Prevalence and functional consequences of chromosomal mosaicism in hESC lines.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00174
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Human embryonic stem cells (hESCs) hold great promise for treating many human diseases because of their ability to become any type of cell in the human body. In order to choose the optimal hESCs for therapeutic use in various diseases, it is critical to characterize fully the safety and potential of different stem cell lines. Chromosomes are large packages of DNA within a cell, and changes to a cell’s chromosomes can affect its function. Large scale chromosome changes include: the loss and gain of whole chromosomes, termed aneuploidy; partial loss of chromosomes, deletion; and movement of large chromosome segments between chromosomes, translocation. As chromosomal changes can dramatically alter the properties of many cell types, it is important to assess and compare the prevalence and consequences of chromosomal changes in hESC lines. Our proposed research will use a technique that fluorescently labels each chromosome pair within a cell with a unique color, to allow us to identify aneuploidy, deletions and translocations within stem cells. We will further observe whether extended culture time in the laboratory can influence the characteristics of chromosomes within stem cells. Once we know what changes are occurring at the chromosome level within a stem cell population, it is crucial to determine how these changes affect their capacity to become different cell types, including normal and/or tumor cells. The second part of our proposal will compare the ability of stem cell populations with or without chromosome changes, to survive, divide and become neurons. The data gained from this proposal will provide key information for future stem cell research regarding the therapeutic potential and safety of hESCs.
Statement of Benefit to California: 
The California Institute of Regenerative Medicine will be funding many research projects designed to use human embryonic stem cells for treating many human diseases, including Parkinson’s and Alzheimer’s diseases, diabetes and cancer. Clearly this research will benefit the lives of hundreds of thousands of Californians who are affected by these diseases, if it leads to safe and effective treatments. However, in order to spend California’s limited research dollars on the most promising research, it is vital to know which stem cell lines have the best therapeutic potential. Our proposal addresses this key issue through assessing genetic changes at the chromosome level that can influence the function and survival of different stem cell populations. Strong California-based expertise in the hESC field will attract biotechnology industries and top academic researchers to the area, bringing more jobs and money into the state of California. San Diego is currently a hotbed for stem cell research with multiple research institutions that have dedicated stem cell research programs, including The Scripps Research Institute and The Burnham Research Institute. The collaborative nature of our research proposal will foster high quality stem cell research in this region that is based on a clear understanding of the basic genomic characteristics of the cells being studied.
Progress Report: 
  • Full clinical potential of human ES (hES) cell therapy can be achieved when one can grow hES cells effectively while maintaining full pluripotency. We have focused on developing stem cell culture media by which we can maintain pluripotency of human ES (hES) cells. It is critical to determine and develop a chemically defined media that are animal product-free and feeder cell-free conditions so that the media can be standardized throughout stem cell research and in clinical situations.
  • One major recombinant protein component we will use in developing chemically-defined media is a set of TGF-beta signaling ligands, receptor domains, and ligand-specific antagonists. We have established a new method of generating a diverse array of these ligands, including BMPs, Activins, inhibin, and their heteromeric ligands of the BMP/Activin class ligands. Some of these heteromeric ligands possess their signaling properties unlike their homodimeric counterparts. These reagents include Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, and their derivatives. These reagents have been engineered by chimeric recombination. They were also further modified by site-specific mutagenesis, and by combinatorial heterodimeric assembly to create and modify protein-specific binding affinity to their binding counterparts. Several of these reagents are now available as recombinant protein in sufficient quantity for large-scale screening for media composition.
  • To establish the functional characteristics and optimal culture combinations using these new reagents, we have used an established hES cell, H9. We have cultured H9 cells in various compositions of culture media containing some of the engineered reagent and followed expression of several differentiation markers to monitor for pluripotency of hES cells, and also for their differentiation-guiding and pluripotency-maintaining abilities. We have first examined effect of aforementioned reagents: Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, BMP3 S28A mutant, in our standard culture media mTeSR condition, which does contain bFGF, for proliferation and differentiation of hES cells. In these assays, hES cell line H9 was cultured and reagents were added at varying concentration (1-100 ng per ml) over 1-5 days culture period. Reagents were added in new media during the course of cell culture. We have used morphological change and the presence of markers as a means to follow the differentiation. Ectoderm markers are Nestin, Cdx2; Mesoderm by Brachyury, HBZ; Endoderm markers by CXCR4, Sox17, Gata4, HBF4 alpha, Gata6, AFP. Two BMPs had pronounced effects in inducing cells to endoderm. We have followed up by analyzing the efficiency using FACS. Up to 60% of cells have undergone to endoderm-marked cells. With the availability of a cell sorter, we evaluated pluripotency by means of proliferation rate, morphology, fluorescent signal in the reporter lines by visual inspection and FACS, then we further characterized the factors by real-time PCR for stem cell markers and karyotyping.
  • It is known that high concentration of FGF can suppress the action of BMPs, so we planned to repeat the experiments in mTeSR media with lowered levels of FGF to re-evaluate the effects of BMPs on cell differentiation abilities. After these tests were completed, we established a protocol performing these assays in high-throughput manner. We are currently in the process of writing this work for publication (Valera et al., in preparation).
  • Towards the development of chemically defined culture media to maintain pluripotency, we have then tested various newly-engineered reagent to replace a protein component in TeSR media. We have established a combination of protein factors known to maintain established hES cells without using nonhuman products except human albumin, which include basic fibroblast growth factors (bFGF), and a bone morphogenetic protein derivative known as AB2008. We have termed this new media as CAV media. We are currently in the process of writing this work for publication (Valera et al., in preparation).

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