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Ex vivo tools for the clonal analysis of zebrafish hematopoiesis

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Abstract

This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3–10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.

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Figure 1: Flowchart of the described experimental procedures.
Figure 2: Gating strategy for the isolation of specific cell populations via FACS.
Figure 3: Dissection of zebrafish kidney.
Figure 4: Number of HSPCs in zebrafish WKM.
Figure 5: Representative images of particular colonies grown in methylcellulose.
Figure 6: Enumeration of colonies from fractionated WKM cells and cd41medium cells.

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Acknowledgements

This work was supported by the Ministry of Education, Youth and Sports—Program NPU I (LO1419), the Czech Science Foundation (16-21024S to P.B.), the Charles University Grant Agency (598712 to O.S.), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH; K01-DK087814-01A1 to D.L.S.).

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Authors and Affiliations

  1. Department of Cell Differentiation, Institute of Molecular Genetics AS CR v.v.i., Prague,, Czech Republic

    Ondrej Svoboda, Olga Machonova & Petr Bartunek

  2. Department of Biological Sciences, California State University Chico, Chico, California, USA

    David L Stachura

  3. Stem Cell Program, Dana-Farber/Boston Children's, Boston, Massachusetts, USA

    Leonard I Zon

  4. Division of Hematology/Oncology, Dana-Farber/Boston Children's, Boston, Massachusetts, USA

    Leonard I Zon

  5. Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, USA

    Leonard I Zon

  6. Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA

    David Traver

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  1. Ondrej Svoboda
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  2. David L Stachura
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  3. Olga Machonova
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  4. Leonard I Zon
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  5. David Traver
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Contributions

O.S., D.L.S., O.M., and P.B. performed the research; O.S., D.L.S., and P.B. designed the research; O.S., D.L.S., and P.B. wrote the manuscript; and L.I.Z. and D.T. provided critical reagents for the work, as well as suggestions on experimental design.

Corresponding author

Correspondence to Petr Bartunek.

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Competing interests

L.I.Z. is a founder and stockholder of Fate, a founder and stockholder of Scholar Rock, and a scientific advisor for Stemgent.

Integrated supplementary information

Supplementary Figure 1 Selection of optimal fish sera that support growth and survival of zebrafish HSPCs.

Representative images of cells, grown in the absence of fish serum (no serum) or in the presence of either bass, carp, pike or trout serum for three days. Suspension cells were visualized using phase contrast microscopy and adherent cells were stained with May-Grünwald Giemsa. Scale bars (left) represent 100 um.

Supplementary Figure 2 Blood collection from euthanized carp.

Needle should be positioned perpendicularly to the ventral surface and in the midline between pectoral fins. Pull the syringe plunger out to collect the blood.

Supplementary Figure 3 Experimental strategy for recombinant protein expression using E. coli or baculovirus expression system.

Amplify the mature coding sequence (red box) using polymerase chain reaction (PCR). Ligate the PCR product into the prokaryotic expression vector pQE. This leads to introduction of 6xHis sequence within the CDS (violet box). Optionally use EcoRI and PstI restriction enzymes to transfer the insert containing 6xHis and mature CDS into the baculovirus expression vector pAcGP67.

Supplementary Figure 4 Plucking a colony from methylcellulose using a fine 30-μl tip.

Find the colony, to be plucked out from the methylcellulose. Use the 30ul tip and reach the bottom of the plate. Move the tip above the colony and suck off the colony inside the tip using the pipetman. Photomicrograph was taken at original magnification x40, scale bar represents 500um.

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Svoboda, O., Stachura, D., Machonova, O. et al. Ex vivo tools for the clonal analysis of zebrafish hematopoiesis. Nat Protoc 11, 1007–1020 (2016). https://doi.org/10.1038/nprot.2016.053

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