Funding opportunities

RUNX1 in maintenance, expansion, and differentiation of therapeutic pluripotent stem cells

Funding Type: 
Basic Biology II
Grant Number: 
Principle Investigator: 
Funds requested: 
$1 371 540
Funding Recommendations: 
Grant approved: 
Public Abstract: 
Recent technical advancements in human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) production have revolutionized their potential applications in regenerative medicine. However, a remaining big hurdle in this process is the need for efficient, effective, and stable generation of specific cell types from such stem cells for therapeutic usage. The ultimate goal of the proposed study is to identify approaches to increase the production of therapeutically useful blood cells from human ESCs and patient-specific iPSCs. Currently, bone marrow transplantation is the best way to cure many blood-related disorders, such as sickle cell anemia, thalassemia, and blood cancers like leukemia. Furthermore, blood transfusion is an effective way to rapidly counteract blood cell loss due to ablative treatments, such as chemotherapy and radiation therapy. Unfortunately, the limiting factor in transplantation and transfusion treatments is the lack of matched donors. The ability to producing unlimited numbers of blood stem cells and/or functioning differentiated blood cells from human ESCs and patient-derived iPSCs will greatly improve the opportunity of such treatments. In this application, we propose experiments to examine how specific factors that control gene expression can promote blood cell formation, expansion, and differentiation from human ESCs and iPSCs. We plan to use experimental tools to control the time and amount of expression of these factors in human ESCs and iPSCs during their growth in the tissue culture. Furthermore, we will study the generation of blood cells based on specific markers present on the blood cell surface. Current technology used to produce iPSCs utilizes retroviruses to introduce genetic material. To avoid the creation of unfavorable mutations due to random insertion of DNA fragments into the genomes of these cells, we will also explore the possibility of delivering cell membrane penetrating versions of these factors in cell culture medium. The proposed studies will provide valuable insight into the control of stem cell differentiation and the therapeutic usage of factors that regulate gene expression, which are highly relevant to the main goals of CIRM.
Statement of Benefit to California: 
Thousands of Californians are suffering from blood-related diseases that may potentially be cured with bone marrow transplantation and/or blood transfusion. However, these life-saving measures are limited by a lack of eligible donors and the necessity of finding correctly matched blood products. Current treatments for some of these conditions can cost patients tens of thousands of dollars per year. Despite these treatments, many patients die from their disease waiting for a bone marrow transplant. Recent technical advancements in human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) production have revolutionized their potential applications in regenerative medicine and have provided enormous hope for these patients. Based on our accumulated knowledge in blood cell-related research, we propose to identify useful tools and methods to enhance the specificity and efficiency in the production of blood cells from human ESCs and iPSCs. Producing therapeutically useful differentiated cells from pluripotent stem cells is a critical step in raising ESCs and iPSCs into the realm of clinical application. Therefore, one long term benefit of the proposed work is to improve the treatment of thousands of Californian patients who need to receive healthy, functioning blood cells to alleviate their disease conditions. In turn, this will benefit California’s financial status in reducing the cost of treating these patients with expensive yet ineffective methods. The proposed research will continue to maintain California’s leadership in the field of stem cell research. Our proposal will provide a better understanding of the mechanisms involved in producing blood stem cells and mature blood cells from ESCs and iPSCs. We will also explore the use of a novel method in producing these cells, and thus enhance the field of stem cell biology. In addition, the involved work will include the training and education of some of California’s bright young minds. This preparation will instill in them an enthusiasm for biomedical research and allow them to become successful scientists in the future.
Review Summary: 
EXECUTIVE SUMMARY The goal of this proposal is to investigate the mechanisms by which the master regulator, RUNX1, contributes to differentiation of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) towards the hematopoietic lineage. To achieve these ends, a series of three Aims has been proposed. First, the applicant will explore the role of RUNX1 in hematopoiesis by creating hESCs with inducible RUNX1 expression. Next, the applicant will conduct similar studies using iPSCs and retroviral expression of RUNX1. For the third and final Aim, the applicant seeks to express cell-penetrating RUNX1 proteins and test their function in the production of hematopoietic stem cells (HSC) from hESCs and iPSCs. The reviewers agreed that this proposal addresses a major unsolved problem in the field of regenerative medicine, an inability to derive sufficient HSCs from pluripotent cells or adult tissues. If successful, this effort could lead to novel mechanistic insights as well as a potential new means for treating and investigating blood disorders. The reviewers praised the innovation and creativity of both the specific molecular hypothesis to be tested as well as the approaches to be exploited, particularly the recombinant protein cell delivery system that could obviate the need for gene transduction. Based on these strengths, the reviewers were highly enthusiastic about the potential for this work to have impact. While they appreciated the clear presentation and logical rationale, the reviewers discussed a number of weaknesses in the research plan. Most importantly, there were no preliminary data generated using hESC or iPSCs, and there was no evidence provided to demonstrate the feasibility of the protein transduction strategy in the hands of the principal investigator (PI), a concern that was exacerbated by a missing letter of support from a key collaborator. The reviewers further criticized that critical data from earlier mouse work supporting the novel hypothesis to be tested were not shown. Reviewers noted that several constructs necessary for the first two Aims have yet to be generated, and some worried that the impetus for Aim 3 would be lost if these earlier approaches were to fail. One questioned why the applicant chose to use different RUNX1 expression systems for the hESC vs. iPSC studies when it might have been useful to compare them directly using the same methodology. The reviewers acknowledged that the PI is a strong leader in his/her field with an outstanding publication record in high impact journals. They did note, however, a limited experience with hESC or iPSC. While they appreciated the complementary expertise of the collaborators, the reviewers were puzzled by what they perceived to be a lack of engagement with the local community of stem cell scientists at the applicant’s institution. The research environment and facilities were considered excellent. In summary, the reviewers were highly intrigued by the innovation and significance of this proposal. While they recognized its potential for impact, they were uncertain of its overall feasibility. PROGRAMMATIC REVIEW A motion was made to move this application into Tier 1, Recommended for Funding. A reviewer reasoned that this proposal fills a programmatic need in the area of hematopoiesis for CIRM. Though the application was judged risky, the proposal was felt to represent a scientific direction with great potential. The review panel discerned that the benefits of studying RUNX1 in humans warranted the risks and outweighed the concerns over feasibility. The motion carried.
  • Ali Brivanlou

© 2013 California Institute for Regenerative Medicine