Public Abstract:
The goal of the proposed study is to generate large numbers of hematopoietic progenitor cells that can be manipulated in order to become red and white blood cells. This is important since large numbers of such blood cells can be provided to patients that suffer from different sets of immune-deficiency disorders. In principle, the gowth of human hematopoietic progenitor cells can be achieved using culture conditions in which human cord blood or embryonic stem cells (hESCs) are differentiated in the presence of the appropriate cytokines and stromal cells. However, the efficiency by which human progenitor cells differentiate from hESCs is currently inefficient at best. Here we propose to develop a novel strategy that would permit the generation of large numbers of progenitors (up to 109) from cord blood or hESCs. To accomplish this objective we would target a critical regulator of early hematopoiesis, named E2A. In previous studies, we have demonstrated that murine E2A-deficient hematopoietic progenitors can be grown indefinitely in culture without significant loss of multipotency. We have recently continued these studies with the ultimate aim to generate long-term cultures of human hematopoietic progenitors. Specifically, we have generated a lentiviral vector carrying Id2, an antagonist of E2A activity. Using this vector we have inactivated the E2A gene in murine hematopoietic progenitors and established a long-term culture of mouse hematopoietic progenitors that can be grown long-term in culture. These progenitors self-renew in vitro and remain pluripotent. In collaboration with {REDACTED} we have initiated studies to generate long-term cultures of human hematopoietic progenitors from human cord blood using the same approach. These studies are now in progress. Here we propose to continue these studies with the ultimate goal of generating long-term cultures of pluripotent hematopoietic progenitors from human embryonic stem cells. We would like to avoid using lentiviruses as a means to suppress E2A activity. To achieve this objective we propose to develop a cell permeable peptide approach, permitting us to inactivate E2A DNA binding activity in a transient manner.
Statement of Benefit to California:
If successful the proposed study would directly benefit patients that are immune-deficient. These include patients that are infected with the HIV virus, patients that suffer from auto-immune disease or cancer. For example, a prominent subset of white blood cells, named CD4 helper T cells, are critical in modulating the immune response against viral and bacterial pathogens. During HIV infection, the CD4 compartment is selectively reduced, suppressing the activity and response of cytolytic CD8 T cells, needed to abolish cells infected with the virus. Pharmaceutical therapies have been developed but they are not consistently effective and multidrug resistant viral strains are increasingly prevalent. Similarly, it is important to establish large cultures of NK and dendritic cells with the ultimate goal for the treatment of auto-immune disease and malignancy.
The efficiency by which human progenitor cells develop into commited lymphoid or myeloid cells is low. Here we propose to develop a novel strategy that would permit the generation of large numbers of human T cell progenitors (up to 109) from both human cord blood and human embryonic stem cells. To accomplish this objective we would target a critical regulator of early hematopoieisis, named E2A.
This strategy is unconventional since it would permit the growth and isolation of large numbers of human hematopoietic progenitors, has not been achieved sofar by conventional culture conditions. If successful the approach would enable clinicians to reconstitute the hematopoietic compartments of patients that are immune-deficient, caused either by genetic abnormalities, virus infection or aging.
Review Summary:
EXECUTIVE SUMMARY
The goal of this project is to capitalize on the applicant’s discovery over 5 years ago that mouse blood cell (hematopoietic) progenitors deficient in the E protein E2A, can be extensively expanded in culture while retaining their reconstituting potential. The proposed work will specifically employ Id2, a protein antagonist of E2A, to inactivate E2A function in human hematopoietic progenitors. It is hoped that this will enable the generation of large numbers of these cells, which could then be used for therapeutic purposes. In Aim 1, the principal investigator (PI) proposes to generate long-term cultures of multipotent hematopoietic progenitors derived from human umbilical cord blood as well as human embryonic stem cells (hESCs), using lentiviral transduction of the Id2 gene into the derived progenitors. The goal of Aim 2 is to circumvent the need for viral transduction by delivering cell-permeable Id2-like peptides and identifying small molecules that transiently induce Id2 activity.
Reviewers acknowledged that the proposed studies have the potential to significantly impact the stem cell field since the ability to substantially expand blood cell progenitors ex vivo is not currently possible and is considered an important bottleneck. However, they differed in their opinion regarding the proposal’s responsiveness to this RFA. While some felt that the pursuit of E protein to Id protein balance addresses a molecular mechanism, others believed that the proposal emphasizes methodology development over questions of basic stem cell biology.
Reviewers identified a number of issues with the research plan that led them to question its feasibility. While the preliminary data were compelling, they were largely focused on mouse cells. Reviewers noted that the applicant did not provide convincing evidence to demonstrate that he/she could successfully transition this type of study to human cells, despite previous attempts to do so. Reviewers further criticized that the PI presents no preliminary data using hESC and that data supporting the cell-permeable peptide and small molecule screening approaches are lacking, further calling into question whether the applicant would be able to accomplish the proposed goals. In addition to these issues, reviewers raised several concerns regarding the experimental design. Only one hESC line was proposed for study, which does not take into account potential line-to-line variation. Furthermore, the rationale for choosing that line, its NIH approval status, was largely discounted, as there are many other lines that could be utilized for this project. For the RNA profiling studies, the PI did not discuss the criteria for determining the similarity of the in vitro differentiated cells to cells from primary human samples. Finally, the description of the small molecule screen lacked important details such as information on the library to be screened and the imaging technology to be employed. While these criticisms were substantial, the reviewers acknowledged the inducible gene expression system as state of the art and appreciated the creativity of the cell-permeable peptide approach.
The PI is a professor with an outstanding track record and strong expertise in E-proteins in hematopoiesis. However, the PI has no prior publications on hESC, and reviewers expressed concern that no collaborators were enlisted to support hESC work and small molecule approaches. The research environment was judged to be suitable for the completion of this project.
In summary, this proposal addresses an important bottleneck in the stem cell field and could have a significant impact if successful. Despite the interesting supporting data from mouse studies, the reviewers lacked confidence that these findings would be readily translated into the human system. Several flaws in the experimental design further undermined their impression of feasibility.
