Tools and Technologies I
Human stem cells (hSCs) have been recognized as an inexhaustible source for regenerative medicine, a promising platform for developing and testing new drugs as well as an invaluable tool to study human development. However, specific tools and technologies of tracking and manipulating hSC’s multiple lineage development potential (also known as pluripotency) and differentiations into different adult cell types in responding to exogenous stimuli are far from clear. The ability to direct track the differentiations of hSCs into specific cell types, e.g. motor neuron progenitor and mature motor neuron, will significantly advance the hESCs research and translation into medical and pharmaceutical applications. Although many traditional techniques have played important roles, rapid progresses of stem cell research are limited because of lack of new tools to follow stem cell fate. One of the tools missing is the comprehensive reporter system for tracking stem cell fates for basic research and pre-clinical development. The current tools and techniques used to track stem cell fates are generally traditional approaches, such as luciferase reporter gene, single fluorescence protein reporter, RT-PCR, immunofluroescnece staining, and immunochemistry staining. These techniques are either destructive or labor intensive, or high technique challenging. Therefore, it will be very valuable and critical to develop non-invasive, image-based, and quantitative marker system to track different stem cell fates in culture conditions and in transplantations. We propose to develop critical basic tools of establishing multi-fluorescent protein markers for stem cell pluripotency and lineage-specific markers in one vector for monitoring stem cell fate simultaneously. Technologies to make the stem cell manipulation and track their fates easier are vital to the further developments in the field. If this proposal is funded by CIRM, it will lead to a new set of technology and products developments. The outcomes of the project will improve our capability of manipulating hESCs for medical and pharmaceutical applications in vitro and in vivo, and particularly facilitate the eventually development of treatments for degenerate diseases like Parkinson disease and diabetes. It will make immediate impact on both cell replacement therapy and drug discovery and development of hESs.
Statement of Benefit to California:
Rapid progresses have been made in human stem cells (hESs) research recently. hESs initially derived from embryos or recently derived from somatic nuclear transfer and combinatorial gene expression, represent a inexhaustible source of precursor cells to treat degenerative, malignant, or genetic diseases, or injury due to inflammation, infection, and trauma. This pluripotent cell has been hold as a possible means for treating Parkinson’s disease, Alzheimer’s, diabetes, spinal cord injury, heart failure, and bone marrow failure. Meanwhile, hSCs are an invaluable research tool to study human development, both normal and abnormal, and can serve as a platform to develop and test new drugs. The proposed project of development of image-based stem cell fate tracking system, when successful, eventually leads to a new set of technology and products developments. The outcomes of the project will enhance our capability of tracking hSCs for medical and pharmaceutical applications in vitro and in vivo, and particularly facilitate the eventually development of treatments for degenerate diseases like Parkinson disease, heart failure and diabetes. Improved function in patients afflicted with these diseases will greatly promote the public health and result in tremendous savings to California in healthcare costs, particularly in the areas of long-term care. Federal constraints on stem cell research create a critical need for non-federal funds to achieve these goals. Funding by the California Institute for Regenerative Medicine improve California’s stem cell infrastructure and speed the translation of basic university research into medical products that change lives in the nearly future. These products, and methods will improve the tax base, create many new jobs, and save billions in healthcare costs in California. Stem cell research is currently a very strong research area. California has initiated the strongest efforts in stem cell research in the world. Therefore, California is viewed as a world leader in basic research, biotechnology, and pharmaceutical research and development of stem cells, and the technology advances in the field made here will immediately contribute to the California’s leading position in these fields.
This proposal focuses on the development of multiple fluorescent protein markers and their expression via a single vector to allow tracking of stem cell (SC) fate. The applicant proposes to use various promoters to drive expression of different fluorescent proteins in several steps of SC fate determination—from pluripotent SC to progenitor cell to terminally differentiated cell. The group plans to transfect human embryonic stem cells (hESCs) with the resulting vectors, stably express the reporter genes and then test their functionality in vitro and in vivo. All reviewers agreed that the impact of this proposal, if successful, would be very high. The ability to label hESCs with different colors for each stage in their development along different lineages would aid research on SC differentiation as well as enhance the ability to track the movement and lineage of transplanted cells. On the other hand, reviewers had many problems with the research design and did not find the proposal to be feasible as written. Specifically, reviewers found the application to be disorganized; lacking clearly defined milestones, with too many aims and without a logical order to experimental plans. Moreover, reviewers felt they were not presented with sufficient data to convince them that the applicants would be able to deliver on the design of the constructs or accurately image multispectral details and temporal dynamics of the fluorescent reporters. One reviewer expressed doubts about the possibility of expressing three reporter gene constructs via a single vector. This reviewer thought it would be very difficult to place all of these elements into one lentiviral construct because of size, and even more difficult to express all three transgenes without insertion site effects. Reviewers also described the proposal as overly ambitious. Several aspects of the experimental design lead reviewers to question the research team’s SC biology background: One reviewer was puzzled by the choice of genes along the cell lineage development (ex. using a neuron-specific marker for cardiac progenitors). This reviewer noted that choice of markers for germ layers was similarly not optimal. A reviewer also pointed out that the differentiation protocols described were developed for mouse rather than hESCs, and will not work in hESCs if just transferred from the mouse literature. While reviewers agreed that the applicant and research team are qualified to perform the vector engineering, they raised questions about their experience with hESCs and recommended collaboration. Reviewers were very confused by the identification of personnel. Specifically, four trainees are included, but one has no name or description of his/her role and should be deleted. Moreover, a reviewer found that two trainees who are listed at 59% effort each, appear to be in charge of the major efforts underpinning the project. Finally the principal investigator (PI) is listed at only 12% effort despite the fact that his/her one source of external support lists the PI at only 5% effort. Given these issues, the budget justification for personnel does not seem realistic. Overall, while this proposal has worthy goals, it is poorly organized, exceedingly complex and has a number of flaws in its experimental design that make it unfeasible as written.