Norman Iscove Professor M.D., Ph.D., University of Toronto Princess Margaret Cancer Research Tower MaRS Centre 101 College Street, Room 8-356 Toronto, ON M5G 1L7 CANADA Phone: (416) 581-7460 Lab Phone: (416) 581-7462, 581-7464 iscove ,,at,, uhnresearch.ca

Self-renewal in normal and leukemic blood stem cells

Sustained ability to self-renew is the signature capacity of stem cells. In adult tissues that contain actively dividing cells, such as the epithelial layers of the gut or skin, or the blood forming system, the stem cells maintain themselves indefinitely, in contrast to the greatly more numerous differentiating progeny that have only finite lifespans. The continuous maintenance of such tissues therefore depends critically on the self-renewal activity of the stem cells. Malignant tumours have also been shown in recent years to be organized in a similar way, depending on rare tumour stem cells for the continued persistence and growth of the tumour.

Research in our lab is focused on core mechanisms of self-renewal in precursor cells of the mammalian blood-forming system:

• A key finding in the lab was the discovery of a distinction between stem cells with permanent blood cell forming capacity - called Long Term Stem Cells - and more advanced stem cells - called Intermediate Term Stem Cells - that in the mouse provide massive numbers of blood cells for only 3 months, after which they stop self-renewing (5). Within this model system, we have been investigating what sustains self-renewal in the Long Term cells, and why initially active self-renewal eventually shuts down in Intermediate Term cells.
• Our work in this system led to the discovery of a role for the Gata3 transcription factor, expressed in Long Term but not Intermediate Term cells, which turned out to downregulate self-renewal in response to stress signalling from the p38 MAPK pathway (3).
• Recent experiments have also highlighted a central role of the clustered Homeobox genes. These turn out to be essential for self-renewal activity at successive stages of differentiation downstream of the Long Term stem cells. We have secured evidence that the eventual loss of self-renewal activity in downstream cells occurs as a direct result of the natural silencing of Hox gene expression in these cells, and can be prevented by enforcing the expression of a Hox transgene (1). The Hox transgene endows lineage-restricted cells with stem cell-like properties of indefinite proliferative capacity and serial transplantability without affecting their lineage restriction or ability to supply mature blood cells in vivo.
• An exciting projection of these findings concerns the beginnings of leukemia. Currently ongoing work tests the notion that leukemia begins with a first mutation in a marrow precursor cell that extends its lifespan, that the persistence of growing leukemia cells depends on the same first mutation, and that disrupting the first mutation could provide a pathway to leukemia cure.
• The lab has an ongoing interest in development of tools needed for the investigation of rare stem cells. We pioneered investigation of gene expression at single cell level via global RT-PCR amplification (4,6,8), a methodology that is currently under further development in the lab to allow for direct analysis of amplified product by RNAseq. We have identified strategies for purification of blood stem cells to absolute homogeneity (3,5,7). The leukemia work is supported by gene transduction approaches allowing for excision of the transgene at any later time. We are also advancing toward an ability to maintain and expand adult blood forming stem cells in culture in response to developmentally important ligands (2), work that is intended to facilitate eventual gene repair and human bone marrow transplantation.

Overall, our research program addresses self-renewal, a central mechanism in normal and cancer systems. The longer term aim is to facilitate development of therapeutic approaches with curative potential in human leukemia via direct targeting of the self-renewal process.

Publications