Funding opportunities

Generating Reprogrammed Pluripotent Human Cell Lines Using a Histone Modification Barcode

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00106
Funds requested: 
$2 569 999
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human embryonic stem cells (hESCs) hold significant promise for the future of cell-based therapeutics through their ability to differentiate into any cell lineage. A major consideration for cell transplantation therapy is to transplant cells that will not be rejected. The most effective way to do this will involve deriving hESC lines from a patients own cells. This is achieved through dedifferentiation of an adult cell back to a pluripotent state in a process known as reprogramming. For successful reprogramming three fundamental problems need to be tackled: 1. Identify an efficient reprogramming strategy; 2. Develop a comprehensive in vitro assay to measure reprogramming success; and 3. Understand the mechanism by which reprogramming occurs in order to prevent reprogramming towards cancerous transformation. In the current proposal we aim to develop a comprehensive in vitro assay to measure reprogramming success, and use this assay to monitor reprogramming of an adult human cell to a pluripotent stem cell. Results from this proposal will generate a sensitive and specific reprogramming diagnostic tool that measures genome-wide nuclear changes that associate with formation of a reprogrammed pluripotent cell type. This will significantly improve success rates of reprogramming, and enhance the methods for generating therapeutically useful cell types.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) hold significant promise for cell-based therapeutics for diseases including HIV/AIDS, Alzheimer’s and neurodegenerative disorders such as Parkinson’s and MS/ALS, type I diabetes, heart attack, stroke, and tissue repair following devastating injury such as damage to the spinal cord. For treatment using cell-based therapies, the derivation of individualized hESC lines would be ideal, since genetic differences between individuals prevents a `one cell line fits all’ approach. The only way to generate individualized hESC lines is through the process of nuclear reprogramming. At the current time, nuclear reprogramming of human cells is extremely inefficient. In this research proposal, we aim to improve the existing methods for monitoring nuclear reprogramming. We also aim to use this information to reprogram human cells without the requirement of a woman’s oocyte for somatic cell nuclear transfer. Research findings from this proposal will provide a much-needed barometer for reprogramming success allowing the generation of individualized human stem cells.
Review Summary: 
SYNOPSIS: This proposal seeks to define an epigenetic “barcode” of pluripotent cells. The applicant will use ChIP on chip, and DNA IP on chip, comparing a wide variety of cell types. These include hESCs, ICM, differentiated cells, PGCs, PGCs induced to pluripotency by multiple means, skin fibroblasts, and fibroblasts into which retroviral expression vectors are introduced carrying genes associated with pluripotency. IMPACT AND SIGNIFICANCE: The idea that epigenetic modifications largely define pluripotency is compelling, since somatic cells can be reprogrammed to pluripotent cells by nuclear transfer, and since the DNA sequence of stem and somatic cells is the same. The methods that are being employed are relatively new but have been introduced to the stem cell arena by other groups. The conceptual framework is descriptive and not particularly original. The proposed research would provide a very detailed picture of the epigenome of pluripotent cells, which in and of itself, would be of some utility. The applicant hopes that the acquisition of this comprehensive set of data will assist efforts to reprogram cells, but this hope seems to be quite optimistic, as histone modifications will be used mostly as a post-hoc assay to judge reprogramming after it is complete. Overall, it is not clear that the proposed work would be likely to yield improved methods to reprogram terminally-differentiated cells. QUALITY OF THE RESEARCH PLAN: This is an overambitious and poorly focused proposal to explore the epigenome of pluripotent cells. As the investigator notes, we know virtually nothing about this key area, yet the approach taken is to evaluate large numbers of cell types and large numbers of marks, hoping that a “barcode” for pluripotency emerges. Antibodies will be used for H4K16, H4K8, H3K9, H3K18, H2AK5, H3K56, H4K20, H4R3 by ChIP on chip, and DNA methylation by MeDIP. There is no discussion of the problems inherent in the methods, including sensitivity and specificity. While good success has been shown by others in euchromatin map marking, heterochromatin is much more difficult. Special methods used by others for amplification of heterochromatin are not addressed. Further, the small numbers of cells is a significant problem. The investigators plan to circumvent this problem using a new method from others involving carrier chromatin, although it was not used for high throughput microarray analysis. The methods are comparable across the aims, which address different cell types. Aim 1 involves a comparison of hESC, the differentiated BJ cell line, and ICMs. They acknowledge the difficulty of obtaining sufficient material from blastocyts and propose the carrier approach described above, but have not shown its feasibility in this context. The numbers of replicates and statistical considerations are not addressed. Aim 2 involves human PGCs isolated from gonads and derivation of EGCs according to published methods. In preliminary data, they demonstrate the ability to culture ESCs but not the derivation of EGCs. They will also activate oocytes for parthenogenetic growth. The aim seems tangential to the application, as it is unclear whether the marks they observe are related to germ cells, treatment, or imprinting defects in parthenogenotes. Aim 3 involves reprogramming an established fibroblast line using known pluripotent markers expressed in retroviral vectors, or combinations thereof. This is a particularly weak aim, as the investigators have not considered the substantial genetic or epigenetic alterations that plague established cell lines. It would be more straightforward to address the changes associated with loss of pluripotency in cells obtained under the previous aims. There is no discussion of validation of the antibodies themselves, which vary greatly in specificity even from lot to lot, or of the results that will be obtained. They also do not explore what a barcode will mean from a statistical standpoint. Given the large number of marks and the limited number of biological replicates, how will they know what a marker for pluripotency is? STRENGTHS: A comprehensive picture of the epigenome of hESCs and EGCs will be determined. This is important because epigenetic modification is likely to be important in understanding pluripotency. Methods to reprogram fibroblasts might be achieved by adapting the successes of Yamanaka to a human system. WEAKNESSES: The proposal lacks focus. The applicant would be better served by taking a narrower focus that would be explored in depth. Potential weaknesses of the approach should be addressed, along with controls that will be done to control for these. The critical issues of antibody sensitivity and specificity are not addressed nor those of validation of methods or results. There are also problems of obtaining sufficient cell numbers as discussed above. Carrier ChIP may be difficult to perform on pools of 3 or so human ICMs. There is a conspicuous lack of a statistical approach in defining what a "bar code" means. It will be difficult to cross-compare the many cell types and experimental conditions. Knowledge of the epigenetic "barcode" of any cell type cannot easily be used as a direct tool to achieve reprogramming. Rather, it provides only a somewhat tedious post-hoc assay of completed reprogramming. It is proposed that knowledge of histone modifications at a genomic level will lead to better methods of reprogramming, but this may be overly optimistic. A better rationale for performing these expensive experiments might be developed for future applications. DISCUSSION: There were no issues raised in discussion other than those above.
Conflicts: 

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