Human ES cells can be used to make healthy neurons to replace the cells that are lost in neurological diseases such as Alzheimer’s and Parkinson’s diseases. This holds great hope for future treatment of these disorders. Our research aims to produce neurons from ES cells more efficiently. During the process of differentiation to form nerve cells, human ES cells first develop into primitive neural progenitor cells before they become mature functional neurons. Our research focuses on how we can expand the neural progenitor cell population.
We have found that a cell surface protein called Wnt can expand neural progenitor cells. However, it is not clear how this protein can make human neural progenitor cells grow. This proposal will address the question. We will determine which surface molecules can recognize Wnt and how these signals control cell growth. This proposed research will help us to find a method to expand neural progenitor cells and thus make more neurons in the future. This will eventually contribute to the clinical application of human embryonic stem cells in the treatment of neurological diseases.
Neurodegenerative diseases, such as Alzheimer’s and Parkinson diseases, and neuronal injuries caused by stroke and trauma, will result in neuronal cell loss. Current treatments cannot replace or recover the neuronal loss. Our proposed research ultimately may lead to the development of an effective treatment for the neuronal cell loss. Once we find a detailed Wnt pathway to control neural progenitor cell expansion, there will be many possible clinical applications. Transplant-ready neuronal tissues, gene therapy, and drugs targeting the Wnt pathway to promote neuronal regeneration are some of the treatments of the future for neurological disorders. Our finding will facilitate the progress of developing those new, promising treatments. The success of our project in neural progenitor cells expansion will greatly improve the quality of health for Californians.
SYNOPSIS: A goal of this proposal is to clarify the role of Wnt signaling in hESC-derived hNPCs. The PI has found that: Wnt-3a induces expansion and maintenance of hESC-dervied NPCs; Wnt-3a can bind to the transmembrane protein Ryk, which is a novel receptor for Wnts; and Ryk plays an important role in the proliferation of ESC-derived NPCs in mice. The PI hypothesizes that Wnt-3a-induced expansion of hESC-derived neural cells is mediated through Ryk protein as a result of cleavage upon Wnt stimulation with translociation of Ryk to the nucleus. In Aim I, the PI will determine if Wnt-3a signals through Ryk protein to regulate proliferation of hESC-derived NPCs by seeing the effect on proliferation in response to Wnt signaling in hNPCs transduced with Ryk siRNA-GFP. In Aim II, the PI will determine if: Ryk functions in NPCs are dependent on or involve Frizzled (a canonical receptor for Wnt proteins). In Aim III, the PI will investigate if cleavage of Ryk protein by Wnt-3a is important for NPC proliferation.
INNOVATION AND SIGNIFICANCE: An understanding of the molecular mechanisms for the viability and differentiation of stem cells is key to realizing their potential. The processes involved in these mechanisms depend on transcriptional programs and signaling networks that regulate gene expression. This proposal aims to identify mechanisms involving the Wnt pathway by which hNPCs expand during the differentiation of hEScs. The hypotheses of this grant and the proposed role of Wnt-3 in hESC-derived neural cells are novel, and the methods that are planned are contemporary and up-to-date ones. The studies will provide insights into the mechanism of signaling by Wnt proteins that may be important in stem regulation in normal cells and cancer.
STRENGTHS: This is a hypothesis-driven and complete project that may shed light on ES cell differentiation into various neural lineages. The PI has interesting preliminary data from Ryk knockout mice that suggest that Ryk is important for NCP proliferation. The preliminary data also suggest that the PI will be able to carry out the specific aims and that the plan is feasible. The results of this proposal may have signficant impacts on understanding the differentiation of hESCs into neural cells and may influence the protocols used in the preparation of these cells. Anticipated problems are noted and potential alternative plans are provided.
WEAKNESSES: One reviewer expressed concern about the presentation of some of the data. Fig. 5 is said to show that Wnt-3a is expressed in both ESCs and NPCs, however, the band corresponding to Wnt3a in NPCs is difficult to see. The amount of the CTF compared to full-length Ryk is very low in Fig. 7B compared to Fig. 7A for unclear reasons. There is an asterisk aligned with a band in all the lanes, but it is not clear what this represents. What is CTF2 in this figure? The figures are not cited in the order in the text, which is a bit confusing. Additionally, the PI is taking a risk in developing this work since Ryk may have little to do with ES cell differentiation. Finally, although the PI has published in outstanding journals, there are no publications listed since 2004.
DISCUSSION: This promising junior investigator has proposed significant and novel work concerning an important differentiation pathway in hESCs. There is general acceptance that Wnt signaling is important in hESCs, and the study of Wnt3a and the new Ryk receptor that shows nuclear accumulation is very novel. The PI is an expert in this field and the work has significance that extends beyond the immediate field of study (i.e., hESCs). Substantial mechanistic studies are proposed with good biology; however, the proposal is risky given that Ryk may have nothing to do with hESC differentiation, and there was a little sloppiness in preparing the text and figures, which lowered reviewers' support.
In addition, a question was raised about why one should do these studies in hESCs, especially since it was noted that Wnt3a causes proliferation of undifferentiated cells (a recent PNAS paper was cited that shows Wnt3a is necessary for proliferation in defined media). Reviewers appreciated that signaling pathways are context-dependent and if Wnt has some role in differentiation or proliferation, it should be studied in hESCs.