A Functional Bioluminescent Zebrafish Screen for Enhancing Hematopoietic Cell Homing

Abstract

SummaryTo discover small molecules that modulate hematopoietic cell homing after adoptive transfer, we created a transgenic zebrafish expressing firefly luciferase downstream of the ubiquitin promoter (ubi:luc) to serve as a hematopoietic donor. Bioluminescence imaging (BLI) was used to detect and follow ubi:luc hematopoietic cells that homed to the marrow as early as 1 day post-transplant. BLI was able to detect the biological effect of prostaglandin E2 on early homing/engraftment of donor hematopoietic cells. This system was utilized in a functional screen of small molecules to enhance homing/engraftment. We discovered a phytosterol, ergosterol, that could increase hematopoietic cell homing in zebrafish and mice. In addition, ergosterol increased CXCR4 expression and promoted expansion of Lin−SCA-1+KIT+ cells in vitro. We have demonstrated the utility of in vivo BLI to non-invasively monitor donor hematopoietic cell activity in adult zebrafish as a functional screen for mediators of cellular homing.

Noninvasive Tracking of Hematopoietic Stem Cells in a Bone Marrow Transplant Model

“…Of the 1694 articles identified in Scopus, after screening, 771 articles were excluded (416 reviews, 106 publications before 2000, 50 publications in other languages, and 199 duplicated in Pubmed search), and after assessing eligibility, 910 articles were excluded (287 reported no data about HSC and 623 reported no data about noninvasive imaging); thus, only 13 articles were included from this database. In total, only 21 nonduplicate full text articles were included in this review [9,11,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34], as depicted in Figure 1.…”

“…The hematopoietic stem cell (HSC) extraction and isolation process used in the selected studies is described in detail in Table 1. Of the 21 selected studies, nine (43%) [16,[18][19][20]23,25,28,32,34] studies used mice as a cell donor for bone marrow transplant (BMT), seven studies (33%) used humans as a cell donors [9,11,21,24,26,27,33], two studies (10%) used rat cells [29,30], and the remaining studies used other cell donors, such as fish [17], axolotl [22], and dogs [31], corresponding 5% for each study. Regarding the source of the cells, the majority of studies (53%) [16,[18][19][20]23,25,[28][29][30][31][32] used bone marrow; however, the study by Ushiki [25] used bone marrow and spleen cells, and the second main source of cells was the umbilical cord used in five studies (24%) [9,11,24,27,33], followed by peripheral blood, which was used in two studies (10%) [21,26].…”

“…Regarding the source of the cells, the majority of studies (53%) [16,[18][19][20]23,25,[28][29][30][31][32] used bone marrow; however, the study by Ushiki [25] used bone marrow and spleen cells, and the second main source of cells was the umbilical cord used in five studies (24%) [9,11,24,27,33], followed by peripheral blood, which was used in two studies (10%) [21,26]. Furthermore, the study by Lopes [22] used liver and spleen cells, the study by Astuti [17] collected the renal marrow cells from zebrafish, and the study by Sweeney [34] obtained cells from embryonic cells. Of eleven studies that used rodents as cell donors, the age of the animals was, on average, 8 weeks [18,20,23,25,30], ranging from 6 [18] to 10 [20] weeks, and the use of male rodents [29][30][31][32] was more prevalent than of female rodents [18], though two studies [23,28] used both genders.…”

“…Concerning the medium and supplementation used during the harvesting of stem cells, two studies [29,30] used Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and heparin. Three other studies [17,22,23] used Phosphate-buffered saline (PBS), and the study by Parada-Kusz [23] supplemented the PBS with 0.5% bovine serum albumin (BSA)/FBS and 2 mM EDTA (Ethylenediaminetetraacetic acid). Furthermore, the study by Lopez [22] used 0.75× PBS supplemented with 5% BFS (APBS, axolotl PBS); the study by Lin [18] used minimum essential medium Eagle alpha modification (α-MEM) supplemented with 10% heparin; the study by Sweeny [34] used StemPro34 medium with a hematopoietic cytokine selection; and in the study by Niemeyer [33], the harvesting of stem cells was performed with citrate buffer.…”

“…To study the dynamics of hematopoietic reconstitution, in which cells localize deep inside hematopoietic organs and other tissues, different modalities of molecular imaging are used for HSC tracking and engraftment, such as (i) optical imaging represented by bioluminescence imaging (BLI) [9,11,[16][17][18][19][20] and fluorescence imaging (FLI) [21][22][23][24][25], (ii) nuclear imaging represented by positron emission tomography (PET) [26][27][28] and single-photon emission computed tomography (SPECT) [29][30][31], and (iii) magnetic resonance imaging (MRI) [32][33][34].…”