Just like cells in a human embryo, embryonic stem cells have the potential to give rise to all cell types and tissues in a human body. That is why it is an exciting prospect to use these cells in tissue repair. But in order to do so, we have to understand how we can guide the differentiation of stem cells. For example, if one wants to use stem cells for replacing defective insulin-producing cells in the pancreas, we have to learn how we can convert stem cells into pancreas cells, or at least precursors to pancreas cells. So the question is then, how do cells in an embryo become different from each other? Research done in animals has shown that there are signaling proteins that instruct cells to change from one type into another. One important group of these signaling proteins are the Wnts. Studied in our lab for along time, Wnts are powerful differentiation factors. To use Wnt proteins as factors under controlled conditions, one has to be able to isolate them. This has been a major problem in the past, but we have solved this recently. We are therefore now in a position to test how Wnt proteins, when added to stem cells, change the state of differentiation of the cells and our preliminary results indicate that there are indeed significant consequences. A second question we want to address is how we can recognize intermediate stages in stem cell differentiation. Going from a stem cell to a pancreatic insulin-producing cell is a step-wise process, following a road map where we know the beginning and the end but not the steps in between. We intend to perform gene-chip experiments to chart those steps and to map the pathways that stem cells follow to differentiated progeny cells. Finally, we will focus our research on promoting the differentiation of stem cells into endoderm, a tissue that is the precursor to pancreas development. We expect this research to contribute significantly to our insights into stem cell behavior, but also to generate new tools to improve the use of stem cells for regenerative medicine.
This research will lead to new tools to control the differentiation pathways of human embryonic stem cells. More specifically, the work aims at generating an important class of differentiation factors, the Wnts, that are known to influence how cells in an embryo differentiate. Despite the potential of Wnts in stem cell research, their practical use has been limited because the proteins were difficult to purify. Our lab has solved this problem and we will therefore explore their use. Our experiments will include adding the factors to stem cells and to test how they differentiate, in particular into endoderm, cells that are the direct precursors of the pancreas, liver, lungs and other medically-important organs. As we have done in the past, the products of the work, including the Wnt factors, will be made available to other researchers. This work will generate fundamental biological insights into how stem cells differentiate. In turn, this new knowledge should accelerate efforts to use HESC for regenerative medicine.
SYNOPSIS: The overall project goal is to study the role of Wnt proteins in directed differentiation of human embryonic stem cells (hESCs) to specific lineages with a particular focus on differentiation towards definitive endoderm. Wnt protein function in human development is particularly relevant because studies in lower organisms have determined that Wnts are important regulators of cell fate decisions and early embryonic development. However, the use of Wnts as reagents for hESC experiments has been limited because these proteins are difficult to handle and purify. The applicant's lab has the unique capacity to isolate several different Wnt proteins in their active form and has a significant degree of experience in this field. The experimental plan centers around four specific aims.
IMPACT AND SIGNIFICANCE: This proposal focuses on the important Wnt pathway in hESC differentiation with emphasis on the formation of endoderm, which is an important step towards generating certain cell types targeted in widespread human disease (such as diabetes). It may define novel regulators of hESC fate determination and define new conditions for the directed differentitation of hESCs into disease-relevant cell types. This outstanding proposal should have great impact for the field of stem cell biology and for the more specific area of endoderm derived organs. Many internal organs (lung, GI tract, liver, and pancreas) originate from endoderm, so knowledge gained will facilitate our ability to manipulate the cells towards endoderm and to identify those cells that are becoming endoderm.
The studies are also likely to generate valuable tools for the hESC investigator community including purified Wnt proteins, Sox17-eGFP expressing hESCs, and transcription profiles of hESCs both at early stages in response to various Wnt proteins as well as of hESCs at various stages of endoderm development. In addition there will be analyses of likely cell surface markers and parameters for sorting unlabeled hESCs. Overall the reagents, the cell signatures and the markers/parameters for cell sorting of intermediates to endoderm will be of major value to the field. Finally, the PI proposes the innovative use of FACS to isolate hESC-derived endodermal progenitors for study. The application of Wnt signaling to hESC cell differentiation is relatively innovative and human Sox17-eGFP reporter lines, if developed, would also be original.
QUALITY OF THE RESEARCH PLAN: This is a very ambitious research plan from a group of well-established investigators who are leaders in their fields. The research proposal is well written, with thoughtful discussions of the literature and their own findings, and the benefit of unique tools/techniques (purification of Wnts, Sox17-eGFP “knock-in” mESCs and mice, and constructs for and previous success with Oct4-GFP for making similar hESCs). The proposal addresses the interesting and medically important question of how hESC fate is determined and how it may be manipulated to promote the generation of disease-relevant endodermal cell types. However, as written, the experimental plan is quite diffuse, and there is relatively little preliminary data supporting the appropriateness of the approaches.
In the first Aim, the investigators will expose hESCs to purified Wnt proteins and assess their effects after 4 hours. It is unclear what insights into the differentiative effects of Wnts will be gained at this short timepoint, and no preliminary data is included to justify this choice. Also, it is unclear what parameters will be used to discriminate lineage choice based on gene expression profile alone. Ultimately, functional analysis will be needed, but they do not provide evidence of a robust system in which they can accomplish this.
In Aim 2, they will generate knockin hESCs to drive GFP under the Sox17 (endodermal) promoter. This is a challenging prospect, although this group has been successful in targeting the Oct4 locus in hESCs previously, and no alternative approaches are suggested if this line cannot be generated in a timely fashion. The Sox17 targeted cells will be differentiated and cell populations sorted for analysis by transplantation under the kidney capsule of NOD/SCID mice. The sensitivity of this assay and criteria for enrichment of endoderm in this assay are not discussed.
Aim 3 proposes more transcriptional profiling, over a timecourse, following endoderm induction by Wnt proteins. However, if Aim 1 fails to demonstrate the ability of Wnts to promote endoderm differentiations, it is unclear what will be done in Aim 3. Also, the purity of the populations to be analyzed will clearly impact the data significantly, and it is not clear how well-purified the profiled cells will be, or how this will be assessed.
Finally, in Aim 4, the investigators will try to identify novel cell surface markers that enable isolation of endodermal populations from differentiating hESCs without the need for Sox17-GFP. Since no strategy is described for the generation of new antibodies, one assumes this relies on known proteins with existing antibodies. Again, the assay that will be used to assess cell purification and function is not clear. Also, since discovery of novel antigens in this aim relies on transcriptional profiling of endodermally differentiated hESCs, this Aim depends on success in Aims 1 and 2.
STRENGTHS: The PI is an established investigator who has made outstanding contributions in the control of development in Drosophila. Recently, interest in Wnt biology has resulted in several important publications. The PI is collaborating with Dr. Wong and Dr. Kim in this proposal. Dr. Wong will be responsible for statistical analysis of microarray data and Dr. Kim’s expertise is in the area of pancreas development and has good experience with ESC biology. Finally, Dr Nusse has already shown the unique ability to purify several Wnt proteins for experimentation. A synergistic collaboration of these three investigators in this proposal is evident with each providing unique expertise.
The PI proposes a thoughtful use of Sox17-eGFP mESC and transgenic mice to facilitate and complement the identification of intermediates of endoderm formation from hESCs. Successful homologous recombination to target EGFP to the Oct 4 locus in hESCs and the creation of Sox17-GFP mouse ESC lines establish some feasibility for the project, but it is likely that targeting of the Sox17 locus in hESCs will be more difficult than they anticipate.
There will be a large database of microarray data at early time points of hESCs exposed to numerous Wnt proteins and varied treatments, so Dr.Wong’s bioinformatics expertise is critical. Comparison to similar data from mESC or embryonic tissues from sox17-eGFP mice being generated from other projects will be informative, particularly for determining cell surface markers for sorting definitive endoderm.
Each aspect of the proposal has reached a maturity so results within the time frame are reasonable. The facilities and resources to perform the proposed experiments are excellent.
WEAKNESSES: This proposal presents a diffuse and vague experimental design with dependent aims, that include shotgun, unfocused approaches. It will generate a lot of transcriptional array data, but is unclear exactly how useful this data will be (the timepoints chosen for analysis may impact the data greatly, and no preliminary data is given to justify the times selected). Important validation of microarrays using quantitative RT-PCR and immunohistochemistry are not included in the proposal. This is relatively simple to do and does not require the Sox17-eGFP reporter line. Moreover, information that Wnt increases Sox17 expression is entirely lacking in the preliminary data nor is it highly suggested by the studies of D’Amour et al. Because mixed cell populations may be derived even with short term culture and Wnts may have effects on each of these different populations, they may in fact get a further heterogeneously mixed population and the microarray results may be difficult to interpret.
The following questions were also posed. What are the potential targets of Wnt signaling in hESCs? What do they anticipate learning from the microarray studies? Will the investigators have enough purified Wnt proteins of various types to perform large volume hESC culture experiments? Also, they state that they will include hESC lines other than H9 in their studies, which ones and why is this not indicated?
While Dr. Nusse is the PI, most of the key personnel to be supported by the grant appear to be located in Dr. Kim's lab.
DISCUSSION: Dr. Nusse brings strength in Wnt and Drosophila melanogaster, and Dr. Kim brings endoderm and beta cell experience. Overall, this is a well-written grant, but the aims are not well integrated. For example, Aim 3 depends on success of Aims 1 and 2, and there is no good description of what they expect to see in the outcomes. The PI didn’t speak to the reagents available (apparently some are commercially available). The gene expression profiling is a shotgun approach, but aim 2 will probably provide the community with a unique resource. In Aim 3, Sox 17 probably won’t be a marker for definitive endoderm because the literature shows that it is expressed in premature endoderm, too. Wnt will have effects in many cell types, not just endoderm. In Aim 4, despite the work of a post-doc in screening 30 antibodies and finding 5 useful ones, there is no preliminary data that gives an indication that this approach will work.
Another reviewer felt that this was among the best proposals. Good cell surface markers for endoderm and intermediates are lacking in the field. The PI is the only person who has shown an ability to get Wnt protein and show that it can be used. The bioinformatics component is important because they will be developing a huge amount of microarray data on early timepoints with different Wnts. This will provide background data for the whole field, and resources and reagents that the field can move on is a big strength. The weakness is that this is somewhat of a shotgun approach and lots of information will be generated.
A third reviewer felt that the investigators are an exceptional group with a good track record and unique skill set, all of which make the questions and concerns that much greater. In using Wnts to get endoderm, they won’t get a homogeneous population, and from this they want to generate lots of transcriptional data. This data will be very messy if they start with a heterogeneous population. There are also questions about how they will identify cell surface receptors, and how they will generate antibodies to these receptors. How will they assay sorting? The antibodies for Aims 3 and 4 depend on the first aims, which is risky. The Sox-GFP knock-in is fantastic and a good resource to have, but what happens if they don’t get it? It is probable that this group will get something, but as written it is diffuse.
Additional comments were contributed by the reviewers. First, Dr. Kim is a world-renowned investigator in beta cells and holds high regard due to his broad background and productivity. The collaboration is a real endorsement for this work. Given the stature of the investigators, the grant proposal is a little disappointing. These are good people addressing an important problem, but plan that is presented is not as good as could be.
Wnts work as morphogens. A discussant posed a question of whether the applicants factored in concentration or time of exposure into their combinatorial approach? This was not addressed and is key since effects appear to be context dependent. Also not addressed are what receptors bind the Wnt ligands, and whether they are expressed in hESCs. The answer to these questions may say that Wnts are not that important (although some people claim that they are). The PI relies on a recent paper rather than presenting preliminary data for the Wnts in hESCs. The only data with hESCs came from knockin studies in mice, where they have an mESC line and a mouse Sox17 knockin line. They appear to be doing parallel studies in mouse so that they can perform the mouse to human comparisons.