Grant Award Details
A suite of engineered human pluripotent stem cell lines to facilitate the generation of hematopoietic stem cells
Our goal is to generate a toolbox of human embryonic stem cell lines to monitor blood forming (hematopoietic) stem cell development in culture. Currently, we lack markers that we can use to pinpoint the developmental stage of human ES cell derived candidate hematopoietic stem cells (HSC), and whether they have established the correct molecular machinery required for proper function. Our studies have revealed that correct pattern of HOXA gene activity is essential for specifying human HSCs and maintaining their function. We have generated fluorescent reporter hESC lines for HOXA9 and HOXA10 genes, and are preparing the constructs to target the earlier HOXA genes. In addition, we have generated hESC lines that express a fluorescent reporter from the regulatory elements of novel HSC genes, MLLT3 and HLF. Our next goal is to determine culture conditions that will generate HSCs that are able to turn on and maintain the expression of these important HSC regulators. This knowledge will be critical for generating HSCs in culture dish for transplantation therapies to treat leukemias and other life-threatening blood disorders.
<p>The major shortcoming in generating functional hematopoietic stem cells (HSC) from pluripotent stem cells is the lack of adequate markers for specification and maintenance of functional human HSCs. To overcome this hurdle, we have generated fluorescent reporter hESC lines for a set of novel human HSC regulators that we have identified. As these factors are localized in the nucleus, it is not possible to observe their expression using conventional methods without killing the cell. We have now finalized the targeting and have progressed with validating the expression of the fluorescent reporters for the HSC regulators. By comparing single cell RNA seq data from hESC derived cells to human early developmental tissues, we have shown that the expression patterns for these genes and their reporters matches with that in early human developmental hematopoietic tissues, with each HSC regulator being expressed in the most undifferentiated HSPC, and selectively in other cell types (HLF also in hepatocytes, MLLT3 and HIF3 also in erythroid cells, HOXA5, HIF3a and MYCT1 also in distinct endothelial cell, HIF3A in mesenchymal stroma). Our functional studies on these factors have revealed distinct roles in HSC biology for each one of them. Our studies also suggest that these hESC reporter lines will become valuable for investigators working on optimizing stem cell differentiation and culture for other tissues, as many of these reporter genes are not only restricted to HSC. Using these reporter lines to monitor the induction of HSC "stemless' program, we are making progress in optimizing differentiation conditions toward generating functional HSCs for transplantation.</p>
Grant Application Details
- A suite of engineered human pluripotent stem cell lines to facilitate the generation of hematopoietic stem cells
Our goal is to develop tools that address major bottlenecks that have prevented the generation of blood forming stem cells in culture for therapeutic use. To help overcome these bottlenecks, we will generate a suite of human embryonic stem cell reporter lines that can be used to monitor key milestones in blood stem cell development. These lines will serve as tools to identify factor combinations to improve the in vitro differentiation of hESCs to functional blood stem cells. Once individual lines have been validated, lines that contain multiple fluorescent reporters will be generated, and a multi factor screen will be performed to optimize conditions that induce these blood stem cell regulators. To track the location and quantity of transplanted cells in recipient small animal model, we will generate hESC lines with in vivo reporter system that combines bioluminescent or PET imaging, and serum-based assay. Our in vivo tracking tools will be broadly relevant and not restricted to studying the in vivo biology of blood forming cells. These tools will help translate the promise of stem cells to cell based therapies to treat human disease.
Statement of Benefit to California:
This project will help improve California economy as many of the vendors used for reagents and supplies are located in California. This project will also help create and maintain jobs for skilled personnel and helps train post-doctoral fellows who will become the next generation of stem cell scientists. The long-term goal of this project is to improve in vitro differentiation protocols to create transplantable blood forming stem cells for therapeutic use. If we, or others who will use our reporter lines generated in this study, achieve this goal, there will be new, theoretically unlimited sources of HLA-matched or patient specific blood stem cells that can be used for treating many serious blood diseases, including leukemias and inherited immunodeficiencies or anemias. Availability of patient specific blood stem cells for transplantation would be a major benefit in California, as there is currently limited availability of suitable bone marrow donors for individuals from mixed ethnic backgrounds.
Source URL: https://www.cirm.ca.gov/our-progress/awards/suite-engineered-human-pluripotent-stem-cell-lines-facilitate-generation