Derivation and Epigenomic Analysis of hES Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00416
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
We propose to derive new human embryonic stem (hES) cell lines, and begin an investigation of epigenetic abnormalities in these lines. Epigenetics refers to changes in the behavior of genes that are not caused by changes in the DNA itself (i.e. mutations) but rather by alterations to protein and chemical (methyl) groups bound to the DNA. The overall goals of this project are to develop the capacity to derive hES cell lines, and to perform pilot projects on the natural history of epigenetic marks in hES cells and the body cells derived from them. The project builds on our efforts to establish the capacity to derive hES lines in collaboration with an IVF facility. In keeping with the goals of CIRM, this proposal is intended to serve as the nucleus for a program of ES cell research; we think that it is important that this program have the ability to derive ES cells. It is often assumed that ES cells do not retain epigenetic marks acquired in previous generations. Some evidence indicates that epigenetic marks can arise in the human germline and cause a disease syndrome; they may also be inherited. Epigenetic inheritance requires that an epigenetic mark be retained in cells of the inner cell mass, and thus presumably in ES cells as well. This raises the possibility that individual ES cell lines carry epigenetic marks, either inherited from the parent or acquired in the derivation process, that are capable of affecting the phenotype of their progeny somatic cells. We propose a pilot project to investigate this hypothesis. To prepare for the derivation of hES cell lines, we have established a collaboration with an IVF facility, which provides us with access to extensive expertise in human embryo manipulation. We have recruited a technician with substantial ES cell experience, which we have extended by further training, and are ready to begin derivation of hES cell lines. We propose to derive new hES cell lines from spare embryos by established methods; these will be used in our own studies, and will also be made available to the wider research community. In the hES lines we derive, we will study the occurrence and natural history of epigenetic abnormalities. We will identify epigenetic marks using antibodies, then choose likely candidates for further analysis. We will study several such marks in multiple hES lines. Because there is a new area of study, it is difficult to predict just what we will find; this is entirely appropriate for a pilot study.
Statement of Benefit to California: 
Human embryonic stem (hES) cells hold tremendous promise for the alleviation of human suffering, because they are able to form new cells that can be used to replace cells that are defective due to inherited or other types of disease, and because by studying them we may learn how to change the behavior of cells within the human body. If cells derived from hES cells are to be useful, we must develop ways to assess not just their efficacy, but their safety, because cellular therapies may require implanted cells to survive indefinitely in a patient’s body. We are aware that one possible source of defects in ES cells are epigenetic changes, which alter the behavior of genes without changing the DNA of those genes. Epigenetic defects can predispose to diseases such as cancer. We need methods to assess hES cells for such defects and understand their natural history and consequences; we are proposing a pilot project that begins to explore ways of detecting and analyzing them. As part of this effort, we also propose to derive new hES cell lines from spare embryos donated for research. These new hES lines will be made available to other investigators for their projects. Thus this project will benefit California by producing new hES lines, which are badly needed to advance stem cell research generally, and by exploring methods to assess the integrity of existing hES cell lines.
Progress Report: 
  • The original goal of this project was to generate oocytes (eggs) from human embryonic stem (hES) cells in cell culture dishes in the laboratory. Such oocytes could be of use as vehicles to reprogram the DNA from cells of patients with life-threatening or debilitating conditions, thereby allowing generation of new lines of hES cells that are immune matched to the patient. The paucity of donated human oocytes precludes research using such material, and production of human oocytes from hES cells in the laboratory would in theory provide a limitless source of material.
  • Since our last progress report another CIRM-funded group, Dr. Renee Reijo Pera’s lab at Stanford University, has published exciting results demonstrating successful production of primordial oocytes from mouse ES (mES) and human ES (hES) cells. Consequently, during the remaining period of the award we propose to use the Reijo Pera methods for production of female germ line cells in our lab using H9 and HUES-9 female hES cells. After accomplishing this, we will introduce human mtDNA containing mutations that cause either a severe or mild reduction in oxidative phosphorylation (energy) production into H9 and HUES-9 hES cells and investigate the impact of the different mtDNA mutations on the ability of hES cells to form cells with characteristics of PGCs, then primordial oocytes in vitro and in vivo. An important related goal of this research is to investigate whether development of oocytes from ES cells could be used as a method to remove deleterious mtDNA mutations from the hES cell population, thereby improving the utility and possibly safety of derived cell types for therapeutic purposes.
  • During this reporting period we have focused on our approved revised research plan. We continued our efforts to generate embryonic female germ cells from human embryonic stem (hES) cells in vitro using methods reported in the literature at the end of 2009. Our revised plan included the new goal of using in vitro developed female embryonic germ cells (oocytes) as a resource to investigate how mitochondrial genomic DNA containing deleterious mutations is segregated during female germ cell development. As well as providing novel information about the biology of germ cell development, this research may provide important information relevant to development of safe methods for therapeutic cloning. We began by using two different female hES cell lines (HUES-6 and H9) to investigate whether we could use the reported methods to develop female germ cells from hES in our lab. A third female hES cell line (HUES-9) we originally intended to use was found to have a high propensity to gain an extra chromosome (become aneuploid) and was therefore not used. Despite following the reported methods that demonstrated in vitro differentiation of hES cells into female germ cells, we were unable to reproduce the previously reported results in our own lab. The reasons for this are currently unclear but may involve subtle, but important, differences in the methodology or materials we used.

© 2013 California Institute for Regenerative Medicine