This proposal is designed to provide the Principal Investigator, Kai-Hsin Chang, with a training experience in the fields of molecular biology to expand the characterization of the commitment and differentiation of hematopoietic/erythroid cells from human embryonic stem cells (hESCs) and to study the expression of beta- locus globin genes at both cellular and molecular levels. The proposed studies are based on the observation that while hESCs can be induced to undergo hematopoietic and erythroid differentiation to help delineate the mechanisms underlying early events of hematopoiesis, the efficiency of generating hematopoietic/erythroid progenitors is low. Furthermore, unlike their fetal liver counterparts that mainly express fetal gamma globin, hESC-derived fetal-like definitive erythroid cells coexpress high levels of embryonic (epsilon) and fetal (gamma) globins with little adult beta globin. Thus, hESC-derived erythroid cells represent a valuable tool to study the activation and silencing of the beta-locus globin genes. The following specific aims are proposed: 1) To study the effect of ectopic expression of homeobox (Hox) transcription factor HoxB4 on the hematopoietic/erythroid differentiation of hESCs. 2) To characterize the cis- and trans-acting elements associated with the unique beta-locus globin expression pattern of hESC-derived erythroid cells. 3) To study the modulation of beta-locus globin gene expression by manipulation of intracellular transcriptional environment via cell fusion. While the applicant's short-term goal is to learn the molecular techniques to investigate hematopoietic/erythropoietic differentiation of hESCs and to study the regulatory mechanisms governing the globin expression. Her long-term goal is to develop an independent research program devoted to the study of erythroid cells, which will contribute to the development of althernative therapy for treating hemoglobinpathies. Dr. Thalia Papayannopoulou's laboratory has an outstanding record of studying the globin regulation using cellular and molecular approaches and thus will provide the applicant with an appropriate and stimulating teaching environment. The applicant will also benefit from the expertise of several other investigators nearby, including Dr. George Stamatoyannopoulos, the co-mentor, and Drs. David Russell, and Xiangdong Fang, the advisory committee members, necessary to accomplish the goal of present application and future objectives.
