Mechanisms of Hematopoietic stem cell Specification and Self-Renewal

Mechanisms of Hematopoietic stem cell Specification and Self-Renewal

Funding Type: 
New Faculty I
Grant Number: 
RN1-00557
Award Value: 
$2,286,900
Disease Focus: 
Blood Cancer
Cancer
Anemia
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Status: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

The goal of this grant is to investigate the cell intrinsic mechanisms that govern hematopoietic stem cell specification and self-renewal. During the second year of this award, we have further elucidated the regulatory mechanisms that dictate hematopoietic fate specification by validating the target genes that Scl/tal1 activates and represses in vivo (Aim 1). We have also shown that loss of Scl results not only results in loss of all blood cells, but also causes defective arterio-venous identity that precludes generation of hemogenic endothelium and hematopoietic stem cells. We have defined the phenotype of hemogenic endothelium and emerging HSCs in both mouse and human embryos (Aim 2), and identified novel markers that can be used to isolate developing HSCs at distinct stages, as well as to purify functional HSCs further (Aim 3). We have also established an inducible lentiviral based expression system that will now be used to test functionally candidate HSC regulators that were identified by comparing gene expression profiles between freshly isolated HSCs and dysfunctional HSCs that were expanded in culture or generated from human ES cells. We hope that these studies will provide better understanding of the key regulatory mechanisms that govern HSC properties, and ultimately lead to development of improved methods for generation of functional HSCs in culture.

Year 2

Our work has focused on defining mechanisms that govern the specification and self-renewal of hematopoietic stem cells during mouse and human development. Using gene targeted mouse ES cells and mouse embryos, we defined the transcriptional programs that are regulated by Scl, the master regulator for blood formation. We discovered that Scl not only establishes the transcriptional programs that are critical for specifying hemogenic endothelium and hematopoietic stem cells, but it also represses heart development. Strikingly, in the absence of Scl, hemogenic endothelium in embryonic hematopoietic tissues becomes converted to cardiogenic fate, and gives rise to fully functional, beating cardiomyocytes. In order to define the key programs that distinguish self-renewing HSCs from their downstream progenitors or the compromised HSPCs (hematopoietic stem/progenitor cells) that were generated in vitro, we performed microarray analysis for human phenotypic HSCs from various sources. We identified novel markers for human HSCs that can be used to purify transplantable HSCs to a higher purity. We have identified key molecular defects in HSCs that are expanded in culture, or generated from human ES cells. We have further validated that dysregulation of certain Hox genes is a major bottleneck for generating functional HSCs from human ES cells. Future studies are focused on establishing methods that would allow correction of the compromised HSC regulatory networks in cultured HSCs.

Year 3

We have defined key regulatory mechanisms that are required for generation and maintenance of blood forming stem cells. We showed that transcription factor Scl is critical for specifying hemogenic endothelium from where blood stem cells emerge, and moreover, we discovered and unexpected repressive function for Scl to suppress cardiomyogenesis; in the absence of Scl, the blood vessels in start to generate beating cardiomyocytes. We have also identified factors that are critical for blood stem cells to maintain the unique properties: to self-renew (make more of themselves) and engraft (interact with the niche cells that support them). We will now continue to define how these key regulators act so that we can design better strategies to generate blood stem cells as well as heart muscle precursors for therapeutic applications.

Year 4

The goal of this grant was to define mechanisms that govern blood stem cell specification and self-renewal. We have completed the studies on hematopoietic fate specification by defining how Scl/tal1 establishes hemogenic endothelium. We documented that, in addition to Scl’s critical function in activating blood cell regulators, Scl also has to repress heart factors to prevent the misspecification of blood precursors to heart muscle. We documented that Scl controls blood and heart regulators through enhancers that have been primed for activation prior to Scl action (Aim 1). We identified a new surface marker that is expressed in hemogenic endothelium and blood forming cells in the yolk sac (Lyve1), which provides new tools to investigate the origin of blood stem and progenitor cells during development (Aim 2). We identified GPI-80 as a novel marker for transplantable blood stem cells during human fetal development (Aim 2, 3). Taking advantage of this new marker for blood stem cells, we narrowed down the critical defects in the dysfunctional blood precursors that are generated from human ES cells, or expanded in culture from fetal liver blood stem cells (Aim 3). We showed that the inability to induce HOXA cluster genes and other novel blood stem cell regulators that cannot be sustained in culture hinder the generation of blood stem cells from pluripotent cells, and further validated these novel regulators using lentiviral knockdown and overexpression. These findings will now be used to develop novel strategies to generate blood stem cells in culture.

© 2013 California Institute for Regenerative Medicine