Genetic dissection of mesodermal commitment to the hematopoietic fates.

Genetic dissection of mesodermal commitment to the hematopoietic fates.

Funding Type: 
New Faculty I
Grant Number: 
RN1-00575
Approved funds: 
$2,150,620
Public Abstract: 
Genetic dissection of mesodermal commitment to hematopoietic fates. Hematopoietic cell transplantation is the gold standard for cell-based therapy and is routinely used to treat a wide variety of blood disorders and cancer. A major limitation exists, however, in finding donors whose immune systems are compatible with those of the patients requiring transplantation. The recent creation of human embryonic stem cell (hESC) lines holds great promise for new cell-based therapies. ES cells can generate all cell types in the body and can be stored indefinitely. Large banks of genetically diverse or genetically engineered hESC cells could thus be used to match donor and host immune systems. For hematopoietic cell transplantation, ESCs must be coaxed to differentiate into hematopoietic stem cells (HSCs). This is currently not possible, due in large part to a lack of understanding of the molecular cues required to generate HSCs during development. In the vertebrate embryo, two waves of blood cell production occur. The first generates only erythroid cells and the second HSCs. Understanding the development of these two waves is important since ES cells have been shown to normally generate only the first. In this application, we will determine the genetic factors necessary to create HSCs from mesoderm by leveraging the unique advantages of the zebrafish system. Zebrafish embryos are transparent, and we have recently created transgenic animals that possess fluorescent HSCs. We will therefore combine genetic analyses with the direct imaging of HSC behavior in living embryos to provide an unprecedented view of HSC development. Many of the genetic pathways used to pattern the early embryo are later used to specify and maintain HSCs. These include pathways controlled by the Notch and Wnt factors. We will focus our efforts on these pathways using in vivo developmental and genetic approaches. Zebrafish possess the same blood cell types as humans, and findings in one system can be readily translated to the other. Understanding the development of HSCs in the vertebrate embryo, and how we can ultimately recapitulate this process in vitro using hESCs, is critical in improving human health since HSCs are the cells responsible for the therapeutic benefits of hematopoietic cell transplantation.
Statement of Benefit to California: 
The therapeutic use of stem cells began decades ago following the advent of bone marrow transplantation (BMT). BMT has routinely been used to cure blood cell disorders, leukemia, and immune deficiencies. BMT is often limited, however, by an inability to find donors that are genetically matched to patients requiring transplantation. The recent creation of human embryonic stem cell (hESC) lines holds great promise for new cell-based therapies, including BMTs. ESCs can generate all cell types in the body and can be stored indefinitely. Large banks of genetically diverse or genetically engineered hESCs could thus be used to match donor and host immune systems. For use in BMTs, however, ESCs must be coaxed to differentiate into hematopoietic stem cells (HSCs), the rare cell type within bone marrow responsible for the long-term, curative effects of BMT. This is currently not possible, due in large part to a lack of understanding of the molecular cues required to generate HSCs during development. The goal of our proposed experiments is to provide a better understanding of the molecules required to generate HSCs in the vertebrate embryo. The results from our powerful in vivo system can easily be translated to the in vitro system of hESCs. Insight into the molecular factors needed to drive mesodermal commitment to HSCs in vivo will be used to provide similar factors at similar timepoints during in vitro culture of hESCs to generate HSCs. Once realized, it will be possible to create genetically characterized banks of diverse hESCs that can be selected based on patient genotypes to generate HSC-based therapies. Our research will thus lead to great improvements in stem cell therapies to better meet the needs of patients in California.
Progress Report: 

Year 1

I am pleased to report that we have continued to make considerable progress on my CIRM New Investigator Award. In the past year, we have helped solve a long-standing controversy in the field of developmental hematopoiesis as to exactly where the first hematopoietic stem cells (HSCs) are born in the embryo. Using the strategies outlined in Aim 2 of this proposal, we demonstrated that HSCs are born from hemogenic endothelial cells lining the ventral floor of the dorsal aorta (Bertrand and Chi et al., Nature 464: 108-111). This new knowledge will be immensely helpful in continuing our genetic dissection of how HSCs are patterned from mesoderm, as we now know precisely when and where HSCs arise, and that a requisite step in their formation is through an endothelial intermediate. In addition, we have made excellent progress on Aims 3 and 4. Under Aim 3, we have recently demonstrated that, of the four independent waves of hematopoietic precursors that arise in the vertebrate embryo, Notch signaling plays a role only in HSC formation (Bertrand and Cisson, Blood, ePub 1/27/10). Furthermore the requirement for Notch signaling in HSCs is absolute, in that no HSCs are formed in the absence of all Notch ligands. These findings are guiding our studies in Year 3. Finally, under Aim 4, we have completed our first set of experiments on the role of Wnt16 in HSC formation and will submit a manuscript to Nature within the month. Wnt16 morphants lack HSCs, and transient induction of Notch signaling in Wnt 16 morphants rescues HSC development. We have demonstrated that Wnt16 lies genetically upstream of DllC and DllD, and shown, of the seven Notch ligands, that these two are necessary for HSC specification. These findings are important, since very little is known regarding the roles of Wnt signaling in HSC formation.

Year 2

Over the past year, we have made excellent progress on my CIRM New Investigator Award. We have demonstrated using direct imaging approaches that hematopoietic stem cells (HSCs) are born from hemogenic endothelial cells lining the ventral floor of the dorsal aorta (Bertrand and Chi et al., Nature 464: 108-111). This finding has allowed us to refine our approaches, knowing now that HSC specification requires transition through an aortic endothelial intermediate. Moving forward, our major goals are to understand how Wnt and Notch signaling integrate to regulate the specification of hemogenic endothelium. Our efforts over the past year have demonstrated that Wnt16 acts as a novel regulator of HSC fate through its regulation of two Notch ligands. Intriguingly, this pathway is non-cell autonomous with respect to HSCs and thus represents one of the earliest known extrinsic regulators of HSC fate specification. Our discovery of this pathway may therefore be a critical link that has been missing in the efforts to instruct HSCs in vitro from pluripotent precursors.

Year 3

Hematopoietic stem cells are an important population of cells that continuously produce and replace all blood and immune system cells throughout life. These rare cells are responsible for the curative effects of bone marrow transplants, which are used to treat a variety of conditions including many forms of blood cancer. Understanding how hematopoietic stem cells are made during embryonic development is important because it could teach us how to make such cells in the laboratory, and possibly allow circumvention of donor immune compatibility issues. In this research we describe a previously unknown set of molecular inputs that are required to make hematopoietic stem cells during embryonic development. Of note, this signaling pathway is required for environmental instruction of hematopoietic stem cells, meaning we are one step closer to understanding how to generate them in vitro. Eventually these findings may help us discover the complete set of molecular controls necessary for making hematopoietic stem cells.

Year 4

Hematopoietic stem cells are an important population of cells that continuously produce and replace all blood and immune system cells throughout life. These rare cells are responsible for the curative effects of bone marrow transplants, which are used to treat a variety of conditions including many forms of blood cancer. Understanding how hematopoietic stem cells are made during embryonic development is important because it could teach us how to make such cells in the laboratory, and possibly allow circumvention of donor immune compatibility issues. In this research we describe a previously unknown set of molecular inputs that are required to make hematopoietic stem cells during embryonic development. Of note, this signaling pathway is required for environmental instruction of hematopoietic stem cells, meaning we are one step closer to understanding how to generate them in vitro. Eventually these findings may help us discover the complete set of molecular controls necessary for making hematopoietic stem cells.

© 2013 California Institute for Regenerative Medicine