Tools and Technologies II
The future success of regenerative medicine depends on our understanding of human stem cell biology. Stem cells are unique because they have the capacity to generate many different cell types. The goal of regenerative medicine is to harness the power of stem cells to repair or replace damaged tissue due to disease, aging, trauma or inherited defects by generating new, functional and transplantable human tissue in the laboratory. To accomplish this, it is imperative to define how tissue specific stem cells (also termed adult stem cells), cells capable of generating each cell type within a specific organ (i.e. blood stem cells generate both red blood cells and white blood cells), develop from conception to adulthood. However, inadequate tools and resources to identify, isolate and purify tissue specific stem cells are major roadblocks in tracking and profiling tissue specific stem cells throughout development. This issue confounds the development of novel therapeutics from pluripotent stem cells, which posses the potential to become any cell in the body, including tissue specific stem cells. We have developed a new systematic approach to isolate and study rare tissue specific stem cell populations. The focus of this project is on two adult stem cell populations, hematopoietic (blood) and hepatic (liver). Our approach includes isolating hematopoietic and hepatic stem cells from human fetal and adult tissues using known cell surface markers, followed by molecular profiling studies to identify new markers that will further enrich for the stem cell population. Monoclonal antibodies (molecules designed to target specific cell surface markers) will be generated to bind and tag the newly identified markers. These antibodies will aid in separating stem cells from other cells. Every novel marker identified will be functionally validated to ensure that real stem cells are isolated. This process is repeated continuously to further enrich for a pure stem cell population. Our findings will not only provide the field with standardized methods and tools to define novel markers for human adult stem cell identification, isolation and purification but will also directly advance efforts in regenerative medicine specifically targeted for blood and liver disorders by providing new reagents for the identification of hematopoietic and hepatic stem cells.
Statement of Benefit to California:
In an era of healthcare reform and economic depression, the growth of the State of California largely depends on the innovation and discovery generated by both its academic and industrial sectors. This project represents a collaboration between academia and industry to advance the translation of stem cell research from bench to bedside. A major roadblock in accomplishing this goal is our limited understanding of the biology of human tissue specific stem cells due to a lack of resources and necessary reagents. Tissue specific stem cells are unique cells capable of regenerating an entire organ. The studies proposed herein will provide the stem cell community with new tools,reagents and standardized methods to identify, isolate, purify, characterize and molecularly interrogate human tissue specific stem cells to advance the development of stem cell-based therapeutics. These new therapies will impact on children and adults suffering from diseases of the blood and liver. The data produced has the potential to directly impact California citizens and their families who have a disease or disorder that could benefit from stem cell therapy.
This proposal is focused on the identification of novel markers of tissue-specific blood and liver stem cells and the development of tools to isolate pure populations of these cells. The applicant argued that the lack of well-defined human tissue-specific cell surface markers is a translational bottleneck, as these markers could aid efforts to define and isolate hematopoietic stem cells (HSCs) or hepatic progenitor cells derived from pluripotent stem cells. There are two Specific Aims: (1) to identify novel cell surface markers of HSCs at different stages of development, generate novel antibodies to these markers, and test the function of purified HSCs in vitro and in vivo; and (2) to perform similar sets of experiments with cells of the hepatic lineage. Reviewers were not convinced that this proposal addresses a translational bottleneck, particularly Aim 1, which is focused on HSCs. They noted that a tremendous effort over many years has been put toward defining surface markers on hematopoietic progenitors. As a result, adult HSCs are one of the best-characterized cell populations, with a large number of markers for prospective isolation already identified. Reviewers did not agree with the applicant’s supposition that the absence of cell surface markers for HSCs is a major roadblock for progress in this system, arguing that the need for better differentiation methods is the rate-limiting step. While there was slightly more enthusiasm among reviewers for Aim 2, as hepatic progenitors have been studied much less intensively, they did note that available antibodies can enrich progenitors roughly one hundred-fold from adult liver. They described the overall approach of focusing on identifying markers for HSC or hepatic stem or progenitor cells at early stages of development as a strength of the proposal, but questioned the hypothesis that in vitro differentiation will necessarily recapitulate differentiation during development. Recent successes in direct reprogramming suggest that this may not be the case. Finally, reviewers noted that the proposal’s approach and methods are not particularly innovative or novel. Reviewers commented that the iterative approach based on global transcriptional profiling and bioinformatic analyses to identify surface markers with the desired specificity appears sound and feasible. However, they raised several concerns with the research plan and its feasibility. They appreciated the strong preliminary data but were not convinced that the applicant could make meaningful advances beyond currently established methods for isolating progenitor populations. Reviewers cautioned that the proposed HSC marker set might not mark HSCs throughout development, as two of the markers are activation markers that may reflect the current cycling status of the cells. They further noted that the proposed in vitro assays for hepatic progenitors may not accurately reflect their stem cell character and that the in vivo animal model has some clear weaknesses, as acknowledged by the applicant. The reviewers did appreciate that pitfalls and alternative approaches are discussed in the research plan. However, they found aspects of the timeline unrealistically optimistic, especially the estimated of only three months required for the generation of novel monoclonal antibodies. Reviewers described the Principal Investigator (PI) as a well-established scientist with an excellent track record in human pluripotent stem cell research. They agreed that the PI has assembled a well-qualified team to conduct the proposed research and described the available facilities and resources as excellent. One reviewer did note that the subcontract budgets for antibody development seemed surprisingly small. Overall, while reviewers were supportive of the research team and certain aspects of Aim 2, they were not convinced that the proposal addressed a significant translational bottleneck or would have a major impact on the field of regenerative medicine.
- A GWG member proposed a motion to assess the level of enthusiasm among the panel for funding Aim 2, focused on hepatic progenitors, to the exclusion of Aim 1, focused on hematopoietic stem cells. Reviewers reiterated their opinion that this proposal will not remove a major roadblock to translation. It was noted that CIRM has already funded a large Early Translational grant focused on generating functional hepatocytes from human embryonic stem cells. The GWG did not support a motion to consider the application further.