Although the hemangioblast population is reasonably well characterized in mice, the same cannot be extrapolated to humans because of several key reasons: 1) undefined, inefficient methods for differentiation of hES/hiPS cells to hemangioblasts, 2) lack of faithful markers for various stages of hemangioblast development, 3) lack of understanding of key developmental stages and molecular events in the generation of hemangioblasts, 4) lack of long-term engraftment of human hematopoietic progenitors (HPCs), 5) early senescence of the isolated progenitors. Identification of human hemangioblasts in combination with our differentiation protocols will open the door for potential gene-correction approaches, thus leading to personalized treatments involving autologous transplantation. Gene-editing technologies could then be applied not only to restore a wild-type version of a mutated gene but also to restore normal levels of soluble factors that might be deficient in the patient. Taking into account the potential of hemangioblasts to differentiate towards endothelial lineages and connect to the pre-existing vasculature, genetic modification and further differentiation of hemangioblasts to vascular cells might allow for personalized cell-engineering leading to secretion of desired soluble factors to the circulation system. Moreover, the lack of knowledge regarding a HPC long-term reconstitution population strongly hampers the potential use of in vitro generated HPCs in a clinical setting and currently limits treatment of hematopoietic diseases to bone marrow transplantation of HLA-matched donors. In this regard, human HPCs have been speculated to be contained in a CD34+ population of cells however the specific nature of real long-term human HPCs in terms of surface markers remains undefined. Several reports have pointed out the possibility for human long-term HPCs to be contained in a CD34+ side population. Even though promising, the current approaches have yet failed to concisely identify and characterize a human HPC long-term reconstitution population. Thus, we believe our methods to generate HPCs by hemangioblast generation and/or transdifferentiation could provide the perfect platform for the generation and developmental study of human HPCs. Of special interest will be the transplantation of hemangioblast for the treatment of ischemic areas in order to restore the normal oxygen supply. Moreover, the information obtained might serve as the basics for hemangioblast-like targeted therapy in solid tumors requiring angiogenesis.
Altogether, we believe that knowledge of the basic biology underlying hemangioblast generation and differentiation will push forward regenerative therapies in human, whether they involve or not gene-editing approaches. Moreover, a deeper understanding of the human hemangioblast biology will surely contribute to the implementation of iPS-hemangioblasts based therapies into the clinic.
Statement of Benefit to California:
In 2006 a major breakthrough was published when researchers discovered the way to exploit the inherent plasticity of somatic cells in order to reprogram adult fibroblasts and skin cells back into a cell type referred to as an induced pluripotent stem cell (iPS), which appears to be indistinguishable from a pluripotent ES cell. This is accomplished without the need for embryo destruction, solving most of the ethical concerns raised by the use of ES cells. The possibility to take somatic cells from the diseased patient opens the unique opportunity for personalized therapy and autologous transplantation. Yet, a major hurdle on iPS technology, as well as with ES cells, is the need for highly efficient and robust protocols for differentiation. Efficient differentiation is of major importance as transplantation of differentiated cells alongside undifferentiated ones could potentially lead to tumor formation. In this study we propose to gain insight into the basic mechanisms of differentiation. We have developed a highly efficient and robust protocol for the differentiation multipotent progenitors of not only ES cells but also up to 12 different iPS lines tested so far. Furthermore, during the course of differentiation we observed different subpopulations of cells that could potentially bear bias differentiation potential towards one or more of the cell lineages derived from this multipotent progenitors or hemangioblast-like cells. Our findings could be used as a basis to enhance new strategies that render the process of directed differentiation more efficient, thus reducing the risk of cancer formation. Rapid generation of an unlimited number of the desired cell types will contribute to the treatment of acute diseases, such as heart failure. Indeed, heart failure could be potentially ameliorated by transplantation of vascular progenitors as shown in animal models. Moreover, understanding the basic mechanisms leading to hemangioblast generation as well as a full molecular characterization can lead to the development of highly specific differentiation and isolation protocols minimizing the risk for cell contamination and uncontrolled differentiation towards undesired cell lineages. We anticipate that our work will generate important contributions to understanding the basic mechanisms of gene regulation, differentiation and the upstream signals regulating cell-fate. This knowledge can be of great importance to uncovering the molecular mechanisms governing pluripotency, cell plasticity and differentiation. We feel confident that the proposal presented here will open the door for highly efficient, exogenous DNA-free, and chemically defined methods of differentiation. Our research will benefit the State of California by generating knowledge that will clearly reinforce the demonstrated scientific leadership of California and that in the long term will inspire strategies applicable to therapeutic approaches that will directly benefit the people of California.