Methods and compositions for producing ovarian support cell co-culture
Patent Information
- Application Number
- HK62026127298
- Authority / Receiving Office
- HK · HK
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2023-03-24
- Filing Date
- 2026-08-10
- Publication Date
- 2026-09-18
- Estimated Expiration
- 2044-03-24
Smart Images
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Abstract
Description
This document describes methods and compositions for in vitro maturation of oocytes. Specifically, this disclosure relates to methods and compositions for promoting oocyte maturation in culture media by engineering various ovarian Sertoli cells (OSCs). Such methods and compositions are particularly useful for assisted reproductive technology (ART) procedures. Abstract
Claims
CLAIMS1 . An ex vivo composition comprising one or more ovarian support cells (OSCs) and one or more diluents or excipients, optionally wherein the composition promotes maturation of one or more oocytes.
2. The composition of claim 1 , wherein the one or more OSCs comprise one or more granulosa cells.
3. The composition of claim 2, wherein the one or more OSCs express FOXL2, AMHR2, CD82 or any combination thereof.
4. The composition of any one of claims 1 -3, wherein the one or more OSCs express one or more genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB.
5. The composition of claim 3 or 4, wherein the one or more OSCs further express one or more genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4.
6. The composition of claim 3 or 4, wherein the one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2.
7. The composition of claim 3 or 4, wherein the one or more OSCs further express genes CDH2 and / or NOTCH2.
8. The composition of any one of claims 1 -7, wherein the one or more OSCs do not exhibit significant expression of RARRES2.
9. The composition of any one of claims 1 -8, wherein the one or more OSCs express NR2F2.
10. The composition of any one of claims 1 -9, wherein the one or more OSCs comprise ovarian stroma cells.11 . The composition of any one of claims 1 -10, wherein the one or more OSCs comprise granulosa cells and ovarian stroma cells.
12. The composition of any one of claims 1 -11 , wherein the one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.
13. The composition of any one of claims 1 -12, wherein the one or more OSCs are obtained by differentiation of a population of iPSCs, optionally wherein the iPSCs are hiPSCs.
14. The composition of claim 13, wherein the hiPSCs express or overexpress one or more of the following transcription factors:(i) RUNX2;(ii) NR5A1 ;(iii) GATA4;(iv) FOXL2;(v) any combination of two of the transcription factors;(vi) any combination of three of the transcription factors; or(vii) a combination of all four of the transcription factors.
15. The composition of claim 13 or 14, wherein the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.
16. The composition of any one of claims 13-15, wherein the hiPSCs are contacted with a Wnt / p- catenin pathway activator.
17. The composition of claim 16, wherein the Wnt / p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.
18. The composition of any one of claims 1 -17, wherein at least one of the one or more OSCs are encapsulated.
19. The composition of claim 18, wherein the one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or any mixture thereof.
20. The composition of claim 19, wherein the one or more OSCs are encapsulated in laminin, optionally wherein the laminin is laminin-521 .21 . The system or composition of claim 19 or 20, wherein the one or more OSCs are encapsulated in vitronectin.
22. The composition of any one of claims 1 -21 , wherein the one or more OSCs have lower expression, or undetectable expression, of one or more genes associated with pluripotency relative to an iPSC.
23. The composition of claim 22, wherein the one or more genes associated with pluripotency comprise NANOG.
24. The composition of claim 23, wherein the one or more genes associated with pluripotency comprise POU5F1 .
25. The composition of any one of claims 1 -24, wherein at least some of the OSCs produce one or more growth factors.
26. The composition of claim 25, wherein the one or more growth factors comprise insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMPs), C-type natriuretic peptide (CNP), or any combination thereof.
27. The composition of claim 25 or 26, wherein at least a portion of the one or more growth factors is secreted.
28. The composition of any one of claims 1 -27, wherein the one or more OSCs produce one or more steroids.
29. The composition of claim 28, wherein the one or more steroids comprise estradiol, progesterone, or a combination thereof.
30. The composition of claim 28 or 29, wherein the one or more steroids are produced in response to hormonal stimulation.31 . The composition of claim 30, wherein the hormonal stimulation comprises FSH, androstenedione treatment, or a combination thereof.
32. The composition of any one of claims 28-31 , wherein at least a portion of the one or more steroids is secreted.
33. The composition of any one of claims 1 -32, wherein the one or more OSCs are cryopreserved.
34. The composition of any one of claims 1 -33, further comprising an in vitro maturation (IVM) media.
35. The composition of claim 34, wherein the IVM media comprises a cell culture media.
36. The composition of claim 34 or 35, wherein the IVM media comprises Medicult-IVM media.
37. The composition of any one of claims 34-36, wherein the IVM media comprises one or more supplements.
38. The composition of claim 37, wherein the one or more supplements comprise:(i) human serum albumin (HSA), optionally at a concentration of about 5 to about 15 mg / mL, further optionally at a concentration of 10 mg / mL;(ii) recombinant follicle stimulating hormone (rFSH), optionally at a concentration of about 70 mIU / mL to about 80 mIU / mL, further optionally at a concentration of 75 mIU / mL;(iii) human chorionic gonadotropin (hCG), optionally at a concentration of about 95 mIU / mL to about 105 mIU / mL, further optionally at a concentration of 100 mIU / mL;(iv) androstenedione, optionally at a concentration of about 495 ng / mL to about 505 ng / mL, further optionally at a concentration of 500 ng / mL;(v) doxycycline, optionally at a concentration of about 0.5 pg / mL to about 1 .5 pg / mL, further optionally at a concentration of 1 pg / mL; or any combination of the one or more supplements.
39. The composition of any one of claims 1 -38, wherein the one or more oocytes are retrieved from a donor subject.
40. The composition of claim 39, wherein the donor subject is from about 19 years old to about 45 years old.41 . The composition of claim 39, wherein the subject is undergoing ovarian stimulation.
42. The composition of claim 41 , wherein the ovarian stimulation comprises treatment with gonadotropin releasing hormone (GnRH).
43. The composition of claim 42, wherein the ovarian stimulation comprises treatment with one or more GnRH analogs.
44. The composition of claim 43, wherein the one or more GnRH analog is a GnRH agonist or antagonist.
45. The composition of any one of claims 41 -44, wherein the ovarian stimulation comprises one or more ovulatory triggers.
46. The composition of claim 45, wherein the one or more ovulatory triggers comprise human chorionic gonadotropin (hCG).
47. The composition of claim 45 or 46, wherein the one or more ovulatory trigger comprise a GnRH agonist, optionally wherein the GnRH agonist is leuprolide.
48. The composition of any one of claims 41 -47, wherein the ovarian stimulation comprises FSH treatment.
49. The composition of any one of claims 41 -47, wherein the ovarian stimulation does not comprise FSH treatment.
50. The composition of claim 48, wherein the FSH treatment comprises 300 international units (IU) to 700 IU of FSH.51 . The composition of claim 50, wherein the FSH treatment comprises 400 IU to 600 IU of FSH.
52. The composition of any one of claims 48, 50, and 51 , wherein the FSH treatment comprises 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment comprises a plurality of injections, wherein each injection comprises a dose of about 100 IU to about 200 IU of the FSH.
53. The composition of any one of claims 41 -52, wherein the ovarian stimulation further comprises clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more injections, optionally wherein each injection comprises a dose of 50 mg to 150 mg.
54. The composition of claim 41 -53, wherein the ovarian stimulation further comprises one or more hCG triggers.
55. The composition of claim 54, wherein the one or more hCG triggers comprises 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.
56. The composition of any one of claims 1 -55, wherein the one or more oocytes are in cumulus oocyte complexes (COCs).
57. The composition of any one of claims 1 -56, wherein the one or more oocytes comprise one or more denuded immature oocytes.
58. The composition of claim 57, wherein all of the one or more oocytes are denuded immature oocytes.
59. The composition of any one of claims 1 -56, wherein the one or more oocytes are not denuded.
60. The composition of any one of claims 1 -59, wherein the one or more oocytes comprise one or more germinal vesicle (GV)-containing oocytes.61 . The composition of any one of claims 1 -60, wherein the one or more of the oocytes comprise one or more oocytes in metaphase I (Ml).
62. The composition of any one of claims 1 -61 , wherein the one or more of the oocytes comprise one or more oocytes in metaphase II (MH).
63. The composition of any one of claims 1 -62, wherein at least a portion of the one or more oocytes comprise one or more previously vitrified oocytes.
64. The composition of any one of claims 1 -63, wherein at least a portion of the one or more oocytes comprise one or more previously cryopreserved oocytes.
65. The composition of any one of claims 1 -64, wherein the one or more oocytes are co-cultured with the one or more OSCs.
66. The composition of claim 65, wherein prior to and / or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV-stage to Mil-stage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Mil-stage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.
67. The composition of claim 66, wherein the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.
68. The composition of claim 66 or 67, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs.
69. The composition of any one of claims 66-68, wherein the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes.
70. The composition of any one of claims 65-69, wherein the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.71 . The composition of any one of claims 65-70, wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours.
72. The composition of claim 71 , wherein the one or more oocytes are co-cultured with the one or more OSCs for about 24 hours to about 28 hours.
73. The composition claim 71 or 72, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more blastocytes following contact with one or more mature sperm cells.
74. The composition of any one of claims 65-73, wherein the one or more oocytes are co-cultured in direct contact with the one or more OSCs.
75. The composition of any one of claims 65-74, wherein the one or more oocytes do not directly contact the OSCs.
76. The composition of any one of claims 65-75, wherein the culture system is a suspension culture.
77. The composition of any one of claims 65-75, wherein the culture system is an adherent culture.
78. A method of cultivating oocytes, wherein one or more oocytes are co-cultured with one or more ovarian supporting cells (OSCs).
79. A method of preparing one or more oocytes that have previously been retrieved from a human subject for use in an assisted reproduction technology (ART) procedure, the method comprising coculturing the one or more oocytes with one or more OSCs.
80. A method of producing a mature oocyte for use in an ART procedure, the method comprising coculturing one or more oocytes that have previously been retrieved from a human subject with a population of ovarian support cells that have been differentiated from one or more iPSCs.81 . A method of inducing oocyte maturation in vitro, the method comprising co-culturing one or more oocytes with a population of ovarian support cells that have been differentiated from one or more iPSCs, optionally wherein the co-culturing is conducted for a period of from about 6 hours to about 120 hours.
82. A method of producing a mature oocyte for use in an ART procedure, the method comprising:(a) differentiating one or more iPSCs to produce one or more OSCs;(b) retrieving one or more immature oocytes from a subject; and(c) co-culturing the one or more oocytes with the one or more OSCs, thereby producing one or more mature oocytes.
83. A method of promoting oocyte maturation for a subject undergoing an ART procedure and that has previously been administered one or more follicular triggering agents during a follicular triggering period, the method comprising:(a) retrieving one or more immature oocytes from the subject;(b) co-culturing the one or more oocytes with one or more OSCs that have been differentiated from iPSCs, thereby producing one or more mature oocytes; and(c) isolating the one or more mature oocytes.
84. The method of any one of claims 78-83, wherein prior to and / or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV- stage to Mil-stage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Milstage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.
85. The method of claim 84, wherein the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.
86. The method of any one of claims 84 or 85, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs.
87. The method of claim 86, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes matured in vivo.
88. The method any one of claims 84-87, wherein the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes.
89. The method of any one of claims 78-87, wherein the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.
90. The method of any one of claims 78-89, wherein the one or more OSCs express FOXL2, AMHR2, CD82 or any combination thereof.91 . The method of any one of claims 78-90, wherein the one or more OSCs express one or more of the genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB.
92. The method of claim 90 or 91 , wherein the one or more OSCs further express one or more of the genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4.
93. The method of claim 90 or 91 , wherein the one or more OSCs further express one or more of the genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2.
94. The method of claim 90 or 91 , wherein the one or more OSCs further express genes CDH2 and / or NOTCH2, with no significant gene expression of RARRES2.
95. The method of any one of claims 78-94, wherein the one of more OSCs comprise granulosa cells.
96. The method of any one of claims 78-90, wherein the one or more OSCs express NR2F2.
97. The method of any one of claims 78-90 and 96, wherein the one or more OSCs comprise ovarian stroma cells.
98. The method of any one of claims 78-97, wherein the one or more OSCs comprise granulosa cells and ovarian stroma cells.
99. The method of any one of claims 78-98, wherein the one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.
100. The method of any one of claims 78-99, wherein the one or more OSCs are obtained by differentiation of a population of iPSCs, optionally wherein the population of iPSCs are hiPSCs.101 . The method of claim 100, wherein the hiPSCs express or overexpress the following one or more transcription factors:(i) RUNX2;(ii) NR5A1 ;(iii) GATA4;(iv) FOXL2;(v) any combination of two of the transcription factors;(vi) any combination of three of the transcription factors; or(vii) a combination of all four of the transcription factors.
102. The method of claim 101 , wherein the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.
103. The method of any one of claims 100-102, wherein the hiPSCs are contacted with a Wnt / p- catenin pathway activator.
104. The method claim 103, wherein the Wnt / p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.
105. The method of any one of claims 78-104, wherein the one or more OSCs are encapsulated.
106. The method of claim 105, wherein the one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or a mixture thereof.
107. The method of claim 106, wherein the one or more OSCs are encapsulated in laminin, optionally wherein the laminin is laminin-521 .
108. The method of claim 106 or 107, wherein the one or more OSCs are encapsulated in vitronectin.
109. The method of any one of claims 78-108, wherein the one or more OSCs have low or undetectable expression of one or more genes associated with pluripotency relative to an iPSC.
110. The method of claim 109, wherein the one or more genes associated with pluripotency comprise NANOG.
111. The method of claim 110, wherein the one or more genes associated with pluripotency comprise POU5F1.
112. The method of any one of claims 78-111 , wherein the one or more of the OSCs produce one or more growth factors.
113. The method of claim 112, wherein the growth factors comprise IGF, SCF, EGF, LIF, VEGF, BMPs, CNP, or any combination thereof.
114. The method of claim 112 or 113, wherein at least a portion of the one or more growth factors is secreted.
115. The method of any one of claims 78-114, wherein the one or more OSCs produce one or more steroids.
116. The method of claim 115, wherein the one or more steroids comprise estradiol, progesterone, or a combination thereof.
117. The method of claim 115 or 116, wherein the one or more steroids are produced in response to hormonal stimulation of the OSCs.
118. The method of claim 117, wherein the hormonal stimulation comprises exposure to FSH, androstenedione, or a combination thereof.
119. The method of any one of claims 115-118, wherein at least a portion of the one or more steroids is secreted.
120. The method of any one of claims 78-119, wherein the one or more OSCs were previously cryopreserved.121 . The method of any one of claims 78-120 further comprising culturing the one or more OSCs with the one of more oocytes in an in vitro maturation (IVM) media.
122. The method of claim 121 , wherein the IVM media comprises a cell culture media.
123. The method of claim 121 or 122, wherein the IVM media comprises Medicult-IVM media.
124. The method of any one of claims 121 -123, wherein the IVM media comprises one or more supplements.
125. The method of claim 124, wherein the one or more supplements comprise:(i) human serum albumin (HSA), optionally at a concentration of about 5 to about 15 mg / mL, further optionally at a concentration of 10 mg / mL;(ii) recombinant follicle stimulating hormone (rFSH), optionally at a concentration of about 70 mIU / mL to about 80 mIU / mL, further optionally at a concentration of 75 mIU / mL;(iii) human chorionic gonadotropin (hCG), optionally at a concentration of about 95 mIU / mL to about 105 mIU / mL, further optionally at a concentration of 100 mIU / mL;(iv) androstenedione, optionally at a concentration of about 495 ng / mL to about 505 ng / mL, further optionally at a concentration of 500 ng / mL;(v) doxycycline, optionally at a concentration of about 0.5 pg / mL to about 1 .5 pg / mL, further optionally at a concentration of 1 pg / mL; or any combination of the one or more supplements.
126. The method of any one of claims 78-125, wherein the one or more oocytes is retrieved from a donor subject.
127. The method of claim 126, wherein the subject is from about 19 years old to about 45 years old.
128. The method of claim 126, wherein the subject is undergoing ovarian stimulation.
129. The method of claim 128, wherein the ovarian stimulation comprises treatment with gonadotropin releasing hormone (GnRH).
130. The method of claim 129, wherein the ovarian stimulation comprises treatment with one or more GnRH analogs.131 . The method of claim 130, wherein the GnRH analog is a GnRH agonist or antagonist.
132. The method of any one of claims 128-131 , wherein the ovarian stimulation comprises one or more ovulatory triggers.
133. The method of claim 132, wherein the one or more ovulatory triggers comprise hCG.
134. The method of claim 132 or 133, wherein the one or more ovulatory triggers comprise GnRH agonist, optionally wherein the GnRH agonist is leuprolide.
135. The method of any one of claims 128-134, wherein the ovarian stimulation comprises FSH treatment.
136. The method of claim 128-134, wherein the ovarian stimulation does not comprise FSH treatment.
137. The method of claim 135, wherein the FSH treatment comprises 300 IU to 700 IU of FSH.
138. The method of claim 137, wherein the FSH treatment comprises 400 IU to 600 IU of FSH.
139. The method of any one of claims 135, 137, and 138, wherein the FSH treatment comprises 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment comprises a plurality of injections, wherein each injection comprises a dose of about 100 IU to about 200 IU of the FSH.
140. The method of any one of claims 128-139, wherein the ovarian stimulation comprises clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, optionally wherein each dose is between 50 mg and150 mg.
141. The method of claim 128-140, wherein the ovarian stimulation comprises one or more hCG triggers.
142. The method of claim 141 , wherein the one or more hCG triggers comprises 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.
143. The method of any one of claims 78-142, wherein the one or more oocytes are in cumulus oocyte complexes (COCs).
144. The method of any one of claims 78-143, wherein the one or more oocytes comprise one or more denuded immature oocytes.
145. The method of claim 144, wherein all of the one or more oocytes are denuded immature oocytes.
146. The method of any one of claims 78-143, wherein the one or more oocytes are not denuded.
147. The method of any one of claims 78-146, wherein one or more oocytes comprise one or more germinal vesicle (GV)-containing oocytes.
148. The method of any one of claims 78-147, wherein one or more oocytes comprise one or more oocytes in metaphase I (Ml).
149. The method of any one of claims 78-148, wherein one or more oocytes comprise one or more oocytes in metaphase II (Mil).
150. The method of any one of claims 78-149, wherein one or more oocytes comprise one or more previously vitrified oocytes.151 . The method of any one of claims 78-150, wherein one or more oocytes comprise one or more previously cryopreserved oocytes.
152. The method of any one of claims 78-151 , wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours.
153. The method of claim 152, wherein the one or more oocytes are cultured with the one or more OSCs for about 24 hours to about 28 hours.
154. The method of claim 152 or 153, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more blastocytes following contact with one or more mature sperm cells.
155. The method of any one of claims 78-154, wherein the one or more oocytes are cultured in direct contact with the one or more OSCs.
156. The method of any one of claims 78-155, wherein the one or more oocytes do not directly contact the one or more OSCs.
157. The method of any one of claims 78-156, wherein the co-culture is a suspension co-culture.
158. The method of claim 78-156, wherein the co-culture is an adherent co-culture.
159. A method of promoting differentiation of one or more induced pluripotent stem cells (iPSCs) to one or more ovarian support cells (OSCs), the method comprising:(a) culturing the one or more iPSCs in vitro;(b) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors comprising FOXL2, NR5A1 , RUNX2, GATA4, or any combination thereof, thereby producing differentiated cells;(c) determining that the differentiated cells resulting from (b) exhibit a gene expression profile that is similar to that of one or more OSCs; and(d) co-culturing the identified one or more OSCs with one or more oocytes previously retrieved from a subject, thereby maturing the one or more oocytes.
160. A method of producing one or more ovarian support cells (OSCs) from one or more induced pluripotent stem cells (iPSCs), the method comprising:(a) culturing the one or more iPSCs in vitro;(b) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors comprising FOXL2, NR5A1 , RUNX2, GATA4, or any combination thereof, thereby producing differentiated cells;(c) determining that the differentiated cells resulting from (b) exhibit a gene expression profile that is similar to that of one or more OSCs; and(d) co-culturing the identified one or more OSCs with one or more oocytes previously retrieved from a subject, thereby maturing the one or more oocytes.161 . A method of preparing a composition comprising one or more ovarian support cells (OSCs), the method comprising:(a) culturing the one or more iPSCs in vitro;(b) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors comprising FOXL2, NR5A1 , RUNX2, GATA4, or any combination thereof, thereby producing differentiated cells;(c) determining that the differentiated cells resulting from (b) exhibit a gene expression profile that is similar to that of one or more OSCs; and(d) co-culturing the identified one or more OSCs with one or more oocytes previously retrieved from a subject, thereby maturing the one or more oocytes.
162. The method of any one of claims 159-161 , wherein the iPSCs are human iPSCs (hiPSCs).
163. The method of any one of claims 159-162, wherein the iPSCs were previously cryopreserved.
164. The method of any one of claims 159-163, wherein the co-culturing is performed in in vitro maturation (IVM) media.
165. The method of claim 164, wherein the IVM media comprises a cell culture media.
166. The method of claim 164 or 165, wherein the IVM media comprises Medicult-IVM media.
167. The method of any one of claims 164-166, wherein the IVM media comprises one or more supplements.
168. The method of claim 167, wherein the one or more supplements comprise:(i) human serum albumin (HSA), optionally at a concentration of about 5 to about 15 mg / mL, further optionally at a concentration of 10 mg / mL;(ii) recombinant follicle stimulating hormone (rFSH), optionally at a concentration of about 70 mIU / mL to about 80 mIU / mL, further optionally at a concentration of 75 mIU / mL;(iii) human chorionic gonadotropin (hCG), optionally at a concentration of about 95 mIU / mL to about 105 mIU / mL, further optionally at a concentration of 100 mIU / mL;(iv) androstenedione, optionally at a concentration of about 495 ng / mL to about 505 ng / mL, further optionally at a concentration of 500 ng / mL;(v) doxycycline, optionally at a concentration of about 0.5 pg / mL to about 1 .5 pg / mL, further optionally at a concentration of 1 pg / mL; or any combination of the one or more supplements.
169. The method of any one of claims 159-168, wherein the induction of iPSCs to OSCs occurs for about 1 day to about 10 days, optionally wherein the induction occurs for about 5 days.
170. The method of any one of claims 159-169, wherein the iPSCs are cultured in a media comprising a matrix.171 . The method of claim 170, wherein the matrix comprises alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or a mixture thereof.
172. The method of claim 171 , wherein matrix comprises laminin, optionally wherein the laminin is laminin-521 .
173. The method of claim 171 or 172, wherein the matrix comprises vitronectin.
174. The method of any one of claims 159-173, wherein the iPSCs are reprogrammed using a transposase method to carry one or more inducible transcription factors.
175. The method of any one of claims 159-173, wherein the iPSCs are transformed via electroporation, liposome-mediated transformation, or viral-mediated gene transfer.
176. The method of any one of claims 159-175, wherein the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.
177. The method of any one of claims 159-176, wherein the iPSCs are contacted with a Wnt / p- catenin pathway activator.
178. The method claim 177, wherein the Wnt / p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.
179. The method of any one of claims 159-178, wherein the one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof.
180. The method of any one of claims 159-179, wherein the one or more OSCs express one or more of the following genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB.181 . The method of claim 179 or 180, wherein the one or more OSCs further express one or more of the genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4.
182. The method of claim 179 or 180, wherein the one or more OSCs further express one or more of the genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2.
183. The method of claim 179 or 180, wherein the one or more OSCs further express genes CDH2 and / or NOTCH2, optionally with no significant gene expression of RARRES2.
184. The method of any one of claims 159-179, wherein the one or more OSCs comprise one or more granulosa cells.
185. The method of any one of claims 159-179, wherein the one or more OSCs express NR2F2.
186. The method of any one of claims 159-179 and 185, wherein the one or more OSCs comprise one or more ovarian stroma cells.
187. The method of any one of claims 159-186, wherein the one or more OSCs comprise granulosa cells and ovarian stroma cells.
188. The method of any one of claims 159-187, wherein the one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.
189. The method of any one of claims 159-188, wherein the one or more OSCs have low or undetectable expression of one or more genes associated with pluripotency relative to an iPSC.
190. The method of claim 189, wherein the one or more genes associated with pluripotency comprise NANOG.191 . The method of claim 190, wherein the one or more genes associated with pluripotency comprise POU5F1 .
192. The method of any one of claims 159-191 , wherein one or more of the OSCs produce one or more growth factors.
193. The method of claim 192, wherein the growth factors comprise IGF, SCF, EGF, LIF, VEGF, BMPs, CNP, or any combination thereof.
194. The method of claim 192 or 193, wherein at least a portion of the one or more growth factors is secreted.
195. The method of any one of claims 159-194, wherein the one or more of the OSCs produce one or more steroids.
196. The method of claim 195, wherein the one or more steroids comprise estradiol, progesterone, or a combination thereof.
197. The method of claim 195 or 196, wherein the one or more steroids are produced in the presence of one or more hormones.
198. The method of claim 197, wherein the one or more hormones comprises FSH, androstenedione, or a combination thereof.
199. The method of any one of claims 194-198, wherein the one or more steroids are secreted.
200. The method of any one of claims 159-199, wherein the one or more oocytes retrieved from the subject are immature oocytes.201 . The method of any one of claims 159-200, wherein the co-culturing the one or more OSCs with one or more oocytes promotes the maturation of the one or more oocytes.
202. The method of any one of claims 159-201 , wherein the method further comprises harvesting the one or more oocytes for an assisted reproductive technology procedure.
203. The method of any one of claims 159-202, wherein the subject is undergoing ovarian stimulation prior to the retrieval of one or more oocytes.
204. The method of claim 203, wherein the ovarian stimulation comprises treatment with gonadotropin releasing hormone (GnRH).
205. The method of claim 203 or 204, wherein the ovarian stimulation comprises treatment with one or more GnRH analogs.
206. The method of claim 205, wherein the one or more GnRH analog is a GnRH agonist or antagonist.
207. The method of any one of claims 203-206, wherein the ovarian stimulation comprises one or more ovulatory triggers.
208. The method of claim 207, wherein the one or more ovulatory triggers comprises hCG.
209. The method of claim 207 or 208, wherein the one or more ovulatory trigger comprises a GnRH agonist, optionally wherein the GnRH agonist is leuprolide.
210. The method of any one of claims 203-209, wherein the ovarian stimulation comprises FSH treatment.211 . The method of any one of claims 203-209, wherein the ovarian stimulation does not comprise FSH treatment.
212. The method of claim 210, wherein the FSH treatment comprises 300 IU to 700 IU of FSH..
213. The method of claim 212, wherein the FSH treatment comprises 400 IU to 600 IU of FSH.
214. The method of any one of claims 210, 212, and 213, wherein the FSH treatment comprises 1 , 2,3, or more injections of FSH, optionally wherein the FSH treatment comprises a plurality of injections, wherein each injection comprises a dose of about 100 IU to about 200 IU of the FSH.
215. The method of any one of claims 203-214, wherein the ovarian stimulation further comprises clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, optionally wherein each dose is between 50 mg and 150 mg.
216. The method of claim 203-215, wherein the ovarian stimulation further comprises one or more hCG triggers.
217. The composition of claim 216, wherein the one or more hCG triggers comprises 2,500 IU to10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.
218. The method of any one of claims 159-217, wherein the one or more oocytes are in cumulus oocyte complexes (COCs).
219. The method of any one of claims 159-218, wherein the one or more oocytes comprise one or more denuded immature oocytes.
220. The method of claim 219, wherein all of the one or more oocytes are denuded immature oocytes.221 . The method of any one of claims 159-217, wherein the one or more oocytes are not denuded prior to or following co-culturing.
222. The method of any one of claims 159-221 , wherein the one or more oocytes comprise one or more germinal vesicle (GV)-containing oocytes.
223. The method of any one of claims 159-222, wherein the one or more oocytes comprise one or more oocytes in metaphase I (Ml).
224. The method of any one of claims 159-223, wherein the one or more oocytes comprise one or more oocytes in metaphase II (MH).
225. The method of any one of claims 159-224, wherein at least a portion of the one or more oocytes comprise one or more previously vitrified oocytes.
226. The method of any one of claims 159-225, wherein at least a portion of the one or more oocytes comprises one or more previously cryopreserved oocytes.
227. The method of any one of claims 159-226, wherein prior to and / or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV-stage to Mil-stage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Mil-stage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.
228. The method of claim 227, wherein the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.
229. The method of claim 227 or 228, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs.
230. The method of any one of claims 227-229, wherein the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes.231 . The method of any one of claims 159-230, wherein the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.
232. The method of any one of claims 159-231 , wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours.
233. The method of claim 232, wherein the one or more oocytes are cultured with the one or more OSCs for about 24 hours to about 28 hours.
234. The method of any one of claims 232 or 233, the method further comprising isolating one or more Mil-stage oocytes from the co-culture comprising the one or more oocytes retrieved from the subject with the one or more OSCs.
235. The method of any one of claims 232-234, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more blastocytes following contact with one or more mature sperm cells.
236. The method of any one of claims 159-235, wherein the one or more oocytes are cultured in direct contact with the one or more OSCs.
237. The method of claim 159-235, wherein the one or more oocytes do not directly contact the one or more OSCs.
238. The method of any one of claims 159-237, wherein the co-culture is a suspension co-culture.
239. The method of any one of claims 159-237, wherein the co-culture is an adherent co-culture.
240. A cell culture system comprising one or more ovarian support cells (OSCs), wherein the system promotes maturation of one or more oocytes.241 . The system of claim 240, wherein the one or more OSCs comprise one or more granulosa cells.
242. The system of claim 241 , wherein the one or more OSCs express FOXL2, AMHR2, CD82 or any combination thereof.
243. The system of any one of claims 240-242, wherein the one or more OSCs express one or more genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB.
244. The system of claim 242 or 243, wherein the one or more OSCs further express one or more genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4.
245. The system of claim 242 or 243, wherein the one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2.
246. The system of claim 242 or 243, wherein the one or more OSCs further express genes CDH2 and / or NOTCH2.
247. The system of any one of claims 240-246, wherein the one or more OSCs do not exhibit significant expression of RARRES2.
248. The system of any one of claims 240-247, wherein the one or more OSCs express NR2F2.
249. The system of any one of claims 240-248, wherein the one or more OSCs comprise ovarian stroma cells.
250. The system of any one of claims 240-249, wherein the one or more OSCs comprise granulosa cells and ovarian stroma cells.251 . The system of any one of claims 240-250, wherein the one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.
252. The system of any one of claims 240-251 , wherein the one or more OSCs are obtained by differentiation of a population of iPSCs, optionally wherein the iPSCs are hiPSCs.
253. The system of claim 252, wherein the hiPSCs express or overexpress one or more of the following transcription factors:(i) RUNX2;(ii) NR5A1 ;(iii) GATA4;(iv) FOXL2;(v) any combination of two of the transcription factors;(vi) any combination of three of the transcription factors; or(vii) a combination of all four of the transcription factors.
254. The system of claim 252 or 253, wherein the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.
255. The system of any one of claims 252-254, wherein the hiPSCs are contacted with a Wnt / p- catenin pathway activator.
256. The system of claim 255, wherein the Wnt / p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.
257. The system of any one of claims 240-256, wherein at least one of the one or more OSCs is encapsulated.
258. The system of claim 257, wherein the OSC(s) are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or any mixture thereof.
259. The system of claim 258, wherein the OSC(s) are encapsulated in laminin, optionally wherein the laminin is laminin-521 .
260. The system or system of claim 258 or 259, wherein the OSCs are encapsulated in vitronectin.261 . The system of any one of claims 240-260, wherein the one or more OSCs have lower expression, or undetectable expression, of one or more genes associated with pluripotency relative to an iPSC.
262. The system of claim 261 , wherein the one or more genes associated with pluripotency comprises NANOG.
263. The system of claim 262, wherein the one or more genes associated with pluripotency comprises POU5F1 .
264. The system of any one of claims 240-263, wherein at least some of the OSCs produce one or more growth factors.
265. The system of claim 264, wherein the one or more growth factors comprise insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMPs), C-type natriuretic peptide (CNP), or any combination thereof.
266. The system of claim 264 or 265, wherein at least a portion of the one or more growth factors are secreted.
267. The system of any one of claims 240-266, wherein one or more of the OSCs produce one or more steroids.
268. The system of claim 267, wherein the one or more steroids comprise estradiol, progesterone, or a combination thereof.
269. The system of claim 267 or 268, wherein the one or more steroids are produced in response to hormonal stimulation.
270. The system of claim 269, wherein the hormonal stimulation comprises FSH, androstenedione treatment, or a combination thereof.271 . The system of any one of claims 267-270, wherein at least a portion of the one or more steroids are secreted.
272. The system of any one of claims 240-271 , wherein the one or more OSCs are cryopreserved.
273. The system of any one of claims 240-272, further comprising an in vitro maturation (IVM) media.
274. The system of claim 273, wherein the IVM media comprises a cell culture media.
275. The system of claim 273 or 274, wherein the IVM media comprises Medicult-IVM media.
276. The system of any one of claims 273-275, wherein the IVM media comprises one or more supplements.
277. The system of claim 276, wherein the one or more supplements comprise:(i) human serum albumin (HSA), optionally at a concentration of about 5 to about 15 mg / mL, further optionally at a concentration of 10 mg / mL;(ii) recombinant follicle stimulating hormone (rFSH), optionally at a concentration of about 70 mIU / mL to about 80 mIU / mL, further optionally at a concentration of 75 mIU / mL;(iii) human chorionic gonadotropin (hCG), optionally at a concentration of about 95 mIU / mL to about 105 mIU / mL, further optionally at a concentration of 100 mIU / mL;(iv) androstenedione, optionally at a concentration of about 495 ng / mL to about 505 ng / mL, further optionally at a concentration of 500 ng / mL;(v) doxycycline, optionally at a concentration of about 0.5 pg / mL to about 1 .5 pg / mL, further optionally at a concentration of 1 pg / mL; or any combination of the one or more supplements.
278. The system of any one of claims 240-277, wherein the one or more oocytes are retrieved from a donor subject.
279. The system of claim 278, wherein the donor subject is from about 19 years old to about 45 years old.
280. The system of claim 278, wherein the subject is undergoing ovarian stimulation.281 . The system of claim 280, wherein the ovarian stimulation comprises treatment with gonadotropin releasing hormone (GnRH).
282. The system of claim 281 , wherein the ovarian stimulation comprises treatment with one or more GnRH analogs.
283. The system of claim 282, wherein the one or more GnRH analog is a GnRH agonist or antagonist.
284. The system of any one of claims 280-283, wherein the ovarian stimulation comprises one or more ovulatory triggers.
285. The system of claim 284, wherein the one or more ovulatory triggers comprises hCG.
286. The system of claim 284 or 285, wherein the one or more ovulatory trigger comprises a GnRH agonist, optionally wherein the GnRH agonist is leuprolide.
287. The system of any one of claims 280-286, wherein the ovarian stimulation comprises FSH treatment.
288. The system of any one of claims 280-286, wherein the ovarian stimulation does not comprise FSH treatment.
289. The system of claim 287, wherein the FSH treatment comprises 300 international units (IU) to 700 IU of FSH.
290. The system of claim 289, wherein the FSH treatment comprises 400 IU to 600 IU of FSH.291 . The system of any one of claims 287, 289, and 290, wherein the FSH treatment comprises 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment comprises a plurality of injections, wherein each injection comprises a dose of about 100 IU to about 200 IU of the FSH.
292. The system of any one of claims 280-291 , wherein the ovarian stimulation further comprises clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, optionally wherein each dose is between 50 mg and 150 mg.
293. The system of claim 280-292, wherein the ovarian stimulation further comprises one or more hCG triggers.
294. The system of claim 293, wherein the one or more hCG triggers comprises 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.
295. The system of any one of claims 240-294, wherein the one or more oocytes are in cumulus oocyte complexes (COCs).
296. The system of any one of claims 240-295, wherein the one or more oocytes comprise one or more denuded immature oocytes.
297. The system of claim 296, wherein all of the one or more oocytes are denuded immature oocytes.
298. The system of any one of claims 240-295, wherein the one or more oocytes are not denuded.
299. The system of any one of claims 240-298, wherein one or more oocytes comprise one or more germinal vesicle (GV)-containing oocytes.
300. The system of any one of claims 240-299, wherein one or more of the oocytes comprise one or more oocytes in metaphase I (Ml).301 . The system of any one of claims 240-300, wherein one or more of the oocytes comprise one or more oocytes in metaphase II (MH).
302. The system of any one of claims 240-301 , wherein at least a portion of the one or more oocytes comprise one or more previously vitrified oocytes.
303. The system of any one of claims 240-302, wherein at least a portion of the one or more oocytes comprise one or more previously cryopreserved oocytes.
304. The system of any one of claims 240-303, wherein the one or more oocytes are co-cultured with the one or more OSCs.
305. The system of claim 304, wherein prior to and / or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV-stage to Milstage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Mil-stage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.
306. The system of claim 305, wherein the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.
307. The system of claim 305 or 306, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs.
308. The system of any one of claims 305-307, wherein the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes.
309. The system of any one of claims 304-308, wherein the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.
310. The system of any one of claims 304-309, wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours.311 . The system of claim 310, wherein the one or more oocytes are co-cultured with the one or more OSCs for about 24 hours to about 28 hours.
312. The system claim 310 or 311 , wherein the one or more oocytes co-cultured with the one or more OSCs form one or more blastocytes following contact with one or more mature sperm cells.
313. The system of any one of claims 304-312, wherein the one or more oocytes are co-cultured in direct contact with the one or more OSCs.
314. The system of any one of claims 304-313, wherein the one or more oocytes do not directly contact the OSCs.
315. The system of any one of claims 304-314, wherein the culture system is a suspension culture.
316. The system of any one of claims 304-314, wherein the culture system is an adherent culture.
317. A kit comprising the composition of any one of claims 1 -77 and a package insert, wherein the package insert instructs a user of the kit to co-culture the population of ovarian support cells with one or more oocytes in accordance with the method of any one of claims 78-158.
318. A kit comprising a vial that contains a population of iPSCs and a package insert, wherein the package insert instructs a user of the kit to differentiate the population of iPSCs to one or more ovarian support cells in accordance with the method of any one of claims 159-239.
319. A kit comprising a vial that contains one or more OSCs and a package insert, wherein the package insert instructs a user of the kit to cultivate the cell culture system of any one of claims 240-316.