Improving efficiency of embryonic stem cell derivation from mouse blastocysts and preblastocysts using modified medium, glucose concentration, and epigenetic modifications
Date of Completion
January 2009
Keywords
Biology, Ecology
Degree
Ph.D.
Abstract
Embryonic stem cells (ES cells) are derived from the inner cell mass of preimplantation stage embryos known as blastocysts, and are unique and very invaluable cells because they are pluripotent, meaning that they are able to differentiate into all derivatives of the three primary germ layers, such as ectoderm, endoderm and mesoderm, including each of the more than 220 cell types in the adult body. Also, they have been proposed for use in regenerative medicine and tissue replacement after injury or disease, because they have potentially unlimited capacity for self-renewal and plasticity. ^ Although the first mouse ES cell lines were derived two decades ago and standard protocols for ES cell derivation are widely used today, the technical difficulty of these protocols still pose a challenge for many investigators attempting to produce large numbers of ES cell lines, and are limited to only a few mouse strains. In order to explore the potential for improvement of ES cell derivation efficiency, I evaluated the use of embryonic material from different developmental stages for efficient ES cell derivation, examined the gene expression profiles of ES cell lines derived from different preimplantation stages, and demonstrated the use of modified media and epigenetic modifications to influence derivation efficiency. ^ I demonstrated that I can enable more efficient derivation of larger numbers of ES cell lines by new media and novel methods with different levels of glucose (1000 vs. 4500 mg/L) and the epigenetic modification reagents 5-aza-2'-deoxycytidine (a methyltransferase inhibitor) and Trichostatin A (a histone deacetylase inhibitor) in mouse strains which have been typically less-efficient for ES cell derivation. Interestingly, epigenetic modifications made the most significant improvement (8.5 fold) in ES cell derivation efficiency using blastocysts with less-efficient media formulations. I have also demonstrated improved derivation efficiency from a different developmental stage, preblastocyst, which results when preimplantation embryos are blocked from progression from the morula to the blastocyst stage. Interestingly, ES cell lines derived from preblastocysts have different characteristics with regard to their nutritional requirements for efficient ES cell line derivation and expression of pluripotency-related genes, compared with ES cell lines derived from blastocysts. By comparison of microarray gene expression profiles of derived ES cell lines from blastocysts and preblastocysts using new media, it was shown that the ES cell lines derived from blastocysts and preblastocysts in different culture conditions were not significantly different from standard lines. Furthermore, teratoma formation assays showed that these lines are pluripotent in vivo, giving rise to all three developmental germ layers. These optimized methods will be able to improve ES cell derivation efficiency to enable large-scale ES cell production in mice, and it raises the exciting possibility that these methods might make human ES cell derivation efficient enough for clinical applications. ^
Recommended Citation
Kim, Chul, "Improving efficiency of embryonic stem cell derivation from mouse blastocysts and preblastocysts using modified medium, glucose concentration, and epigenetic modifications" (2009). Doctoral Dissertations. AAI3351335.
https://digitalcommons.lib.uconn.edu/dissertations/AAI3351335