Redefining the Hematopoietic Microenvironment
Abstract
Hematopoietic stem cells (HSC) and their progeny reside in specialized niches of the microenvironment (ME) in the bone marrow. The ME niches control HSC self-renewal, differentiation and maturation. The ME niche cells were derived from non-hematopoietic cells, including fibroblasts, osteoblastic and endothelial cells. Macrophages, which are hematopoietic in origin, are also a critical component of the ME, and can influence the function of the ME niche cells. I hypothesize that the macrophages can acquire defects that may compromise ME function and lead to bone marrow failure. To test this hypothesis, I proposed to develop a new in vivo model that allows the inducible depletion of the macrophages in dogs, followed by the documentation of marrow failure, and subsequent therapeutic interventions. At this period of the grant support, I achieved 4 goals: (1) Optimize culture conditions for generating dog macrophages, (2) Optimize transduction efficiency of a macrophage specific CD163 promoter construct in dog CD34+ HSC, and test its macrophage-specific expression, (3) Establish a Luciferase-reporter assay to test the macrophage-specific promoter activity, and (4) Generate multiple lentiviral vectors containing the dog/human CD163promoter, iCasp9 and p140MGMT constructs. I am currently in the final step to generate a lentiviral construct that incorporates both a constitutive selectable marker (p140MGMT) that will increase the proportion of the transduced cells in marrow, and an inducible macrophage-specific promoter that will drive the expression of a suicide gene (iCasp9). In the original grant application, I proposed to use the CD68 promoter sequence to drive the macrophage specific expression of the inducible iCasp9 suicide gene. The CD68 promoter, however, is intronic and clones into a reverse orientation in the lentiviral vector to avoid splicing during retroviral packaging.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 2012
- Accession Number
- ADA573826
Entities
People
- Mineo Iwata
Organizations
- Fred Hutchinson Cancer Center