It is expected that research funded by the California Institute of Regenerative Medicine will result in the development of many new human embryonic stem cells. The properties of these cell lines will have to be verified. One of the main attributes of embryonic stem cells is their capacity to differentiate toward an unlimited number of cell fates. This is what will make them a powerful tool in future regenerative medicine. Therefore, we need new methods capable of quickly evaluating the response of many cell lines to many different environments. In this proposal, we will develop and test a new test bed to evaluate the capabilities of potential human embryonic stem cells: the chicken embryo. Since chicken embryos develop outside the body in an egg, they are highly accessible to experimental manipulations. This enables us both to introduce reagents (ie., human embryonic stem cells) and to visualize the response of those reagents to their local environments as they happen. We propose to introduce human embryonic stem cells to six different organ systems during several stages of development. This will test the ability of these cells to respond to a large number of different environmental stimuli. Since different embryonic cell lines may have different capacities, we will test the abilities of seven different human embryonic stem cells. We will compare their response with that of partially differentiated cells that should have more limited differentiation capabilities. Transplanted cells will be fluorescently tagged so their migration can be traced by fluorescence microscopy. Antibodies and probes of molecular expression will be used to assess the response of these cell lines to different environments. Their origin (human or chicken) can also be confirmed with these methods using different antibodies and probes. This will help us to develop a set of formal criteria that to assess the response capability of hESC as they progressively become more differentiated. To further understand molecular aspects of the cellular response, we will begin to characterize changes in molecular expression that take place as cells progress toward specific cell fates. This profile will enable us to begin to understand molecular factors which regulate cellular differentiation, so they can be harnessed for effective future regenerative medical applications. This last goal will serve to show the power of this technology, but will have to await a later stage of funding to be completed.
Statement of Benefit to California:
Human embryonic stem cells offer tremendous potential toward significant advances in the new age of regenerative medicine. These cells can be induced to differentiate along many different cell fates, providing the promise of tissue and/or organ replacement or supplementation. This approach offers great hope toward improving health care especially where tissues are damaged due to disease or injury. Ultimately, this approach could reduce health care costs and increase the well being of the general population. We expect that many new embryonic stem cell lines will be derived with support from the California Institute for Regenerative Medicine. We would not be surprised if the ability of these putative stem cells to differentiate toward specific cell fates differed from cell line to cell line. Additionally, some cell lines may lose their differentiative capacity as they are kept in in vitro culture conditions. Our research proposal aims to provide an easy and effective assay to test the pluripotentiality of these new putative human embryonic stem cell lines. In order for these cell lines to live up to their full potential and be useful in curing human diseases, we must understand their pluripotential properties. To date, assays of pluripotentiality have depended on 1) in vitro assays with limits in ascertaining the true developmental potential and 2) transplantation to mouse embryos which do not facilitate the high throughput analysis, essential to screen the myriad of generated cell lines. The latter assay also involves causing pain in a sentient being (the mother). Chicken embryos develop in an egg, outside of the body. Hence it is easily accessible to experimentation including the delivery and observation of putative embryonic stem cells. The chicken embryo is a classic model of development and has been very well documented through years of research. It offers a myriad of developmental microenvironments which can be utilized to test the responsiveness of these new cell lines. This research would contribute to the progress of stem cell research which ultimately could improve health care for everyone, worldwide. Since California is one of the first states to implement support for human embryonic stem cell research, our findings could also contribute to major economic advantages to the citizens of the state.
SYNOPSIS: The principle investigator (PI) proposes to develop a chick embryo model system to assess differentiation of hESC. The applicant will characterize the microenvironments that lead to the differentiation of hES cell types, specifically neural crest, cardiac neural crest, somites, olfactory placode, and limbs at different stage of fetal development. Working with Dr. Martin Pera, who will provide the cells and facilities for hESC studies, the applicant will transplant cells into the chick embryonic environment at different sites and times. Initially mouse cells will be used and then both NIH-approved and other human ESC lines. The applicant will use this model system to assess the differentiation potential of hESC lines and assess gene expression of hESC lines as they progress towards different lineages. It was unclear to one reviewer whether the PI thinks the gene expression studies will be accomplished within the time frame of the SEED grant or whether it is just there to let the reviewers know where the work would go. INNOVATION AND SIGNIFICANCE: One reviewer noted that although the concept of using chick embryo to assess differentiation potential of hESC is neither novel nor innovative, the experimental approach proposed is important. Another reviewer noted that no one is putting hESC into this potent developmental model, thus the innovation of the grant is high. STRENGTHS: The PI has a strong developmental biology background and the necessary collaborations, experience and equipment to study hESC in chick embryos. The use of a suitable microenvironment may well unmask unknown potential of hESC, and be useful in the study the expression patterns of now difficult-to-study differentiation pathways. The limitation of the study is that the chick embryo will send relatively ‘normal’ developmental cues and is not the hostile, pathologic environment to which stem cells will mostly be implanted clinically. Nonetheless, this is a good idea for pushing hESC in a way that will benefit the field. The team is likely to get interesting results of importance in understanding not only differentiation efficiencies of hESC, but their heterogeneity. Recent work by Kulesa showed that human malignant melanomas injected into the neural crest migratory stream can become non-tumorlike in their behavior and contribute to many neural crest derivatives. These studies are a testament to the use of the normal embryonic environment for the kind of work proposed by the investigators. WEAKNESSES: The experimental details are not well explained. The PI does not seem to be aware of more sophisticated techniques to assess the micro-environment and the response of the cells to the microenvironment. For example, it is now possible to use a laser dissecting microscope to cut out individual groups of transplanted cells or surrounding cells for Q-RT-PCR, microRNA, or other analyses of the cells. The PI does not mention but should emphasize that it is possible to take advantage of the differences between chick and human molecules to carry out differential analyses of the transplanted cell versus the host chick cells. The PI should also be able to take advantage of viral constructs to label the transplanted cells or use transgenic chicks that overexpress certain factors or do not express certain factors. A weakness is the idea that murine ES cells are a good way to get the work going, if it is hard at first with hESC. Rather, effort should be expended making the various hESC lines amenable to these kinds of experiments, and gaining expertise in their manipulation. The various lines are certain to have some individual handling issues (some are easier to get into single cell suspensions), but there is no reason to suspect that the PI will need to resort to murine cells. DISCUSSION: The proposed research plan and analysis techniques are weak but the model is important. A reviewer noted that there was not a single paper describing use of human cells in this model. At least two reviewers believed that this model was a potentially very significant model for in vivo studies and should be made more broadly available.