Derivation of hESC lines from multi-pronuclei zygotes
Human embryonic stem cells (hESCs) have the potential to become a wide range of cells in the human body including nerve cells. Moreover, hESCs can be expanded in culture plates into a large quantity, thus serving as an ideal, renewable source for cell transplantation for clinical use. However, the federally-approved, existing hESC lines are not useful for clinical therapies because they are grown in the presence of mouse feeder cells using culture medium containing animal products. As a consequence, these cell lines cannot pass the regulations stipulated by the US Food and Drug Administration (FDA) for cell-based therapy. Therefore, generating new lines of clinical grade hESCs is highly important. However, the production of new lines of hESCs requires donation of early embryos from patients that undergo in vitro fertilization (IVF) treatment and the source of donated early embryos is very limited. During normal IVF procedures, doctors routinely discover a portion of abnormal embryos (5-15%) that carry extra sets of chromosomes at the stage of fertilization. These abnormal embryos are formed either due to the entrance of two sperms into one egg or due to the retention of two copies of egg chromosomes plus one sperm, and thus contain three sets of chromosomes instead of two. These abnormal embryos are routinely isolated immediately after fertilization and discarded as medical waste. However, it is now possible to use a microsurgical procedure to remove one set of chromosomes at the stage of fertilization to produce an embryo containing two copies of chromosomes (or diploid genome). By chance, we will obtain either normal embryos with a copy of paternal and maternal genome each or mutant embryos that carry two copies of sperm (paternal) chromosomes or egg (maternal) chromosomes. Our proposal will make a proof-of-principle that we can make use of those routinely discarded abnormal embryos and produce three types of hESCs, including normal hESCs as well as mutant hESCs that contain two copies of paternal or maternal chromosomes. All hESCs generated will be of great interests to either regenerative medicine or basic human genetic research. In fact, the obtained mutant cells lines will exhibit characteristic features of known human genetic disorders such as Angelman and Prader-Willi Syndromes. Our study will pave the way for the future use of genetically abnormal IVF embryos as a source of normal hESCs benefiting regenerative medicine, as well as producing mutant hESCs that are model systems of human genetic diseases.
Human embryonic stem cells (hESCs) hold great promise for regenerative medicine. Among different types of stem cells available, hESCs remain the gold standard for lineage-specific cell differentiation in tissue repair, or for studying disease mechanism with genetic mutations, as well as for drug screening and toxicology studies. For example, recent studies of stem cell differentiation and cell transplantation are paving the ways for using nerve cell derivatives of hESCs in the anticipated clinical trials of spinal cord injury and eye diseases. Currently, there is still a great need to produce new cell lines that can be used in clinical settings. Our proposed project will demonstrate a new avenue to obtain hESCs for regenerative medicine and for understanding pathological mechanism of certain human genetic disorders. The experiments in this grant application will also produce novel cell lines that model genetic disorders due to uniparental sources of chromosome segments. Such uniparental chromosomal diseases include the mental retardation disorders, Angelman and Prader-Willi Syndromes. Therefore, our proposed studies will of great benefit to the people of the State of California through the generation of new, ethically diverse lines of human embryonic stem cells for clinical trials and increasing our knowledge of the underlying mechanism of several mental retardation disorders caused by genetic mutations.