Maturation of Human Oocytes for SCNT and Embryonic Stem Cell Derivation

The Stanford Fertility and Cancer Program preserved ovarian tissues from cancer patients before chemo- and radiation therapy to avoid damages. We have obtained patients' consent and are promoting the growth of arrested small follicles from ovaries of cancer patients with specific activators to allow the development of large follicles containing mature eggs. The surplus eggs can be used to re-program cell nuclei from patients, thus allowing the generation of patient-compatible pluripotent stem cells for the treatment of diverse degenerative diseases. We are also improving the procedures for the reprogramming of somatic cell nuclei by using electro-fusion, micro-injection, and Sendi virus envelop fusion. After identifying key embryonic hormonal factors important for the optimal growth of human early embryos in the test tube, we will promote the development of re-programmed cells with hormone-enriched culture media for the generation of patient-compatible stem cell lines. In addition to providing pluripotent stem cells for future generation of diverse cells in the body (neurons, muscles, pancreatic insulin-producing cells, etc), the present generation of mature oocytes could benefit cancer patients and diverse infertile women who still have arrested small follicles but are unable to respond to the present gonadotropin therapy.

We are continuing our effort to perform somatic cell nuclear transfer in order to obtain immune-compatible embryonic stem cells for future regenerative medicine application in patients. Due to the limited availability of human oocytes, we obtained human ovarian cortical fragments from patients with ovarian tumor and designed an in vitro activation protocol to promote the growth of dormant ovrain follciles, followed by grafting into immune-deficient mice to derive mature oocytes. We have substantially improved the follicle activation approach and shortened the duration needed to generate mature oocytes. We are continuing to reprogram somatic cell nuclei using mature oocytes and further increase the efficiency of embryonic stem cell derivation.

We continue to obtain ovarian cortical fragments from cancer patients for activating dormant primordial follicles using xenografting in immune-deficient mice. In addition to the activation of primordial follicles, we discovered that the growth of isolated secondary follicle from mice could also be promoted following treatment with PTEN inhibitor and PI3K (phosphatidyl inositol-3 kinase) stimulators to activate the Akt pathway. In addition, ovarian fragmentation could disrupt the ovarian Hippo signaling system and promote secondary follicle growth. Following Akt activation together with ovarian fragmentation to disrupt the Hippo signaling pathway, we found additive increases in ovarian graft weights and secondary follicle growth in mice. Of particular interest, human secondary follicles, following Akt stimulation and Hippo signaling disruption, could grow into the antral stage in four weeks in xeno-grafts. We are using this approach to obtain more human mature oocytes for re-programming and embryonic stem cell derivation. For Aim 2, we have collected immature and failed-to-be-fertilized oocytes from IVF patients in addition to mature oocytes obtained after xeno-transplantation. We found that it is easier to first perform ICSI (intra-cytoplasmic sperm injection) followed by removing female pronuclei from injected oocytes to derive early embryos. We, therefore, concentrated our studies to derive these early embryos using in vitro matured oocytes and mature oocytes from xenografts as recipients. For Aim 3, we have established mouse in vitro culture models to generate mature oocytes. We further demonstrated that mouse ovarian explants containing secondary follicles were stimulated by CCN growth factors downstream of Hippo signaling disruption, leading to the promotion of follicle growth. We are checking if combined Akt stimulation and CCN2 growth factor treatment can lead to cumulative promotion of human secondary follicle growth.