A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models

Abstract

Haematopoietic stem cell (HSC) transplantation is a critical therapy for haematopoietic malignancies and immune disorders. Incomplete or delayed engraftment of HSCs in the host results in increased risk of infection and morbidity. The mechanisms of HSC engraftment are poorly understood and understanding these processes will increase transplantation success on many levels. Current animal models are immunocompromised 'humanised' mice transplanted with human HSCs. Harmful procedures include genetic manipulations and irradiation to ablate the mouse immune system, and opaque mouse tissues make visualisation of the early steps of HSC engraftment impossible. There is a need for new models to offer alternatives to humanised mice in the study of HSC transplantation. Here we described a detailed method for transplantation of human HSCs into zebrafish, before the onset of adaptive immunity. Human HSCs were purified from whole blood by enrichment of the CD34 cell population using a positive magnetic selection and further purified using an anti-CD34 antibody and cell sorting. Sorted CD34 cells were transplanted into the blood stream of 52 hour old zebrafish larvae. Human HSCs home into the zebrafish haematopoietic niche, where they engage with endothelial cells and undergo cell division. Our model offers the opportunities to image in vivo human HSC engraftment in a transparent organism, without the myeloablative strategies used in mice, and provides a unique system to understand the dynamic process of engraftment and replace current murine models. This technique can be applied to current engraftment protocols to validate the viability and efficiency of cryofrozen HSC grafts. This humanised zebrafish model will be instrumental to develop the 3Rs values in stem cell transplantation research and our detailed protocol will increase the chances of uptake of this zebrafish model by the mouse community.

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.…”

“…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]. 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.…”

“…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].…”

“…The NIRF disadvantages are the same as that of the BLI modality (low spatial resolution and low tissue penetration), and also as that of autofluorescence [40,41]. Most of the studies used this technique for early HSC engraftment evaluation in the mouse models, but mainly fishes [21][22][23][24][25].…”

Animal models of leukodystrophy: a new perspective for the development of therapies

“…Zebrafish develop transparent embryos ex utero with no adaptive immunity until 2 weeks of age, allowing transplant of foreign cells without the need of harmful irradiation procedures. Recent research has demonstrated that zebrafish and human primary stem cells are able to engraft within the haematopoietic niche of transplanted zebrafish embryos [83][84][85][86]. Hence, zebrafish models may provide a simpler alternative to their murine counterparts in exploring the potential efficacy of HSCT in embryos (Fig.…”