Funding opportunities

Development of Induced Pluripotent Stem Cells for Modeling Human Disease

Funding Type: 
New Cell Lines
Grant Number: 
Principle Investigator: 
Funds requested: 
$1 737 720
Funding Recommendations: 
Recommended if funds allow
Grant approved: 
Public Abstract: 
Human embryonic stem cells (hESC) hold great promise in regenerative medicine and cell replacement therapies because of their unique ability to self-renew and their developmental potential to form all cell lineages in the body. Traditional techniques for generating hESC rely on surplus IVF embryos and are incompatible with the generation of genetically diverse, patient or disease specific stem cells. Recently, it was reported that adult human skin cells could be induced to revert back to earlier stages of development and exhibit properties of authentic hES cells. The exact method for “reprogramming” has not been optimized but currently involves putting multiple genes into skin cells and then exposing the cells to specific chemical environments tailored to hES cell growth. While these cells appear to have similar developmental potential as hES cells, they are not derived from human embryos. To distinguish these reprogrammed cells from the embryonic sourced hES cells, they are termed induced pluripotent stem (iPS) cells. Validating and optimizing the reprogramming method would prove very useful for the generation of individual cell lines from many different patients to study the nature and complexity of disease. In addition, the problems of immune rejection for future therapeutic applications of this work will be greatly relieved by being able to generate reprogrammed cells from individual patients. We have initiated a series of studies to reprogram human and mouse fibroblasts to iPS cells using the genes that have already been suggested. While induction of these genes in various combinations have been reported to reprogram human cells, we plan to optimize conditions for generating iPS cells using methods that can control the level of the “reprogramming” genes, and also can be used to excise the inducing genes once reprogramming is complete; thus avoiding unanticipated effects on the iPS cells. Once we have optimized the methods of inducing human iPS cells from human fibroblasts, we will make iPS cells from patients with 2 different neurological diseases. We will then coax these iPS cells into specific types of neurons using methods pioneered and established in our lab to explore the biological processes that lead to these neurological diseases. Once we generate these cell based models of neural diseases, we can use these cells to screen for drugs that block the progress, or reverse the detrimental effects of neural degeneration. Additionally, we will use the reprogramming technique to study models of human blood and liver disease. In these cases, genetically healthy skin cells will be reprogrammed to iPS cells, followed by introduction of the deficient gene and then coaxed to differentiate into therapeutic cell types to be used in transplantation studies in animal models of these diseases. The ability of the reprogrammed cell types to rescue the disease state will serve as a proof of principle for therapeutic grafting in
Statement of Benefit to California: 
It has been close to a decade since the culture of human embryonic stem (hES) cells was first established. To this day there are still a fairly limited number of stem cell lines that are available for study due in part to historic federal funding restrictions and the challenges associated with deriving hES cell lines from human female egg cells or discarded embryos. In this proposal we aim to advance the revolutionary new reprogramming technique for generating new stem cell lines from adult cells, thus avoiding the technical and ethical challenges associated with the use of human eggs or embryos, and creating the tools and environment to generate the much needed next generation of human stem cell lines. Stem cells offer a great potential to treat a vast array of diseases that affect the citizens of our state. The establishment of these reprogramming techniques will enable the development of cellular models of human disease via the creation of new cell lines with genetic predisposition for specific diseases. Our proposal aims to establish cellular models of two specific neurological diseases, as well as developing methods for studying blood and liver disorders that can be alleviated by stem cell therapies. California has thrived as a state with a diverse population, but the stem cell lines currently available represent a very limited genetic diversity. In order to understand the variation in response to therapeutics, we need to generate cell lines that match the rich genetic diversity of our state. The generation of disease-specific and genetically diverse stem cell lines will represent great potential not only for CA health care patients but also for our state’s pharmaceutical and biotechnology industries in terms of improved models for drug discovery and toxicological testing. California is a strong leader in clinical research developments. To maintain this position we need to be able to create stem cell lines that are specific to individual patients to overcome the challenges of immune rejection and create safe and effective transplantation therapies. Our proposal advances the very technology needed to address these issues. As a further benefit to California stem cell researchers, we will be making available the new stem cell lines created by our work.
Review Summary: 
Executive Summary This is a proposal to improve somatic cell reprogramming in order to generate induced pluripotent stem cells (iPS cells) for use in cell-based disease models. In Aim 1, the applicant proposes to test both inducible vectors and a system that will allow excision of genes after they have induced reprogramming. Successful methods will be used in Aim 2 to generate disease-specific iPS cells in order to study the biological basis of Rett syndrome and amyotrophic lateral sclerosis (ALS). Finally, the applicant proposes to induce iPS cells into hematopoietic or hepatic lineages in Aim 3, in order to explore therapy of blood and liver diseases. This is an application from a highly accomplished investigator who proposes to collaborate with a second outstanding scientist to improve somatic cell reprogramming. The inducible method described is novel and, according to the preliminary data described, has already been accomplished. The application provides good preliminary data, and the in vitro model of Rett syndrome would be a great asset to understanding this complex disorder. However, reviewers commented that the application was overly ambitious and unfocused. They also commented that the proposal itself was not well-crafted and Aim 3 was only superficially thought-out. A major strength of this proposal is that the two groups involved in the experiments are recognized as leaders in the field and have the experience and the technical know-how to carry out the work described. The proposed method for generating iPS cells is novel and would be a significant advance in iPS cell technology. The cell lines to be generated through reprogramming will be appropriately tested for their pluripotency. Each part of Specific Aim 1, if accomplished, would advance the field. In Specific Aim 2, the major advance would be in the development of an in vitro system of the complex disorder Rett syndrome. This would be significant and novel, and the applicant is an expert in many aspects of the investigation as proposed. It will be important to determine whether the Rett syndrome results obtained in mice are repeatable in human iPS cells. The contribution of the ALS work is less clear, and although the applicant does not cite them, there are recent publications of similar results in a mouse model of ALS. Reviewers were unclear that anything new would be gained from the proposed ALS experiments. In Aim 3, the applicant moves away from neuroscience and proposes to investigate blood disorders and hemophilia, moving into mouse models for cell therapy. The applicant does not have much expertise in this area. Reviewers commented, for instance, that Factor VIII is not produced by hepatocytes as stated by the applicant but mostly in large vessel endothelial cells in the liver. This aim was a distraction and suggested a lack of focus. During programmatic review, reviewers discussed this application’s principal investigator, the potential importance of the new techniques for generating iPS cells, and the fact that the proposal addresses an important disorder (Rett syndrome). Overall, however, reviewers remained concerned at the lack of focus of the proposal and therefore did not make a motion to move it into the funding category. Reviewer One Comments Significance: Methods to remove integrating genes in iPS cells are clearly required, and new technologies to do this need to be developed. The proposed use of 1) self deleting viruses and 2) a single virus with all pluripotency genes is a good idea that would, if it worked, have a significant impact on the field. Equally, the use of iPS cell derived hepatocytes or HSCs represent an attractive way to treat hemophilia or diseases such as SCID respectively. Feasibility/Research Plan. The strengths of this application are the PIs, the novelty and the significance. There are however a number of weaknesses The plan is very poorly focused – to examine 4 diseases is unrealistic and is accompanied by a lack of depth in the analysis. This superficiality is exemplified by the section on Rett syndrome. The interesting cross-genotype transplant experiments described in the aims are not detailed in the research plan. None of the translational applications are described in enough detail to be confident that progress could be made There is no plan B for the self-deleting viruses should they fail, and no preliminary data to support their use. Preliminary data is provided for Tet-inducible systems, but these are not a major focus of the proposal. In the single virus experiments, how will any necessary changes in the balance of expression be achieved? Responsiveness to RFA: Good Reviewer Two Comments Feasibility: Strengths: 1- The approach. PI will develop two new viral vector systems, much improved than the ones described by Yamanaka and Thomson’s groups. In one system, the authors use tetracycline inducible vectors. Upon the addition of tetracycline MEF and human cells are induced to de-differentiate. This is novel and according with the preliminary data described, has already been accomplished. The second approach will rely on the use of cre-lox system to remove the vectors after they have been used to dedifferentiate cells 2- The principal investigator. Dr Gage has a long record of accomplishments and will certainly produce most of the results proposed. 3- The collaborator. Dr Verma’s laboratory has demonstrated the capacity to built lentiviral vectors of all kinds as well as other viral systems. Weaknesses 1- Aim 2 and 3 are dependent on the success of aim 1. Regardless, the authors have already partially shown that they can generate iPS cells in their own laboratory. 2- Rett syndrome results obtained in mice may not be repeatable in human iPS cells (Aim 2). Unfortunately the text in aim 2 has the wrong numbers on the references. Responsiveness to RFA: Yes Reviewer Three Comments Significance: This proposal has significance to stem cell research in that it will 1) develop new techniques of reprogramming human fibroblasts to iPS cells using conditional vectors that will allow the excision of transgenes involved in the dedifferentiation of the cells. 2) They will generate iPS cells from patients with Rett Syndrome and ALS and then differentiate them into the appropriate neurons and glia. These cells could then be used to model the target disease. If this were accomplished, this would be a significant advance. 3) They will also induce iPS cells into hematopoietic or hepatic lineages to explore therapy of blood and liver diseases. Feasibility: If the work detailed in Specific Aim 1 is successful, this would be a significant advance in the iPS technology. A major strength of this proposal is that the two groups involved are both recognized as leaders in the field and have the experience and the technical know-how to carry it off. The cell lines generated will be appropriately tested for their pluripotency. Each part of Specific Aim 1 if accomplished, would advance the field. In Specific Aim 2, the major advance would be in the development of an in vitro system of the complex disorder Rett syndrome. This would be very novel and the PI is an expert at many aspects of the investigation as proposed. In contrast the ALS work is not so novel as two other groups have already published here and identified the potential role of mutant astrocytes in motor neuron death. While iPS cells can potentially be differentiated toward any cell type, I feel that the work described in Specific Aim 3 detracts from the overall focus. In my view it would have been a stronger and more focused proposal if it had been limited to the first two Specific Aims, perhaps choosing another neurodegenerative disease. Also while they state that they have differentiated hES cells to endoderm in the preliminary results, no actual data is shown. I was slightly disappointed with the preparation of this proposal. There were places where data should have been referenced (e.g. p5 – “A mouse model of the disease…”), a number of grammatical and/or spelling mistakes and inadequate figure labeling (Fig. 3). This figure also should really be in the Preliminary Results. To reiterate however, a huge strength of this proposal is the investigators themselves and the institution where they are located. Responsiveness to RFA: Yes on both counts.

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