Maturation of Human Oocytes for SCNT and Embryonic Stem Cell Derivation

Maturation of Human Oocytes for SCNT and Embryonic Stem Cell Derivation

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01553
Award Value: 
$1,309,018
Disease Focus: 
Fertility
Stem Cell Use: 
SCNT
Cell Line Generation: 
SCNT
Status: 
Closed
Public Abstract: 
A major issue for tissue and cell therapy in regenerative medicine is the immune rejection of grafts originated from a non-compatible individual. Mature eggs contain factors essential for the re-programming of cell nuclei from patients to allow the establishment of patient-compatible pluripotent stem cells for the treatment of diverse degenerative diseases. Although up to half a million dormant small follicles are present in young women reaching puberty, only 400 of them developed to the large follicle stage and release mature eggs during a women’s reproductive life. The present application proposes to overcome the major obstacle dealing with the shortage of human mature oocytes for the generation of patient-compatible pluripotent stem cells. The {REDACTED} preserved ovarian tissues from cancer patients before chemo- and radiation therapy to avoid damages. We have obtained patients' consent and propose to promote the growth of arrested small follicles from ovaries of cancer patients with specific activators to allow the development of hundreds 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. 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 will benefit cancer patients and diverse infertile women who still have arrested small follicles but are unable to respond to the present gonadotropin therapy.
Statement of Benefit to California: 
Because human ovaries contain thousands of arrested small follicles and only a few of them grow into large follicles containing mature eggs, we devised a new approach to promote the growth of these dormant follicles to generate a large surplus of mature oocytes. Future adaptation of this procedure for the re-programming of the nuclei of cells from patients with degenerative diseases will allow the generation of patient-compatible pluripotent cell lines. Because these cells can be induced into diverse cell types of the body, establishment of optimal culture conditions for the derivation of these cells would allow new approaches for assisted reproductive technologies and provide new treatment modalities for diverse patients with degenerative diseases. We will submit patent applications on our findings to protect intellectual property rights according to {REDACTED} guidelines. In addition to applications in regenerative medicine, the present findings are also expected to substantially broaden the patient population for and improve the success rate of in vitro fertilization procedures that are presently used for an estimated one million treatment cycles per year worldwide. We anticipate the successful completion of the present proposal could benefit patients with degenerative diseases and infertility in California and throughout the world. The P.I. already has a pending patent application on the use of brain-derived neurotrophic factor in the promotion of early embryo development. We expect future findings will lead to additional patent applications and licensing agreements that would benefit the State of California.
Progress Report: 

Year 1

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.

Year 2

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.

Year 3

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.

© 2013 California Institute for Regenerative Medicine