Transplanted hematopoietic stem cells (HSCs) home to the marrow space, engraft and self-renew. The process of engraftment is difficult to study in most vertebrates because it is impossible to visualize the engraftment directly. Here we have developed competitive transplantation of fluorescent donor marrow samples into a completely transparent adult zebrafish called casper. This allows the direct visualization of engraftment and the process of HSC competition. Transplantation of zebrafish whole kidney marrow (WKM) cells, containing HSCs, progenitors and mature blood cells, can rescue lethally irradiated recipient fish and repopulate all the blood lineages. We used different GFP-/DsRed-labeled transgenic fish as donors in WKM transplantation, such as the myeloid specific Tg(mpo:GFP), or the ubiquitous expression of Tg(β-actin:GFP), and Red GloFish® to study the kinetics of engraftment. Engraftment can be visualized with a simple fluorescent microscope, and was evident in the recipient kidney as early as 2 weeks post transplant (wpt). By 4 wpt, mature hematopoietic cells differentiated from donor HSCs or progenitors can be observed in the recipient circulation as well as in epidermis as tissue residential macrophages. At 8 wpt, donor-derived lymphoid cells also repopulate the recipient immune system. The fluorescence intensity of engrafted cells was quantified by image analysis software and positively correlated with the engraftment efficiency quantified by FACS. Using Red GloFish® as competitor donor and Tg(β-actin:GFP) as test donor, we were able to see the competition between the two donors both by fluorescence intensity analysis and FACS analysis. To test the sensitivity of this competitive transplant system to the alteration of the ratios between the two colors, we increased the ratio of the test vs. competitor donor from 1:3 to 1:2 to 1:1. At 4wpt, in the 1:3 ratio group, only 14% of the recipients had stronger GFP intensity than the red (n=7), while the rest had weaker or equivalent GFP intensity than the red; however, this percentage increased to 43% and 50% in the 1:2 (n=7) and 1:1(n=8) groups respectively. Therefore, the relative engraftment ratio can be fast detected in vivo by direct fluorescent visualization with the transparent zebrafish recipients. Next, we treated the test donor cells with 16,16-methyl prostaglandin E2 (dmPGE2) or DMSO for 2 hours in vitro at room temperature before transplant. The engraftment efficiency of the dmPGE2 group dramatically increased by at least 2 folds compared with the DMSO group. The colors of competitor and test donors were switched, and the increase of engraftment efficiency by dmPGE2 treatment of the test donor was still evident. In conclusion, direct visualization of competitive marrow repopulation greatly facilitates the developmental, physiological or pathological study of HSCs and progenitors and should provide a new platform for the assay of specific genes or chemicals that could alter homing, engraftment or self-renewal.

Disclosures: No relevant conflicts of interest to declare.

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