Application of in vitro regenerated platelets in the treatment of premature ovarian insufficiency

Abstract

The present invention belongs to the field of medical preparations. Specifically, the present invention provides a pharmaceutical composition comprising in vitro regenerated platelets and / or culture supernatant of the in vitro regenerated platelets, wherein the in vitro regenerated platelets exhibit the following characteristics: significantly increased levels of FGF and / or significantly increased levels of VEGF; the pharmaceutical composition is used to promote ovarian regeneration, improve ovarian endocrine function, improve ovarian reserve function, and / or improve reproductive outcomes in mice.

Description

Technical Field

[0001] The present invention belongs to the field of medical preparations, and in particular relates to the application of in vitro regenerated platelets in treating premature ovarian insufficiency. Background Art

[0002] Premature ovarian insufficiency (POI) refers to a clinical syndrome of ovarian dysfunction in women before the age of 40, characterized by menstrual abnormalities (amenorrhea, oligomenorrhea), follicle-stimulating hormone (FSH) > 25 U / L, and fluctuating estrogen levels.

[0003] The incidence of POI ranges from 1% to 4%, with a global analysis showing a prevalence of 3.7%. Common causes of POI include genetic, immune, infectious, iatrogenic, and environmental factors. However, the cause of POI in more than half of patients remains unknown, resulting in idiopathic POI.

[0004] During long-term diagnosis and treatment, experts have discovered that POI exhibits a progressive course. Domestic guidelines have introduced the concept of "subclinical POI," which refers to FSH levels between 15 and 25 IU / L. These patients are at high risk of developing POI. The diagnostic criteria for POI are two elevated FSH levels (>25 IU / L) within four weeks. When FSH levels exceed 40 IU / L twice, it progresses to premature ovarian failure (POF), signifying the advanced stage of POI. This not only affects sexual health and can lead to infertility, but can also cause long-term complications such as cardiovascular disease, osteoporosis, neurocognitive impairment, and even premature death.

[0005] Because the cause of POI remains unclear, there is still a lack of effective, specific treatments. Currently, the primary approach is symptomatic treatment, with the principles of regulating menstruation, relieving symptoms, improving quality of life, resolving fertility issues, and reducing long-term health problems and complications.

[0006] Therefore, it is urgent to develop POI treatment drugs with excellent effects, good stability, relatively clear ingredients and wide sources, and it is urgent to develop efficient and stable POI treatment methods. Summary of the Invention

[0007] The present invention provides an in vitro regenerated platelet and its application in treating premature ovarian insufficiency.

[0008] In the first aspect of the present invention, a pharmaceutical composition for treating premature ovarian insufficiency in a subject is provided, wherein the pharmaceutical composition comprises in vitro regenerated platelets and / or the culture supernatant of the platelets, and the in vitro regenerated platelets have the following characteristics: a significantly increased FGF level and / or a significantly increased VEGF level.

[0009] In another preferred embodiment, the "significantly increased FGF level" means that the FGF level X1 of the platelet is compared with the FGF level X0 of human platelets, X1 / X0≥1.1, preferably, X1 / X0≥1.3, more preferably, X1 / X0≥1.5, and optimally, X1 / X0≥2.

[0010] In another preferred embodiment, the "significantly increased VEGF level" means that the VEGF level Y1 of the platelet is compared with the VEGF level Y0 of human platelets, Y1 / Y0≥1.1, preferably, Y1 / Y0≥1.3, more preferably, Y1 / Y0≥1.5, and optimally, Y1 / Y0≥2.

[0011] In another preferred embodiment, the TGF-β level, EGF level, BDNF level, FGF level, VEGF level, PDGF-AB level, PDGF-BB level, and / or IGF level of the in vitro regenerated platelets do not show significant changes compared with those of human platelets.

[0012] In another preferred embodiment, the lack of significant change means there is no statistically significant difference.

[0013] In another preferred embodiment, the content of the in vitro regenerated platelets in the pharmaceutical composition is 0.2-3E9 / mL, preferably 1E9 / mL, and most preferably 2E9 / mL.

[0014] In another preferred embodiment, the composition is in the form of a liquid.

[0015] In another preferred embodiment, the culture system for in vitro regenerated platelets includes a first stage culture medium, a second stage culture medium, a third stage culture medium and / or a fourth stage culture medium.

[0016] Wherein, the first stage culture medium includes basal culture medium, BMP4, VEGF, activin A, and CHIR-99021;

[0017] The second stage culture medium includes StemSpan™-ACF Erythroid Expansion Medium, BMP4, VGEF, and bFGF;

[0018] The third stage culture medium includes basal culture medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, and PVA;

[0019] The fourth stage culture medium includes basal culture medium, TPO, SCF, and PFHM-II.

[0020] In another preferred embodiment, the method for preparing in vitro regenerated platelets comprises the following steps:

[0021] (S1) Culture and expand ESC / iPSC cells using stem cell culture medium;

[0022] (S2) performing mesodermal differentiation culture on the cells obtained in step (S1) using the first stage culture medium;

[0023] (S3) using the second stage culture medium to culture the cells obtained in step (S2) for differentiation into hemogenic endothelial cells and hematopoietic stem and progenitor cells;

[0024] (S4) continuing to culture the cells obtained in step (S3) with the third stage culture medium to obtain megakaryocytes;

[0025] (S5) Continuing to culture the cells obtained in step (S4) with the fourth stage culture medium to obtain platelets.

[0026] In a second aspect of the present invention, there is provided a use of the pharmaceutical composition according to the first aspect of the present invention for preparing a kit for treating premature ovarian insufficiency in a subject.

[0027] In a third aspect of the present invention, a kit for treating premature ovarian insufficiency in a subject is provided, the kit comprising:

[0028] (i) a test kit for premature ovarian insufficiency; and

[0029] (ii) The pharmaceutical composition according to the first aspect of the present invention.

[0030] In another preferred embodiment, the kit further comprises an instruction manual, wherein the instruction manual directs the use of the kit for treating premature ovarian insufficiency in a subject.

[0031] In a fourth aspect of the present invention, there is provided a method for regenerating platelets in vitro for preparing a pharmaceutical composition for one or more of the following uses:

[0032] (Z1) Treatment of premature ovarian insufficiency in subjects;

[0033] (Z2) promoting ovarian regeneration in subjects;

[0034] (Z3) Improve the estrous cycle of the subjects;

[0035] (Z4) Improve the sex hormone levels of the subjects;

[0036] (Z5) Improve the ovarian reserve function of the subjects;

[0037] (Z6) Improve reproductive outcomes in subjects.

[0038] In another preferred embodiment, the subject is a subject with subclinical premature ovarian insufficiency; a subject with clinical premature ovarian insufficiency; a subject with premature ovarian failure; a subject with spontaneous premature ovarian insufficiency; a subject with iatrogenic premature ovarian insufficiency; a subject with idiopathic premature ovarian insufficiency; a subject with immune premature ovarian insufficiency; or a subject with congenital ovarian dysgenesis syndrome (Turner syndrome).

[0039] In another preferred embodiment, the in vitro regenerated platelets are platelets differentiated in vitro from human pluripotent stem cells (ESCs) / iPSCs.

[0040] In another preferred embodiment, the in vitro regenerated platelets are platelets differentiated in vitro from hematopoietic stem cells HSPCs.

[0041] In another preferred embodiment, the HSPCs are peripheral blood-derived HSPCs and / or umbilical cord blood-derived HSPCs.

[0042] In another preferred embodiment, the hematopoietic stem cells include hematopoietic stem cells, multipotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells or a combination thereof.

[0043] In another preferred embodiment, the hematopoietic stem cells are CD34 + cell.

[0044] In another preferred embodiment, the in vitro regenerated platelets are platelets differentiated in vitro from megakaryocytes MK and / or megakaryocyte progenitors MKP.

[0045] In another preferred embodiment, the in vitro differentiation system includes a first stage culture medium, a second stage culture medium, a third stage culture medium and / or a fourth stage culture medium.

[0046] Wherein, the first stage culture medium includes basal culture medium, BMP4, VEGF, activin A, and CHIR-99021;

[0047] The second stage culture medium includes StemSpan™-ACF Erythroid Expansion Medium, BMP4, VGEF, and bFGF;

[0048] The third stage culture medium includes basal culture medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, and PVA;

[0049] The fourth stage culture medium includes basal culture medium, TPO, SCF, and PFHM-II.

[0050] In another preferred embodiment, the first stage culture medium includes STEMdiff™ APEL™ 2 medium, 30 ng / mL BMP4, 50 ng / mL VEGF, 25 ng / mL activin A, 1.5 uM CHIR-99021, and 1% PS.

[0051] In another preferred embodiment, the second stage culture medium comprises StemSpan™-ACF Erythroid expansion medium containing 30 ng / mL of BMP4, 50 ng / mL of VEGF, 50 ng / mL of bFGF, and 1% PS.

[0052] In another preferred example, the third stage culture medium includes STEMdiff™ APEL™ 2 medium, 50 ng / mL TPO, 50 ng / mL SCF, 25 ng / mL Flt3, 10 ng / mL IL3, 10 ng / mL IL6, 5% PFHM-II, 100 ug / mL PVA, and 1% PS.

[0053] In another preferred embodiment, the fourth stage culture medium includes STEMdiff™ APEL™ 2 medium, 50 ng / mL TPO, 50 ng / mL SCF, 5% PFHM-II, and 1% PS.

[0054] In another preferred embodiment, the treatment of premature ovarian insufficiency in the subject includes preventing premature ovarian insufficiency in the subject, improving the symptoms of premature ovarian insufficiency in the subject, maintaining the degree of premature ovarian insufficiency in the subject, slowing down the rate of deterioration of the symptoms of premature ovarian insufficiency in the subject, treating premature ovarian insufficiency in the subject, or a combination thereof.

[0055] In another preferred embodiment, the promoting of ovarian regeneration of the subject means that the ovarian weight A1 of the subject after administration of the pharmaceutical composition is compared with the ovarian weight A0 of the subject before administration of the pharmaceutical composition, A1 / A0≥1.05, more preferably, A1 / A0≥1.10.

[0056] In another preferred embodiment, A1-A0 is ≥ 0.005% of the mass of the subject, preferably ≥ 0.01% of the mass of the subject.

[0057] In another preferred embodiment, the improving the estrous cycle of the subject means that after administration of the pharmaceutical composition, the estrous period of the subject is significantly increased and / or the diestrus period of the subject is significantly reduced.

[0058] In another preferred example, the significant increase in the estrus of the subject means that the estrus B1 of the subject after administration of the pharmaceutical composition is compared with the estrus B0 of the subject before administration of the pharmaceutical composition, B1 / B0≥1.2, more preferably, B1 / B0≥1.5, and most preferably, B1 / B0≥2.

[0059] In another preferred embodiment, the significantly reduced estrus of the subject means that the estrus C1 of the subject after administration of the pharmaceutical composition is compared with the estrus C0 of the subject before administration of the pharmaceutical composition, C1 / C0≤0.9, more preferably, C1 / C0≤0.8, and most preferably, C1 / C0≤0.6.

[0060] In another preferred embodiment, improving the subject's sex hormone level includes: improving the subject's AMH level, improving the subject's estrogen level, improving the subject's FSH level, improving the subject's LH level, or a combination thereof.

[0061] In another preferred example, the improvement of the AMH level of the subject means that the AMH level D1 of the subject after administration of the pharmaceutical composition is compared with the AMH level D0 of the subject before administration of the pharmaceutical composition, D1 / D0≥1.2, more preferably, D1 / D0≥1.5, and most preferably, D1 / D0≥2.

[0062] In another preferred embodiment, the improvement of the estrogen level of the subject means that the estrogen level E1 of the subject after administration of the pharmaceutical composition is compared with the estrogen level E0 of the subject before administration of the pharmaceutical composition, E1 / E0≥1.1, more preferably, E1 / E0≥1.3, and most preferably, E1 / E0≥1.5.

[0063] In another preferred example, the improvement of the subject's FSH level means that the subject's FSH level F1 after administration of the pharmaceutical composition is compared with the subject's FSH level F0 before administration of the pharmaceutical composition, F1 / F0≤0.9, more preferably, F1 / F0≤0.8, and most preferably, F1 / F0≤0.6.

[0064] In another preferred example, the improvement of the FSH level of the subject means that the LH level G1 of the subject after administration of the pharmaceutical composition is compared with the LH level G0 of the subject before administration of the pharmaceutical composition, G1 / G0≤0.95, more preferably, G1 / G0≤0.9, and most preferably, G1 / G0≤0.85.

[0065] In another preferred embodiment, the improvement of the ovarian reserve function of the subject refers to a significant increase in the total number of follicles and / or a significant increase in the number of growing follicles in the subject.

[0066] In another preferred example, the significant increase in the total number of follicles in the subject means that the total number of follicles H1 of the subject after administration of the pharmaceutical composition is compared with the total number of follicles H0 of the subject before administration of the pharmaceutical composition, H1 / H0≥1.3, more preferably, H1 / H0≥1.5, and most preferably, H1 / H0≥2.

[0067] In another preferred example, the significant increase in the number of growing follicles in the subject means that the number of growing follicles J1 of the subject after administration of the pharmaceutical composition is compared with the number of growing follicles J0 of the subject before administration of the pharmaceutical composition, J1 / J0 ≥ 1.3, more preferably, J1 / J0 ≥ 1.5, and most preferably, J1 / J0 ≥ 2.

[0068] In another preferred embodiment, the improvement of the reproductive outcome of the subject includes: a significant increase in the number of ovulations of the subject, a significant decrease in the number of deformed eggs, a significant increase in the number of MII stage eggs, and a significant increase in the number of two-cell embryos.

[0069] In another preferred example, the significant increase in the number of ovulations of the subject means that the number of ovulations K1 of the subject after administration of the pharmaceutical composition is compared with the number of ovulations K0 of the subject before administration of the pharmaceutical composition, K1 / K0≥1.3, more preferably, K1 / K0≥1.5, and most preferably, K1 / K0≥2.

[0070] In another preferred example, the significant reduction in the number of deformed eggs means that the number of deformed eggs L1 of the subject after administration of the pharmaceutical composition is compared with the number of deformed eggs L0 of the subject before administration of the pharmaceutical composition, L1 / L0≤0.2, more preferably, L1 / L0≤0.1, and most preferably, L1 / L0≤0.05.

[0071] In another preferred example, the significantly reduced number of deformed eggs comprises comparing the number of deformed eggs L1 of the subject after administration of the pharmaceutical composition with the number of deformed eggs L0 of the subject before administration of the pharmaceutical composition, L1 / L0=0.

[0072] In another preferred example, the significant increase in the MII stage eggs of the subject means that after the administration of the pharmaceutical composition, the MII stage eggs M1 of the subject are compared with the MII stage eggs M0 of the subject before the administration of the pharmaceutical composition, M1 / M0 ≥ 2, more preferably, M1 / M0 ≥ 3, and most preferably, M1 / M0 ≥ 4.

[0073] In another preferred example, the significant increase in the number of two-cell embryos of the subject means that the number N1 of the two-cell embryos of the subject after administration of the pharmaceutical composition is compared with the number N0 of the two-cell embryos of the subject before administration of the pharmaceutical composition, N1 / N0≥1.5, more preferably, N1 / N0≥2, and most preferably, N1 / N0≥3.

[0074] In another preferred embodiment, the culture system for in vitro regenerated platelets includes a first stage culture medium, a second stage culture medium, a third stage culture medium and / or a fourth stage culture medium.

[0075] Wherein, the first stage culture medium includes basal culture medium, BMP4, VEGF, activin A, and CHIR-99021;

[0076] The second stage culture medium includes StemSpan™-ACF Erythroid Expansion Medium, BMP4, VGEF, and bFGF;

[0077] The third stage culture medium includes basal culture medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, and PVA;

[0078] The fourth stage culture medium includes basal culture medium, TPO, SCF, and PFHM-II.

[0079] In another preferred embodiment, the basal culture medium includes E8 culture medium, complete culture medium, StemSpan TM SFEM Hematopoietic Medium, StemPro-34, STEMdiff™ APEL™2 Medium, and / or BPEL.

[0080] In another preferred embodiment, the basal culture medium is STEMdiff™ APEL™2 medium.

[0081] In another preferred embodiment, the first stage culture medium includes STEMdiff™ APEL™ 2 medium, 20-40 ng / mL BMP4, 40-60 ng / mL VEGF, 20-30 ng / mL activin A, and 1-3 uM CHIR-99021.

[0082] In another preferred embodiment, the second stage culture medium comprises StemSpan™-ACF Erythroid Expansion Medium containing 20-40 ng / mL of BMP4, 40-60 ng / mL of VEGF, and 40-60 ng / mL of bFGF.

[0083] In another preferred embodiment, the third stage culture medium includes STEMdiff ™ APEL™ 2 culture medium, 40-60 ng / mL TPO, 40-60 ng / mL SCF, 20-30 ng / mL Flt3, 5-15 ng / mL IL3, 5-15 ng / mL IL6, 1%-10% PFHM-II, 50-200 ug / mL PVA.

[0084] In another preferred embodiment, the fourth stage culture medium includes STEMdiff™ APEL™ 2 medium, 40-60 ng / mL TPO, 40-60 ng / mL SCF, and 1%-10% PFHM-II.

[0085] In another preferred embodiment, the culture medium comprises 0.5%-3% PS.

[0086] In another preferred embodiment, the first stage culture medium includes STEMdiff™ APEL™ 2 medium, 30 ng / mL BMP4, 50 ng / mL VEGF, 25 ng / mL activin A, 1.5 uM CHIR-99021, and 1% PS.

[0087] In another preferred embodiment, the second stage culture medium comprises StemSpan™-ACF Erythroid expansion medium containing 30 ng / mL of BMP4, 50 ng / mL of VEGF, 50 ng / mL of bFGF, and 1% PS.

[0088] In another preferred example, the third stage culture medium includes STEMdiff™ APEL™ 2 medium, 50 ng / mL TPO, 50 ng / mL SCF, 25 ng / mL Flt3, 10 ng / mL IL3, 10 ng / mL IL6, 5% PFHM-II, 100 ug / mL PVA, and 1% PS.

[0089] In another preferred embodiment, the fourth stage culture medium includes STEMdiff™ APEL™ 2 medium, 50 ng / mL TPO, 50 ng / mL SCF, 5% PFHM-II, and 1% PS.

[0090] In another preferred embodiment, the culture system includes M1 medium, which contains StemSpan™ SFEMII medium, IL-3, IL-6, SCF, TPO and FLt.

[0091] In another preferred embodiment, the culture system includes M2 culture medium, which contains IMDM culture medium, N2B27, NEAA, glutamic acid, ITS, ascorbic acid, IL-6, SCF, and TPO.

[0092] In another preferred example, the M1 medium contains StemSpan™ SFEM II medium, 5-50 ng / ml IL-3, 5-50 ng / ml IL-6, 5-50 ng / ml SCF, 5-50 ng / ml TPO and 5-50 ng / ml FLt.

[0093] In another preferred embodiment, the M2 culture medium comprises IMDM culture medium, N2B27, NEAA, glutamic acid, ITS, ascorbic acid, 5-50 ng / ml IL-6, 5-50 ng / ml SCF, and 5-50 ng / ml TPO.

[0094] In another preferred embodiment, the culture system includes a stem cell culture medium.

[0095] In another preferred embodiment, the stem cell culture medium is mTeSR1 medium.

[0096] In another preferred embodiment, the method for preparing in vitro regenerated platelets comprises the following steps:

[0097] (S1) Culture and expand ESC / iPSC cells using stem cell culture medium;

[0098] (S2) performing mesodermal differentiation culture on the cells obtained in step (S1) using the first stage culture medium;

[0099] (S3) using the second stage culture medium to culture the cells obtained in step (S2) for differentiation into hemogenic endothelial cells and hematopoietic stem and progenitor cells;

[0100] (S4) continuing to culture the cells obtained in step (S3) with the third stage culture medium to obtain megakaryocytes;

[0101] (S5) Continuing to culture the cells obtained in step (S4) with the fourth stage culture medium to obtain platelets.

[0102] In another preferred embodiment, the stem cell culture medium includes: mTesR1 culture medium containing Y27632 or mTesR1 culture medium not containing Y27632.

[0103] In another preferred embodiment, step (S1) comprises: culturing ESC / iPSC cells with mTesR1 medium containing Y27632 for 12-24 hours; and then continuing to culture with mTesR1 medium not containing Y27632 until the confluence reaches 70%-80%.

[0104] In another preferred embodiment, the culturing time in step (S2) is 2 days.

[0105] In another preferred embodiment, the culturing time in step (S3) is 5 days.

[0106] In another preferred embodiment, the culturing time in step (S4) is 7 days.

[0107] In another preferred embodiment, the culture time in step (S5) is 5-8 days, preferably 7 days, and most preferably 6 days.

[0108] In another preferred embodiment, the method for preparing in vitro regenerated platelets comprises the following steps:

[0109] (Q1) culturing hematopoietic stem cells with the M1 medium to obtain megakaryocyte progenitor cells;

[0110] (Q2) Continuing to culture the megakaryocyte progenitor cells obtained in step (Q1) using the M2 medium to obtain platelets.

[0111] In another preferred embodiment, the hematopoietic stem cells are CD34 + cell.

[0112] In another preferred embodiment, the culturing time in step (Q1) is 7 days.

[0113] In another preferred embodiment, the culturing time in step (Q2) is 10-12 days.

[0114] In another preferred embodiment, the in vitro regenerated platelets have the following characteristics: a significantly increased FGF level and / or a significantly increased VEGF level.

[0115] In another preferred embodiment, the "significantly increased FGF level" means that the FGF level X1 of the platelet is compared with the FGF level X0 of human platelets, X1 / X0≥1.1, preferably, X1 / X0≥1.3, more preferably, X1 / X0≥1.5, and optimally, X1 / X0≥2.

[0116] In another preferred embodiment, the "significantly increased VEGF level" means that the VEGF level Y1 of the platelet is compared with the VEGF level Y0 of human platelets, Y1 / Y0≥1.1, preferably, Y1 / Y0≥1.3, more preferably, Y1 / Y0≥1.5, and optimally, Y1 / Y0≥2.

[0117] In a fifth aspect of the present invention, an in vitro regenerated platelet for treating premature ovarian insufficiency in a subject is provided, wherein the in vitro regenerated platelet has the following characteristics: a significantly increased FGF level and / or a significantly increased VEGF level.

[0118] In another preferred embodiment, the "significantly increased FGF level" means that the FGF level X1 of the platelet is compared with the FGF level X0 of human platelets, X1 / X0≥1.1, preferably, X1 / X0≥1.3, more preferably, X1 / X0≥1.5, and optimally, X1 / X0≥2.

[0119] In another preferred embodiment, the "significantly increased VEGF level" means that the VEGF level Y1 of the platelet is compared with the VEGF level Y0 of human platelets, Y1 / Y0≥1.1, preferably, Y1 / Y0≥1.3, more preferably, Y1 / Y0≥1.5, and optimally, Y1 / Y0≥2.

[0120] In another preferred embodiment, the culture system for in vitro regenerated platelets includes a first stage culture medium, a second stage culture medium, a third stage culture medium and / or a fourth stage culture medium.

[0121] Wherein, the first stage culture medium includes basal culture medium, BMP4, VEGF, activin A, and CHIR-99021;

[0122] The second stage culture medium includes StemSpan™-ACF Erythroid Expansion Medium, BMP4, VGEF, and bFGF;

[0123] The third stage culture medium includes basal culture medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, and PVA;

[0124] The fourth stage culture medium includes basal culture medium, TPO, SCF, and PFHM-II.

[0125] In another preferred embodiment, the method for preparing in vitro regenerated platelets comprises the following steps:

[0126] (S1) Culture and expand ESC / iPSC cells using stem cell culture medium;

[0127] (S2) performing mesodermal differentiation culture on the cells obtained in step (S1) using the first stage culture medium;

[0128] (S3) using the second stage culture medium to culture the cells obtained in step (S2) for differentiation into hemogenic endothelial cells and hematopoietic stem and progenitor cells;

[0129] (S4) continuing to culture the cells obtained in step (S3) with the third stage culture medium to obtain megakaryocytes;

[0130] (S5) Continuing to culture the cells obtained in step (S4) with the fourth stage culture medium to obtain platelets.

[0131] In the sixth aspect of the present invention, a method for treating premature ovarian insufficiency is provided, the method comprising administering to a subject the in vitro regenerated platelets described in the fourth aspect of the present invention, the pharmaceutical composition described in the first aspect of the present invention, or the kit described in the second aspect of the present invention.

[0132] In another preferred embodiment, the subject is a human.

[0133] In another preferred embodiment, the administered dose is 0.2E9-6E9 per ovary, preferably 1E9-4E9 per ovary.

[0134] In another preferred embodiment, the subject is a non-human mammal.

[0135] In another preferred embodiment, the subject is a mouse.

[0136] In another preferred embodiment, the administered dose is 0.1E7-5E7 per ovary, preferably 0.5E7-2E7 per ovary.

[0137] In another preferred embodiment, the administration cycle is ≥21 days, preferably ≥28 days.

[0138] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1 The data show the adaptive feeding period of each group of mice to the end of the sampling period ( Figure 1 A) Weight changes and ( Figure 1 B) Endpoint ovarian weight (n=6).

[0140] Figure 2 The changes in estrous cycle of mice in each group are shown (n=6).

[0141] Figure 3 The serum hormone levels of mice in each group are shown. Specifically, the serum hormone levels of mice at the end of the first estrous cycle are shown. Figure 3 A)AMH, Figure 3 B) E2, ( Figure 3 C) FSH, ( Figure 3 D) LH content; and the serum level of mice at the end of the fourth estrous cycle ( Figure 3 E)AMH, Figure 3 F) E2, ( Figure 3 G) FSH, ( Figure 3 H) LH content; biological n = 3, technical replicate n = 3.

[0142] Figure 4 The gross pathological appearance of the mouse ovary and the follicle counts are shown. Figure 4 A shows a scanned image of a mouse ovarian tissue section stained with HE; Figure 4 B shows the total follicle count (including primordial follicles, primary follicles, secondary follicles, antral follicles and atretic follicles), n = 3; Figure 4 C shows the number of growing follicles (including primary follicles, secondary follicles, and antral follicles), n = 3.

[0143] Figure 5 Mouse ovulation and two-cell embryos are shown. Figure 5 A shows the morphology of oocytes, MII stage oocytes and two-cell embryos of mice in each group; Figure 5 B shows the total number of ovulations in each group of mice, n = 3; Figure 5 C shows the number of MII stage eggs in each group of mice, n = 3; Figure 5 D shows the counts of two-cell embryos of mice in each group, n=3.

[0144] Figure 6 It was shown that the platelets of the present invention protect against apoptosis of POI ovarian cells by releasing cytokines. DETAILED DESCRIPTION

[0145] After extensive and in-depth research, the inventors unexpectedly discovered, for the first time, a novel in vitro differentiated platelet system. This in vitro differentiated platelet system significantly improves ovarian development and function in women with POI, including ovarian volume, texture, follicular development, hormone levels, and reproductive outcomes. Furthermore, it offers advantages such as clear composition, high safety, and simple preparation. Furthermore, by constructing an in vitro cell model and treating it with cytokines released by PLTs, the inventors observed a significant anti-apoptotic effect of the platelets. This work was completed on this basis.

[0146] the term

[0147] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0148] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0149] As used herein, the terms "comprises," "includes," and "comprising" are used interchangeably to encompass not only closed definitions but also semi-closed and open definitions. In other words, the terms encompass "consisting of," "consisting essentially of."

[0150] Where a numerical range is provided, it is understood that every intervening integer of that value, every tenth of each intervening integer of that value, between the upper and lower limits of that range, and any other intervening values ​​in the stated range are encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any expressly excluded limits in the stated range. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," and the like.

[0151] As used herein, the terms "in vitro regeneration" and "in vitro differentiation" are used interchangeably and refer to the in vitro differentiation of hematopoietic stem cells HSPCs, megakaryocytes MKs, and / or megakaryocyte progenitors MKPs into platelets.

[0152] As used herein, the level of TGF-β, EGF, BDNF, FGF, VEGF, PDGF-AB, PDGF-BB, and / or IGF in the in vitro regenerated platelets compared to human platelets is not significantly changed, which means that the level of one or more cytokines in the in vitro regenerated platelets is increased, but not more than 1.05 times, or decreased, but decreased to not less than 95%, compared to human platelets, and there is no statistically significant difference overall. For example, the TGF-β level P1 of the in vitro regenerated platelets is compared to the TGF-β level P0 of the human platelets, and 0.95≤P1 / P0≤1.05, preferably 0.97≤P1 / P0≤1.03, and most preferably 0.99≤P1 / P0≤1.01.

[0153] Premature ovarian insufficiency (POI)

[0154] Premature ovarian insufficiency (POI) refers to a clinical syndrome of ovarian dysfunction in women before the age of 40, characterized by menstrual abnormalities (amenorrhea, oligomenorrhea), follicle-stimulating hormone (FSH) > 25 U / L, and fluctuating estrogen levels.

[0155] Over the long term, POI exhibits a progressive course. "Subclinical POI" refers to FSH levels between 15 and 25 IU / L, placing these patients at high risk for progression to POI. The diagnostic criteria for POI are two elevated FSH levels (>25 IU / L) within four weeks. When FSH levels exceed 40 IU / L twice, it progresses to premature ovarian failure (POF), marking the advanced stage of POI. This not only impacts sexual health and leads to infertility, but can also lead to long-term complications such as cardiovascular disease, osteoporosis, neurocognitive impairment, and even premature death.

[0156] In the present invention, the "premature ovarian insufficiency" includes "subclinical POI", "primary and secondary POI" and "premature ovarian failure".

[0157] Symptoms and common treatments of POI

[0158] Treatment for POI currently includes mental health management, hormone therapy, fertility treatment, and treatment of other complications and health management. Symptomatic treatment is generally the primary approach, and specific treatments are lacking. Multiple treatments, such as mental health management, fertility treatment, hormone therapy, and non-hormonal therapies, are often used in parallel.

[0159] Hormone therapy. The current "European Society of Human Reproduction and Embryology Clinical Diagnosis and Treatment Guidelines" and the domestic "Clinical Diagnosis and Treatment Expert Consensus" point out that unless there are absolute contraindications, all POI patients should receive estrogen therapy to reduce the risk of osteoporosis and cardiovascular disease. However, there is currently limited research on the optimal regimen for hormone replacement therapy, and there are still large differences in clinical practice in terms of administration route, dosage, and type of estrogen and progesterone preparations. Hormone replacement therapy recommends continuous medication, which should be started at the time of diagnosis until the average age of natural menopause, in order to induce sexual development, maintain secondary sexual characteristics and bone mass, improve the quality of life of patients, and reduce the incidence and mortality of long-term complications.

[0160] Treatment and health management of other complications. Patients with related systemic complications should receive personalized treatment and management, including those addressing bone health, cardiovascular and metabolic health, neurological health, genitourinary health, and sexual health. Because POI is the primary cause of these complications, treatment remains focused on first-line treatment for POI, with regular monitoring of bone density.

[0161] Non-hormonal treatments. For POI patients who have contraindications to hormone replacement therapy or are temporarily unwilling or unsuitable for hormone replacement therapy, non-hormonal agents can be used to alleviate symptoms of hypoestrogenism. However, research data on the benefits of non-hormonal therapies (such as phytoestrogens and traditional Chinese medicine) for bone health and cardiovascular and metabolic health in POI patients is limited, and no consensus has yet been reached.

[0162] Mesenchymal stem cell (MSC) therapy. Previous studies have demonstrated efficacy in animal models of POI using MSCs derived from various sources, including bone marrow, placenta, and umbilical cord. The mechanism of action is primarily paracrine stem cell effects, including the release of various cytokines, promotion of neovascularization, anti-inflammatory, anti-apoptotic, anti-fibrotic, and immunomodulatory effects. In the context of POI, conditioned medium derived from stem cells has been observed to protect the ovaries from age-related damage, highlighting the key role of paracrine mechanisms in improving ovarian function. Further studies have shown that MSCs can upregulate the release of hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-8, IL-10, IL-11, and IL-15, while reducing the secretion of tumor necrosis factor-alpha (TNFα) and IL-6. However, the effects of stem cell differentiation on patients with POI remain uncertain. It is currently unknown whether MSCs can differentiate into oocytes, although previous animal studies have shown that MSCs can differentiate into granulosa cells, suggesting their potential to restore ovarian endocrine function and folliculogenesis in rodents. It is worth noting that the possibility of stem cells becoming malignant cannot be ignored.

[0163] Platelet-Rich Plasma (PRP)

[0164] PRP, also known as autologous conditioned plasma, is a platelet-rich protein concentrate extracted from fresh whole blood. It is centrifuged to remove red blood cells, thereby exerting its anti-inflammatory and regenerative properties. Over the past two decades, PRP has gradually attracted significant attention from medical professionals for its potential to enhance regenerative processes. Numerous clinical studies have demonstrated the potential of PRP for tissue regeneration and repair in various areas, such as dermatology, osteoarthritis, intervertebral disc degeneration, and infertility.

[0165] Intrauterine injection of PRP promotes all aspects of follicular maturation and has a positive impact on pregnancy outcomes in patients with unexplained recurrent implantation failure. Furthermore, PRP treatment also has positive implications for improving ovarian physiological structure and reserve function.

[0166] PRP is believed to improve ovarian function by enhancing the vitality of primary and preantral follicles and promoting the development of isolated human primordial and primary follicles to the preantral stage. Another mechanism of action is believed to be the abundant presence of PLTs and α-granules in PRP. Activated PLTs release various cytokines, such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), VEGF, IGF-1, thrombospondin 1 (THBS1), EGF, and HGF. These cytokines promote the restoration of the ovarian microenvironment by reducing oxidative stress and promoting angiogenesis. A third mechanism suggests that PRP's restorative effects on the ovaries are achieved through sphingosine-1-phosphate (S1P). PLT α-granules are rich in S1P, which is released upon activation. Measurements of S1P levels per 1×10 7 The S1P content in each PLT exceeds 300 nM. If there is a linear relationship between S1P concentration and PLT count, it is estimated that approximately 9 μM of S1P was delivered in studies of activated PRP infusion into the ovaries. Currently, PRP treatment for POI has been clinically tested on a small scale as a non-first-line treatment. Evidence-based medical evidence suggests that PRP injection (intravenous / intraovarian) can improve ovarian reserve and function.

[0167] However, despite extensive animal models and clinical evidence demonstrating the effectiveness of PRP for POI treatment, current PRP preparation and injection protocols vary between centers. Due to heterogeneity in PRP sources and variations in administration methods, the extent to which intraovarian PRP injection improves ovarian function remains controversial, and there is no clinical consensus on the optimal regimen. Therefore, the development of PRP alternatives with clearly defined ingredients is urgently needed for standardized clinical application.

[0168] In summary, although PRP has achieved certain results in clinical trials, its heterogeneity, accessibility, and differences in preparation methods have led to unstable clinical treatment effects. Since PRP is isolated from the patient's own peripheral blood, on the one hand, if the patient is older (for example, ≥35 years old), the patient's own PRP is also at risk of aging, which will affect the treatment effect on POI. On the other hand, if the patient also has other underlying diseases and is not suitable for collecting the peripheral blood required for PRP preparation, the accessibility of PRP is limited. Thirdly, the preparation method of PRP has not yet been standardized, resulting in uncertainty in the dosage (such as the number of PLTs per milliliter of PRP), which leads to unstable treatment effects.

[0169] Platelets of the present invention

[0170] The platelets of the present invention are the in vitro regenerated platelets provided by the present invention. The starting cells for the in vitro platelet regeneration of the present invention are hematopoietic stem / progenitor cells (HSPCs) or human ESC / iPSCs isolated from mobilized peripheral blood of healthy individuals that meet the inclusion criteria.

[0171] This invention applies in vitro regenerated PLT to the treatment of POI patients, aiming to address the issues not addressed by PRP. The in vitro regenerated PLT described in this invention is administered intraovarianally under ultrasound guidance at a dose of (1-4) E9 per ovary. Single or regular injections are administered, depending on the therapeutic objective and effectiveness.

[0172] The main advantages of the present invention include:

[0173] (a) The present invention provides a use of in vitro regenerated platelets in the treatment of premature ovarian insufficiency, providing a potential therapeutic drug for premature ovarian insufficiency that is highly effective, stable, safe, and has no obvious side effects.

[0174] (b) The in vitro regenerated platelets provided by the present invention are not limited by the variability of the patient's autologous platelet-rich plasma, have a clear dosage, are simple to prepare, have uniform properties, stable performance, safety, and strong accessibility, which can make up for the disadvantages of autologous platelet-rich plasma in clinical applications.

[0175] (c) Upon activation, regenerated PLTs in vitro release a rich array of cytokines, including but not limited to PDGF, TGF-β, bFGF, VEGF, IGF-1, THBS1, EGF, and HGF. These cytokines are crucial for regulating the repair of the ovarian tissue microenvironment. Regenerated PLTs produced through a specific differentiation process can release a clearly controllable amount of cytokines, making it easy to manage dosage in clinical practice.

[0176] (d) The present invention provides a safe, efficient, stable, controllable, easy-to-operate, and easily-implemented treatment method for premature ovarian insufficiency based on the in vitro regenerated platelets provided by the present invention.

[0177] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally performed under conventional conditions, such as those described in Qiao Zilin et al., "Animal Cell Culture Engineering Experimental Guide", 2024, or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0178] Example 1: Experimental method.

[0179] First, a POI model was established in NOD-SCID mice by intraperitoneal injection of 1.2 mg / kg busulfan and 12 mg / kg cyclophosphamide for 7 days. The model group was then divided into groups and administered according to the schedule shown in Table 1.

[0180] Table 1 Experimental plan

[0181]

[0182] The positive control (positive) sample is PRP isolated from donor peripheral blood that meets the same inclusion criteria as the test sample. The isolation method refers to previous studies, and the absolute number of PLTs after counting is consistent with that of the test sample. The donor is a young and healthy subject. However, in actual autologous platelet-rich plasma, due to the influence of patients' older age and underlying diseases, the number and quality of PLTs in PRP are often significantly inferior to the PRP used in the examples of the present invention.

[0183] Among them, the PLT preservation solution is a phosphate buffer solution that meets the standards for pharmaceutical excipients.

[0184] Superovulation and co-housing of male and female mice: On the 21st day of modeling, 10 IU PMSG and 10 IU hCG were injected at 48 h intervals for superovulation, with 3 female mice and 1 male mouse in each group.

[0185] Humane endpoints for experimental animals: During the experiment, if mice showed abnormal movement, paralysis, or respiratory abnormalities, they were euthanized.

[0186] Body weight test: Body weight test was performed once before modeling, and twice a week during modeling and treatment.

[0187] Clinical observation: Clinical observation was conducted once a day during the adaptive feeding period and the experimental period. The observation contents included but were not limited to the animal's mental state, diet, etc.

[0188] Estrous Cycle Monitoring: Vaginal smears and Giemsa staining were used to monitor the estrous cycle of mice for 21 days after dosing. During the proestrus stage, vaginal smears were dominated by nucleated epithelial cells with a small number of leukocytes. During the estrus stage, vaginal smears were dominated by anucleated keratinized squamous cells, which were large, flat, and had irregular margins. During the metestrus stage, the keratinized epithelium decreased, and numerous nucleated epithelial cells and leukocytes appeared. During the diestrus stage, leukocytes predominated.

[0189] Serum hormone level detection: At the end of the first estrus and 21 days after administration, venous blood was collected from mice to separate serum, which was frozen and stored. Reproductive hormone (FSH, LH, E2, AMH) levels were then detected according to the kit instructions.

[0190] Endpoint sampling and processing: 48 hours after cohabitation, mice were killed and their ovaries were removed, and blastomeres in the oviduct were collected for counting and statistics. The remaining mice underwent oviduct retrieval on the day after hCG injection for morphological observation and statistics, and eggs, granulosa cells, and ovaries were collected. The ovaries were fixed in 4% paraformaldehyde for paraffin section preparation and HE staining.

[0191] Statistics of MII stage oocytes and two-cell embryos: The number of first polar bodies released and two-cell embryos in the collected oocytes was counted.

[0192] Ovarian tissue pathology: Paraffin sections were prepared from fixed ovarian tissues. Each section was 3 μm thick. After HE staining and scanning, the ovarian morphology and histological changes were observed. The number of follicles and corpora lutea at each level was counted.

[0193] Statistical Analysis: Results are expressed as mean ± SEM. P < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0194] Example 2: Effects of the PLT of the present invention on the body weight and ovary weight of mice.

[0195] There were no abnormalities in the activity and eating status of the experimental animals during the experiment, and all mice reached the end point of the experiment.

[0196] The mouse body weight results were as follows Figure 1 As shown in Figure A, compared with the control group, the model group had a significant decrease in body weight, and the body weight increased after treatment with PLT of the present invention. The weight change indicates that the health of the mice has improved after treatment.

[0197] Ovarian weight results Figure 1 As shown in Figure B, by weighing the ovaries of the endpoint mice, it was found that the ovarian weight of the POI model mice decreased significantly. After treatment with the PLT of the present invention and the positive control, the ovarian weight was significantly increased, indicating that the drug promotes the regeneration of ovarian tissue.

[0198] Example 3: Effects of the PLT of the present invention on the estrous cycle of mice.

[0199] The results are as follows Figure 2 As shown, the results showed that compared with the control group, the estrus stage of the model mice was significantly shortened, while the diestrus stage was significantly prolonged.

[0200] After treatment with PLT of the present invention, the number of days in estrus significantly increased to more than 2 times that of the model group, while the diestrus period was significantly shortened to 2 / 3 of the model group. The improvement in estrus cycle indicates that ovarian function has been significantly improved after treatment.

[0201] The effect of the positive control on the estrous cycle of mice is basically equivalent to that of the PLT of the present invention, indicating that the same amount of PLT of the present invention and PLT derived from PRP of healthy young people have equivalent effects on ovarian function.

[0202] Example 4: Effects of the PLT of the present invention on hormone levels in mice.

[0203] At the end of the first estrous cycle after drug administration, the levels of reproductive hormones in mice were detected. Figure 3 A- Figure 3 As shown in D.

[0204] The results showed that the AMH and estrogen (E2) levels of the model group mice were significantly reduced, and the FSH and LH levels were significantly increased. After treatment with the PLT of the present invention and the positive control product, the hormone levels of the mice were improved. Specifically, after treatment, AMH ( Figure 3 A) and E2 ( Figure 3 B) levels increased significantly, while FSH ( Figure 3 C) and LH ( Figure 3 D) levels were significantly decreased.

[0205] On the 21st day after administration, the sex hormone levels of mice in each group were tested again. Figure 3 E- Figure 3 As shown in H.

[0206] Discovery of AMH ( Figure 3 E), E2 ( Figure 3 F), FSH ( Figure 3 G) and LH ( Figure 3 H) all continued to improve, indicating that the endocrine function of the ovary was improved.

[0207] The positive control group showed a more significant improvement in sex hormone levels in mice compared to the PLT of the present invention. On the one hand, the effectiveness of autologous PRP is limited by the patient's own health, and its improvement in sex hormones is likely inferior to that of the positive control group of the present invention. Therefore, the improvement in sex hormone levels by the PLT of the present invention and that by autologous PRP may be comparable. On the other hand, PRP contains a variety of factors and other beneficial ingredients in addition to PLT. Therefore, further optimization and addition of other factors to the PLT of the present invention could lead to even better improvements in sex hormone levels.

[0208] Example 5: Effect of the PLT of the present invention on mouse follicle development.

[0209] The follicle development of mice in each group is shown in Figure 4 shown.

[0210] like Figure 4 As shown in A, the ovaries of the mice in the model group were obviously fibrotic, solid and atrophic. After the PLT treatment of the present invention, the texture became obviously loose.

[0211] Further, if Figure 4 As shown in B, the total number of follicles increased significantly after the PLT treatment of the present invention, and was more than that of the positive control group.

[0212] Among them, the results of growing follicles are as follows Figure 4 As shown in C, the number of growing follicles was significantly increased, which was also more than that of the positive control group.

[0213] In summary, the results of this example confirm that after the PLT treatment of the present invention, the tissue structure of the ovary is restored, indicating that the ovarian reserve function is improved.

[0214] In addition, although there are certain individual differences in the number of follicles in mice, the overall improvement effect of the PLT of the present invention on the tissue structure of the ovary and the ovarian reserve function is better than that of the positive control group.

[0215] Example 6: Effects of the PLT of the present invention on the reproductive outcomes of mice.

[0216] The reproductive outcomes of mice in each group were as follows Figure 5 As shown in the figure, the ovulation status of mice was observed by superovulation.

[0217] Compared with the control group, the number of ovulations in the model group was significantly reduced ( Figure 5 B), the number of deformed eggs is large ( Figure 5 A), MII stage egg ( Figure 5 C) and the number of two-cell embryos ( Figure 5 D) significantly decreased.

[0218] After being treated with the PLT of the present invention, the number of ovulations of mice increased by about 2 times, the number of deformed eggs decreased, the number of MII eggs increased by about 4 times, and the number of two-cell embryos increased by about 3 times.

[0219] The positive control substance has a substantially equivalent effect on mouse ovulation as the PLT of the present invention, indicating that the same amount of PLT of the present invention and PLT derived from PRP of healthy young adults have comparable effects on reproductive outcomes.

[0220] The results of this example show that the PLT treatment of the present invention effectively improves the reproductive outcomes of mice.

[0221] Example 7: Anti-apoptotic effect of the present invention in an in vitro cell model.

[0222] In order to verify the functional effects of the product of the present invention in vitro, a POI cell model was constructed using the human ovarian granulosa cell line KGN.

[0223] The results are as follows Figure 6 The results showed that KNG cells treated with cyclophosphamide (CTX) underwent significant apoptosis. Subsequently, the model cells were treated with the collected PLT culture supernatant containing a large amount of cytokines, and it was found that the cell apoptosis state was significantly restored, indicating that the product of the present invention can combat POI ovarian cell apoptosis by releasing cytokines.

[0224] In summary, a POI mouse model was established through chemotherapy-induced injury. Treatment with in vitro regenerated platelet-derived human HSPCs significantly improved ovarian development and function in POI patients in terms of ovarian volume, texture, follicular development, hormone levels, and reproductive outcomes. Furthermore, by establishing an in vitro cell model and treating the cells with cytokines released by the platelet-derived cells, a significant anti-apoptotic effect of the product was observed. Furthermore, it was concluded that the platelet-derived cells of the present invention are superior to, or substantially equivalent to, the effects of autologous platelet-rich plasma on improving premature ovarian insufficiency. Testing of the levels of various cytokines in the platelets of the present invention, including TGF-β1, EGF, BDNF, FGF, VEGF, PDGF-AB, PDGF-BB, and IGF, revealed that the levels of FGF and VEGF in the platelets of the present invention were significantly higher than those in platelet-derived cells isolated from human peripheral blood. Specifically, the bFGF content in the induced platelets of the present invention was 2-fold higher than that in hPLs derived from peripheral blood, and the VEGF content was 2-fold higher. In the induced hPL of the present invention, the levels of other cytokines generally do not change significantly compared with the levels of corresponding cytokines derived from peripheral blood.

[0225] The mechanisms by which the product of the present invention exerts its effects include: 1) promoting ovarian tissue regeneration and development through cytokines released after PLT activation; 2) regulating the ovarian microenvironment through "cross-talk" between PLT and ovarian granulosa cells; 3) promoting oocyte maturation and granulosa cell development through other molecular mechanisms.

[0226] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of in vitro regenerated platelets in the preparation of a pharmaceutical composition for treating premature ovarian insufficiency in a subject, wherein: The pharmaceutical composition comprises in vitro regenerated platelets as the sole active ingredient, and the in vitro regenerated platelets have the following characteristics: FGF levels increased significantly and VEGF levels increased significantly; Wherein, the "significant increase in FGF level" means that the FGF level X1 of the platelets is compared with the FGF level X0 of human platelets, X1 / X0≥1.5; The “significant increase in VEGF level” means that the VEGF level Y1 of the platelets is compared with the VEGF level Y0 of the human platelets, and Y1 / Y0 is ≥1.5; Furthermore, the content of the in vitro regenerated platelets in the pharmaceutical composition is 0.2-3E9 / mL; The culture system for in vitro regenerated platelets includes a first-stage culture medium, a second-stage culture medium, a third-stage culture medium, and a fourth-stage culture medium. Wherein, the first stage culture medium comprises basal culture medium, BMP4, VEGF, activin A, and CHIR-99021; The second stage culture medium includes StemSpan™-ACF Erythroid Expansion Medium, BMP4, VGEF, and bFGF; The third stage culture medium includes basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, and PVA; The fourth stage culture medium includes basal medium, TPO, SCF, and PFHM-II; Furthermore, the in vitro regenerated platelets are prepared by a method comprising the following steps: (S1) Human ESC / iPSC cells were cultured and expanded using stem cell culture medium; (S2) performing mesodermal differentiation culture on the cells obtained in step (S1) using the first stage culture medium; (S3) using the second stage culture medium to culture the cells obtained in step (S2) for differentiation into hemogenic endothelial cells and hematopoietic stem and progenitor cells; (S4) continuing to culture the cells obtained in step (S3) with the third stage culture medium to obtain megakaryocytes; and (S5) Continuing to culture the cells obtained in step (S4) with the fourth stage culture medium to obtain platelets.

2. The use according to claim 1, characterized in that The levels of TGF-β, EGF, BDNF, FGF, VEGF, PDGF-AB, PDGF-BB, and IGF in the in vitro regenerated platelets were not significantly changed compared with those in human platelets.

3. The use according to claim 1, characterized in that The pharmaceutical composition treats premature ovarian insufficiency by one or more of the following methods: (Z2) promoting ovarian regeneration in subjects; (Z3) Improve the estrous cycle of the subjects; (Z4) Improve the sex hormone levels of the subjects; (Z5) Improve the ovarian reserve function of the subjects; (Z6) Improve reproductive outcomes in subjects.

4. The use according to claim 3, characterized in that Improving the estrous cycle of the subject means that after administration of the pharmaceutical composition, the estrous period of the subject is significantly increased and / or the diestrus period of the subject is significantly reduced.

5. The use according to claim 3, characterized in that The improvement of the ovarian reserve function of the subject refers to a significant increase in the total number of follicles and / or a significant increase in the number of growing follicles in the subject.

6. The use according to claim 5, characterized in that The significant increase in the total number of follicles of the subject means that the total number of follicles H1 of the subject after administration of the pharmaceutical composition is compared with the total number of follicles H0 of the subject before administration of the pharmaceutical composition, and H1 / H0 is ≥1.

5.

7. The use according to claim 5, characterized in that The significant increase in the number of growing follicles in the subject means that the number of growing follicles J1 of the subject after administration of the pharmaceutical composition is compared with the number of growing follicles J0 of the subject before administration of the pharmaceutical composition, and J1 / J0 is ≥1.

5.

8. The use according to claim 3, characterized in that The improvement of the reproductive outcome of the subject includes: a significant increase in the number of ovulations of the subject, a significant decrease in the number of deformed eggs, a significant increase in the number of MII stage eggs, and / or a significant increase in the number of two-cell embryos.

Images