{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T09:42:23Z","timestamp":1767174143383,"version":"build-2238731810"},"update-to":[{"DOI":"10.12688\/f1000research.14507.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2018,12,23]],"date-time":"2018-12-23T00:00:00Z","timestamp":1545523200000}}],"reference-count":29,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2018,12,23]],"date-time":"2018-12-23T00:00:00Z","timestamp":1545523200000},"content-version":"unspecified","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["MR\/M004864\/1"],"award-info":[{"award-number":["MR\/M004864\/1"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["G0700091"],"award-info":[{"award-number":["G0700091"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008731","name":"Association Europ\u00e9enne contre les Leucodystrophies","doi-asserted-by":"publisher","award":["ELA2016-012F4"],"award-info":[{"award-number":["ELA2016-012F4"]}],"id":[{"id":"10.13039\/501100008731","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100010757","name":"National Centre for the Replacement Refinement and Reduction of Animals in Research","doi-asserted-by":"publisher","award":["NC\/M001490\/1"],"award-info":[{"award-number":["NC\/M001490\/1"]}],"id":[{"id":"10.13039\/501100010757","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Wellcome Trust","award":["GR077544AIA"],"award-info":[{"award-number":["GR077544AIA"]}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns4:p>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.<\/ns4:p>\n                  <ns4:p>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.<\/ns4:p>\n                  <ns4:p>\n                    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\n                    <ns4:italic>in vivo<\/ns4:italic>\n                    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.\n                  <\/ns4:p>\n                  <ns4:p>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.<\/ns4:p>","DOI":"10.12688\/f1000research.14507.2","type":"journal-article","created":{"date-parts":[[2018,12,23]],"date-time":"2018-12-23T09:10:06Z","timestamp":1545556206000},"page":"594","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":16,"title":["A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models"],"prefix":"10.12688","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3299-9133","authenticated-orcid":false,"given":"No\u00e9mie","family":"Hamilton","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ian","family":"Sabroe","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1790-1641","authenticated-orcid":false,"given":"Stephen A.","family":"Renshaw","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"2560","published-online":{"date-parts":[[2018,12,23]]},"reference":[{"key":"ref-30","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1186\/s12885-017-3919-8","article-title":"Improving zebrafish embryo xenotransplantation conditions by increasing incubation temperature and establishing a proliferation index with ZFtool.","volume":"18","author":"P Cabezas-Sainz","year":"2018","journal-title":"BMC Cancer."},{"key":"ref-1","doi-asserted-by":"publisher","first-page":"734-739","DOI":"10.1101\/gad.1629408","article-title":"Foxn4 directly regulates tbx2b expression and atrioventricular canal formation.","volume":"22","author":"N Chi","year":"2008","journal-title":"Genes Dev."},{"key":"ref-2","doi-asserted-by":"publisher","first-page":"481-501","DOI":"10.1146\/annurev.genet.39.073003.095931","article-title":"Use of the zebrafish system to study primitive and definitive hematopoiesis.","volume":"39","author":"J de Jong","year":"2005","journal-title":"Annu Rev Genet."},{"key":"ref-3","doi-asserted-by":"publisher","first-page":"309-315","DOI":"10.1016\/j.transci.2005.11.012","article-title":"Cryopreservation of hematopoietic and non-hematopoietic stem cells.","volume":"34","author":"K Fleming","year":"2006","journal-title":"Transfus Apher Sci."},{"key":"ref-4","doi-asserted-by":"publisher","first-page":"389-400","DOI":"10.1016\/j.devcel.2005.01.010","article-title":"Hedgehog signaling is required for adult blood stem cell formation in zebrafish embryos.","volume":"8","author":"M Gering","year":"2005","journal-title":"Dev Cell."},{"key":"ref-5","article-title":"Dataset 1 in: A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models.","author":"N Hamilton","year":"2018a","journal-title":"F1000Research."},{"key":"ref-6","article-title":"Dataset 2 in: A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models.","author":"N Hamilton","year":"2018b","journal-title":"F1000Research."},{"key":"ref-7","article-title":"Dataset 3 in: A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models.","author":"N Hamilton","year":"2018c","journal-title":"F1000Research."},{"key":"ref-8","article-title":"Dataset 4 in: A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models.","author":"N Hamilton","year":"2018d","journal-title":"F1000Research."},{"key":"ref-9","doi-asserted-by":"publisher","first-page":"1109-1121","DOI":"10.1016\/j.cell.2005.05.026","article-title":"SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.","volume":"121","author":"M Kiel","year":"2005","journal-title":"Cell."},{"key":"ref-10","doi-asserted-by":"publisher","first-page":"112-115","DOI":"10.1038\/nature08761","article-title":"Blood stem cells emerge from aortic endothelium by a novel type of cell transition.","volume":"464","author":"K Kissa","year":"2010","journal-title":"Nature."},{"key":"ref-11","doi-asserted-by":"publisher","first-page":"1147-1156","DOI":"10.1182\/blood-2007-07-099499","article-title":"Live imaging of emerging hematopoietic stem cells and early thymus colonization.","volume":"111","author":"K Kissa","year":"2008","journal-title":"Blood."},{"key":"ref-12","doi-asserted-by":"publisher","first-page":"7369-7374","DOI":"10.1073\/pnas.0402248101","article-title":"In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish.","volume":"101","author":"D Langenau","year":"2004","journal-title":"Proc Natl Acad Sci U S A."},{"key":"ref-14","doi-asserted-by":"publisher","first-page":"533-543","DOI":"10.1038\/ncb2730","article-title":"Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment.","volume":"15","author":"C Nombela-Arrieta","year":"2013","journal-title":"Nat Cell Biol."},{"key":"ref-15","article-title":"Zebrafish: A practical approach","author":"C N\u00fcsslein-Volhard","year":"2002"},{"key":"ref-16","doi-asserted-by":"publisher","DOI":"10.1101\/216895","article-title":"Generation of Mouse-Zebrafish Hematopoietic Tissue Chimeric Embryos for Hematopoiesis and Host-Pathogen Interaction Studies.","author":"M Parada-Kusz","year":"2017","journal-title":"bioRxiv."},{"key":"ref-17","doi-asserted-by":"publisher","first-page":"1014-1025","DOI":"10.1182\/blood-2017-03-770164","article-title":"NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival.","volume":"130","author":"L Prince","year":"2017","journal-title":"Blood."},{"key":"ref-18","doi-asserted-by":"publisher","first-page":"38-47","DOI":"10.1242\/dmm.007138","article-title":"A model 450 million years in the making: zebrafish and vertebrate immunity.","volume":"5","author":"S Renshaw","year":"2012","journal-title":"Dis Model Mech."},{"key":"ref-19","doi-asserted-by":"publisher","first-page":"770-790","DOI":"10.1038\/bmt.2011.185","article-title":"Haematopoietic SCT in severe autoimmune diseases: updated guidelines of the European Group for Blood and Marrow Transplantation.","volume":"47","author":"J Snowden","year":"2012","journal-title":"Bone Marrow Transplant."},{"key":"ref-20","doi-asserted-by":"publisher","first-page":"360-365","DOI":"10.1089\/scd.2015.0195","article-title":"Visualizing Human Hematopoietic Stem Cell Trafficking In Vivo Using a Zebrafish Xenograft Model.","volume":"25","author":"F Staal","year":"2016","journal-title":"Stem Cells Dev."},{"key":"ref-21","doi-asserted-by":"publisher","first-page":"382-6","DOI":"10.1046\/j.1365-2141.1997.d01-2032.x","article-title":"Long-term follow-up of leukaemia patients after related cryopreserved allogeneic bone marrow transplantation.","volume":"96","author":"M Stockschl\u00e4der","year":"1997","journal-title":"Br J Haematol."},{"key":"ref-22","doi-asserted-by":"publisher","first-page":"S48-56","DOI":"10.1016\/j.bbmt.2009.10.034","article-title":"Hematopoietic cell transplantation for autoimmune disease: updates from Europe and the United States.","volume":"16","author":"K Sullivan","year":"2010","journal-title":"Biol Blood Marrow Transplant."},{"key":"ref-23","doi-asserted-by":"publisher","first-page":"620-627","DOI":"10.1038\/nature03728","article-title":"Treatment of severe autoimmune disease by stem-cell transplantation.","volume":"435","author":"M Sykes","year":"2005","journal-title":"Nature."},{"key":"ref-24","doi-asserted-by":"publisher","first-page":"241-252","DOI":"10.1016\/j.cell.2014.12.032","article-title":"Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.","volume":"160","author":"O 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Zaucha","year":"2001","journal-title":"Blood."}],"container-title":["F1000Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/f1000research.com\/articles\/7-594\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/7-594\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/7-594\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2020,5,20]],"date-time":"2020-05-20T12:33:17Z","timestamp":1589977997000},"score":1,"resource":{"primary":{"URL":"https:\/\/f1000research.com\/articles\/7-594\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,12,23]]},"references-count":29,"URL":"https:\/\/doi.org\/10.12688\/f1000research.14507.2","relation":{},"ISSN":["2046-1402"],"issn-type":[{"value":"2046-1402","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,12,23]]},"assertion":[{"value":"Indexed","URL":"https:\/\/f1000research.com\/articles\/7-594\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.15792.r34036, Owen J. Tamplin, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA, 04 Jun 2018, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/7-594\/v1#referee-response-34036","order":0,"name":"referee-response-34036","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Noemie Hamilton<\/b>; \n<i>Posted: 18 Dec 2018<\/i>; We thank Professor Owen Tamplin for his positive review and encouraging comments for revisions. We have uploaded a revised manuscripts and updated figures addressing all of his minor comments. We have responded to his suggestions and additional discussion points below:Some suggestions for revisions are listed below:  We have not tested different dyes for this study and we have stuck to using a green dye to go alongside the red Tg(kdrl:mcherry) line. What happens to the model after 5dpf is a crucial experiment to validate the assay and ensure the human cells have truly engrafted. This is already part of the discussion, mentioning other studies looking at the different lineages emerging from the engrafted human HSCs. However, our current experiments end at 5dpf&nbsp; and raising chimera animals past this age would require an amendment of our project licence which is not within the scope of this study. A very recent study (Cabezas-Sainz et al., 2018) has explored the effect of raising the incubation temperature of zebrafish to accommodate for human cells and we have added to the&nbsp; \u2018Limitations\u2019 section of our discussion.&nbsp; We have not tried other routes of injection and we are grateful for the RO injection suggestion which will be tested. We have not transplanted zebrafish CD41 cells and we would expect them to engraft better than human cells. This is a great suggestion and this is how we hope this model will be used in the future: To find new molecules that could improve engraftment success.  &nbsp;Possible additional \u201cDiscussion\u201d points:  We have added the automated injection to our \u2018Limitations of our current protocol\u2019 section to go alongside sourcing CD34 from different sources to scale up the pt The limitations of using a non-isogenic recipient and an \u2018foetal-like niche\u2019 are very valid points. We hope that our zebrafish embryonic model will be used instead of mice to optimise protocols initially. We fully expect that mice will still be used to corroborate results from zebrafish studies before being used in patients. We agree that \u2018lodge\u2019 would be a more suitable description for our model until we show long term engraftment of human cells. This was replaced in the result section.&nbsp; Replacing the adult mouse with the zebrafish embryo as a transplant recipient for human HSPCs switches the system from an adult bone marrow niche to a developmental \u201cfetal-like\u201d niche (i.e., CHT). How should this be considered when interpreting the results?  &nbsp;Minor points related to the manuscript:  1+2+3: Figure 4: we apologise for the confusion in the quantification from Figure 4C. We have changed the text and figure legend to be more consistent and emphasise that the absolute number of cells was the number of cells lodged within the CHT at 1hpt. We subsequently quantified the number of cells at 5hpt, 9hpt and 13hpt and plotted them in percentage. We do not find GFP positive cells in any other tissues after transplantation, so we can conclude that the cells initially visualised in the CHT could be debris or healthy cells that have died and be removed by innate immune cells. Figure 5b and 5C are all single plane, we have specified this in the figure legend We have modified the text on page 3 to make sure readers do not think we are referring to neonatal transplant. \u2018These immunodepleted mice must be grown to adulthood in order to assess engraftment success. They live their entire lives undergoing severe procedures with high maintenance requirements, since they need to be homed in sterile rooms and fed sterile food to avoid fatal infection due to defective immunity.\u2019 Publications using immune compromised mice were counted using a PubMed search, reading articles and averaging how many mice were used per study. We have added this to the text to make it clearer.","URL":"https:\/\/f1000research.com\/articles\/7-594\/v2#referee-comment-4317","order":1,"name":"referee-comment-4317","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.15792.r34964, Stuart M. Allan, Siobhan Crilly, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, UK, 19 Jun 2018, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/7-594\/v1#referee-response-34964","order":2,"name":"referee-response-34964","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Noemie Hamilton<\/b>; \n<i>Posted: 18 Dec 2018<\/i>; We thank Prof Allan and Miss Crilly for their positive comments. We have uploaded an improved version of the manuscript including all the minor corrections they suggested. Their recommended revisions have also been added to the manuscript and detailed below points by points:  We have added this to the methods. The antibody and the beads were not been removed, however we have been advised by the company selling the CD34 MicroBead kit that the beads naturally detach within 24 hours.&nbsp; We thank you for this suggestion and we hope that the innate immune cellular response to the engraftment of transplanted cells can be part of another study. We have added your suggestion as part of the \u2018future validation assays\u2019 of our discussion. A very recent study (Cabezas-Sainz et al., 2018) has explored the effect of raising the incubation temperature of zebrafish to accommodate for human cells and we have added to the&nbsp; \u2018Limitations\u2019 section of our discussion.&nbsp; We have not investigated whether the engraftment of new cells had an impact on the development of endogenous zebrafish HSCs, nor the integrity of the CHT (point 5). However, we have added this to our discussion as an important future validation assay. Included in point 4 above. What happens to the model after the development of the mature adaptive immune system in the zebrafish after 2 weeks has already been mentioned in the validation assay part of the discussion. We agree that this is a crucial experiment to perform to investigate the presence of different lineages emerging from the engrafted human HSCs. However, our current experiments end at 5dpf&nbsp; and raising chimera animals past this age is not within the scope of this study. We have added the full details of statistical analyses to our figure legends, results sections and have uploaded updated figures. &nbsp;","URL":"https:\/\/f1000research.com\/articles\/7-594\/v2#referee-comment-4318","order":3,"name":"referee-comment-4318","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This study was supported by a NC3Rs grant to SAR and IS (NC\/M001490\/1). SAR is supported by a MRC Programme Grant (MR\/M004864\/1) and NH is supported by a European Leukodystrophy Fellowship (ELA 2016-012F4). Imaging was carried out in the Wolfson Light Microscopy Facility, supported by an MRC grant (G0700091) and a Wellcome Trust grant (GR077544AIA).","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Data associated with the article are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).","order":0,"name":"copyright-info","label":"Copyright"}]}}