The hematopoietic microenvironment is made up of a number of niches that regulate hematopoietic stem cell (HSC) function in health and under conditions of stress. Sinusoidal endothelial cells comprise a niche that is necessary for HSC recovery after myelotoxic chemotherapy and engraftment after hematopoietic stem cell transplantation (HSCT). A sinusoidal endothelial cell niche is transiently present in the caudal hematopoietic territory (CHT) of the developing zebrafish (Danio rerio), where it supports HSC survival and expansion before hematopoiesis shifts to the adult kidney marrow. The overarching goal of this proposal is to identify new factors that are important for HSC-endothelial cell interactions within the CHT and to understand how these interactions affect HSC expansion, HSC clonal diversity and the biology of the niche itself.
The first aim addresses how HSCs and other blood cells use the angiogenic cytokine, CXCL8, to stimulate the growth of the CHT and induce expression of molecules that favor HSC retention, survival and proliferation. In gain-of-function experiments, CXCL8 will be expressed at high levels in HSCs and other blood lineages using specific gene promoters which have been previously identified and cloned. An endothelial cell-specific CRISPR-based system will be used to knock out the putative CXCL8 receptors, CXCR1 and CXCR2, in order to understand the receptor specificity for CXCL8 within the CHT. HSC engraftment, mitotic rate, and cell-cell interactions within the CHT as well changes in the size and function of the niche itself will be quantified in the gain- and loss-of-function experiments using confocal microscopy and digital image analysis. The goal of the second aim is to identify new extracellular signaling factors that specifically mediate HSC-endothelial cell interactions within the CHT and to determine how the niche affects HSC clonality via these factors. Interacting HSCs and endothelial cells expressing a photoconvertible protein within the CHT will be identified and permanently labeled using UV irradiation. Single photoconverted cells will be sorted and gene expression profiles specific for interacting HSCs and endothelial cells will be generated by RNA sequencing and comparing to gene expression profiles from non-interacting, control cells. The functional role of factors specific to the HSC-endothelial cell interaction will be tested using gain- and loss-of-function experiments as described in the first aim. In addition, the role of these factors in regulating the clonal diversity of the HSC pool will be assessed in gain- and loss-of-function experiments using an optical barcoding approach. These studies have the potential to identify new therapeutic targets for improving HSC numbers and clonal diversity in patients with blood disorders and those who are recovering from chemotherapy or HSCT.
Blood stem cells (hematopoietic stem cells, HSCs) must interact with the supporting cells in the microenvironment of the bone marrow in order to survive, self-renew, and produce the appropriate number of mature blood cells to meet the demands of the organism. Using the developing zebrafish as an experimental model to observe and perturb these interactions, I will find new genes that are involved in HSC-microenvironment communication and identify how they impact HSC biology. These genes may ultimately be useful drug targets for increasing the number and diversity of HSCs in patients with blood disorders and those who are recovering from chemotherapy or hematopoietic stem cell transplantation.
|Blaser, Bradley W; Zon, Leonard I (2018) Making HSCs in vitro: don't forget the hemogenic endothelium. Blood 132:1372-1378