Human embryonic stem cells (HESCs) are capable of giving rise to a variety of differentiated human cell types that in principle could be used therapeutically to treat tissue damage that arises in human disease. The promise of HESCs is still quite limited because of technical limitations in our ability to propagate these cells in culture, while retaining their potency to become many different types of cells, and to guide them to become the right type of cell needed for clinical use. The proposed work will develop the tools to address these issues, by focusing on the Notch signaling pathway. Studies of the Notch pathway in model organisms like mice has shown that it plays a pivotal role in regulating the development of embryonic cells, by activating critical target genes that maintain cells in a proliferative, undifferentiated state. The proposed experiments will examine the activity of the Notch pathway in HESCs, as they are experimentally induced to form the precursors to nerve cells. The long-term goal of this work is to develop the information and tools needed to manipulate HESCs in culture via the Notch pathway, allowing one to better control their proliferation and differentiation into defined cell types.
Statement of Benefit to California:
The goal of the proposed research is to develop tools that can be used to manipulate human embryonic stem cells, thus allowing them to be more effectively used as therapeutic agents. The process we are studying will help define optimal procedures to encourage human embryonic stem cells to produce homogeneous populations of specific neural cell types that are needed to replace damaged neural tissues for patients with Parkinson’s and other neural diseases.
SYNOPSIS: This proposal is for basic investigations of notch signaling in hES cells as differentiation to neural progenitors proceeds. The project seeks to translate data derived from cancer cell lines to the hES system. INNOVATION AND SIGNIFFICANCE: The proposed research is important, fundamental, not quite perfect, but will advance the field as a foundation for other work. STRENGTHS: The PI is an outstanding biochemist who studies transcription factors. The labs involved (PI and co-PI) are solid labs, with proven productivity and rigor, addressing an important element of future hES cell control. The logic and rationale are well described. In previous work in cancer cell lines, the PI showed that notch cooperates with AP2 to induce Erb2, while in normal development both notch and erb2 are essential for radial glial cells. WEAKNESSES: The major issue is that the basic protocol for generation of neural progenitors in vitro is not developed. The PI indicates that the laboratory of Dr. Gage has developed protocols that will be adapted, but there is no indication of consideration for the heterogeneity of the cultures, which will be essential to address in order to interpret the meaning of gene expression levels, etc. Notch of course does many things, so how will it be possible to determine that correlative effects relate to neural progenitor development rather than some other lineages? It can be challenging to translate data from homogeneous tissue culture cells to a complex heterogeneous ES culture, and this needs to be addressed. Will subsets of cells be sorted and if so by what means? Will only start and end points be evaluated? Also, it is not clear that there are not many such pathways that might differ substantially for forebrain vs. more caudal identities, to name just two. This weaknesses could be minimized by adjusting to rapidly advancing knowledge in the field, as well as by incorporating the world's input on ES cell to neural fates, rather than one local protocol only. The project is a collaboration between Drs. Jones and Kinter. While these PIs bring expertise to notch and neural development, they have no experience with hESC work. Dr. Gage, who must supply the protocols, is not involved in any manner. The role of Dr. Kinter is not clear, outside of general advice, since he works in Xenopus. DISCUSSION: This proposal is based on long standing expertise in both labs (PI and co-PI) in notch signaling pathway. It is a 'transition proposal' to apply notch signaling expertise to hESC. This is an important signaling pathway. It is not clear if the involved labs have the relevant experience with hESC and neurogenesis. The protocols for making neural progenitors are poorly developed. Reference is made to a neuroprogenitor differentiation assay by an investigator (Fred Gage) who does not appear to be invoved in the proposed research. The source of hESCs is unclear and it can be challenging to go from working with relatively homogeneous mouse cancer lines to heterogeneous hESC lines. Also, this pathway could be studied well in mice.