The Role of TBX3 in Human ES Cell Differentiation

PURPOSE OF STUDY The purpose of this study is to understand what triggers stem cells to differentiate into one type of tissue or another. The study of TBX3, a protein called a transcription factor that controls the expression of other genes, shows that it is a downstream mediator of the protein BMP4, a known key regulator for hES cell differentiation.

Hypothesis and Aims

To understand the role of TBX3 in hES cell differentiation, we hypothesize that TBX3 is a downstream mediator of BMP4 and play an important role in hES cell differentiation. We propose the following specific aims:

1. To examine TBX3 function in hES cell differentiation. NIH approved hES cells will be cultured in a conditioned medium with BMP4 to induce differentiation and TBX3 expression. TBX3 will be knocked down with siRNA technology. The cell morphology and biochemical changes will be examined for the effects of TBX3 knockdown on hES cell differentiation, including testing human ß-CG estradiol and progesterone. The gene expression profile will also be examined with Affymetrix gene expression chip and verified with real-time RT-PCR.
2. To identify the genome-wide downstream targets directly bound by TBX3 in hESC with Chromatin immunoprecipitation-guided ligation and selection (CHIP-GLAS). To identify the targets of TBX3 in BMP4-induced hES cells, we will perform ChIP-GLAS analysis. CHIP-GLAS is a novel high-throughput technique with combination of chromatin precipitation and oligonucleotide-based microarray. This technology will allow us to identify the genome-wide downstream target genes directly bound by TBX3 in hES cells.

We expect that the proposed experiments will identify a key regulator for hES cell differentiation and open a new avenue to understanding the function of TBX3 in hES cells. The CHIP-GLAS promoter array provides us a cutting edge tool and allows us to examine the broad function of TBX3. We anticipate that the study will significantly enhance our understanding of TBX3 function in hES cell differentiation.

STUDY DESIGN NIH-approved cell lines will be available at the Sue and Bill Gross Stem Cell Research Core Facility at the University of California, Irvine. Non-NIH-approved cell lines will be obtained from Sue \& Bill Gross Stem Cell Core at UC Irvine.

Environment: Huang Lab is currently supported by the Helen and Larry Hoag Foundation and the Susan Komen Foundation. There are no active grants from the federal government. Previously, Dr. Huang was supported by K23 and R03 grants. They were completed in 2004 and 2006, respectively. No equipment was purchased with the funds from the federal grants. As all equipment currently in Huang lab was purchased with the UCI startup fund or with private funds, there is no conflict with government regulations.

At University California, Irvine, Sue and Bill Gross Stem Cell Research Center will be a key component of the Stem Cell Core Facility. This facility provides stem cell technology and resources to the UCI faculty (Please see attached letter). By merging technical and scientific expertise in a number of essential disciplines, the Core harbors a diverse tool kit for the exploration of multiple aspects of stem cell biology. The Core also provides a knowledge base for differentiating, genetically modifying and assaying human embryonic stem cells (hESCs) in support of multiple stem cell projects on campus and in the surrounding area. All services and procedures are performed in accordance with good quality assurance and record keeping practices pursuant to standard operating procedures and good laboratory practices.

The Sue and Bill Gross Stem Cell Research Center The human embryonic stem (hES) cell core facility of the Sue and Bill Gross Stem Cell Research Center opened in April 2006. It occupies approximately 10,000 square feet of space in the research park immediately adjacent to the University of California, Irvine campus. Approximately $1.7 million were committed by the UCI to fully equip the facility for all aspects on hES cell research. Further gifts totaling $236,687 from the Fuji Foundation and the Nichols Foundation provided additional major equipment and supplies that are available for all investigators. The facility was set up as a Federal-free zone in which investigators can to carry out all aspects of hES cell research. Individual rooms are equipped with tissue culture biosafety cabinets, CO2 incubators, an inverted microscope, a dissecting microscope, digital imaging equipment, a centrifuge, a water bath and a dedicated refrigerator and freezer. Ten such tissue culture set-ups as well as standard lab bench space are available to UCI investigators and visitors.

The facility also has major pieces of equipment needed for hES cell analysis such as a Zeiss 510 Meta Two-Photon Confocal Microscope. The facility will shortly have a BD LSR II Flow cytometer and a BD FacsAria cell sorter. Pretested reagents will be available to investigators through the core facility. These will include medium components, growth factors, feeder cells, gelatin, Matrigel, antibodies and primers to assay stem cell marker expression. In addition, the core facility will assist in karyotype analysis and tumor formation tests of hES cells.