Basic Biology I
Hematopoietic cells are responsible for generating all cell types present in the blood and therefore critical for the provision of oxygen and nutrients to all the tissues in the body. Blood cells are also required for defense against microorganisms and even for the recognition and elimination of tumor cells. Because blood cells have a relatively short life-span, our bone marrow is constantly producing new cells from hematopoietic progenitors and responding to the relative needs to our tissues and organs. Blood cancers (leukemias), as well as other disorders or treatments that affect the production of blood cells (such as chemotherapy or radiation therapy) can significantly jeopardized health. Transfusions are done to aid the replacement of blood cell loss, but pathogens and immunological compatibility are significant and frequent roadblocks. In this grant application, we present experiments to further understand how another cell in the body, the endothelium, located in the inside wall of all our vessels, can be coax to produce large numbers of hematopoietic cells with indistinguishable immunological properties from those in the bone marrow of each individual. Endothelial cells are easily obtained from skin biopsies or from umbilical cord and they can be expanded in Petri dishes. The experiments outlined were designed to further understand how endothelial cells are capable of generating blood cells during development. This information will be used to entice endothelial cells to generate hematopoietic cell progenitors in vitro. The impact of this research is broad because of its clinical applicability and because of its potential to decipher the mechanisms used by endothelial cells to undergo normal reprogramming and generate undifferentiated progenitor cells of a distinct lineage. Adult cell reprogramming is one of the fundamental premises of stem cell research and thus, highly relevant to the main goals identified by the CIRM program.
Statement of Benefit to California:
Technology developed from this grant application has the potential to be translated directly to clinical settings. This technology is extremely likely to engender interest by the big pharma which can potentially license the information from the University of California or purchase the patent for the invention / technology. Naturally this will bring revenues and recognition to the state of California. Furthermore, California will remain ahead of the technological wave that takes advantage of stem cell technology and implements innovative medical treatments in the entire country and abroad. In addition, the execution of this proposal will immediately provide employment to four individuals, two of these trainees in stem cell research. Indirectly, the grant will also support salaries of employees at the university associated with research, animal care and administration.
In this proposal the applicant seeks to define the transcription factors that endow embryonic endothelial (blood vessel) cells with hemogenic (blood forming) potential and to exploit this knowledge to confer hemogenic potential to adult human endothelial cells. The endothelial origin of hematopoietic stem cells (HSCs) in the embryo has only recently been established. This proposal focuses on the mechanisms responsible for the spatiotemporal restriction in the hemogenic potential of endothelial cells. In Aim 1, the applicant proposes to characterize the origin, properties and transcriptional profile of hemogenic endothelial cells using lineage tracing in mice as well as transcriptional and epigenetic profiling. In Aim 2, knockdown and overexpression constructs will be used to test whether transcription factors identified in Aim 1 alter the hemogenic capacity of endothelium in vivo. Finally, in Aim 3, transcription factors identified in previous aims will be expressed in adult human endothelial cells in an attempt to reprogram them into hemogenic cells. Reviewers agreed that this proposal could have a significant impact and possibly translational applications if successful. The limited availability of HLA-matched HSC donors as well as the shortcomings of umbilical cord blood transplantation highlight the need to identify alternative sources of HSCs for a range of therapeutic applications. The current lack of robust, reproducible methodologies to specify HSC derivation from pluripotent stem cells compounds this problem. The idea of using the mechanisms by which hemogenic potential is conferred on embryonic endothelial cells to reprogram adult endothelium to achieve the same result is innovative and represents an exciting alternative strategy to generate HSCs. Reviewers appreciated that this project is based on a sound and logical rationale and is focused on the mechanisms responsible for the unique hemogenic properties of endothelial cells in early development. Reviewers raised a number of issues with the research plan that led them to doubt its feasibility. They criticized Aim 1 as constituting a "fishing expedition" but still found it feasible and original. One reviewer described Aim 1 as poorly designed, noting that no clear strategy had been articulated for narrowing down the genes that will come out of the transcriptional and epigenetic profiling screens. This reviewer also worried that the anticipated use of 4-5 animals to validate each target gene by RT-PCR methods could be prohibitively expensive and time-consuming if 100(s) of genes require follow up. Furthermore, this approach may not be sufficiently informative; thus, the use of RNA in situ hybridization to provide in vivo localization of gene expression was suggested instead. Reviewers pointed out that most of the proposed experiments will be performed in mouse, and only the last aim uses human cells. This raises questions regarding the proposal's responsiveness to the RFA, which was limited to the study of human cells but allowed use of model organisms for groundbreaking reprogramming studies. A reviewer also complained that the proposal was very poorly written, with numerous typographical errors. Reviewers described the applicant as an outstanding established investigator who consistently publishes in high quality journals. They noted that s/he has been very productive recently and has made important contributions to the field of vascular biology, including data that endothelial cells are direct precursors to HSCs. They agreed that the research team has the appropriate expertise to carry out the proposed research. Overall, reviewers appreciated the significance and potentially high impact of the scientific problem addressed by the proposal and its innovative approach but they had concerns about the feasibility of the research plan. PROGRAMMATIC REVIEW A motion was made to move this application into Tier 1 - Recommended for Funding. The maker of the motion argued that this proposal has groundbreaking potential (reprogramming of adult endothelium to HSC) and would broaden CIRM's portfolio. Embryonic hemogenic endothelial cells are the only cells known to be able to reprogram in vivo and thus are important to study. Some panelists reemphasized concerns regarding the proposal's feasibility, whereas others noted that the proposal may be "high-risk" but has the potential for very high reward. The motion to move this application to Tier 1 carried.