Using modern proteomic and molecular tools to identify novel MSC and hESC lineage specific membrane markers: Development of new antibodies for live cell imaging and isolation
Tools and Technologies I
In order to move stem cell research closer to the clinic and fully realize the potential these cells have in bettering the human condition, researchers must learn as much as possible about how these cells replicate and differentiate into adult tissue. They must be able to identify cells at specific stages in complex mixtures and have the ability to isolate them for detailed analysis. The most useful set of tools for accomplishing this are antibodies which recognize specific proteins that are expressed at certain stages in the cell's path toward the fully differentiated state. There currently exists a broadly known set of markers and antibodies that have been used in the field for some time. Many of these markers are proteins that reside inside the cell which essentially requires killing the cells to allow the antibodies to recognize their target. Up to now, there have been few concerted efforts to identify new markers and develop antibodies to them. Key to isolation of specific populations of cells for basic research, diagnostics, or therapy will be to have a robust set of antibodies that recognize specific lineage markers that reside in the cell membrane and are accessible to antibodies without killing the cells one is trying to isolate. The project proposed in this application will use the most modern cell culture, proteomic, and molecular tools to create a workflow for identification of novel lineage-specific membrane protein markers. These markers will in turn be used to create new antibody tools that will allow identification and isolation of live cells from cultures and complex tissue preparations. These new antibodies will fill a necessary (and currently somewhat empty) toolbox for fast detection and gentle isolation of cells at specific points in differentiation both for study and eventually for diagnostic and clinical use.
Statement of Benefit to California:
The work proposed here will push the boundaries of stem cell research by providing a new set of modern tools for identification and isolation of cells at specific points as they differentiate to adult tissue. These tools will be developed by a California company known to lead the field of research reagents that will ensure rapid and efficient development and broad distribution of the new tools to the research community. By partnering California tax payer money for this development and the reagent tools powerhouse of a known California-bases reagent provider, the taxpayers can be assured that repayment of their investment in the form of agreed upon royalties on sales of these tools would be forthcoming. Further, successful development of this new set of stem cell tools in California, by Californians will increase the stature of this state as a center of expertise for stem cell research.
This proposal focuses on the identification of novel, lineage-specific membrane markers for mesenchymal stem cells (MSCs) and human embryonic stem cells (hESCs). These markers will be validated by the development of novel antibodies that will allow the identification, isolation and tracking of stem cells and their various progeny. Using MSCs and their in vitro differentiated progeny as proof-of-principle, the Principal Investigator (PI) proposes to use a proteomic approach to identify differentially expressed cell surface proteins and generate monoclonal antibodies to epitopes on these molecules. The utility of these antibodies will be validated in part by attempts to isolate MSCs from complex tissue sources. A final component of the project is to use the same proteomic approach to identify membrane markers for neural precursor cells derived from hESCs. The reviewers agreed that this proposal addresses a significant roadblock to stem cell research. However, they noted that its strategy is high-risk and raised concerns about feasibility. They praised the PI and research team as qualified to carry out the proposed research. The reviewers agreed that this proposal could have a broad impact on the field. There is a pressing need to identify membrane markers that distinguish stem cells from their differentiated progeny. If this proposal were successful, it would yield valuable antibody reagents for the stem cell research community. One reviewer did question the significance of the proposal, noting that there are at least ten different commercially available antibodies for MSCs. But other reviewers commented that most of these antibodies are generic and confirmed that this proposal addresses an important need. These reviewers also noted that MSC populations are often heterogeneous and poorly defined. They thought the PI could have made a stronger case for the value of MSC antibodies that distinguish subpopulations within a culture. The reviewers’ main concern with this proposal was its feasibility. The PI acknowledges that the strategy is high-risk and may identify as few as one unique marker, although they hope for ten. Reviewers felt that the preliminary data was not strong enough to justify this high-risk approach. They also raised some concerns about the research design. One reviewer noted that the antibody enrichment criteria set in the second aim of the proposal may not be high enough to obtain meaningful results. Preliminary data presented in the proposal further support the view that substantial further optimization of the protocol will be required. The lack of stronger preliminary data is a weakness of the proposal. Another reviewer found the third aim to be overly ambitious, describing collection and analysis of proteomic data from cells derived from number of donors, as well as induced differentiation products. It was unclear to this reviewer why differentiated progeny have value if the goal is to discriminate MSCs from alternate tissue sources. Overall, this reviewer felt that the third aim could be dropped in favor of the fourth, which represents the greatest value in the proposal by developing lineage-specific tools to discriminate hESC-derived progenitors from each other and their differentiated progeny. The reviewers praised the research team and found it well qualified to carry out the proposed research. They noted that the PI has a very strong background in stem cell biology and biomarkers, the co-investigator has complementary expertise in proteomics and mass spectrometry and the research technician is experienced in stem cell research. The reviewers also appreciated the PI’s committed effort for this project. They felt the budget was appropriate but one reviewer commented on the lack of details on the consultant’s role and time commitment on this project. Overall, the reviewers felt this was a strong proposal from an excellent team that could have a broad impact on stem cell research. However they described the approach as high-risk and questioned its feasibility as written. PROGRAMMATIC REVIEW A motion was made to move this proposal from Tier 2 to Tier 1 based on the need for additional valuable immunoreactive reagents to identify, track and isolate cells. Other panel members noted that other proposals in the biomarker category had been recommended for funding and that programmatically the biomarker category was adequately represented. The motion failed to move this application from Tier 2 to Tier 1 failed.