Embryonic stem cells are isolated from the early developing embryo and are capable of forming the different cell types found in the body. Understanding how these cells develop and maintain their specialized state is critical to understanding how they function. We hypothesize that embryonic stem cells acquire a unique metabolic state that aids them in maintaining their specialized state. Using two distinct mouse pluripotent states, embryonic stem cells (ESC’s) and epiblast stem cells (EpiSC’s) representing cells isolated from the inner cell mass of the blastocyst and the epiblast, a later stage in embryonic development, we found that EpiSC’s are much more glycolytic in terms of their energy usage compared to ESC’s as measured by oxygen consumption and extracellular acidification rates. Furthermore, using quantitative polymerase chain reaction experiments, we found that genes key to regulating and maintaining high rates of glycolysis are significantly upregulated in EpiSC’s compared to ESC’s. We showed that the overexpression of HIF1α, a known key regulator of these glycolytic genes, in ESC’s is able to shift the ESC’s towards a more glycolytic metabolic state and cellular morphology reminiscent of EpiSC’s. In addition, transition of ESC’s to EpiSC’s using the known chemical inducers Activin and Fibroblast Growth Factor 2 revealed that changes in the expression of metabolic genes occur prior to changes in expression of cell fate markers, which suggest that a metabolic transition in the ESC’s may be key in its transition to an EpiSC-like state.

MicroRNAs (miRNAs) are small noncoding RNA molecules that post-transcriptionally regulate gene expression. MiRNA biogenesis involves two key ribonucleases, Drosha and Dicer. I am investigating the Drosha enzyme. Previous research from the Ruohola-Baker lab reveals that reducing levels of Drosha in stem cells significantly reduces levels of some miRNAs, while only slightly reducing levels of another group of miRNAs. This surprising result led us to conduct a bioinformatics analysis to identify characteristics that differentiate these two groups of miRNAs. We discovered structural differences, specifically that bulges in the central region of miRNAs were more frequent in heavily reduced miRNAs than in miRNAs that were not affected. My mentor, Henrik Sperber and I propose a model explaining why miRNAs with mismatches (bulges) in the central region of the sequence are more sensitive to reduced levels of Drosha than rigid miRNAs with no mismatches in that region. To test whether a bulge causes differential miRNA processing when Drosha is the limiting factor, I am adding and removing bulges to primary miRNA transcripts, overexpressing them in cells with low Drosha levels, and comparing their mature levels using qPCR. In addition, I am testing whether in nature cells vary Drosha levels as a mode of regulation. A previous study shows that T cells at specific stages of differentiation reduce Drosha expression by as much as 10 fold. My own bioinformatics analysis reveals that certain tissues in mice have an 8-fold reduction of Drosha. To see whether reduction of Drosha levels correlates with differential microRNA processing, I am using microarray data to find subsets of microRNAs that have dramatically reduced levels when Drosha expression is significantly low. With my research, I hope to discover a potentially novel mechanism of regulation in which cells alter Drosha levels to selectively process a subgroup of miRNAs.

Understanding stem cell maintenance is crucial for delineating conditions such as tissue loss, aging, and cancer. Using the Drosophila female germ-line stem cell (GSC) model to observe stem cells in vivo, several extrinsic and intrinsic factors have been demonstrated to regulate maintenance of Drosophila GSCs; they have also been found to be conserved in mammalian systems. The search is on for novel regulatory factors of stem cell maintenance with the hope of extending our understanding to stem cells in mammalian systems. My project involves the characterization of the molecular function of Pineapple eye gene (Pie), a novel regulator of GSC maintenance. Through a loss-of-function screen in GSCs, it has been demonstrated that mutations of pie result in GSC maintenance failure. The amino acid sequence of pie revealed that it contains ring-domains suggesting possible E3 ubiquitin ligase function. Such proteins are key components of the ubiquitination pathway, which are involved in protein tagging and degradation. To test pie’s function as an E3 ligase, I am using a novel assay that employs the fact that bacteria do not have endogenous ubiquitination pathway and therefore utilizes Escheria coli as an in vivo system to express the components of the ubiquitination pathway and detect the formation of ubiquitin chains, a key indicator of E3 Ligase activity. I have demonstrated that the expression of pie’s ring-domains without a substrate is not sufficient for the detection of ubiquitin chains. I will now test whether the full length pie is necessary for proper ubiquitination. Further, I will also test whether pie’s functional domain needs a substrate in order to function effectively. Successful characterization of pie can reveal new pathways to target for conditions involving stem cell maintenance.

Differentiated human embryonic stem cells (hESCs) demonstrate the potential for regenerative medicine and drug discovery utilizing rationally assigned cell fate. But hESC are limited by the technical difficulty of recapitulating an ever growing set of cellular phenotypes using a finite set of pluripotent cell lines. The resulting molecular deficiencies limit the ability of differentiated cells to integrate into host tissue, and diminish the translational potential of findings based on differentiated hESC. Induced pluripotent stem cells (iPSCs) can be derived from patients with or without genetic disorders, and have much greater use for drug discovery and basic research, and patient derived iPSCs are less prone to rejection by host tissue. However, iPSC generated by insertion of genes into the host genome, increasing risk of rejection and limiting maturation of iPSC-derived cells. RNA-induced pluripotent stem cells (RiPSC) offer a solution to problems of genomic modification by inducing pluripotency exclusively through the transfection of mRNA. MicroRNA (miRNA) exhibit widespread regulation of mRNA translation and degradation, and do not directly alter the genome. A bioinformatics study curating publicly available microarray data for components of the miRNA biogenesis pathway, pluripotency factors, and miRNA expression will generate hypotheses about the functions of miRNA and relevant proteins, with a focus on differentiation and neural cell fate. Quantification of miRNA and mRNA expression in hESC and RiPSC during retinal differentiation will support or disprove hypothesized relationships between protein and miRNA expression. Transfection of miRNA will be used to determine the ability of these miRNA to enhance differentiation and maturation of retinal cells. Additionally, the resulting retinal progenitors will be implanted into mouse retina, directly testing the potential of miRNA to improve clinical outcomes of stem cell based therapies for the nervous system.