Study of TBX3 Function in human Embryo Stem (hES) Cell Differentiation and Identification of Genome-Wide TBX3 Promoter Binding-Sites with the CHIP-GLAS Promoter Array

Funding Type: 
SEED Grant
Grant Number: 
RS1-00381
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Stem cells can develop into every cell, every tissue and every organ in the human body, e.g. they can make any kind of cells in the human body. Stem cells reproduce themselves many times over and over. Their almost limitless potential has made stem cells a significant focus of medical research. But before scientists can use stem cells for medical purposes, they must first learn how to harness their power. They can't treat disease until they learn how to manipulate stem cells to get them to develop into specific tissues or organs. We would like to be able to grow a particular type of cell in the laboratory and then inject it into a patient, where it would replace diseased tissue. But stem cells are not yet being used to treat disease because we still haven't learned how to direct a stem cell to differentiate into a specific tissue or cell type (brain vs. liver, for example) and to control that differentiation once the cells are injected into a patient. We know that turning genes on and off is crucial to the process of differentiation, so we can add some factor into the culture dish and observe stem cells to differentiate into specific types of cells. But some sort of signal is needed to actually trigger the stem cells to differentiate. We are still searching for that signal. If we can ultimately learn how to direct stem cells to differentiate into one type of tissue or another, they can use them to treat the patients. In this proposal, we will first examine. We propose a novel approach to understanding differentiation of human embryo stem (hES) cells, by studying TBX3, a protein called a transcription factor that controls the expression of other genes. In humans, the loss of function of TBX3 causes Ulnar-Mammary Syndrome, a genetic disorder that can pass from one generation to the next. Our studies show that TBX3 can prevent the aging of mouse cells, so that the cells can keep growing. Furthermore, our preliminary results show that TBX3 is downstream mediator of another protein, BMP4. BMP4 is a known key regulator for hES cell differentiation. Thus, TBX3 is an attractive candidate as a downstream mediator of BMP4 in hES cell differentiation. We will test TBX3 effects on hES cell differentiation if down-regulate TBX3 in hES cells with a technology called siRNA knockdown. We will identify the genes controlled by TBX3 with a recently invented powerful technology called CHIP-GLAS. This technique allows us to examine thousands of genes on a small chip in a single experiment. We expect that the innovative experiments proposed here will open a new avenue to understanding the signal of hES cell differentiation.
Statement of Benefit to California: 
In this propose, we will collaborate with AVIVA SYSTEMS BIOLOGY (ASB). ASB is a start-up biotech company in California to develop comprehensive mapping of DNA/Transcription factor interactions on a genomic scale. ChlP-GLAS (chromatin immunoprecipitation-guided ligation and selection) technology is the major product of the company. We have completed the pilot study. The preliminary results are very interesting and demonstrate the feasibility of the assay. ChlP-GLAS was invented by Dr. Xiangdong Fu at the University of California, San Diego (UCSD) and licensed exclusively to ASB. ASB has spent the last two years developing ChlP-GLAS technology. ChlP-GLAS utilizes an oligonucleotide microarray containing unique 40-mer regions of 20,000 human promoters. Corresponding to the 20,000 unique regions, a pool of sequence-specific oligos is allowed to anneal to the ChIP sample DMA. These oligos also contain common overhangs for subsequent amplification and labeling. As a result of this selection process only the DNA sequences of interest are amplified. Nonspecific and repeat-containing elements are removed, greatly increasing the sensitivity and specificity of the assay. Additionally, the amplified DNA molecules are all of uniform size, improving amplification and hybridization conditions. Therefore, ChlP-GLAS technology may represent a new generation of ChlP-on-chip which offers increased sensitivity with much less starting material required. On of the aim is validate application of CHIP-GLAS in stem cell research. As the manufacturer of ChlP-GLAS products and supplier of ChlP-GLAS services, ASB will able to capitalize this invention, which will have a significant benefit for the economy of California State. In addition, California as a largest state in US, the proposal study will benefit many of the patients. The aim of this proposal is ultimately to learn how to direct stem cells to differentiate into different type of tissues or cells, so we can use them for medical purposes. For example, stem cells could also be used to repair cells or tissues that have been damaged by disease or injury. This type of treatment is known as cell-based therapy. One potential application is to inject embryonic stem cells into the tissue Stem cells may also one day be used to repair brain cells in patients with Parkinson's disease. Eventually, we might even be able to grow entire organs in a laboratory to replace ones that have been damaged by disease. Growth factors specific to the organ would be added to guide the organ's development.
Progress Report: 
  • CIRM Grant – Public Abstract:
  • Non-invasive imaging techniques for an in vivo tracking of transplanted stem cells offer real-time insight into the underlying biological processes of new stem cell based therapies, with the aim to depict stem cell migration, homing and engraftment at organ, tissue and cellular levels. We showed in previous experiments, that stem cells can be labeled effectively with contrast agents and that the labeled cells can be tracked non-invasively and repetitively with magnetic resonance imaging (MRI) and Optical imaging (OI). The purpose of this study was to apply and optimize these labeling techniques for a sensitive depiction of human embryonic stem cells (hESC) with OI and MRI.
  • Experimental Design: hESC were labeled with various contrast agents for MRI and OI, using a variety of labeling techniques, different contrast agent concentrations and different labeling intervals (1h – 24h). The cellular contrast agent uptake was proven by mass spectrometry (quantifies the iron oxides) and fluorescence microscopy (detects fluorescent dyes). The labeled hESC underwent imaging studies and extensive studies of their viability and ability to differentiate into specialized cell types.
  • Imaging studies: Decreasing numbers of 1 x 10^5 - 1 x 10^2 contrast agent-labeled hESC and non-labeled controls were evaluated with OI and MRI in order to determine the best contrast agent and labeling technique as well as the minimal detectable cell number with either imaging technique. In addition, samples of hESC were investigated with OI and MRI at 1 min, 2 min, 5 min, 1h, 2h, 6h, 12h, 24h and 48 h in order to investigate the stability of the label over time. Viability and differentiation assays of the hESC were performed before and after the labeling procedure in order to prove an unimpaired viability and function of the labeled cells.
  • Results: The FDA-approved contrast agents ferumoxides and indocyanine green (ICG) provided best results for MR and optical imaging (OI) applications. The cellular load with these labels was optimized towards the minimal concentration that allowed for detection with MR and OI, but did not alter cell viability or differentiation capacity. The ferumoxides and ICG-labeled hESCs as well as stem cell derived cardiomyocytes and chondrocytes provided significantly increased MR and OI signal effects when compared to unlabeled controls. ICG labeling provided short term labeling with rapid excretion of the label from the body while ferumoxides labeling allowed for cell tracking over several weeks.
  • Significance: The derived data allowed to establish and optimize hESC labeling with FDA approved contrast agents for a non-invasive depiction of the labeled cells with MR and OI imaging techniques. Our method is in principle readily applicable for monitoring of hESC -based therapies in patients and allows for direct correlations between the presence and distribution of hESC-derived cells in the target organ and functional improvements. The results of this study will be the basis for a variety of in vivo applications and associated further grant applications.

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