Funding Agency: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Challenge Area - (11) Regenerative Medicine Specific challenge topic - 11-DK-101 Promote regeneration and repair in the digestive system, liver, pancrease, kidneys, hematologic, and urological system. 3. Project Title: Slit2-mediated expansion of primitive hematopoietic stem cell populations for transplantation and hematopoietic recovery Hematopoietic Stem Cells (HSCs) integrate signals from the niche with cell intrinsic regulatory programs to maintain the endogenous HSC pool. Regulation of stem cell-niche interactions influences the entire lymphohematopoietic system and ultimately the organism, making characterization of these pathways fundamental to the pursuit of novel therapeutic interventions involving adult stem cells. Based on natural variation in absolute numbers of HSCs in inbred mice, we initiated a forward genetic study that revealed a quantitative trait locus on chromosome 5 (Chr5) linked to the determination of HSC numbers in C57B/L6 (B6) and DBA/2 (D2) mice. Phenotypic analysis of strains congenic for the chromosome 5 QTL confirmed that D2 alleles in that region produce a 2.4-fold increase in HSC numbers while B6 alleles confer a 1.7-fold decrease in HSCs. We performed microarray and RT-PCR analysis to identify candidate HSC-regulatory genes in the Chr5 QTL region based on gene expression patterns. We found that a single transcript, Slit2, was differentially expressed at a significant level and positively correlated to HSC numbers. A retrovirus was used to stably infect B6 bone marrow cells, which normally do not express Slit2, with a Slit2-containing GFP vector. Infected cells were transplanted into B6 hosts and expanded in vivo for 12 weeks ensuring reconstitution of the complete hematopoietic hierarchy within the GFP+ fraction. In vitro quantification demonstrated that ectopic expression of Slit2 produces a 4-fold increase in HSC numbers relative to an empty vector control. These results provide evidence that Slit2 is a novel regulator of murine hematopoiesis and a potential target for therapeutic intervention in the treatment of a variety of human diseases. Given the pivotal role of Slit/Robo signaling in cell-cell interactions, and known antagonism of the CXCL12/CXCR4 axis we predict that Slit2- mediated regulation of HSCs is accomplished by alterations in HSC-niche interactions. Proposed studies will determine how Slit2 expression influences HSCs within the niche to promote expansion of the HSC pool and how Slit2 expression is regulated in HSCs. We propose three aims: The first is to determine the mechanisms by which SLIT2 regulates stem cell number. Self-renewal is of particular interest. The second is to determine how the differential expression of the Slit2 gene is regulated, with particular focus on transcription factor binding.
The third aim will examine the therapeutic potential of modulating Slit2 expression. A mouse knockout model will be employed. The effects on stem cell localization within the bone marrow will be determined using imaging techniques.
This proposal investigates the role of Slit2 in regulating the number and location of hematopoietic stem cells within the bone marrow. In three specific aims we will investigate the mechanism(s) by which Slit2 regulates stem cell number, with special attention to self-renewal. In the second aim we will investigate the mechanism(s) by which Slit2 expression is regulated in hematopoietic cells. In the third aim the therapeutic potential of altering Slit2 expression will be investigated in a knockout mouse model and using imaging techniques to localize stem cells in the bone marrow.
