Modulators of HSC Engraftment Found in A Competitive Marrow Transplantation Screen in Adult Zebrafish.

Abstract 2430

Poster Board II-407

After transplantation, hematopoietic stem/progenitor cells (HSPCs) home to the marrow, where they engraft and self-renew. To explore the mechanism of this multi-step and dynamic repopulation process, we performed the first in vivo adult vertebrate chemical screen aimed at identifying novel chemical modulators of HSPC repopulation using a novel competitive marrow transplantation assay in zebrafish. To distinguish between the donors, we used ubiquitous GFP or DsRed2 transgenic fish, Tg(β-actin:GFP) and Red GloFish®, for marrow cell isolation. 20,000 GFP+ cells were treated with a chemical and mixed with 80,000 untreated DsRed2+ marrows. This pool of cells was injected retro-orbitally into a transparent adult zebrafish. After a recovery period, the fish was then anesthetized and the region of the kidney (the adult site of hematopoiesis) was examined by fluorescence microscopy. The competition between the two donors was determined by analyzing the ratios of GFP and DsRed2 fluorescence intensity with ImageJ software. Using this assay, we demonstrated that dmPGE₂ and/or GSK-3β inhibitor treatment of GFP+ marrows for 3 hrs could dramatically increase repopulation in fish. A chemical library of 480 chemicals with known bioactivities was screened using this in vivo assay. GFP+ marrows were incubated with different chemicals for 3 hrs and ten recipient fish were transplanted for each chemical. By examining engraftment at 4 weeks, we found 10 chemicals that improved HSPC repopulation. Based on the known bioactivity, these chemicals were categorized into several signaling pathways, including prostaglandin metabolism and retinoic acid pathways. Several of the compounds also increased HSC formation in zebrafish embryos, indicating that some pathways might be shared by different developmental stages. To examine whether the bioactivities of these hits are conserved in mammals, CD45.1 mouse whole bone marrow cells were treated with hit compounds for 3 hrs and competitively transplanted into CD45.2 recipients. Peripheral blood was sampled at 3, 6, 12, and 20 week post transplant. Several hits were confirmed to increase long-term chimerism in mice. The retinoic acid pathway has been shown to play an important role in hematopoiesis. Among the six retinoic acid receptor (RAR) agonists in the chemical library, which includes all-trans retinoic acid (ATRA), only two structurally highly related compounds, AM-580 and TTNPB scored positive in the screen. These two compounds have distinctive chemical moieties from ATRA. This structural difference likely leads to stronger agonistic effects on RAR than ATRA and resists degradation. In conclusion, the in vivo chemical screening using zebrafish competitive marrow transplantation provides a successful example of phenotypic screening in whole adult vertebrates. The discovery of novel repopulation modulators should provide a better understanding of signaling events that regulate homing and self-renewal, and may have clinical application in marrow or cord blood transplantation.