UC Berkeley

A decade after the sequencing of the human genome, there are many biological functions that cannot be discerned from the genetic code. Post-translational modifications that regulate biological functions, such as glycosylation, have become the focus of biological research to help fill in the gaps not filled by genomics. Glycosylation is the process by which oligosaccharides, termed glycans, are appended onto membrane and secreted proteins as well as lipids. Glycans on the cell surface make up the majority of the cellular glycome, and are essential for many biological processes, including cell development and differentiation, cell-cell or cell-matrix communication, and pathogen-host recognition. In addition, they can be powerfully harnessed as cellular markers for distinct cell populations. This thesis focuses on the efforts made to characterize the cell surface glycome of human embryonic stem cells (hESCs) and changes in glycosylation during neural differentiation for

Human embryonic stem cells are derived from human embryos with the capacity to differentiate into any somatic cell type. Since the derivation of hESC lines, there has been an enormous amount of excitement surrounding their use in regenerative medicine and as a model for human development. In the last decade, the properties and behavior of stem cells have been extensively studies. There are still several challenges, however, that remain in understanding hESC biology, such as characterization of the glycome and the functional role of glycans. Glycans and glycoconjugates are ideal candidates for cell surface markers of stem cells as they are easily identified by a wide array of lectins and antibodies. The few stem cell surface markers used are antigens identified, thus, glycome profiling will be extremely useful for specific selection and enrichment of hESCs at various stages of development and for defining the functional roles of glycans in the process.

Because of the heterogeneity and complexity of many glycans, they have been traditionally hard to study. This complexity has presented a significant challenge for obtaining structural information about glycans at the molecular level, particularly in contexts that are relevant to native cellular physiology. Technological advances in the last few decades have allowed for better characterization of these glycans, a major challenge in the past. One such approach is the use of fourier transform (FTICR) mass spectrometry.

(MS), the most sensitive ion detection method with almost unlimited resolution, surpassing commonmass spectrometric methods. Chapter 2 describes the development of a method using FTICR MS and analytical tools to analyze membrane glycans on hESCs. This method is the first to provide structural detail and quantitation of glycans on hESCs membranes and to reveal an unusual glycosylation pattern not commonly found on mammalian cell surface.

The development of this method allowed for analysis of glycans on different cell types. Since changes in glycans during stem cell differentiation have important roles in self-renewal and differentiation, Chapter 3 focuses on characterizing the changes in the glycosylation pattern of embryoid bodies and neural stem cells derived from hESCs. By analyzing glycans on hESC during neural development, new potential stem cell and cell stage markers were identified as well as the identification of high mannose glycans in all cell stages. These findings have elicited a new investigation into the function of high mannose glycans on hESCs and mammalian cells.

In summary, glycosylation is a fundamental post-translational modification that is important in all cells. The wealth of information contained in the glycome of a cell has yet to be discovered. With existing technological advances, and efforts to optimize methods, various part of the glycome can be characterized. hESCs differentiated along the neural lineage was used as a model system to further understand hESC biology and to find novel biomarkers for the specific cell types. Toward this end, several N-glycans were identified on hESCs, embryoid bodies and neural stem cells by developing an optimized method using state-of-the -art mass spectrometry. Characterization of the hESC glycome should contribute to our understanding of hESC biology, and help to accelerate their use in regenerative medicine.