Funding opportunities

Development of Blood and Liver Stem Cells from Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00422
Funds requested: 
$498 704
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
The aim of this research project is to develop a new supply of transplantable blood and liver cells from embryonic stem cells. These cells would have wide application in treating birth defects, cancer and viral diseases by cellular transplantation. To achieve this goal, efficient methods of generating blood and liver cells from embryonic stem cells must be devised. The approach used in this proposal is to apply knowledge gained from the study of how stem cells normally develop to devise methods that can be used for the production of stem cells for human transplantation. The hypothesis being pursued is that tissue stem cells generated from embryonic stem cells are more similar to fetal stem cells than adult stem cells. The implications, if true, is that knowledge of the growth properties of fetal stem cells can be used to devise the best technology for growing tissue stem cells from embryonic stem cells. This project proposes to determine the best conditions for growing blood and liver stem cells from human embryonic stem cells. Various protein growth agents known to play a role in the early stages of development of blood and liver tissues will be tested. A number of different embryonic stem cell lines will be tested under the best growth conditions for their ability to form blood and liver cells. Understanding the variability in the growth of stem cell lines is important in determining the feasibility of using different cell lines to treat patients. Lastly, another goal is to compare how similar tissue stem-cells grown from embryonic stem cells are to normal blood and liver stem cells. Gene expression and other changes can occur to cells grown outside the body and it is important to understand these differences and if they pose any danger to patients treated with cultured cells.
Statement of Benefit to California: 
This research project is aimed at developing a new supply of blood and liver cells from embryonic stem cells that can be transfused or transplanted into patients for a variety of disorders. Blood diseases such as sickle cell anemia and thalassemia as well as liver diseases caused by viral infection, drugs or inherited disease affect many thousands of Californians. Often, transplanting healthy cells offers treatment or a cure for many of these diseases, but a lack of available or suitable donor tissue prevents such therapy in many cases. Embryonic stem cells offer the hope of generating a sufficient supply of tissues for cellular therapy. To achieve this goal we are studying the factors involved in growing embryonic stem cells and turning them into stem cells that form blood or liver tissues. The successful outcome of this work will offer new hope to many Californians suffering from blood or liver diseases. This will improve lives and save money on long-term health care costs associated with these diseases. Development of the technologies and expertise to bring these novel forms of therapy from the laboratory bench to hospital bedside will also keep California in the forefront of the biotechnology industry, will attract talented scientists and clinicians to California and will create high-paying jobs.
Review Summary: 
SYNOPSIS: The objective of this proposal is to establish methods for the development of hematopoietic (HEM) and hepatic (HEP) human embryonic stem cells (hESC) for eventual use in clinical transplantation to correct inherited and acquired diseases. hESC lines will be cultured with relevant molecular factors and in various combinations to generate HEM and HEP sublines which will then be compared to HEM and HEP cell populations derived from midterm fetal livers for the pattern of gene expression and other phenotypic markers. The possibility of differentiating and defining distinct subpopulations from cultures of hESC, upon diverse culture conditions, may allow isolation schemes that perform similarly across hES cell lines. The Principal Investigator (PI) will receive assistance from several collaborators. SIGNIFICANCE AND INNOVATION: A better understanding of hESC differentiation into the hematopoietic system and hepatic lineage will certainly help enhance the prospect of the clinical applications of stem cells. A combination of methods that produce reproducible gene expression patterns starting from different ES cell lines would be a practical contribution of great importance for the future of hESC utilization in research and clinical therapy. More specifically, the patterns of gene expression of HEM and HEP stem cells derived from fetal liver will be used to define the most desirable cells in hESC cultures by comparison to those of early fetal liver cells. The definition of HEP cell characteristics would be a real contribution, as these patterns are not as well known as those for hematopoietic stem cells. Midterm fetal liver-derived HEM and HEP cells appear to be the most efficient in generating additional HEM and HEP cells, and are even hypothesized to be superior to adult and neonatal stem cells and even to fetal liver cells. If gene expression differences between fetal liver HEM and HEP cells from different developmental stages are confirmed and associated with functional differences, and similar differences in hESC cultures could be identified, identification and analysis of the corresponding cells in hESC cultures would importantly facilitate their research and clinical utilization. Expression patterns will be compared across different hESC lines and culture conditions and may serve to classify hESC-derived stem cells to generate cloned cell sublines with optimal HEM and HEP characters. Particular gene expression patterns may define hES cell subpopulations that are especially suited for the treatment of specific diseases. While not particularly innovative, other than with respect to the HEP cell characterization in hESC cultures, the proposal’s Specific Aims and experimental approaches will allow a systematic exploration of fetal liver cell gene expression patterns as indicators of the cultured hES cells’ ability to differentiate into HEM and HEP sublines, and determination of their growth and survival characteristics. The proposal is largely straightforward testing and screening, but its potential pay off is substantial. STRENGHTS: The PI has extensive experience in hematopoietic system development, and has also worked on hepatic progenitor cells. The PI has published numerous research papers since his/her graduate fellowship. This team and collaborators should be able to perform all experiments listed. For example, one collaborator, a recognized expert in this area, will provide training on the generation and maintenance of hESC lines. The specific molecular marker used for fetal liver is considered a strength because this PI has contributed to characterizing this factor. This marker may facilitate the definition of the cultured hESC phenotypes and gene expression patterns that corresponds to fetal liver HEM and HEP stem cells. Also a strength is the assay used to measure differences in growth capabilities. This assay, which has been developed by the PI’s group, is a semi-quantitative method for comparing the expansion of hESC. Although tedious, and while it only captures snapshots of otherwise time-dynamic events, this assay is certain to generate useful data. Specific Aim 2 will be very interesting, and these studies will compliment very well with the gene expression studies. If accomplished, this work will shed much new light on not only the differentiation potential of different stem cells but also the comparative “identity” or “similarities” of differentiated cells derived from different stem cells. WEAKNESSES: Although the proposal work is logical and well laid out, the innovative aspects that give the team a competitive advantage are not very clear. The PI seems to underestimate the difficulty of hESC differentiation to hepatic and hematopoietic lineages. This might due to the lack of direct experience with hESC. The PI does not present a plan of action for the circumstance in which the approaches proposed may provide inconsistent observations. For example, the desired fetal liver-like gene expression pattern may not be constant in culture, or a particular pattern may be simply the average of cells whose individual patterns differ after cloning a hESC with a particularly desirable type. Importantly, there was no description of the cloning and subcloning experiments. Similarly, although the PI is aware that results may vary with the time in culture for some or most of the hESC lines, there are no specific proposals for investigating the effect of the number of culture passages or the consequences of different derivation or maintenance techniques. More importantly, despite the embryonic origin of the cell lines that may be developed using the proposed approaches, these cell lines express HLA genes. Transplantation of expanded cultures of HEM and HEP hES cells would undoubtedly be subject to immune recognition and rejection by allogeneic recipients. The proposal fails to describe methods for avoiding the consequent rejection response. A few details regarding the experimental approach also raised concern. First, obtaining sufficient quantities of differentiated cells for comparing to those obtained from fetal liver will be difficult. Getting sufficient viable cells for the gene expression study may be hard, too. Second, the multi-cell lineage gene expression study may not be straightforward. The analysis will not be a simple statistical analysis of identifying differentially expressed genes, but will need some pattern recognition. DISCUSSION: This work is relatively obvious, and thus not very innovative, but the generation of reproducible gene expression patterns is of great practical importance. The growth capability assay is "time-consuming" and rather "boring", but will yield a much better understanding of the differences in culture conditions. In extended discussion, reviewers felt that the degree of heterogeneity is likely to be so considerable in the analysis population that the gene expression patterns may be simply not relevant. No cloning or subcloning of the population is proposed to get around this problem.
Conflicts: 

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