retinoic acid in the medium can be from 0.1 μM to 10 μM, preferably from 0.3 μM to 7 μM, more preferably from 0.5 μM to 5 μM, and even more preferably from 1 μM to 3 μM. When the concentration of retinoic acid is within the above range, differentiation into intermediate mesoderm can be more efficiently achieved.

[0051] The MEK inhibitor used in step 2 is a compound having inhibitory activity against mitogen-activated extracellular signal-related kinase. Examples of such compounds include, but are not limited to, PD0325901 (CAS No.: 391210-10-9), trametinib (trade name "Mekinist", CAS No.: 871700-17-3), selumetinib (CAS No.: 606143-52-6), MEK162 (CAS No.: 606143-89-9), and CH4987655 (CAS No.: 874101-00-5). Among these, PD0325901 is preferred.

[0052] The concentration of the MEK inhibitor in the medium is preferably 0.1 μM to 5 μM, more preferably 0.5 μM to 3 μM, and even more preferably 0.8 μM to 1.2 μM. When the concentration of the MEK inhibitor is within the above range, NR5A1-positive cells can be induced more efficiently.

[0053] When human pluripotent stem cells are used and plate culture is performed in step 1, it is preferable that the medium used in step 2 further contains a ROCK inhibitor. This makes it possible to more effectively suppress cell death when the cells obtained in step 1 are re-seeded as single cells and subjected to suspension culture in step 2. As the ROCK inhibitor, those exemplified in step 1 can be used.

[0054] When the medium used in step 2 contains a ROCK inhibitor, the concentration can usually be 1 μM to 20 μM, preferably 5 μM to 15 μM, and more preferably 8 μM to 12 μM. When the concentration of the ROCK inhibitor is within the above range, cell death in step 2 can be more effectively suppressed.

[0055] When human pluripotent stem cells are used, it is preferable that the medium used in step 2 further contains a hedgehog signal activator, which allows for more efficient induction of NR5A1-positive cells. On the other hand, when using cynomolgus monkey pluripotent stem cells, it is preferable that the medium used in step 2 does not contain a hedgehog signal activator, which allows more efficient differentiation into NR5A1-positive cells.

[0056] Examples of the hedgehog signal activator used in step 2 include, but are not limited to, sonic hedgehog (SHH), smoothened agonist (SAG; CAS number: 364590-63-6), etc. Among these, SHH is preferred.

[0057] When the medium used in step 2 contains a hedgehog signal activator, the concentration of the hedgehog signal activator in the medium can be 1 ng / mL or higher, preferably 5 ng / mL or higher, more preferably 15 ng / mL or higher, and even more preferably 25 ng / mL or higher. On the other hand, the concentration of the hedgehog signal activator in the medium can be 50 ng / mL or lower, preferably 45 ng / mL or lower, more preferably 40 ng / mL or lower, and even more preferably 35 ng / mL or lower. By keeping the concentration of the hedgehog signal activator within the above range, NR5A1-positive cells can be induced more efficiently.

[0058] The medium used in step 2 may or may not contain EGF (epidermal growth factor).

[0059] In step 2, the additives described above can be added to the basal medium. The basal medium can be the same as those exemplified in step 1. In addition, the other additives added to the medium can be the same as those exemplified in step 1.

[0060] The culture in step 1 can be carried out, for example, by seeding primate pluripotent stem cells in a known cell non-adhesive or low-adhesive culture vessel and culturing them. The culture conditions are not limited to the following, but can be, for example, in an atmosphere of 1 v / v% to 10 v / v% carbon dioxide and 90 v / v% to 99 v / v% air. The culture temperature is about 30°C or higher and 40°C or lower, preferably about 37°C. The culture period is preferably 2 days or more, more preferably 3 days or more.

[0061] Whether the cells obtained in step 2 have differentiated into intermediate mesoderm can be confirmed by analyzing the expression levels of marker genes such as WT1 and NR5A1 using known methods such as immunostaining, FACS analysis, quantitative PCR, and RNAseq.

[0062] [Process 3] In step 3, the cells obtained after step 2 are cultured in a basal medium to obtain ovarian somatic cell-like cells.

[0063] The basal medium used in step 3 may be the same as that exemplified in step 1.

[0064] When using human pluripotent stem cells, the basal medium preferably does not contain additives such as BMP4, FGF signal activators, and MEK inhibitors. The FGF signal activators referred to here include FGF family proteins, such as, but not limited to, FGF9, bFGF, and FGF4.

[0065] That is, when human pluripotent stem cells are used, the basal medium preferably used in step 3 includes Advanced DMEM or Advanced DMEM / F12 containing KSR (preferable concentration: 6.0 v / v% to 9.0 v / v%), 2-mercaptoethanol (preferable concentration: 0.01 mM to 0.5 mM), penicillin and streptomycin (preferable concentration: 50 U / mL to 150 U / mL), and GlutaMax (registered trademark) (preferable concentration: 0.5 mM to 5.0 mM).

[0066] When human pluripotent stem cells are used, examples of basal media that are more preferably used in step 3 include Advanced DMEM / F12 containing KSR (preferable concentration: 7.0 v / v% to 8.0 v / v%), 2-mercaptoethanol (preferable concentration: 0.05 mM to 0.15 mM), penicillin and streptomycin (preferable concentration: 80 U / mL to 120 U / mL), and GlutaMax (registered trademark) (preferable concentration: 1.5 mM to 2.5 mM).

[0067] The culture in step 3 can be carried out, for example, by seeding primate pluripotent stem cells in a known cell non-adhesive or low-adhesive culture vessel and culturing them. The culture conditions are not limited to the following, but can be, for example, in an atmosphere of 1 v / v% to 10 v / v% carbon dioxide and 90 v / v% to 99 v / v% air. The culture temperature is about 30°C or higher and 40°C or lower, preferably about 37°C. The culture period is preferably one week or longer.

[0068] On the other hand, when using cynomolgus monkey pluripotent stem cells, in order to further improve the efficiency of inducing NR5A1-positive cells, it is preferable to use a medium further containing a MEK inhibitor, preferably for at least one day, more preferably for about two days (e.g., 48 hours ± 12 hours, preferably 48 hours ± 6 hours) from the start of induction in step 3.

[0069] That is, when cynomolgus monkey pluripotent stem cells are used, step 3 preferably includes the following steps 3-1 and 3-2. Step 3-1: culturing the cells obtained after step 2 in a medium containing a MEK inhibitor; Step 3-2: culturing the cells obtained after step 3-1 in a basal medium to obtain ovarian somatic cell-like cells.

[0070] Examples of MEK inhibitors include those exemplified in the above step 2. The MEK inhibitor can be added to the basal medium exemplified in the above step 1.

[0071] In step 3-1, the concentration of the MEK inhibitor in the medium is preferably 0.1 μM to 5 μM, more preferably 0.5 μM to 3 μM, and even more preferably 0.8 μM to 1.2 μM. When the concentration of the MEK inhibitor is within the above range, the induction efficiency of NR5A1-positive cells can be further improved.

[0072] The medium used in step 3-1 is preferably a basal medium supplemented with only a MEK inhibitor and containing no other additives. That is, when using cynomolgus monkey pluripotent stem cells, a medium preferably used in step 3-1 includes, for example, GMEM containing a MEK inhibitor (preferably at a concentration of 0.8 μM to 1.2 μM), KSR (preferably at a concentration of 7.0 v / v% to 8.0 v / v%), 2-mercaptoethanol (preferably at a concentration of 0.05 mM to 0.15 mM), NEAA (preferably at a concentration of 0.05 mM to 0.15 mM), pyruvate (preferably at a concentration of 0.5 mM to 1.5 mM), penicillin and streptomycin (preferably at a concentration of 80 U / mL to 120 U / mL), and L-glutamine (preferably at a concentration of 1.5 mM to 2.5 mM).

[0073] Furthermore, the medium used in step 3-2 is preferably a basal medium that does not contain other additives. That is, when cynomolgus monkey pluripotent stem cells are used, examples of the basal medium that is preferably used in step 3-2 include GMEM containing KSR (preferably at a concentration of 7.0 to 8.0 v / v%), 2-mercaptoethanol (preferably at a concentration of 0.05 to 0.15 mM), NEAA (preferably at a concentration of 0.05 to 0.15 mM), pyruvate (preferably at a concentration of 0.5 to 1.5 mM), penicillin and streptomycin (preferably at a concentration of 80 to 120 U / mL), and L-glutamine (preferably at a concentration of 1.5 to 2.5 mM).

[0074] The culture conditions for steps 3-1 and 3-2 are not limited to the following, but can be, for example, performed in an atmosphere of 1 v / v% to 10 v / v% carbon dioxide and 90 v / v% to 99 v / v% air. The culture temperature is about 30°C or higher and 40°C or lower, preferably about 37°C. The culture period may be any number of days so long as the total culture period of steps 3-1 and 3-2 is the culture period of step 3. Step 3-1 is preferably one day or more, and preferably about two days (e.g., 48 hours ± 12 hours, preferably 48 hours ± 6 hours). The culture period of step 3-2 can be the remaining period of the culture period of step 3 minus the culture period of step 3-1.

[0075] Whether the cells obtained in step 3 have differentiated into ovarian somatic cell-like cells can be confirmed by analyzing the expression levels of marker genes such as FOXL2 and FDGFRα using known methods such as immunostaining, FACS analysis, quantitative PCR, and RNA sequencing.

[0076] The ovarian somatic cell-like cells obtained by the production method of this embodiment are preferably cells having properties equivalent to those of fetal ovarian somatic cells, i.e., fetal ovarian somatic cell-like cells, and have the property of differentiating into granulosa cells that form follicles in the future, interstitial cells, etc.

[0077] Furthermore, as shown in the examples described below, the ovarian somatic cell-like cells obtained by the manufacturing method of this embodiment using cynomolgus monkey pluripotent stem cells contain a high proportion of cells with the characteristics of granulosa cells, and therefore this method can also be said to be a method of inducing differentiation of cynomolgus monkey pluripotent stem cells into granulosa cells.

[0078] The ovarian somatic cell-like cells obtained by the production method of this embodiment can be aggregated and cultured with primate PGCs or PGCLCs induced from pluripotent stem cells to form spheroids that reproduce the developing ovary. By culturing these spheroids or transplanting them into a living body, it may be possible to reproduce germ cell maturation, such as the progression of attenuation division, and even follicle formation, which have not previously been reproducible in vitro. In other words, it may be possible to provide a method for ovarian regeneration, oocyte production, or infertility treatment, which includes aggregate co-culture of ovarian somatic cell-like cells obtained by the above-described production method with primate PGCs or PGCLCs. [Example]

[0079] EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0080] <Test to investigate differentiation induction from human iPS cells to ovarian somatic cell-like cells> [material] 1. Human iPS cell lines To easily observe the expression of representative genes at each developmental stage, we genetically engineered the human iPS cell line 1390G3 to generate DNA sequences that emit red fluorescence (tdTomato) with expression of the OSR1 gene, green fluorescence (EGFP) with expression of the NR5A1 gene, and red fluorescence (tdTomato) with expression of the FOXL2 gene (see Figure 1; after chromosomal introduction, the "PGKp-Neo(or Puro)-pA" portion was deleted using Cre). Two iPS cell lines were generated: one transfected with the OSR1 and NR5A1 DNA sequences (hereafter referred to as "OTNG"), and the other transfected with the NR5A1 and FOXL2 DNA sequences (hereafter referred to as "NGFT"). The OTNG line was used in most experiments in this study. Note that NR5A1 and SF1 are the same gene.

[0081] 2. Media and Drugs The supplier and model numbers of the culture medium and chemicals are shown in the table below.

[0082] [Table 1]

[0083] [Table 2]

[0084] [method] 1. Fluorescent immunostaining and observation by fluorescence microscope The antibodies used for immunostaining are as follows: Rat anti-EGFP antibody (Nacalai Tesque, 04404-84, dilution: 250) Rabbit anti-FOXL2 antibody (Abcam, ab246511, dilution: 200) Rabbit anti-laminin antibody (Abcam, ab11575, dilution: 200)

[0085] In addition, cells at each stage were observed using a fluorescence microscope (ZEISS confocal laser scanning, LSM780).

[0086] 2.FACS(Fluorescence activated cell sorting analysis) Cells at each stage were analyzed using a flow cytometer (BD FACSAria III).

[0087] 3. Quantitative PCR The relative expression levels of each marker gene were confirmed using the following primers and conditions using a PCR device (BIO-RAD CFX384 Real-Time System). In the table, the PPIA and RPLP0 genes were used as housekeeping genes. The qPCR difference is the difference from the average value calculated by "(PPIA + RPLP0) / 2". The ERCC gene was also used as an absolute control between samples.

[0088] [Table 3]

[0089] [Table 4]

[0090] [Table 5]

[0091] [Example 1] Figure 2 shows a protocol for inducing differentiation of mouse ES cells into ovarian somatic cell-like cells. In Figure 2, bFGF stands for basic fibroblast growth factor (FGF2), BMP4 stands for bone morphogenetic protein 4, CHIR stands for CHIR99021, a GSK3 inhibitor that activates the WNT pathway, EGF stands for epidermal growth factor, RA stands for retinoic acid, PD03 stands for PD0325901, a MEK inhibitor that suppresses the FGF pathway, SHH stands for sonic hedgehog, and FGF9 stands for fibroblast growth factor 9. These abbreviations will be used hereinafter.

[0092] In mice, embryonic stem cells (ESCs) are first differentiated into EpiLCs by culturing them on fibronectin-coated culture dishes for 42 hours (step 0).

[0093] The cells are then detached, the colonies broken up, and the cell count counted. These are then cultured on low-adhesion plates in batches of 30,000 cells each, where they adhere to each other in a floating state and form cell aggregates. During this process, BMP4, the small molecule CHIR99021 that activates WNT signaling, and EGF are used to differentiate the cells into mesoderm and intermediate mesoderm (step 1).

[0094] Next, the medium is changed to one containing the small molecule PD0325901, which suppresses BMP4, RA, and FGF signaling, SHH, and EGF, and over a period of two days, the cells are induced to develop from the gonadal coelomic epithelium into the genital ridge (step 2).

[0095] The medium is then replaced with one containing BMP4 and FGF9, and within 1 to 2 days, it is observed that the cells separate into the stroma and pregranulosa lineages (step 3).

[0096] Monkey ES cells and human iPS cells are thought to be slightly more differentiated than mouse ES cells and closer to EpiLCs. Therefore, in this study using monkey and human pluripotent stem cells, we decided to skip step 0 and start with step 1. Unlike mouse EpiLCs, human and monkey pluripotent stem cells undergo cell death when colonies are broken down to the single-cell level. The ROCK inhibitor Y27632 is known to be effective in preventing this. Therefore, we added Y27632 to step 1. We used 96-well V-bottom plates as culture plates and seeded the cells at 10,000 cells per well.

[0097] 1. Trial with the same duration as mice FIG. 3A shows a protocol for inducing differentiation of ovarian somatic cell-like cells using human iPS cells. For the human cell experiments, the basal medium was changed from GMEM (used for mouse experiments) to DMEM containing 7.5% v / v KnockOut® Serum Replacement (KSR). The basal medium also contained 0.1 mM 2-mercaptoethanol, 100 U / mL penicillin / streptomycin, and 2 mM GlutaMax®. However, these additives and concentrations are fixed and omitted from the experiments in "2." and subsequent sections. Steps 1 and 2 were performed for the same period as in the mouse protocol, and the culture was continued under the conditions of step 3. The medium was generally replaced in its entirety every two or three days. When subculturing iPS cells, 30,000 cells were seeded onto low-attachment plates and cultured as clumps.

[0098] Expression of the OSR1 fluorescent reporter was observed on day 4 (d4) at the end of step 2 (see Figure 3B). However, even after 34 days had passed since moving on to step 3, OSR1 continued to be expressed, but NR5A1 was not (see Figure 3B and Figure 3C).

[0099] 2. Optimizing cell number in step 1 If the initial number of cells is too large, the signal may not penetrate deep into the cells, so we attempted to reduce the number of cells per cell aggregate. A detailed protocol is shown in Figure 4A.

[0100] As a result, NR5A1 expression was not observed with this differentiation method, but OSR1 expression was uniformly elevated when the cell number was 10,000 or less, and it was revealed that at 30,000 cells, many cells expressed neither OSR1 nor NR5A1 (see Figure 4B). From now on, when using human iPS cells, we decided to start with 10,000 cells.

[0101] 3. Optimizing the period of step 1 Since it was predicted that a shorter period for step 1 would lead to differentiation into tissues closer to the head, and a longer period would lead to differentiation into tissues closer to the tail, we investigated the duration of step 1 by varying it between 2, 3, and 4 days (total durations for step 1 and step 2 were 4, 5, and 6 days). The detailed protocol is shown in Figure 5A.

[0102] As a result, when the period of step 1 was extended to 3 days, a small number of NR5A1-positive cells were observed on the 17th day, and when extended to 4 days, an even larger number of cells became NR5A1-positive (see Figures 5B and 5C).

[0103] FIG. 5D shows the results of examining the time course of OSR1 and NR5A1 expression when the period of step 1 was set to 4 days. As shown in Figure 5D, NR5A1 expression began on day 4 of step 3, and cells gradually shifted to NR5A1-positive cells. Eventually, OSR1-positive and NR5A1-negative cells decreased. Furthermore, OSR1 expression was slightly reduced in cells that were both OSR1 and NR5A1-positive.

[0104] 4. Optimization of the period of step 1 and the effect of combining other drugs in step 1 Next, we investigated what would happen if the duration of step 1 were further extended. In this experiment, we varied the duration of step 1 to 3, 4, 5, and 6 days (total durations of step 1 and step 2 were 5, 6, 7, and 8 days). We also evaluated the effect of Ly294002 (a phosphatidylinositol-3 kinase (PI3K) inhibitor, abbreviated as "Ly" in the figure), a small molecule that may suppress differentiation into cephalic tissues. The detailed protocol is shown in Figure 6A.

[0105] Evaluation on day 18 revealed that cells expressing both OSR1 and NR5A1 were obtained whether step 1 was performed for 3 or 6 days (Fig. 6B). However, FACS analysis revealed that the number of cells expressing both OSR1 and NR5A1 was highest when step 1 was performed for 4 days (C4R2F12) (Fig. 6C). Furthermore, the addition of Ly294002 did not increase the induction efficiency, and in fact, reduced the number of harvestable cells (Fig. 6C). Based on these results, we decided to set the protocol so that step 1 lasted for 4 days without adding Ly294002.

[0106] 5. Optimizing the period of step 2 Next, the duration of step 1 was fixed at 4 days, and the duration of step 2 was varied to 2, 3, and 4 days (total durations of step 1 and step 2 were 6, 7, and 8 days). The detailed protocol is shown in Figure 7A.

[0107] As a result, the percentage and actual number of NR5A1-positive cells obtained were higher when the duration of step 2 was 3 days (R3) than when it was 2 days (R2) (see Figures 7B and 7C). On the other hand, when the duration of step 2 was 4 days (R4), there was little change compared to when the duration of step 2 was 3 days (see Figure 7C). Based on these results, the duration of step 2 was set to three days.

[0108] 6. Combination effect of other drugs in step 1 After extending step 2 to 3 days, we again varied the concentration of CHIR99021 in step 1 and evaluated whether the efficiency could be increased by adding bFGF. The detailed protocol is shown in Figure 8A.

[0109] As a result, it was found that the percentage of NR5A1-positive cells was higher when the concentration of CHIR99021 was 15 μM than when it was 10 μM, and that the efficiency of differentiation induction was better in the absence of bFGF (see FIGS. 8B and 8C).

[0110] 7. Optimization of RA concentration in step 2 The test was conducted under the same conditions as above, except that the duration of step 1 was 4 days, the duration of step 2 was 3 days, the RA concentration in step 2 was 0.3, 1, 3, and 10 μM, and the PD0325901 concentration was 1 and 0 μM (see Figure 9A).

[0111] Evaluation on day 15 of induction revealed that no NR5A1-positive cells were observed without PD0325901 (see Figures 9A-9D). Regarding RA concentrations, while both 0.3 μM and 10 μM induced high levels of NR5A1-positive cells, concentrations of 1 μM to 3 μM, similar to the conditions in mice, were particularly favorable for both the percentage and number of NR5A1-positive cells (see Figures 9A-9D).

[0112] 8. Optimization of KSR and PD0325901 concentrations in step 2 Next, we compared the concentrations of KSR and PD0325901 by adjusting them in detail. KSR was added at 7, 8, 9, and 10% (v / v), and PD0325901 was added at 0.4, 0.6, 0.8, and 1.0 μM. The cells were cultured and evaluated on day 15. The detailed protocol is shown in Figure 10A.

[0113] When KSR was concentrated or PD0325901 was diluted, NR5A1-positive cells did not appear (see Figure 10B). Similar results were obtained by FACS analysis (see Figure 10C).

[0114] The most efficient differentiation induction was achieved when the KSR concentration was 7 v / v% and the PD0325901 concentration was 1.0 μM. However, we next attempted to change the basal medium, aiming to obtain more than 1,000 cells, starting from 10,000 cells.

[0115] 9. Basal medium considerations 1 Five types of media were used: DMEM, Advanced DMEM, Advanced DMEM / F12, Advanced MEM, and Advanced RPMI, with KSR concentrations of 5.0 and 7.5% (v / v). The culture periods were four days for step 1 and three days for step 2, with evaluation on day 23 of induction. This study used a different iPS cell line than previous studies. Specifically, a fluorescent reporter cell line (NGFT line) carrying FOXL2-tdTomato and NR5A1-EGFP was used instead of the fluorescent reporter cell line (OTNG line) carrying OSR1-tdTomato and NR5A1-EGFP.

[0116] As a result, it was found that a KSR concentration of 7.5% (v / v) was superior in differentiation induction efficiency, and that Advanced DMEM and Advanced DMEM / F12 were particularly superior in differentiation induction efficiency (see Figures 11A to 11D). Note that in Figure 11A, there is no photograph of DMEM with a KSR concentration of 5% (v / v), as the cells died.

[0117] 10. Basal medium considerations 2 Five media types (DMEM, Advanced DMEM, Advanced DMEM / F12, Advanced MEM, and Advanced RPMI) were examined under the same conditions as in Section 9 above, except that the KSR concentration was fixed at 7.5% (v / v) and the OTNG strain was used. Evaluations were performed on days 15 and 23 of induction.

[0118] As a result, Advanced DMEM, Advanced DMEM / F12, and Advanced RPMI had better differentiation induction efficiency than DMEM (see Figures 12A to 12D). On the other hand, it was found that although the number of cells increased in Advanced MEM, they were less likely to become NR5A1 positive (see Figures 12A to 12D). In this test, Advanced DMEM had better differentiation induction efficiency than Advanced DMEM / F12, but subsequent studies showed that Advanced DMEM / F12 had similar differentiation induction efficiency (not shown).

[0119] 11. Basal medium study 3 Five media types were examined: DMEM, Advanced DMEM, Advanced DMEM / F12, Advanced MEM, and Advanced RPMI, under the same culture conditions as in "10.", except that SHH was added during the first six days of step 3 (i.e., from day 7 to day 13 of induction). Evaluations were performed on days 15 and 23 of induction.

[0120] As a result, almost no NR5A1-positive cells were observed in Advanced RPMI (see FIGS. 13A to 13D). On the other hand, the number of NR5A1-positive cells obtained was greater in Advanced DMEM and Advanced DMEM / F12 than in DMEM.

[0121] These results suggest that the addition of SHH throughout the culture period from step 2 to step 3 tends to result in better differentiation induction efficiency.

[0122] 12. Optimization of SHH concentration in step 2 Next, we investigated the optimization of the SHH concentration in step 2 and reexamined the effect of Shh administration at the early stage of step 3. The SHH concentration in step 2 was varied between 0, 30, 50, and 100 ng / mL, and the SHH concentration in step 3 was varied between 0 and 30 ng / mL. The SHH addition period in step 3 was fixed at 3 days, and the concentrations of other drugs were unchanged. The basal medium used was DMEM containing 7.5% v / v KSR. The detailed protocol is shown in Figure 14A.

[0123] As a result, the appearance of NR5A1-positive cells was observed even without adding SHH in step 2, and increasing the SHH concentration did not significantly change the cell number or differentiation induction efficiency. On the other hand, adding SHH in step 3 tended to increase the induction efficiency, but did not significantly increase the cell number.

[0124] These results suggest that although SHH is not essential for the induction of gonadal somatic cells, its addition at the beginning of step 3 rather than step 2 tended to increase the number of NR5A1-positive cells obtained.

[0125] 13. Examination of marker genes As a result of the investigation up to "12." above, differentiation induction was performed using the protocol shown in Figure 15A. However, during culture in step 3, the size of the embryoid bodies decreased over time, and many dead cells were observed. Furthermore, qPCR analysis of NR5A1 (SF1)-positive cells on days 15, 22, and 29 of induction revealed expression of the ACTH receptor MC2R, a marker gene for adrenal cells, an anterior organ, and high expression of GATA3, a marker gene for the cephalic (anterior) organ (see Figure 15B). This indicates a high possibility that the cells possess adrenal properties. However, expression of FOXL2, a marker gene for granulosa cells, was not observed (see Figure 15B).

[0126] Based on these results, we believe that the marker gene for the evaluation of the caudal (posterior) position will be in line with previous reports (Reference 3: Dolle P et al., "Two gene members of the murine HOX-5 complex show regional and cell-type specific expression in developing limbs and gonads," The EMBO Journal, Vol. 8, No. 5, pp. 1507-1515, 1989; Reference 4: Zubair M et al., "Two-step regulation of Ad4BP / SF-1 gene transcription during fetal adrenal development: initiation by a Hox-Pbx1-Prep1 complex and maintenance via autoregulation by Ad4BP / SF-1," Mol Cell Biol., Vol. 26, No. 11, pp. 4111-4121, Based on this study (2006), we selected HOXD9 and HOXD10 as marker genes for ovarian somatic cells, and HOXA7 and HOXB9 as marker genes for adrenal cells, and decided to evaluate them subsequently.

[0127] 14. Step 1: Examination of the presence or absence of bFGF and Step 2: Optimization of RA concentration Evaluation was performed on day 15 of induction under the same conditions as the protocol shown in Figure 15A, except that the bFGF concentration in step 1 was changed to 0 and 5 ng / mL, and the RA concentration in step 2 was changed to 0.1 (100 nM), 0.5 (500 nM), and 1 μM.

[0128] As a result, the expression of marker genes for posteriorization was not observed even when bFGF was added in step 1 or when the RA concentration was reduced in step 2 (see Figures 16A and 16B). Furthermore, the size of the embryoid bodies also decreased over time, indicating that these culture conditions are likely not appropriate for promoting posteriorization.

[0129] 15. Optimizing the period of step 13 Because a shorter period for step 1 was expected to lead to differentiation into tissues closer to the head, and a longer period for step 1 was expected to lead to differentiation into tissues closer to the tail, the period for step 1 was set to 4 days and 7 days (total periods for step 1 and step 2 were set to 7 days and 10 days), respectively. Except for this, the test was performed under the same conditions as the protocol shown in Figure 15A. The OTNG cell line was used.

[0130] As a result, expression of HOXB9, which is thought to be expressed in the adrenal gland but not in the ovarian corpus, was observed (see FIG. 17). Furthermore, even when the duration of step 1 was extended or when the use of other drugs was considered as in "14.", HOXB9 expression did not become negative.

[0131] 16. Optimization of the culture method in step 1 In step 1, we hypothesized that the early generation of embryoid bodies would result in uneven stimulation of cytokine signals. To address this issue, we attempted to use plate culture instead of suspension culture on V-bottom plates to more efficiently and uniformly deliver cytokines for posteriorization to the cell population. We also investigated the use of plate coating agents and other agents in combination. A detailed protocol is shown in Figure 18A. The OTNG cell line was used.

[0132] As a representative example, the culture in step 1 was performed by flat culture using a fibronectin-coated plate, and Activin A (10 ng / mL) was added between days 2 and 4 of induction (conditions marked with an asterisk in Figure 18A). Figure 18B shows the time course of changes in embryoid bodies (observed images on days 3, 9, 11, 13, 17, and 19 of induction).

[0133] As shown in Figure 18B, in the sample cultured in step 1 using fibronectin-coated plates and supplemented with Activin A (10 ng / mL) between days 2 and 4 of induction, the size of the embryoid bodies increased over time. Furthermore, in the sample cultured in vitronectin using plates and supplemented with Activin A (10 ng / mL) between days 2 and 4 of induction, the size of the embryoid bodies increased over time, similar to the sample cultured with fibronectin (not shown). On the other hand, in the samples cultured without other agents, with the addition of bFGF, or with the addition of A830-01 between days 2 and 4 of induction, the embryoid bodies were in poor condition (died).

[0134] In addition, on day 15 of induction, the expression levels of marker genes were measured by FACS analysis and quantitative PCR. The results are shown in Figure 18C (FACS analysis) and Figure 18D (quantitative PCR). In Figures 18C and 18D, "conventional" refers to the culture conditions in the protocol shown in Figure 15A. "Fibronectin plate w / o drug" refers to the culture conditions in which step 1 of the culture was performed by flat culture on a fibronectin-coated plate, with no other drugs added between days 2 and 4 of induction. "Fibronectin plate with Activin A" refers to the culture conditions in which step 1 of the culture was performed by flat culture on a fibronectin-coated plate, with Activin A (10 ng / mL) added between days 2 and 4 of induction. In Figure 18D, "P7" refers to OSR1- (negative) and SF1+ (positive) cells, and "P11" refers to OSR1+ (positive) and SF1+ (positive) cells.

[0135] As shown in Figure 18C, when step 1 was performed as a plate culture and no other agents were added between days 2 and 4 of induction, the percentages of OSR1+ (positive) and SF1+ (positive) cells increased. Furthermore, when Activin A (10 ng / mL) was added between days 2 and 4 of induction, the percentages of OSR1- (negative) and SF1+ (positive) cells further increased.

[0136] As shown in Figure 18D, when step 1 was performed using plate culture on fibronectin-coated plates and Activin A (10 ng / mL) was added between days 2 and 4 of induction, GATA3 and HOXB9 expression decreased. Furthermore, when step 1 was performed using plate culture on vitronectin-coated plates and Activin A (10 ng / mL) was added between days 2 and 4 of induction, quantitative PCR results were similar to those of the above samples using fibronectin (not shown).

[0137] The culture in step 1 was performed on a fibronectin-coated plate by flat culture, and Activin A (10 ng / mL) was added between days 2 and 4 of induction. Immunostaining was performed on the 22nd day of induction using an anti-FOXL2 antibody. Immunostaining images are shown in Figure 18E (magnification: 20x) and Figure 18F (magnification: 63x).

[0138] As shown in Figures 18E and 18F, the central part of the embryoid body was filled with dead cells, as evidenced by the absence of DAPI staining. The outermost layer was positive for SF1 (NR5A1), with FOXL2 expression from the innermost layer.

[0139] Furthermore, the culture in step 1 was performed on fibronectin-coated plates using flat culture. Activin A (10 ng / mL) was added between days 2 and 4 of induction. For samples cultured under these conditions, FACS analysis and quantitative PCR were performed on day 27 of induction. The results are shown in Figure 18G (FACS analysis) and Figure 18H (quantitative PCR). In Figure 18G and Figure 18H, "P7" refers to OSR1- (negative) and SF1+ (positive) cells, and "P10" refers to OSR1+ (positive) and SF1+ (weakly positive) cells. "P7" and "P10" have the same meanings in the following figures. In Figure 18H, ovarian somatic cells collected from a living subject were also examined for marker gene expression as a control.

[0140] As shown in Figures 18G and 18H, in step 1, the culture was performed using fibronectin-coated plates as flat culture, and Activin A (10 ng / mL) was added between days 2 and 4 of induction. FOXL2 expression was observed at levels roughly equivalent to that observed in ovarian somatic cells collected from living organisms. At "P7" compared to "P10," OSR1- (negative) and SF1+ (positive) cells showed lower FOXL2 expression. These cells form the outermost layer in the immunostained images of Figures 18E and 18F, suggesting that there are many FOXL2-negative cells.

[0141] 17.Reproducibility confirmation test The reproducibility test was performed twice under the culture conditions described in "16." The OTNG cell line was used. On day 21 or 22 after induction, the expression levels of marker genes were measured by quantitative PCR. The results are shown in Figure 19A.

[0142] As shown in Figure 19A, FOXL2 expression was confirmed in both of the two reproducibility tests, and a decrease in HOXB9 expression was considered to be an indicator of posteriorization.

[0143] In addition, in a reproducibility test, FACS analysis was performed on days 4, 10, 16, and 21 after induction. The results are shown in Figure 19B.

[0144] As shown in Figure 19B, on day 4 of induction, there were many OSR1+ (positive) and SF1(NR5A1)- (negative) cells. On day 10 of induction, the number of OSR1+ (positive) cells decreased, and the number of SF1(NR5A1)+ (positive) cells began to increase. On day 16 of induction, the number of OSR1- (negative) and SF1(NR5A1)+ (positive) cells increased. On day 21 of induction, the number of OSR1- (negative) or OSR1+ (positive) and SF1(NR5A1)+ (positive) cells increased.

[0145] 18. Optimization of the culture method in step 1, 2, and the combined effect of other drugs in step 3 We investigated the presence or absence of Activin A in step 1, the culture method (flat culture on fibronectin-coated plates or suspension culture on V-bottom plates), and the presence or absence of BMP4 and FGF9 in step 3. The specific protocol is shown in Figure 20A. The OTNG cell line was used. Images of embryoid bodies under each culture condition on day 21 of induction are shown in Figure 20B.

[0146] As shown in Figure 20B, in step 1, the culture was performed using a fibronectin-coated plate as a flat culture, Activin A (10 ng / mL) was added between days 2 and 4 of induction, and in step 3, BMP4 and FGF9 were not added. In the cultured samples, embryoid bodies grew large.

[0147] In step 1, the culture was performed in suspension culture using a V-bottom plate. Activin A (10 ng / mL) was added from day 2 to day 4 of induction, and in step 3, BMP4 and FGF9 were not added. Since large embryoid bodies were also obtained in the cultured samples, immunostaining was performed on day 22 of induction. However, expression of FOXL2, SF1 (NR5A1-EGFP), and OSR1 (OSR-tdTomato) was not observed (data not shown).

[0148] On the other hand, in the sample cultured in step 1 using fibronectin-coated plates, with Activin A (10 ng / mL) added between days 2 and 4 of induction, and without BMP4 or FGF9 in step 3, embryoid bodies with ovarian somatic cell-like structures (soma-like), mushroom-like structures (kinoko-like), and black dot structures (black) were observed on day 17 of induction (see Figure 20C). Embryoid bodies with each structure were analyzed by FACS on day 22 of induction. The results are shown in Figure 20D.

[0149] As shown in FIG. 20D, the FACS plots showed the same tendency for Soma-like and Kinoko-like embryoid bodies.

[0150] The results of immunostaining on day 17 of Kinoko-like embryoid body induction are shown in FIG. 20E.

[0151] As shown in Figure 20E, the outermost layer was positive for SF1 (NR5A1), and FOXL2 was expressed from the inner layer, similar to the immunostained images of the embryoid bodies described in "16." (See Figures 18E and 18F).

[0152] Furthermore, the expression levels of marker genes were measured by quantitative PCR on day 22 of induction, and the results are shown in Figure 20F.

[0153] As shown in Figure 20F, FOXL2 expression was barely observed in the samples cultured in suspension on V-bottom plates in step 1. GATA3 expression was low, but HOXB9 expression was high, indicating that the cells were not differentiated into ovarian somatic cells.

[0154] The FOXL2 expression patterns in kinoko-like embryoid bodies at P7 and P10 were almost identical to those in soma-like embryoid bodies at P7 and P10, respectively, suggesting that soma-like and kinoko-like embryoid bodies possess the properties of ovarian somatic cells. On the other hand, FOXL2 expression was also observed in Black embryoid bodies, but the expression pattern of HOXB9 was different from that of Soma-like and Kinoko-like embryoid bodies.

[0155] Based on the above results, the following test was carried out based on the protocol shown in Figure 20G.

[0156] 19. Confirmation of PDGFRα expression Ovarian somatic cells consist of granulosa cells and stromal cells, and FOXL2 is thought to be expressed in granulosa cells, while PDGFRα is expressed in stromal cells. Therefore, changes in PDGFRα expression were confirmed by FACS in cells obtained using the protocol shown in Figure 20G (the OTNG cell line was used) (Figure 21 and Table 6). In Figure 21 and Table 6, "P11" indicates PDGFRα+ (positive) cells. The anti-PDGFRα antibody used in the FACS analysis was APC anti-human CD140a (PDGFRα) antibody (No. 323512) manufactured by Biolgend. The protocol consisted of single cell isolation, followed by incubation with anti-PDGFRα antibody (incubation on ice at 4°C for 30 minutes), followed by two washes.

[0157] [Table 6]

[0158] As shown in FIG. 21, an increase in the proportion of cells expressing PDGFRα was observed around 30 days after induction.

[0159] 20. Examination of long-term culture in step 3 Before examining whether mature oocytes could be obtained by co-culturing the ovarian somatic cell-like cells obtained by the protocol shown in Figure 20G with PGCLCs, we investigated whether the ovarian somatic cell-like cells could maintain the properties of ovarian somatic cells during long-term culture, since co-aggregation culture requires a long period of time. Specifically, long-term culture was performed while maintaining the culture conditions of step 3 of the protocol shown in Figure 20G. The OTNG cell line was used.

[0160] Observation images of embryoid bodies on days 22 and 37 of induction (left) and the results of FACS analysis (right) are shown in Figure 22A.

[0161] As shown in Figure 22A, embryoid bodies on day 37 of induction were larger than those on day 22 of induction, and there was a tendency for SF1- (negative) cells to increase among OSR1+ (positive) cells.

[0162] Immunostaining was also performed on days 37, 57, and 86 after induction. The results are shown in Figure 22B (day 37), Figure 22C (day 57), and Figures 22D to 22F (day 86).

[0163] On day 37 of induction, FOXL2 expression was well maintained (see Figure 22B). On day 57 of induction, a population of OSR1+ (positive) and FOXL2+ (positive) cells formed in the center, and FOXL2+ (positive) cells were also enriched in the center (see Figure 22C). On day 86 of induction, the distribution of SF1+ (positive) and FOXL2+ (positive) cells was consistent (see Figure 22D), and laminin was present surrounding the SF1+ (positive) cells (see Figure 22E). Granulosa cells formed islands surrounded by laminin, confirming that the structure resembled ovarian cords in vivo at day 86 of induction.

[0164] 21. Confirmation of SF1 (NR5A1) expression and cell status in step 3 Because embryoid bodies are not successfully formed when the cell number is low, there is a possibility that the number of viable cells is significantly low in step 2. Therefore, we checked the cell status. Specifically, we used the OTNG cell line and cultured them according to the protocol shown in Figure 20G. FACS analysis was performed on days 7, 8, 9, 10, 11, 12, 14, and 15 of induction. In the FACS analysis, dead cells were removed using DAPI, and OSR1 and SF1 were plotted (see Figures 23A and 23B).

[0165] As shown in Figures 23A and 23B, the population of live cells at P1 tended to increase from day 7 to day 15 of induction. Furthermore, the number of DAPI-positive cells also tended to gradually decrease. From day 12 of induction, an increase in SF1-positive cells was observed.

[0166] <Test to investigate differentiation induction from cynomolgus monkey ES cells to ovarian somatic cell-like cells> [material] 1. Cynomolgus monkey ES cell line To maintain the quality of the female cynomolgus monkey ES15XRi cell line and its knock-in reporter line, only colonies with good morphology were manually picked and subcultured. Alternatively, high-quality ES cells were enriched and subcultured using the pluripotency markers SSEA-4 and TRA 1-60 by flow cytometry. Other subculture methods were performed as previously reported (Reference 5: Sakai Y et al., "Induction of the germ cell fate from pluripotent stem cells in cynomolgus monkeys.", Biology of Reproduction, Vol. 102, Issue 3, pp. 620-638, 2020). The number of cells seeded during subculture was adjusted appropriately.

[0167] To easily monitor the expression of representative genes at each developmental stage, we used Cas9 nickase and gene targeting to delete the stop codons of TBXT, OSR1, NR5A1, FOXL2, WT1, and GATA4 in the female cynomolgus monkey ES15XRi cell line. We then inserted 2A and EGFP (TBXT, NR5A1), tdTomato (OSR1, FOXL2, WT1), and 2A and mTagBFP2 (GATA4), followed by a loxP-flanked drug resistance cassette (see Figures 24A-F; in Figures 24A-F, "PGK" indicates the PGK promoter). The drug resistance cassette was then removed using Cre to generate the following five double reporter cell lines: (1) TBXT-EGFP / OSR1-tdTomato (hereinafter sometimes abbreviated as "TGOT strain"); (2) NR5A1-EGFP / FOXL2-tdTomato (hereafter sometimes abbreviated as “NGFT strain”); (3) NR5A1-EGFP single reporter strain (hereinafter referred to as the "NG-Puro strain." Note that this strain has not undergone Cre-mediated removal of the drug resistance cassette); (4) WT1-tdTomato / GATA4-mTagBFP2 / NR5A1-EGFP triple reporter strain (hereafter sometimes abbreviated as "WTGBNG strain"); (5) WT1-tdTomato / NR5A1-EGFP (hereinafter sometimes abbreviated as "WTNG strain").

[0168] 2. Media and Drugs The media, drugs, etc. used were those listed in Tables 1 and 2 above.

[0169] [method] 1. Fluorescent immunostaining and observation by fluorescence microscope The antibodies used for immunostaining are as follows: (Primary antibodies: dilution ratio 250x) Rabbit anti-FOXL2 antibody (ab246511) from Abcam Goat anti-tdTomato antibody (AB8181-200) from SICGEN Rat anti-GFP antibody (04404-84) manufactured by Nacalai Tesque (Secondary antibodies: dilution ratio 500x) ThermoFisher Donkey anti-rat Alexa Fluor 488 (A21208) Donkey anti-goat Alexa Fluor 568 (A11057) manufactured by ThermoFisher ThermoFisher Donkey anti-rabbit Alexa Fluor 647 plus (A32795)

[0170] In addition, cells at each stage were observed using a fluorescence microscope (ZEISS confocal laser scanning, LSM780).

[0171] 2.FACS(Fluorescence activated cell sorting analysis) Cells at each stage were analyzed using a flow cytometer (BD FACSAria III).

[0172] 3. Quantitative PCR The relative expression levels of each marker gene were determined using the following primers and conditions using a PCR system (BIO-RAD CFX384 Real-Time System). The primers for PAX3, OSR1, PAX2 #3, EMX2 #1, WT1 #2, and LHX9 #2 in the table were used only in the experiment shown in Figure 31C. The primers for OSR1 #10, CXCL12 x4, BMP2, and BMP4 were used only in the experiment shown in Figure 57C. The GAPDH and PPIA genes were measured as housekeeping genes, and the relative values ​​are plotted relative to the control value, which was the average of the two Ct values. Although not shown in the graph, ERCC-00096, ERCC-00009, ERCC-00022, and ERCC-00025 were also used as controls in all experiments except for the experiment shown in Figure 31C.

[0173] [Table 7]

[0174] [Table 8]

[0175] [Table 9]

[0176] [Table 10]

[0177] [Table 11]

[0178] 4.RNA-seq The base sequences of all transcripts were determined using a next-generation sequencer (NGS) (Illumina, NextSeq 550), and then the expression levels of specific genes were calculated.

[0179] [Example 2] 1. Examination of ROCK inhibitors in step 1 For induction, ES cells were cultured on mouse embryonic fibroblasts (MEFs) as feeder cells, then treated with a proteolytic enzyme solution (CTK: collagenase, trypsin, and KSR) and dissociated to single cells using TrypLEselect. Next, 30,000 cells were seeded into low-attachment 96-well U-bottom culture plates (Step 1 was initiated in the same manner unless otherwise noted). The TGOT No. 25 cell line was used. The presence or absence of a ROCK inhibitor (Y-27632) was examined in Step 1. A detailed protocol is shown in Figure 25A. In Figure 25A, the composition of the basal medium GK7.5 is GMEM, 7.5% (v / v) KSR, 0.1 mM 2-ME, 0.1 mM non-essential amino acids (NEAA), 1 mM sodium pyruvate, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine. Step 1 was performed for 2 days, and step 2 for 2 days. PD173074 was used in step 2 instead of PD0325901, which is used in mice. PD173074 is an FGF inhibitor, while PD0325901 is a MEK inhibitor, which is downstream of the FGF signaling pathway.

[0180] As with mice, induction was performed without Y-27632 in step 1, but the efficiency of cell clump (embryoid body) formation was significantly low even on day 2 of induction (see the left photograph in Figure 25B). Therefore, subsequent experiments were performed with the addition of Y-27632 (see the right photograph in Figure 25B). Unless otherwise noted, Y-27632 was removed on day 1.

[0181] As shown in the fluorescence photograph on the right side of Figure 25B, EGFP was expressed in step 1 (d1 and d2), tdTomato was expressed in step 2 (d3 and d4), and it was confirmed that TBXT and OSR1 were expressed.

[0182] FIG. 25C shows the results of FACS analysis, which clearly show that these genes are expressed, even when compared with the wild type analyzed as a control.

[0183] 2. Optimization of CHIR99021 and BMP4 concentrations in step 1 Next, the period in step 1 was fixed at 2 days, and the concentrations of CHIR99021 (hereafter abbreviated as "CHIR") and BMP4 required for TBXT and OSR1 expression were examined (see Figure 26A). CHIR concentrations were varied at 0, 1, 3, 8, 14, and 20 μM, and BMP4 concentrations were varied at 0, 1, 3, and 10 ng / mL, and evaluation was performed by FACS analysis on day 2 of induction. The TGOT No. 25 cell line was used.

[0184] As a result, it was considered that TBXT could be expressed with high efficiency when the CHIR99021 concentration was 8 μM or higher (see Figure 26B).

[0185] 3. Optimization of CHIR99021 and BMP4 concentrations in step 1 2 Based on the results of "2," the CHIR99021 concentration was varied to 3, 8, and 14 μM, and the BMP4 concentration to 1, 3, and 10 ng / mL. Step 1 was performed for 2 days, and step 2 for 4 days (see Figure 27A). In subsequent experiments, PD0325901 was used in step 2, as in mice. The TGOT No. 25 cell line was used.

[0186] From the results of "2," it was thought that TBXT could be expressed efficiently when the CHIR99021 concentration was 8 μM or higher, but when moving on to step 2, it was thought that OSR1 could be expressed efficiently when the CHIR99021 concentration was 14 μM or higher (see Figures 27B and 27C). Furthermore, it was confirmed that OSR1 expression was enhanced when the CHIR99021 concentration was 14 μM or higher and the amount of BMP was increased (see Figures 27B and 27C).

[0187] 4. Optimization of CHIR99021 and BMP4 concentrations in step 13

[0188] Based on the results of "3," in order to investigate the optimal lower limit of CHIR for OSR1 expression, the CHIR concentration was fixed at 10 μM, and the BMP4 concentration was varied between 1, 3, and 10 ng / mL. Step 1 was performed for 2 days and step 2 for 4 days (see Figure 28A). As a positive control, a BMP4 concentration of 1 ng / mL and a CHIR concentration of 14 μM were also used. FACS analysis was performed on days 2, 4, and 6 of induction. The TGOT No. 25 cell line was used.

[0189] Regardless of the BMP4 concentration, OSR1 expression was confirmed at a CHIR concentration of 10 μM (see Figure 28B). Furthermore, an increase in OSR1 expression due to the amount of BMP4 added was observed even at a CHIR concentration of 10 μM (see Figure 28B). From the above, it was considered that the optimal concentration of CHIR for intermediate mesoderm induction was 10 μM or higher.

[0190] 5. Study of bFGF and Activin A addition in step 1 1 Previous studies have reported that the addition of bFGF or Activin A induces intermediate mesoderm, and we investigated the effect of these factors in step 1. Experiments were performed under the following conditions: bFGF was added at 50 ng / mL throughout step 1; Activin A was added at 10 ng / mL for only one day from day 1 of induction; or no Activin A was added. The TGOT No. 25 cell line was used. The results of FACS analysis on days 2, 4, and 6 of induction are shown in Figure 29B.

[0191] As shown in Figure 29B, the addition of bFGF and Activin A both acted to enhance OSR1 expression, and was considered to be useful for inducing intermediate mesoderm.

[0192] 6. Examination of the addition of BMS493 and Y-27632 in step 1 RA provides information for embryo anteriorization and regulates the anterior-posterior direction of the embryo. Therefore, we investigated whether the use of 1 μM of BMS493, an RA inhibitor, in step 1, which involves posteriorization of the embryo, would have any effect (see Figure 30A). We also examined the addition of Y-27632 for one day only or for two days. The TGOT No. 25 cell line was used. CHIR from two manufacturers (Tocris and BioVision) was also tested. The results of FACS analysis on days 2, 4, and 6 of induction are shown in Figures 30B and 30C.

[0193] As shown in Figures 30B and 30C, there was no significant difference in marker gene expression between CHIR manufacturers, so we decided to continue using Tocris products. Regarding BMP493, increased OSR1 expression was observed in step 2.

[0194] 7. Consideration of LDN addition in step 1 Because BMP concentration is known to determine the mesoderm compartment, we evaluated OSR1 and FOXF1 expression using the BMP inhibitor LDN193189 in step 1 (see Figure 31A). FOXF1 is a marker of lateral plate mesoderm and was evaluated by qPCR. BMP4 concentrations were 1 ng / mL and 0 ng / mL, and LDN193189 concentrations were 30 nM, 100 nM, and 1000 nM. The TGOT No. 25 cell line was used.

[0195] It was confirmed that increasing the LDN concentration reduced OSR1 expression at the end of step 1 (day 2.2), and the increase in OSR1 expression weakened even as we progressed to step 2 (days 4 and 6) (see Figure 31B).

[0196] As expected, qPCR confirmed that expression of FOXF1, a marker of lateral plate mesoderm, was almost completely abolished in the absence of BMP4 and with the addition of LDN (see Figure 31C). In addition, HOXD9, which is expressed in the gonad, was expressed (see Figure 31C), suggesting that the embryos were well posteriorized. The FACS results showed that at BMP4 concentrations of 0 ng / mL or higher, almost all cells were OSR1 positive (see Figure 31B), but unexpectedly, expression of PAX3, a marker of paraxial mesoderm, was observed (see Figure 31C).

[0197] 8. Induction of NR5A1-positive cells The TGOT No. 25 and NGFT No. 69 cell lines (NR5A1-EGFP / FOXL2-tdTomato double reporter lines) were used for induction for 14 days (see Figure 32A). Step 1 lasted 2 days, step 2 lasted 2–8 days, and the remaining period was designated step 3. For the TGOT No. 25 line, step 2 was continued for 12 days. The results of FACS analysis are shown in Figure 32B. In Figure 32B, the top row shows the results of flow cytometry performed every two days on the TGOT No. 25 line. In the FACS plot in Figure 32B, the horizontal axis represents EGFP (TBXT or NR5A1 (SF1)) expression, and the vertical axis represents tdTomato (OSR1 or FOXL2) expression. The second and subsequent rows show the results for the NGFT No. 69 line. The second row shows the NGFT No. 69 line, which was subjected to step 2 for 12 days without proceeding to step 3.

[0198] As shown in Figure 32B, in the second row, when NGFT No. 69 was cultured for 12 days without proceeding to step 3, no NR5A1 expression was observed. On the other hand, when step 3 was performed for 10 days, NR5A1-expressing cells (arrows) began to appear around day 10 of induction and were clearly visible by day 14. When step 2 was performed for 6 days or longer (step 3 was performed for 6 days or less), NR5A1-positive cells were no longer observed.

[0199] 9. Induction of FOXL2-positive cells Because FOXL2 expression could not be confirmed from the results of "8.", the induction period was extended to 22 days (see Figure 33A). Step 1 lasted for 2 days, step 2 for 2 to 8 days, and the remaining period was designated step 3. The cell line used was NGFT No. 69. FACS analysis was performed on days 17, 20, and 22 after a 2-day period in step 2. Cells that clearly expressed FOXL2 were observed on day 17, suggesting successful induction of ovarian somatic cells (see Figure 33B).

[0200] 10. Optimization of CHIR99021 and BMP4 concentrations in step 14 To evaluate the effects of CHIR and BMP4 concentrations on NR5A1 and FOXL2 expression in step 1, the duration of steps 1 and 2 was fixed at 2 days. The CHIR concentration was varied at 10, 14, and 20 μM, and the BMP4 concentration was varied at 1, 3, and 10 ng / mL, and the cells were cultured for up to 17 days (see Figure 34A). The cell line used was NGFT No. 69.

[0201] Although many NR5A1-positive cells were observed at a CHIR concentration of 20 μM, 20 μM was a fairly high concentration, and the embryoid bodies tended to become very small, raising concerns about cytotoxicity (see Figures 34B and 34C). Therefore, we decided to examine a CHIR concentration of 14 μM. Furthermore, a similar trend was observed when BMP4 was administered in excessive amounts (see Figures 34B and 34C).

[0202] 11. Optimization of BMP4 and LDN concentrations in step 1 Since the expression levels of OSR1 and FOXF1 were known to vary depending on BMP concentration, we investigated which conditions were most conducive to the emergence of NR5A1-positive cells when the BMP inhibitor LDN193189 was used in step 1 (see Figure 35A). BMP concentrations were 1 ng / mL and 0 ng / mL, and LDN193189 concentrations were 30, 100, and 1000 nM. The NGFT No. 69 cell line was used.

[0203] FACS analysis was performed on days 14, 17, and 21 after induction, and the addition of LDN showed almost no improvement in the percentage of NR5A1-positive cells (see FIG. 35B).

[0204] 12.Examination of the addition of BMP4, LDN, and BMS493 in step 1 We investigated whether the addition of LDN and BMS493 in step 1 contributes to the induction of NR5A1-positive cells (see Figure 36A). Step 1 lasted for 2 or 3 days (when step 1 lasted for 3 days, only LDN was used at a concentration of 30 nM). Six conditions were examined: BMP4 concentrations of 1 ng / mL and 0 ng / mL, with or without BMS493 (1 μM). Step 2 lasted for 2 days, and cells were fixed. FACS analysis was performed on day 14 of induction (see Figure 36B). NGFT No. 69 cell line was used.

[0205] As shown in Figure 36B, there was almost no induction of NR5A1-positive cells, suggesting that there was no particular advantage to adding LDN or BMS.

[0206] 13. Study on the addition of bFGF and Activin A in step 1 2 In step 1, we investigated whether the addition of 50 ng / mL bFGF and 10 ng / mL Activin A (only from day 1) improved the induction of NR5A1-positive cells (see Figure 37A). The duration of both steps 1 and 2 was 2 days, and FACS analysis was performed on days 14 and 21 of induction (see Figure 37B). The cell line used was NGFT No. 69.

[0207] As shown in Figure 36B, the addition of bFGF and Activin A did not improve the induction of NR5A1-positive cells.

[0208] In addition, we sorted weakly and strongly NR5A1-positive cells on day 14 of induction using FACS analysis and performed qPCR. Expression of gonadal markers WT1, GATA4, and NR5A1 was observed, as well as expression of HOXD9 and HOXD10, which are expressed in mouse ovaries (see Figure 37C), findings consistent with their identification as progenitor cells of gonadal somatic cells. Lateral plate mesoderm markers FOXF1 and HAND1 were negative (see Figure 37C).

[0209] 14. Optimization of BMP4 concentration in step 2 and consideration of the addition of LDN and FGF9 Previous studies have used FGF9 and LDN for intermediate mesoderm induction. Based on this, we investigated whether the addition of LDN (100 nM) and FGF9 (100 ng / mL) would improve the induction efficiency of NR5A1+ cells in step 2, using BMP4 concentrations of 0, 1, and 10 ng / mL (see Figure 38A). The NGFT No. 69 cell line was used. Step 1 was performed for 2 days, and step 2 was performed for 2 days. Results were evaluated by FACS analysis on day 14 of induction (see Figure 38B).

[0210] As shown in Figure 38B, none of the conditions improved the induction efficiency of NR5A1-positive cells.

[0211] 15. Optimization of cell number and culture plate in step 1 Next, we investigated whether the induction efficiency would change by changing the cell number and culture plate (96-well V-bottom) (see Figure 39A). We also examined the number of cells to be seeded. The duration of both step 1 and step 2 was 2 days, and step 1 was performed with a CHIR concentration of 14 μM and a BMP4 concentration of 1 ng / mL. FACS analysis was performed on days 14 and 21 of induction, and the state of the embryoid bodies was observed on day 14 of induction. The NGFT No. 69 cell line was used. It was difficult to culture a cell number of 300.

[0212] Although there was little overall induction, no improvement was observed by using a V-bottom (see Figure 39B). From the perspective of the number of viable cells (cell count at P1 gate) and the appearance of NR5A1-positive cells, a U-bottom cell count of 30,000 was considered appropriate (see Figures 39B and 39C). In Figure 39B, the number of cells at P1 gate is calculated as "(total number of cells) x (proportion of P1)". For example, the number of cells at P1 gate when using a U-bottom cell count of 30,000 is calculated as 25195 x 0.727.

[0213] 16. Optimization of bFGF concentration, BMP4 concentration, and culture period in step 1, and culture period in step 1 Based on the results obtained so far, we considered the possibility that further improvement in induction efficiency would not be expected if step 1 were performed for 2 days and step 2 were performed for 2 days. Therefore, we investigated conditions in which step 1 was performed for 2, 3, and 4 days, BMP4 concentrations were set to 0, 1, 5, and 10 ng / mL, and bFGF concentrations were set to 0, 5, 10, and 20 ng / mL, and step 2 was performed for 2 and 3 days (see Figure 40A). Results were evaluated by FACS analysis on day 16 of induction (see Figures 40B–40G). In Figure 40B, B indicates BMP4 and F indicates bFGF. The NGFT No. 69 cell line was used. Hereafter, due to the complexity of the conditions, the durations of step 1, step 2, and step 3 will be referred to as 2-2-10 (days). Unless otherwise noted, Y-27632 was added at 10 μM on day 1 of step 1.

[0214] As shown in Figure 40B, when the periods for step 1, step 2, and step 3 were 2-2-12 days, NR5A1-positive cells were observed, as in the previous cases. There was no significant change due to the addition of bFGF.

[0215] As shown in FIG. 40C, when the periods of step 1, step 2, and step 3 were 3-2-11 (days), no significant changes were observed.

[0216] As shown in Figure 40D, when the periods for step 1, step 2, and step 3 were 4-2-10 (days), a decrease in viable cells was observed when the period for step 1 was 4 days or longer.

[0217] As shown in FIG. 40E, when the periods of step 1, step 2, and step 3 were 2-3-11 (days), no significant changes were observed.

[0218] As shown in Figure 40F, when the duration of steps 1, 2, and 3 was 3-3-10 days, the addition of bFGF in step 1 significantly improved the efficiency of NR5A1-positive cell induction. The number and percentage of positive cells were highest at a BMP4 concentration of 0 ng / mL and a bFGF concentration of 5 ng / mL.

[0219] As shown in Figure 40G, when the periods for step 1, step 2, and step 3 were 4-3-9 (days), a decrease in viable cells was observed when the period for step 1 was set to 4 days.

[0220] From these results, out of the 96 culture conditions tested, only four were considered to be highly effective, with the duration of steps 1, 2, and 3 being 3-3-10 days, no BMP4 added, and a bFGF concentration of 5 ng / mL being considered the best.

[0221] The duration of steps 1, 2, and 3 was 3-3-10 days, and step 3 was added for one more day (day 17 of induction) without BMP4 and with 5 ng / mL bFGF. NR5A1-negative cells (P10 in Figure 40H), NR5A1-positive and FOXL2-negative cells (P8 in Figure 40H), and NR5A1-positive and FOXL2-positive cells (P9 in Figure 40H) were sorted by FACS analysis and subjected to qPCR. The results are shown in Figures 40I to 40K. In Figures 40I to 40K, "NC" indicates NR5A1-negative cells, "SF1" indicates NR5A1-positive and FOXL2-negative cells, and "FOXL2" indicates NR5A1-positive and FOXL2-positive cells.

[0222] As shown in Figures 40I-40K, NR5A1- and FOXL2-positive cells were FOXF1-negative and WT1-, GATA4-, NR5A1-, and AMHR2-positive, indicating gene expression patterns consistent with gonad cells. FOXL2-positive cells were RSPO1-, FOXL2-, WNT6-, and KITL-positive, indicating pregranulosa cell differentiation. NR5A1-positive cells were WNT5a-positive and expressed PDGFRα and TCF21, indicating they represent gonad somatic cells and their precursor cells. However, it was not possible to determine whether they differentiate into interstitial cells or granulosa cells.

[0223] Furthermore, embryoid bodies containing 0 ng / mL BMP4 and 10 ng / mL bFGF were immunostained on day 17 of induction (see Figure 40L, scale bar is 50 µm at 10x magnification and 10 µm at 63x magnification).

[0224] As shown in Figure 40L, we confirmed that FOXL2 was expressed as a reporter gene using tdTomato, and that pre-granulosa cell markers were expressed at the protein level. We also found that gonad somatic cells develop predominantly in the periphery.

[0225] 17. Addition of Activin A in step 1 and examination of the duration of step 1 Since the addition of bFGF without BMP4 in step 1 was the best condition, we conducted an experiment to see if adding Activin A from day 1 onward in step 1 would further improve the system (see Figure 41A). We also examined conditions in which the duration of step 1 was varied from 2.5 days to 3.5 days, with half-day intervals. The NGFT No. 69 cell line was used. FACS analysis was performed on days 16 and 28 of induction.

[0226] As shown in Figure 41B, the addition of Activin A did not improve the results, and a 3-day period for step 1 was considered to be particularly effective. Furthermore, by day 28 of induction, the FOXL2-positive and -negative populations were clearly separated. These FOXL2-positive cells were sorted and subjected to qPCR (see Figure 41C). The results indicated that although ovarian somatic cells have the ability to synthesize sex steroids, FOXL2-positive cells appear to have initiated transcription of genes required for steroid synthesis.

[0227] 18. Optimizing the period of step 2 Next, in step 1, the BMP4 concentration was 0 ng / mL, the bFGF concentration was 5 ng / mL, and the duration of step 1 was 3 days. The duration of step 2 was 3, 4, 5, and 6 days. The induction was continued until day 16 (see Figure 42A). The cells were evaluated by FACS analysis (see Figure 42B). The NGFT No. 69 cell line was used.

[0228] As shown in Figure 42B, the induction efficiency was not very good, but no improvement in the induction efficiency was observed by extending the period of step 2.

[0229] 19. Optimization of PD032901 concentration in step 2 PD032901 is a MEK inhibitor. PD032901 was added at concentrations of 3, 1, 0.3, and 0 μM, or FGF9 was added at a concentration of 20 ng / mL (see Figure 43A). Images of embryoid bodies were taken on day 6 of induction (see Figure 43B). The induction efficiency was evaluated by FACS analysis on day 16 of induction (see Figure 43C). The NGFT No. 69 cell line was used.

[0230] As shown in Figure 43B, the size at the end of step 2 improved more the more PD0325901 was reduced and the more FGF9 was added. Furthermore, as shown in Figure 43C, a PD0325901 concentration of 1 µM was considered optimal, as in the past.

[0231] 20. Optimization of BMP4 and SHH concentrations in step 2 Next, the BMP4 and SHH concentrations were varied in step 2. The BMP4 concentration was varied between 0, 1, 5, and 10 ng / mL, and the SHH concentration was varied between 0, 30, 60, and 100 ng / mL (see Figure 44A). The NGFT No. 69 cell line was used.

[0232] On day 16, FACS analysis revealed that the induction efficiency was optimal at a BMP4 concentration of 1 ng / mL and an SHH concentration of 100 ng / mL (see Figure 44B). Furthermore, the number of FOXL2- and NR5A1-positive cells increased in a SHH concentration-dependent manner, and the number of embryoid bodies also increased (see Figure 44B).

[0233] 21. Optimization of RA and SHH concentrations in step 2 Next, we investigated the RA and SHH concentrations in step 2. We also investigated whether SHH could be replaced by the small molecule SAG. RA concentrations were varied at 0, 0.3, 1, 3, and 10 μM, SHH concentrations at 30, 100, and 200 ng / mL, and SAG at 100 nM (see Figure 45A). Cells were induced at 23,333 cells / well. The NGFT No. 69 cell line was used.

[0234] On day 16, the induction efficiency was evaluated by FACS analysis. However, the induction efficiency of NR5A1-positive cells was particularly high at RA concentrations of 3 μM or higher. Furthermore, it was suggested that SAG may be able to replace SHH.

[0235] Based on these results, at this point in the experiment, an RA concentration of 3 μM, a PD032901 concentration of 1 μM, a BMP4 concentration of 1 ng / mL, and an EGF concentration of 50 ng / mL were considered optimal for step 2. Furthermore, although sufficient induction was sometimes achieved with an SHH concentration of 30 ng / mL, there was a tendency for the induction efficiency to improve with increasing SHH doses.

[0236] 22. Examination of MEF removal using gelatin-coated dishes To minimize the introduction of MEFs into the culture medium on feeders, ES cells were seeded onto gelatin-coated dishes, and the supernatant was collected after 30 minutes to initiate induction. The NGFT No. 69 cell line was used. Induction was continued for up to 16 days from cells induced normally and those treated with gelatin-coated dishes (see Figure 46A), and the results were evaluated by FACS analysis (see Figure 46B).

[0237] As shown in Figure 46B, removal of MEFs did not improve induction efficiency.

[0238] 23. Examination of MEF removal using collagenase type 4 To minimize the introduction of MEFs into the culture on feeders, we attempted to reduce the introduction of MEFs by recovering ES cells using collagenase type 4. We compared the results of conventional induction with those of ES cell recovery using collagenase type 4 after attempting to remove MEFs (see Figure 47A). Because embryoid bodies tended to grow larger after MEF removal, we also varied the number of cells per well to 30,000, 20,000, and 10,000. The NGFT No. 69 cell line was used.

[0239] When the induction efficiency was evaluated by FACS analysis on day 16 of induction, the induction efficiency was better when the MEF removal treatment was not performed (see Figure 47B).

[0240] 24. Optimization of BMP4 and FGF9 concentrations in step 3 In step 3, we investigated the BMP4 and FGF9 concentrations. Induction was performed at BMP4 concentrations of 0, 1, 20, and 50 ng / mL and FGF9 concentrations of 0, 2, 10, and 20 ng / mL (see Figure 48A), and evaluation was performed by FACS analysis on day 16 (see Figure 48B). The NGFT No. 69 cell line was used.

[0241] As shown in Figure 48B, the conditions that most induced NR5A1-positive cells were a BMP4 concentration of 1 ng / mL and an FGF9 concentration of 10 ng / mL. Since BMP4 induces GATA4, which is essential in gonadal progenitor cells, we expected that a BMP4 concentration of 50ng / mL would result in better induction, but the opposite result was obtained.FGF9 is a factor known for its effect on testis formation, and the result that increasing its concentration was effective was also an interesting discovery.

[0242] Although the induction efficiency was not the best, the number of cells obtained was the highest in previous experiments. This is thought to be partly due to the improved dissociation efficiency achieved by changing the cell dissociation reagent from trypsin to Accumax and by enlarging the reaction system.

[0243] 25. Introduction of the 4-step induction method and verification of the necessity of BMP4, SHH, and FGF9 in step 3 Based on the results obtained so far, we believe that SHH may contribute to induction efficiency, that BMP4 contributes to the expression of GATA4, a marker of the reproductive ridge, and that FGF9 is used to induce intermediate mesoderm in the kidney region. Therefore, we investigated the effects of these cytokines after step 2. The cell line used was the NGFT No. 69 line. These cytokines were added from day 6 of induction until around day 10, when NR5A1 expression is expected (the period of step 3). We then further investigated the concentrations of BMP4 and FGF9 from day 10 onward (see Figure 49A).

[0244] When the induction efficiency was evaluated by FACS analysis on day 16 of induction, the addition of BMP4 was unnecessary during the period examined, and in fact, the induction efficiency was better without its addition (see Figures 49B and 49C). Regarding FGF9, the addition of 2 ng / mL slightly increased the number of induced cells, but did not show a consistent trend, and the effect was somewhat unclear (see Figures 49B and 49C).

[0245] Regarding SHH, except for the conditions of adding SHH, BMP4 at a concentration of 1 ng / mL, and no FGF9, the induction efficiency was higher when no FGF9 was added, suggesting that SHH may have an inhibitory effect, contrary to previous tests (see Figures 49B and 49C).

[0246] 26.Verification of the necessity of SHH in step 2 and FGF9 in step 4 Since it was suggested that SHH may have an inhibitory effect, we examined the presence or absence of SHH and the possibility of substituting it with the small molecule SAG in step 2. In addition, since the effect of FGF9 was unclear in "25.", we examined what would happen if PD0325901 was added in step 3. To confirm that BMP4 is not essential from step 3 onward, five conditions were tested in step 2: SHH concentrations of 0 and 30 ng / mL, or SAG concentrations of 10, 30, and 100 ng / mL (see Figure 50A). For these five conditions, from step 3 onward, a total of 20 conditions were tested: FGF9 (2 ng / mL) alone, BMP4 (20 ng / mL) and FGF9 (2 ng / mL), PD0325901 (1 μM) for 4 days followed by no addition, or PD0325901 (1 μM) and BMP4 (20 ng / mL) for 4 days followed by BMP4 (20 ng / mL) alone (see Figure 50A). The cell line used was the NGFT No. 69 cell line.

[0247] Evaluation of induction efficiency by FACS analysis on day 16 of induction revealed that SHH was unnecessary in step 2, and that induction efficiency was better without SHH (see Figure 50B). Regarding FGF9, inhibition of MEK downstream of FGF with PD0325901 also resulted in better induction efficiency (see Figure 50B).

[0248] 27. Verify useful factors in step 3 We investigated whether any cytokines from step 2, other than PD0325901, could be used in step 3. In step 3, RA (3 μM), PD0325901 (1 μM), BMP4 (1 ng / mL), and SHH (30 ng / mL) were added or not in addition to EGF, for a total of 16 patterns. Controls included no addition from step 3 onward, and addition of BMP4 (20 ng / mL) and FGF9 (2 ng / mL) in step 3. The cell line used was NGFT No. 69. Figure 51B shows FACS plots on day 16 of induction for two controls: no addition from step 3 onward (CK-), and addition of BMP4 (20 ng / mL) and FGF9 (2 ng / mL) from step 3 onward, as well as other conditions.

[0249] As shown in Figure 51B, the induction efficiency was extremely high when no cytokines were added from step 3 onwards.

[0250] From step 3 onwards, we compared the results with a control without cytokines (CK-) to examine what would happen if EGF (E) or one of the other cytokines was added in step 3 (see Figure 51C). Although the addition of BMP4(B) increased the number of induced cells, the induction efficiency decreased, and the effect was unclear (see Figure 51C). Furthermore, as in the previous results, the addition of SHH(S) was detrimental, and the addition of PD0325901(P) decreased the number of cells, but further increased the induction efficiency, suggesting its potential usefulness (see Figure 51C). The addition of 3 μM retinoic acid (R) significantly decreased the induction efficiency (see Figure 51C).

[0251] The effects of adding each cytokine were examined in detail (see Figures 51D to 51G).

[0252] When BMP4 was added to each condition, the number of induced cells decreased only when BMP4 was added to EGF+RA (ER+B) (see Figure 51D). This suggests that adding a small amount of BMP4 may increase the number of induced cells. In the above "25.", BMP4 (concentration 1 ng / mL) was added until day 16 of induction, so it was considered that it might be better not to add BMP4 from day 10 of induction onwards.

[0253] When SHH was added, the number of induced cells decreased under all conditions, suggesting that SHH acts inhibitoryly (see Fig. 51E).

[0254] Regarding the addition of PD03, the trend was not consistent, and although the number of cells induced by MEK inhibition decreased, it was thought that the addition of PD03 had the effect of improving the induction efficiency itself (see Figure 51F).

[0255] The addition of RA consistently reduced the number of induced cells.

[0256] Based on the above results, the use of PD0325901 and BMP4 (concentration 1 ng / mL) was considered as candidate factors for step 3.

[0257] Gene expression of SF1 and FOXL2 positive cells induced by the 28.3 step method The results of "27." demonstrated that FOXL2-positive cells were efficiently induced without the addition of any cytokines in step 3. RNA sequencing data was measured during this induction. On day 16 of induction, FOXL2-negative cells (P7 gate, hereafter abbreviated as "d16SF1+") and FOXL2-positive cells (P8 gate, hereafter abbreviated as "d16FOXL2+") that strongly expressed SF1 were sorted by FACS analysis (see Figure 52B) and subjected to RNA sequencing (see Figure 52C). In the heat map of Figure 52C, the left side (black) of the "Color Key" indicates no gene expression, while the right side (white) indicates high gene expression.

[0258] All cells strongly expressed genital ridge marker genes, and the expression of WNT6, a marker of the granulosa cell lineage, was also observed in SF1-single-positive cells (see Figure 52C). Furthermore, the stromal cell markers TCF21 and MAFB were decreased in FOXL2-positive cells (see Figure 52C).The expression of SOX11 and NR2F1, markers of relatively immature genital ridge somatic cells, was also observed (see Figure 52C), suggesting that the differentiation process from genital ridge epithelium to granulosa cells is underway.

[0259] 29. Verification of the addition period of PD0325901 in step 3 The results so far suggest that the addition of PD0325901 may reduce the number of cells induced, but may improve the induction efficiency. We investigated the effects of shortening the treatment period of PD0325901 using the NG-Puro cell line. In step 3, we tested the effects of adding PD03 for 48 hours, 96 hours, or no addition (see Figure 53A).

[0260] The results suggest that the optimal period for adding PD03 in step 3 may be 48 hours (see Figure 53B).

[0261] 30. Verification of the impact of PD0325901 in step 3 To examine the effect of adding PD03 in step 3 on induction, we used the WTGBNG No. 1 cell line. To confirm that SHH is not required for the induction system, we also tested the addition or absence of PD0325901 in step 3, as well as the addition or absence of SHH in steps 2 and 3 (see Figure 54A).

[0262] Figure 54B shows FACS plots for the condition without PD03 addition in step 3. In Figure 54B, the four leftmost columns of FACS plots have GATA4-mTagBFP2 on the horizontal axis and WT1-tdTomato on the vertical axis, ordered from left to right: d8, d10, d12, and d16. The rightmost column has SF1-EGFP on the horizontal axis and APC on the vertical axis, and is an expanded FACS plot for d16. The top row shows the addition of SHH in step 2 (+) and the addition of SHH in step 3 (+). The second row shows the addition of SHH in step 2 (+) and the absence of SHH in step 3 (-). The third row shows the absence of SHH in step 2 (-) and the presence of SHH in step 3 (+). The fourth row shows the absence of SHH throughout the entire period (-).

[0263] As a result, SF1-positive cells were induced even without the addition of SHH (see Figure 54B). Furthermore, at day 8, almost all cells were WT1-positive, and some cells had already begun to express GATA4 (see Figure 54B), indicating that they were in the process of differentiating into genital ridge epithelium.

[0264] Figure 54C shows the FACS plot under the condition of adding PD03 in step 3. The vertical and horizontal axes and the conditions of each row in Figure 54C are the same as those in Figure 54B. In Figure 54C, compared with the FACS plot in Figure 54B, the proportion of cells with low WT1 expression was reduced overall at day 10, and almost all cells were GATA4-positive by day 16. However, the number of induced cells itself was reduced, and the induction efficiency was highest when PD0325901 was added, especially in the absence of SHH.

[0265] These findings suggest that the addition of PD0325901 in step 3 without adding SHH may contribute to stabilizing the induction system, although the yield of SF1-positive cells may be reduced.

[0266] Furthermore, the fluorescence of cell clusters was observed under a microscope when SHH was not added in step 2 and when PD0325901 was added in step 3 (see Figure 54D). As shown in Figure 54D, WT1 expression was observed throughout the cell clusters on day 8, and GATA4 and SF1 expression gradually began to appear from the periphery of the cell clusters, confirming the formation of the genital ridge. This process resembled the thickening of the genital ridge epithelium and the formation of the genital ridge.

[0267] 31. Optimization of RA concentration in step 3 Because we knew that continued use of 3 μM RA dramatically reduced induction efficiency, we investigated what would happen if a small amount of RA were added in step 3, or conversely, what would happen if RA signaling were inhibited. The WTGBNG No. 1 cell line was used. RA was added for 48 hours, matching the time period for PD03. RA concentrations of 3, 1, 0.1, and 0 μM were used, as well as the RA signaling inverse antagonist BMS493 (1 μM) (see Figure 55A). Cell proliferation was assessed by FACS analysis on days 8, 10, 12, 14, and 16 after induction (see Figure 55B and Figure 55C).

[0268] In the FACS plot in Figure 55B, the vertical axis represents gene expression of WT1-tdTomato and the horizontal axis represents gene expression of GATA4-mTagBFP2, and each row represents the number of days of induction. As shown in Figure 55B, after step 3, almost all cells were positive for WT1, suggesting that intermediate mesoderm was efficiently induced under both conditions. When RA signaling was inhibited with BMS493, WT1 expression was slightly reduced. Furthermore, on day 8 of induction, some cells expressed GATA4, suggesting that they were gradually differentiating into genital ridge epithelium. Under RA-supplemented conditions, no significant differences in WT1 and GATA4 expression were observed.

[0269] The FACS plot in Figure 55C shows APC plotted on the vertical axis and SF1-EGFP plotted on the horizontal axis. Because SF1 expression was not observed on day 8 of induction, data from days 10, 12, 14, and 16 of induction are presented. As shown in Figure 55C, by day 16 of induction, the medium without RA was the most efficient at inducing SF1-positive cells. Furthermore, the addition of BMS493 slightly increased SF1-EGFP expression, an interesting phenomenon, although the induction of SF1-positive cells was less efficient. It has been reported that WT1 acts as an inhibitor of SF1 expression, suggesting that the addition of BMS493 may be a signaling mechanism for this effect.

[0270] 32. Confirmation of earlier WT1 expression After step 2, we examined the types of cells that had been induced. A detailed protocol is shown in Figure 56A. The WTNG No. 43 cell line was used. Cells were evaluated by FACS analysis on days 6, 8, 10, and 14 of induction (see Figure 56B).

[0271] It has been previously found that at the end of step 2, almost all cells express OSR1, a marker of intermediate mesoderm or lateral plate mesoderm, and as shown in Figure 56B, WT1-positive cells appeared already on day 6 of induction.

[0272] 33.Consideration of why WT1-positive cells differentiate into the reproductive ridge Gene expression in WT1-positive cells was measured. A detailed protocol is shown in Figure 57A. The WTNG No. 43 cell line was used. WT1-positive cells on day 8 of induction, as well as WT1-single-positive and WT1- and SF1-copositive cells on day 10 of induction, were sorted by FACS analysis (see Figure 57B), and gene expression was confirmed by qPCR (see Figure 57C).

[0273] In Figure 57C, the bar graphs (from left to right) show WT1-positive cells on day 8 of induction, WT1-single-positive cells on day 10 of induction, and WT1- and SF1-copositive cells on day 10 of induction. The vertical axis of the graph shows the dCT value relative to PPIA / GAPDH. As shown in Figure 57C, OSR1 expression was already significantly reduced by day 8 of induction, and gene expression of all cells showed high expression of genital ridge markers WT1 and GATA4. On the other hand, WT1-positive and SF1-negative cells on days 8–10 of induction expressed BMP4, which may promote GATA4 expression (GATA4 is downstream of BMP4) and lead to differentiation into the genital ridge. Furthermore, gene expression of FOXF1 was also reduced in SF1-positive cells, suggesting that this may also be due to the reduced BMP4 expression.

[0274] 34. Detection of stromal cells The ovary consists of FOXL2-positive granulosa cells and PDGFRα-positive stromal cells. In the mouse ovarian somatic cell induction method developed by Yoshino et al., SF1-positive cells differentiate into FOXL2-positive granulosa cell lineages and SF1- and PDGFRα-positive stromal cells. To verify this in monkeys, we performed FACS periodically from around day 12 of induction, when FOXL2 expression begins, to monitor the expression of SF1, PDGFRα, and FOXL2. A detailed protocol is shown in Figure 58A. The cell line used was the NGFT No. 69 line. A FACS plot is shown in Figure 58B. In Figure 58B, the upper panel shows FACS plots for days 12, 14, 16, 18, and 22 of induction, plotted with FOXL2-tdTomato on the vertical axis and SF1-EGFP on the horizontal axis. The bottom left shows FACS plots of SF1-EGFP-positive cells on days 12 and 14 of induction, plotted on the vertical axis as PDGFRα-APC and the horizontal axis as SF1-EGFP. The bottom right shows FACS plots of SF1-EGFP-positive cells on days 16, 18, and 22 of induction, plotted on the vertical axis as PDGFRα-APC and the horizontal axis as FOXL2-tdTomato. As shown in Figure 58B, on day 14 of induction, some cells with relatively low SF1-EGFP levels expressed PDGFRα (arrows), and because these cells were FOXL2-negative based on the expression level of SF1, these were considered to be stromal cells. However, there were few clear stromal cell-like signals in the other FACS plots, suggesting that further investigation of conditions is necessary for the induction of stromal cells.

[0275] 35.Verification of the necessity of EGF in step 1 and step 2 The usefulness of EGF in reproductive ridge development was unknown, and its function was poorly understood. Therefore, aiming to further simplify the induction system and reduce costs, induction was performed without the addition of EGF in steps 1 and 2. A detailed protocol is shown in Figure 59A. The cell line used was the NGFT No. 69 line. FACS analysis was performed on day 16 of induction.

[0276] As a result, the effect of EGF was unclear and it was thought that it was not essential for induction (see Figure 59B).

[0277] 36. Summary These results demonstrate that we have established a method for efficiently and reproducibly inducing FOXL2-positive granulosa cell lineage cells from cynomolgus monkey ES cells (see Figure 60A). Furthermore, the induction method is a simple protocol that uses relatively inexpensive small molecules, with the exception of small amounts of bFGF and BMP4. During the induction process, we successfully induced gastrulation, as seen in TBXT expression from ES cells, which correspond to the epiblast immediately after implantation; OSR1 and WT1 expression, which indicate intermediate mesoderm differentiation; GATA4 and SF1 expression, which indicate differentiation into genital ridge epithelium; and FOXL2 expression, which indicates differentiation into the granulosa cell lineage, in a manner mimicking the in vivo and mouse induction methods (see Figure 60B; a protocol diagram was created based on Non-Patent Document 1). We also observed the emergence of a small number of cells thought to be precursors of interstitial cells, which later play an important role in sex steroid production. In the mouse induction system, granulosa and interstitial cell lines were induced to the same extent, while in the monkey system, the protocol appears to selectively induce FOXL2-positive granulosa cell lineage cells. Methods for improving the efficiency of interstitial cell induction remain a challenge. [Industrial Applicability]

[0278] According to the production method and differentiation induction method of this embodiment, ovarian somatic cell-like cells at any developmental stage can be efficiently obtained from primate pluripotent stem cells.

Claims

1. A method for producing ovarian somatic cell-like cells, comprising: Step 1: culturing primate pluripotent stem cells in a medium containing a GSK3 inhibitor and a ROCK inhibitor; Step 2: culturing the cells obtained after step 1 in a medium containing BMP4, retinoic acid, and a MEK inhibitor; Step 3: culturing the cells obtained after step 2 in a basal medium to obtain ovarian somatic cell-like cells; A manufacturing method comprising:

2. The method according to claim 1, wherein the culture in step 1 is carried out for 3 days or more.

3. The method according to claim 1 or 2, wherein the culture in step 2 is carried out for 3 days or more.

4. The method according to any one of claims 1 to 3, wherein the culture in step 3 is carried out for one week or more.

5. The method according to any one of claims 1 to 4, wherein the culture medium in step 1 further contains BMP4.

6. The step 1 is Step 1-1: culturing primate pluripotent stem cells in a medium containing a GSK3 inhibitor and a ROCK inhibitor; Step 1-2 of culturing the cells after step 1-1 in a medium containing a GSK3 inhibitor and Activin A; The method of any one of claims 1 to 5, comprising:

7. The method according to any one of claims 1 to 6, wherein the culture in step 1 is plate culture.

8. The method according to claim 7 , wherein a container coated with a cell scaffold material is used in the flat culture.

9. The method according to claim 7 or 8, wherein the medium in step 2 further contains a ROCK inhibitor.

10. The method according to any one of claims 1 to 9, wherein the culture medium in step 1 further contains bFGF.

11. The method according to any one of claims 1 to 10, wherein the medium in step 2 further contains a hedgehog signal activator.

12. Step 3 is Step 3-1: culturing the cells after step 2 in a medium containing a MEK inhibitor; Step 3-2 of culturing the cells obtained after step 3-1 in a basal medium to obtain ovarian somatic cell-like cells; The method of any one of claims 1 to 11, comprising:

13. The method of any one of claims 1 to 12, wherein the ovarian somatic cell-like cells are fetal ovarian somatic cell-like cells.

14. The method according to any one of claims 1 to 13, wherein the primate pluripotent stem cells are derived from a cynomolgus monkey or a human.

15. A method for inducing differentiation of primate pluripotent stem cells into ovarian somatic cell-like cells, comprising: A method comprising inducing differentiation from primate pluripotent stem cells into ovarian somatic cell-like cells using the production method according to any one of claims 1 to 14.

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