$2 509 437
Human embryonic stem (hES) show great promise in both therapeutics and basic research. They are a potential source of cells to treat conditions such as diabetes, spinal cord injury, and cardiomyopathy. Several roadblocks remain before the potential of hES cells can be realized. One roadblock is the poor growth of hES cells. Growth of hES cells requires a balance between survival, proliferation and differentiation signals. Neurotrophins (NTs) were recently identified as potent survival factors for hES cells. NTs cause a dramatic increase in survival of single hES cells from about 6% to about 30%. In this project, we will further define the effects of NTs on the derivation and maintenance of hES cell lines. HES cells are often grown in the presence of mouse feeder cells. These cells provide factors that support the growth of hES cells, however, efforts are being made to reduce the exposure of hES cells to animal products. We will determine whether NTs promote survival of hES cells in the absence of mouse feeder cells. Further, we will determine whether NTs will improve the efficiency of deriving new hES cells lines from embryos and the genetic stability of the new lines. HES cells sometimes acquire additional chromosomes. NTs may improve culture conditions and reduce the incidence of acquisition of extra chromosomes in hES cells. Lastly, we will use hES cell lines that have extra chromosomes to identify genes that improve the growth of hES cells. Chromosomal abnormalities are thought to alter gene expression leading to improved cell growth in suboptimal culture conditions. Any hES cell line with an abnormal karyotype represents a powerful tool to identify genes that affect the growth of hES cells. Identifying novel genes that promote hES cell growth and using those factors along with NTs to promote cell survival will improve our ability to maintain and derive hES cells. This ability will have a significant impact on the use of hES cells in the future.
Statement of Benefit to California:
Human embryonic stem (hES) cells are derived from the pluripotent stem cells of the inner cell mass (ICM) of the blastocyst-stage embryo. They retain many properties of the ICM cells including the dual abilities to self-renew and differentiate. HES cells show great promise as both research and therapeutic reagents. They provide an excellent opportunity to study disease processes in vitro and to produce differentiated cells for use as therapeutic agents to treat conditions such as diabetes, neurological disorders, and cardiomyopathy. Several roadblocks remain before the potential of hES cells can be realized. One of these roadblocks is the inability to control the proliferation, survival, and differentiation of hES cells. Insight into the factors that control the survival of hES cells will aid in the production of relatively pure populations of either undifferentiated cells or specific differentiated cell types. The studies described in this proposal will further the knowledge of the factors that control survival of hES cells. A second roadblock to the realizing the potential of hES cells for therapy is the occurrence of chromosomal abnormalities in hES cultures. These abnormalities can affect the growth properties of the cells and potentially increase the tumorigenicity of the cells. The results of this study will determine whether the addition of specific growth factors and growth factor combinations affects the accumulation of chromosomal abnormalities in hES cell cultures. Information from this study will improve the efficacy and safety of the therapeutic uses of hES cells.
SYNOPSIS: The PI proposes a study of neurotrophins on survival and proliferation of human embryonic stem cells (hESCs). Specifically, the PI will determine whether neurotrophins improve survival without serum or feeder cells, whether neurotrophins improve efficiency of establishing hESC lines, and whether they improve genomic stability. The PI will also examine aneuploidal cell lines, to see if these cells would reveal novel genes that regulate proliferation, survival, or differentiation of hESC lines. IMPACT AND SIGNIFICANCE: This is an important area of research. The PI has decided to focus on the effects of neurotrophins on hESCs. The idea to study aneuploidal cell lines to identify novel genes responsible for proliferative, differentiation, or other behavior of the stem cells is very interesting and the work has potential to yield insights into the biology of hESCs. The PI has shown that neurotrophins are potent survival factors for hESCs. The application contains preliminary data showing that hESC lines express a receptor for neurotrophins, TRK-B, particularly in those lines that also express OCT4. There is a need to understand neurotrophins and their roles in nuclear programming in stem cells, and also to better understand how the ESC genome and altered gene expression affects growth, survival, and controlled differentiation of hESCs. The present proposal could provide very important and significant data on the normal and abnormal genome of hESCs that results in distinctive ESC behaviors. QUALITY OF THE RESEARCH PLAN: One reviewer thought the research plan for the first three aims were quite straightforward and simplistic and was surprised that the studies have not yet been done. For example, it was not clear that neurotrophins alone to support hESC survival and growth in the absence of serum and feeder cells has not been investigated by the many studies of culture conditions. The fourth specific aim was considered to be a bit more risky. Overall, the quality of the research is reasonably high and the PI has experience with the techniques proposed. Another reviewer felt that the proposal involved an ambitious series of experiments proposed for a timetable where Aim 4 is the most troublesome because of how much effort will be needed to characterize genes from Aim 3 that are affected by long term growth factor-augmented culturing of hESCs. That, in addition to the effort needed for long term hESC culturing. The microarray screening studies in Aim 4 are quite demanding with regard to time and effort. A third reviewer thought that no detail was provided for alternative strategies, for means of identifying candidate genes in a rational and efficient manner, for considering the problems of whether genes of interest are proto-oncogenes, or other problems of these general categories. The only additional strategy proposed is is to use chromosome region-specific microarray chips. STRENGTHS: This is clearly an important area of research, that is, to solve the problem of generating hESCs in a manner that makes them more suitable for clinical development. Neurotrophins are amongst the most intensely studied of all cell growth factors and the PI has recently discovered that neurotrophins promote survival and proliferation of hESCs and proposes straightforward studies to ascertain the effects of neurotrophins on hESCs in the presence and absence of serum and feeder cells. The point is true that there is a pressing need for new feeder-free growth protocols for studies of hESC lines, eliminating animal products in the growth media. Since mouse feeder layers do have neurotrophins, the addition of particular and measured amounts of neurotrophins as proposed should have a very profound effect on hESC growth and survival. In the fourth aim, the PI proposes to focus on several common aneupoloidy conditions found in hESCs and to examine the genes that are affected by the chromosomal abnormalities, using overexpression and siRNA to suppress these genes to assess the effects on the cells. This is again a novel idea that the PI is quite well equipped to investigate. The PI has strong letters of collaboration from Jeanne Loring, Mahendra Rao, and Hans Keirstead. There are not that many groups looking at abnormal karyotype of hESC lines as a way to access genes involved in both normal and abnormal hESC growth. The use of aneuploid hESCs to uncover genes with altered expression leading to altered growth characteristics is potentially important. WEAKNESSES: Several weaknesses were noted for this proposal. First, no preliminary data is provided for the specific proposed experiments. Second, the track record of the applicant is relatively modest. Third, the proposed work plan is not clear for studying roles of neutrophins and ploidy in hESC proliferation, survival and differentiation. The inclusion of Drs. Loring, Rao, and Kierstead does help the cause of getting the correct cellular and molecular reagents in place in order to better understand genomic stability of different hESCs. The PI is advised to design a clearer set of studies that provide convincing rational for screening different neurotrophins in different growth bioassay settings, and focusing the gene profiling studies in a way that clearly outlines how reasonable data sets can be exploited toward understanding normal and abnormal hESC growth and differentiation. DISCUSSION: This PI has a lot of experience in genomics, but no NIH grants. In 2006, the PI showed that neurotrophins increase hESC survival. The proposed experiments are straightforward and perhaps a little simplistic. The third aim appears to be tacked on. One reviewer felt that the PI needs to write a proposal with a clear rationale for studying neurotrophins and how the data will clearly teach us something. Aim 4 was very troublesome because the characterization of genes in Aim 3 will take a lot of effort, and the array studies in Aim 4 are very demanding. Another reviewer noted that at every stage there are no alternative hypotheses proposed. Given the importance of these observations, why is there a paucity of preliminary data? The preliminary data show that the applicant can grow hESCs and make them survive on feeder layers, but there is no preliminary data for these specific experiments. This reviewer could not be enthusiastic without the appropriate supporting experiments. The reviewer also felt that Aim 4 was a weak genomic fishing expedition.