Advisor(s)

Carol M. Warner (1946-)

Contributor(s)

Mansoor M. Amiji, H. William Detrich, Jacqueline M. Piret, Ann A. Kiessling

Date of Award

2008

Date Accepted

12-2008

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

College of Arts and Sciences. Department of Biology.

Keywords

Biology, Embryonic stem cells, Nanomedicine

Subject Categories

Major histocompatibility complex, Mice, Embryonic stem cells, Immunogenetics

Disciplines

Other Cell and Developmental Biology

Abstract

Major histocompatibility complex (MHC) class I proteins are crucial in both the adaptive and innate immune responses. These proteins may present antigens to T cells, interact with natural killer cells, mediate graft rejection following transplantation, and also have functions outside of the immune system. The experiments described here address the role of MHC class I proteins and their chaperone molecules in two related cell types, mouse preimplantation embryos and mouse embryonic stem cells. In the course of this research, we utilized several types of nanoparticles for cell labeling, imaging and small molecule delivery. The preimplantation stage of embryo development occurs between fertilization of the oocyte and implantation of the blastocyst into the uterus. Preimplantation mouse embryos express both classical (class Ia) and nonclassical (class Ib) MHC class I proteins and yet they are not rejected by the maternal immune system. Although the function of the embryonic MHC class Ia proteins is unknown, one MHC class Ib protein, Qa-2, the product of the preimplantation embryo development (Ped) gene, actually enhances reproductive success. Similar in structure to MHC class Ia proteins, Qa-2 protein is a trimer of the alpha (heavy) chain, ß2 microglobulin and a small (approximately 9 amino acid) bound peptide. Studies on the folding, assembly, and trafficking of MHC class Ia molecules to the cell surface have revealed that this process is dependent on multiple protein chaperone molecules, but information on the role of chaperone molecules in Qa-2 expression is incomplete. In our first set of experiments, we hypothesized that MHC class I antigen processing chaperones would be expressed in preimplantation embryos and that Qa-2 would be dependent on these chaperones for proper cell surface expression. Using real time RT-PCR, we detected mRNA for four chaperone molecules (TAP1, TAP2, calnexin and tapasin) in preimplantation embryos. We next focused on the role of the MHC-dedicated chaperone, tapasin, on Qa-2 protein expression. Using a novel quantum dot nanoparticle imaging protocol, we analyzed Qa-2 cell surface expression in embryos from wild-type and tapasin knockout mice. Our study showed that optimal cell surface expression of Qa-2 is dependent on tapasin in both T cells and preimplantation embryos. These data provide insight into the regulation of antigen presentation by MHC class I proteins in early embryos, an important step toward understanding how embryos evade the maternal immune system. In our second set of experiments, we extended this research to a therapeutically relevant cell type, embryonic stem cells. Embryonic stem (ES) cells are pluripotent cells, typically derived from preimplantation stage embryos, with the potential to differentiate into cells or tissues that may be used for transplantation therapy. Parthenogenetic ES (pES) cells, lacking paternal contribution from sperm, have been recently derived from both mouse and human oocytes and hold promise as a cell source which is histocompatible to the oocyte donor. However, the therapeutic potential of pES cells compared to ES cells derived from normal fertilized embryos (fES cells) has not yet been established. Due to the importance of MHC proteins in mediating tissue rejection or acceptance, we hypothesized that fES and pES cells should have similar expression patterns of MHC class I proteins in order for pES cells to be therapeutically relevant. Therefore we examined levels of mRNA and protein expression of MHC class I proteins, as well as several MHC class I antigen processing and presentation chaperones, in mouse ES cells derived from both fertilized and parthenogenetic embryos. We found that H-2K, Qa-2, TAP1, TAP2 and tapasin mRNAs were all expressed at low levels in undifferentiated and differentiating ES cells, and were significantly upregulated in response to interferon-? (IFN-?) treatment following 14 days of differentiation. Likewise, expression of H-2Kb and H-2Kk proteins were upregulated to detectable levels by IFN-? after 14 days of differentiation, but Qa-2 protein expression remained low or absent. We also found that MHC class I, TAP1, TAP2, and tapasin mRNAs were all expressed at very low levels in ES cells compared to T cells, suggesting transcriptional regulation of these genes in ES cells. Overall, embryonic stem cells derived from fertilized embryos and parthenogenetic embryos displayed remarkably similar patterns of gene expression at the mRNA and protein levels. The similarity between the fES and pES cell lines in regard to expression of MHC class I and antigen processing machinery provides evidence for the potential usefulness of parthenogenetic ES cells in transplantation therapy. In a third set of experiments, we tested various nanoparticles for use in embryonic stem cell research. Gold nanoparticles and quantum dot nanoparticles were successfully used to label and image live embryonic stem cells using two-photon microscopy. This application of nanoparticles may be extended for tracking and monitoring of different stem cell populations in vitro. We also tested different types of polymeric nanoparticles for their ability to deliver short interfering RNA (siRNA) to embryonic stem cells. The nanoparticles tested, gelatin nanoparticles and PMMA-PEI nanoparticles, were not able to successfully transfect ES cells with siRNA. However, modification of these nanoparticles or modification of siRNA may improve their function. The nanoparticles used in this study may be further functionalized for drug or nucleic acid delivery, in combination with cellular tracking and imaging. Therefore, the burgeoning field of nanomedicine should play a large role in fulfilling the therapeutic potential of pluripotent stem cells.

Document Type

Dissertation

Rights Holder

Paula Welter Lampton


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