IPSCs Transplantation Restores Follicular Maturation And Steroid Hormones Production In A Mouse Model Of Chemotherapy-induced Ovarian Failure

Premature ovarian failure (POF) is a disorder characterized by amenorrhea, infertility, estrogen deficiency and elevated gonadotropin levels in women younger than 40 years.Although the incidence of cancer increases each year, earlier detection and more effective treatments have reduced cancer-related mortalities and improved chances for long-term survival of cancer patients. Given this success, clinical concern in good-prongosis malignancies can now also focus on the long-term adverse effects of chemotherapy. The main problems associated with chemotherapy in female survivors include, oligomenorrhea, amenorrhea, secondary sexual characteristics growth retarda-tion, infertility, early menopause, cardiovascular disease, osteoporosis, and so on, which seriously reduced the quality of life of the patients. Therefore, preservation of the ovarian function following adjuvant chemotherapy has become a major concern of both patients and clinician-scientists.Several options have been put forward for preserving female fertility. But unfortunately, none of the suggested methods is ideal, and none guarantees future fertility in survivors. As to the ability of self-renewing, pluripotentiality and homing, stem cell has been used to treat many diseases and achieved remarkable results. Previous researches had showed that ESCs or MSCs can differentiate into germ cell and can repair the ovarian function in mice. However, considering the source and ethical issues of these stem cells, we focus on induced pluripotent stem cells, which also have the ability of self-renewal and multiple differentiation potential. Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that were firstly discovered by Shinya Yamanaka’s lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes could convert adult cells to pluripotent stem cells.IPS cells avoid both the ethical problem of using human fetal tissue and the possibility of immunological rejection, which has broad application prospects in the fields of regenerative medicine.In this study, we will induce iPS cells to differentiate into germ-like cells by co-culture with granulose cells (GCs) in vitro and transplant the iPS cells into the ovaries of chemotherapy-treated mice through the tail veins, expecting that iPS cells can restores follicular maturation and steroid hormones production in vivo.Part Ⅰ Differentiation of iPSCs into granulosa cell-like cells in vitroObjective:This study was to investigate the differentiation potential of iPS cells into granulosa cell-like cells in vitro.Method:In this study, we induced pluripotent stem cells (iPSCs) using the granulosa cell conditioned medium (GS-CM), and then observed the morphological change, analyzed the level of estradiol (E2) in culture supernatants, and tested the expression of germ cells markers by RT-PCR(Real-time polymerase chain reaction).Results:Compared with controls, the morphology of the iPS cells became similar to that of granulosa cells (GCs).The level of estradiol (E2) analyzed by Elisa showed that the E2 concentration of the culture supernatant of iPS cells cultured in GS-CM increased in a time-dependent manner, compared with the no-treated iPS cell group. RT-PCR was performed to analyze the expression level of specific germ cells markers genes, including FSHR and DAZL, which were significantly up-regulated in iPS cells cultured in GC-conditioned medium comparison with cells that were not co-cultured. However, the stem cell mark OCT-4 gene was significantly decreased in iPS cells with GC-CM.Conclusion:Our results suggest that iPS cells possess the potency to differentiate into granulosa cell-like cells in vitro, which provide the possibilities for cell therapy of premature ovarian failure (POF).Part Ⅱ Establishment and evaluation of mice model of chemotherapy-induced ovarian failureObjective:To evaluate the treatment results of iPS cells therapy, we should establish and evaluate a effective and stable mice model of chemotherapy-induced ovarian failure firstly.Method:Based on the references, the most widely used cisplatin and clear ovarian toxicity cyclophosphamide were chased for chemotherapy. Female C57BL/6 mice were intraperitoneally injected with cisplatin and cyclophosphamide respectively. The bodyweight, estrous cyclicity, survival, concentration of gonadal hormones, ovarian weight, histopathology and follicle count were determined and compared with those of the control group.Results:After cisplatin and cyclophosphamide injection, the mice body weight gradually decreased and characterized by estrous cycle disorders compared with normal group. At day 14 after treatment termination, the mice in cisplatin group only tow mice survive, five were already dead, but there were no one dead in other groups. The ovarian volume and wet weight significantly decreased in each group of chemotherapy with wrinkled surface. Meanwhile chemotherapy significantly reduced the E2 levels and elevated the FSH levels after two weeks of injection. Histology of the ovaries observed that inflammatory cell infiltrated obviously with an acuolated appearance in cisplatin group, however cyclophosphamide induced ovarian fibrosis significantly. Moreover, follicle count showed a marked loss of follicles at all developmental stages and increased number of atretic follicles in the chemotherapy-induced mice compared with normal group.Conclusion:Our study demonstrated that either cisplatin or cyclophosphamide intraperitoneal administration was successful to establish a mice model of chemotherapy-induced ovarian failure. Compared to the cisplatin which high mortality, cyclophosphamide intraperitoneal administration was the better method, which could be useful models for study of treatment of POF.Part III Transplantation of iPS cells to treat premature ovarian failure in mouse model.Objective:This study aimed to determine whether transplant the iPS cells into the chemotherapy-induced mice model of POF through the tail veins can restores follicular maturation and steroid hormones production in vivo.Method:Female C57BL/6 mice were divided into four groups based on the treatment they were given:the normal control group (no treatment); the PBS group (injected PBS following CTX-treated); the iPS group (injected iPS cells by tail following CTX-treated); the MEF group (injected MEF cells by tail following CTX-treated). Then the general condition of mice, ovarian function, tracking of iPS cells were determined.Meanwhile housing with males, the number of pregnancies was recorded to evaluate long term fertility.Result:After cyclophosphamide and busulfan injection, the mice body weight, the ovarian weight, and the number of normal follicles decreased in the iPS-transplanted group as compared with the normal group, but increased over time compared with the PBS and MEF groups. Plasma hormone level tested by Elisa showed that in the iPS-transplanted group the E2 level decreased and the FSH level increased compared with normal group, but compared with PBS and MEF group, the E2 level increased and the FSH level decreased significantly. Immunostaining revealed that the graft PKH26 labeled-iPS cells survived and migrated to mouse ovary, meanwhile some of these cells expressed the primordial germ cell marker MVH (Mouse vasa homolog, Mvh). Ovaries of the iPS-transplanted group expressed iPS cells mark (Tet-on gene), but it cannot be detected in other groups by PCR. In the first month mating trial,80% (8 of 10) non-treated females achieved successful (live-birth) pregnancies,10% (1 of 10) in PBS and MEF group, but 30% (3 in 10) in iPS-transplanted group. In the next two month,100% (8 of 10) non-treated females achieved successful (live-birth) pregnancies,50% (5 in 10) successful (live-birth) pregnancies in iPS-transplanted group, but only 10%(1 in10) in PBS and MEF group. The appearance of the offspring mice showed no abnormalities, and no deformity was observed. Interestingly, Tet-on gene was tested in part of the offspring of mice in iPS-transplanted group by PCR but could not be tested in other groups.Conclusion:Intravenously injected iPS cells can reach and survive in the ovaries of chemotherapy-treated mice and differentiated into grem cell-like cells. IPS cells transplantation restores follicular maturation and steroid hormones production in a mouse model of chemotherapy-induced ovarian failure.