Funding opportunities

Regulation of Self Renewal and Lineage Commitment in Human Embryonic Stem Cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00478
Funds requested: 
$2 740 001
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human embryonic stem cells provide an indefinitely renewable source of any type of healthy human cell for use in research, or in transplantation therapy to treat a wide range of intractable, debilitating disorders characterized by cell loss or injury. To realize the promise of embryonic stem cell research, we must first learn to propagate stem cells on a large scale and to produce pure populations of desired cell types, for example cardiac muscle cells to treat heart disease. For some conditions we may require more than a billion cells to treat one patient. While this level of cell production is an attainable goal, at present our ability to grow stem cells on a large scale, or to obtain large numbers of specialized cells from them, is limited. This is because we do not fully understand the factors and conditions that provide for stem cell growth, or specialization into different types of body cells, and because under current conditions, stem cells can be unstable when grown in the laboratory. This research is aimed at achieving a better understanding of how stem cells multiply, and how they specialize to embark on the pathway to the formation of cells such as muscle, liver or brain cells. We will use new technical approaches to study the stem cell population in detail and to understand how to better control it. These studies also aim at ensuring that when we grow stem cells on a large scale in the laboratory, we avoid generating abnormal cells with genetic changes similar to those seen in cancer cells, to ensure safe use of stem cell derivatives in the clinic. The outcome of this work will be improved understanding of the molecular control of stem cells and improved technology for application of stem cells in research and medicine.
Statement of Benefit to California: 
Human embryonic stem cells can in principle provide an indefinitely renewable source of any type of normal tissue for use in research, including the development of new pharmaceuticals, and in regenerative therapy, to treat a wide range of debilitating disorders caused by cell injury or loss. The scope of the applications of human embryonic stem cell technology is enormous, and the major potential benefits to the economy and to public health that may follow from such research are widely recognised. The State of California is poised to take a leading role in this area internationally. In this field, as in other areas of research and development, platform technologies are a critical component of competitiveness. This project will accelerate the application of human embryonic stem cell technology by advancing our knowledge of how cultured stem cells grow and how they turn into specialized cell types. This basic knowledge will be essential to the development of methodology for large scale production of particular cell types from human embryonic stem cells. The Principal Investigator has experience in commercialization of stem cell technologies and our {REDACTED} at the {REDACTED} is well integrated into a medical school environment with excellent translational and clinical research capability. The Principal Investigator is also involved in a number of international initiatives and collaborations and his work will help to integrate stem cell research in California into these global efforts. Thus, we are well positioned to move the discoveries from our laboratory to the biotechnology sector, and into the clinic.
Review Summary: 
SYNOPSIS: The proposal intends to refine our understanding of the phenotype of hESCs and the characteristics of cells representing their immediate differentiation descendants. The underlying premise is that hESC cultures are heterogeneous, even under conditions that provide for stem cell maintenance, and that cell interactions between these varied cell types can destabilize the stem cell state and drive differentiation to specific fates. The project attempts to define and characterize the dynamic interactions that mimic the paracrine signaling that occurs in the early embryo. It is hoped that these studies will improve our basic understanding of the control of pluripotency in the early phases of stem cell commitment. To achieve the overall objective, the proposal is broken down into four aims: 1) to develop a means for isolation of the most primitive cell in ES cell cultures, the putative ultra stem cell, in order to purify it and separate it from the heterogeneous environment, 2) to study the response of this cell to candidate regulators of stem cell maintenance and differentiation, 3) to study the fate of cells that have embarked on the road to lineage commitment, and 4) to understand how genetic or epigenetic adaptation affects the population dynamics, differentiation and survival of hES cells. IMPACT AND SIGNIFICANCE: This proposal addresses significant issues of hESC biology - the efficient self renewal of the stem cell pool and determining the optimal conditions for the culture of cells in a pluripotent state; and the tendency of the cells to differentiate and to acquire karyotypic abnormalities in sub-optimal conditions. If the ultra stem cell hypothesis is upheld, it may provide a higher resolution analysis of the early stages of hESC differentiation and commitment and develop methods to ensure maintenance of hESCs in culture in an undifferentiated state. However, recent advances in the field have already provided improved methods for undifferentiated hESC culture. The prospects for generating new information by new marker identification from the heterogenous hESC population are hampered by the same problem found in hematopoietic stem cell isolation and characterization - the expression of cell-surface markers does not isolate a homogeneous population of stem cells. However, the use of multiple markers could move the field forward incrementally as it has in hematopoiesis. One benefit of the proposal is that it directly addresses differences in the required culture methods for the cells primarily studied by the Pera lab compared with other cell lines that do not require manual passaging under standard culture conditions, and with one that is routinely cultured using conditions less optimized for supporting self renewal. This characterization would yield valuable information. The importance of the preliminary findings remains a question based on insufficient preliminary data to demonstrate the existence of an ultra stem cell, and given the distinct possibility that they may simply be observing cultures that have begun to partially differentiate. Based on the aforementioned considerations, the potential impact of this proposal is deemed to be limited. QUALITY OF THE RESEARCH PLAN: This ambitious project aims to fractionate populations of human ESC based on cell surface marker expression that correlate with the stem cell/differentiation status of the cells, and to identify new markers that further identify undifferentiated, reversibly differentiated, differentiation-primed and fully differentiated cells. The PI hypothesizes that commitment to differentiation is not a binary choice, but rather that the hierarchy in hES cell differentiation is a continuum and that lineage priming – the activation of lineage specific transcription in stem cells prior to silencing of the molecular circuitry that maintains pluripotency - is a key part of the commitment process. Aim 1 proposes to isolate and characterize separately ultra stem cells and their early lineage progenitors. The PI proposes to fractionate cell populations based on cell surface markers and preliminary data is provided here on cell fractionation into 4 different populations based on GCTM-2 and TG30 antibody recognition. Putative additional ultra stem cell markers, TDGF-1, versican, 6 transmembrane epithelial antigen of the prostate, ACTRIIB, bamacan, opticin, and GP96 have been identified in this way. Using these and other candidate markers identified for the early differentiated cells, the PI proposes to further isolate subpopulations and characterize them by gene expression analysis of 96 genes. In parallel they will attempt to raise monoclonal antibodies to these new early stage linage markers. Development of monoclonal antibodies is a risky and expensive way to fractionate cells, however, novel useful antibodies may be produced by this method. The PI’s lab has developed some of the antibodies they propose to use and these monoclonal antibodies provide useful tools for the field. There is concern with the strength of the supportive preliminary data. The PI performed gene expression analysis of pluripotency genes for the 4 different populations (described above)) and for many of these genes it is not clear that the populations are substantially different from one another. In addition, the three populations that are not the putative ultra stem cell population appear to be similar with respect to extraembryonic endoderm markers and some neural markers. Statistical analysis of differences is not provided. Thus, some of these intermediate populations may represent cells in transition from one state to another or may be partially differentiated. Specific Aim 2 will study the response of the ultra stem cell to candidate regulators. By studying purified cells rather than mixed population, the hope is to define conditions that maintain this cell population in pure form. The PI cites Smith's data that holding cells in their most primitive state may simply represent the isolation of cells from differentiation signals. This aim, which proposes to use a combination of magnetic bead depletion and positive selection for surface markers to purify pluripotent cells, is highly dependent on the tools and methodologies defined and developed in aim 1. If the FACS methods proposed do not work, the PI also proposes an alternative method for the marking of the prospectively identified cells within an existing culture that will allow clonal analysis of specific cells, which is required for the rigorous analysis of the potential for self renewal and pluripotency, and culture conditions that support these properties. Following isolation of the putative ultra stem cell, the PI will examine the growth of these cells under a range of defined culture conditions particularly utilizing serum free conditions in conjunction with PDGF and sphingosine 1 phosphate, as well as testing new candidate maintenance factors GDF-3, activin and April. The PI also proposes to translate and integrate optimal maintenance culture systems, once available from the International Stem Cell Initiative Project, into this proposal. What this will add in terms of new knowledge is not clear. Finally, in this aim, they will examine the potential of the ultra stem cell for directed differentiation with growth factor signals. The hypothesis is that by isolating this cell, free from potential inducers of differentiation elaborated by other cell types, they will be able to obtain a more restricted population of lineage progenitors with particular growth factors. Unfortunately there is no proof of concept here and this is a series of undirected studies. In addition, the panel of genes to be assayed by quantitative RT-PCR is not indicated for each germ layer and quantitative PCR at the population level is not adequate technology to demonstrate population purity. In Aim 3, the PI proposes to isolate subpopulations of primed cells. How they will do this is not precisely described, but presumably they will use some combination of cell surface markers and FACS separation. They will evaluate differentiation into various lineages in vitro; and test whether placing cells in the most optimal ultra stem cell conditions will revert them to puipotency and assess their potential to form teratomas following implantation into immunocompromised mice. Flow cytometry will necessarily identify mixed populations of cells, preventing identifying reversal of differentiation status, so previously described marking studies may be required. Aim 4 is based on the observation that hESC develop characteristic cytogenetic abnormalities that render cells less sensitive to apoptosis. Previous work of the PI suggests that the appearance of these abnormalities is associated with expression and activation of a cell surface receptor CD30. All diploid lines lack CD30, while those with alterations have CD30 expression, and reduced apoptosis. The PI will test the hypothesis that activation of CD30 results in suppression of stem cell differentiation. They will study cell lines developed under conditions that express CD30 with lines that do not as well as introduce CD30 into those cell lines and compare to various other lines (e.g., the Melton lines). STRENGTHS: All the reviewers noted the PI’s expertise and extensive experience in the hESC field. This PI has worked on stem-like cells from human for 19 years, and claims to be the second group to isolate hESC and the first to report their in vitro somatic differentiation. The PI has extensive experience in the area of hESC self renewal and differentiation. There is also strong synergism among the collaborators - Sean Grimmond for transcriptional profiling and genome-wide SNP analysis; Ernst Wolvetang in Melbourne, Australia for CD30 work; and Andrew Laslet for the development of monoclonal antibodies. There is a real need to learn more about culturing cells in the optimal state. The PI has developed a number of novel antibodies to hESC. A strength is the CD30 work which has been extended in preliminary data although with a limited number of cell lines. WEAKNESSES: This is a high risk hypothesis. Much of the work rests on the underlying notion of the "ultra stem cell", and it is not certain whether this is a sustainable concept. Although cultures may be heterogeneous due to different "compartments", it is possible that heterogeneity reflects the quality of the lines as they were isolated and limitations in the methodology of isolation of new lines and their handling. Partial lineage priming may simply represent the initial proclivity of cells to differentiate in suboptimally maintained and partially differentiated cultures. This alternate hypothesis has not been disproved. The repeated use of flow cytometry to isolate and characterize hESC presents some technical problems. First, as stated in the proposal, the viability of the cells will be reduced by the dissociation of the cultures. Equally likely is the possibility that the dissociation (or removal of clumps of cells from the dish) will alter the differentiation status and gene expression patterns of the cells. These issues may lead to incorrect conclusions, and will require careful confirmation in normal culture conditions. Enthusiasm also is dampened because the experiments are not clearly laid out and the methodologies used to test the hypothesis are suboptimal. There are some discrepancies between various statements in the grant, and it is quite confusing in places, thus it is not clear in many sections what the applicants are proposing to do nor what the expected outcome is intended to be. It is not clear that this will advance the science of stem cell biology. In fact, the applicant states that they will use multiple different hESC lines, but a compelling rationale for using so many different lines is not provided. These studies could easily be done with NIH registry approved lines alone. DISCUSSION: The hypothesis is that there is an ultra stem cell that goes on to divide, while the presence of heterogeneity in a population inhibits the ability to guide differentiation to specific lineages (due to paracrine effects, etc.). The hypothesis was considered untenable as it was not upheld by preliminary data. The PI does not show morphology or histology data to exclude the possibility that this is simply a result of culture conditions on differentiation. In fact, one reviewer commented that the lack of morphology and histology in support of the hypothesis is the fatal criticism for this proposal. The PI has good experience in field, and has a number of novel antibodies, but the proposal is poorly written with a number of discrepancies. One reviewer, who claims to be more familiar with this topic, feels that while the work is high risk the results could be valuable and interesting. The difficulties with the clarity of the proposal may have arisen because this investigator is new to U.S. and grant writing techniques. Still, this is risky work that uses old techniques. The novelty of this investigator’s work is the novel markers against cell surface molecules that are not functionally characterized. Correlating the markers to functional characteristics is difficult but they can be used to eventually identify (and isolate) eventually get cleaner and cleaner population of cells as happened with hematopoietic stem cells. Reviewers feel that CIRM should support the development of novel markers, but here not enough attention was paid here to properly developing those markers. There was discussion about the PI’s specific hESC lines, and whether they are truly different. Reviewers say that these have differences in the growth and differentiation but they are pluripotent. It might be an advantage to compare these unique cells with other hESC lines under the conditions that maintain other cell lines as undifferentiated.
Conflicts: 

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