Name: development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos
Uploaded: 2017-11-30T15:00:00
Description: Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Funding was provided by the National Institutes of Health (NIH: K01-DK087814-01A1 to D.L.S.), the California State University Program for Education &amp; Research in Biotechnology (CSUPERB: Molecular Control of the Vertebrate Hematopoietic Niche to D.L.S.) and from the Graduate Studies Office at California State University Chico (to A.C.B.).

The Institutional Animal Care and Use Committee (IACUC) advisory board at California State University, Chico, approved all methods described below.
1. Bleach 48 hpf Zebrafish Embryos
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	<li>With 15 cm (w) x 15 cm (l) x 7 cm (h) plastic containers create 3 wash stations: 1) with&#160;1 mL of 5% bleach in 1000 mL&#160;embryo medium (E3; see Table 1), 2) with sterile E3, and 3) with sterile E3. Ensure that each container has 500 mL of solution to submerge the embryos in.<br ...

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E2+regulates+vertebrate+haematopoietic+stem+cell+homeostasis.">Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis.</a> Nature. 447, 1007-1011 (2007).</li><li>Astuti, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Bioluminescent+Zebrafish+Screen+for+Enhancing+Hematopoietic+Cell+Homing.">A Functional Bioluminescent Zebrafish Screen for Enhancing Hematopoietic Cell Homing.</a> Stem Cell Reports. 8, 177-190 (2017).</li><li>Kiel, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121, 1109-1121 (2005).</li><li>Spangrude, G. J., Heimfeld, S., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+mouse+hematopoietic+stem+cells.">Purification and characterization of mouse hematopoietic stem cells.</a> Science. 241, 58-62 (1988).</li><li>Morrison, S. J., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-term+repopulating+subset+of+hematopoietic+stem+cells+is+deterministic+and+isolatable+by+phenotype.">The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.</a> Immunity. 1, 661-673 (1994).</li><li>Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+lymphohematopoietic+reconstitution+by+a+single+CD34-low/negative+hematopoietic+stem+cell.">Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.</a> Science. 273, 242-245 (1996).</li><li>Goodell, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+efflux+studies+suggest+that+hematopoietic+stem+cells+expressing+low+or+undetectable+levels+of+CD34+antigen+exist+in+multiple+species.">Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.</a> Nat Med. 3, 1337-1345 (1997).</li><li>Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J Exp Med. 183, 1797-1806 (1996).</li><li>Gansner, J. 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The zebrafish model system has become an efficient, effective, and inexpensive model for studying primitive and definitive vertebrate hematopoiesis. Generation of assays that are fast, inexpensive, and present few technical difficulties can be utilized for testing small molecules, analyzing mutant embryos, and elucidating molecular pathways important for HSPC biology. In vitro plating of HSPCs from adult zebrafish is an effective method to study mutagenesis, cytokines, and hematopoietic defects. This protocol bu...

Hematopoiesis is the process of making the multitude of mature blood cells required for an organism&#39;s survival. It is a key developmental process that involves the differentiation of hematopoietic stem cells (HSCs) into a variety of developmentally restricted cell types that comprise mature blood. These HSCs must self-renew so that the system is never exhausted and they must persist from early embryonic development until death. In vertebrates, constant differentiation and proliferation of hematopoietic stem and progenitor cells (HSPCs) are needed to adequately replenish the majority of blood cells that are post-mitotic and being recycled every day. HSCs generate mature blood cells by first differentiating into subsets of restricted progenitor cells; common lymphoid progenitors (CLPs)1, which eventually produce T, B, and NK cells, and common myeloid progenitors (CMPs)2 that generate granulocytes, erythrocytes, macrophages, and megakaryocytes. These progenitors are committed to generating specific cell lineages, and further differentiate into more developmentally restricted progenitor cells such as myeloid erythroid progenitors (MEPs) that generate erythrocytes and platelets, or granulocyte macrophage progenitors (GMPs) that generate basophils, eosinophils, neutrophils, and macrophages2. Identifying and isolating these progenitors allows the identification of important molecular pathways involved in hematopoietic differentiation and many hematopoietic diseases such as leukemia arise when these progenitor cells fail to properly differentiate.
Over the past few decades, the zebrafish (Danio rerio) model system has become a key research tool for embryonic and adult hematopoietic studies. Zebrafish are amenable to genetic analysis and are the phylogenetically lowest vertebrate model species that have a similar vasculature and hematopoietic system to humans. Zebrafish embryos develop ex utero, and within 48 hours post fertilization (hpf) generate HSPCs3,4,5,6,7,8. Zebrafish are also highly fecund, with females laying over 100 eggs in a single clutch, allowing for large sample sizes and experimental replication. Zebrafish embryos are optically transparent, allowing for microscopic visualization of the hematopoietic system. Several fluorescent transgenic lines of zebrafish marking HSCs such as runx1:EGFP fish9, cd41:EGFP fish10, and kdrl:mCherry; cmyb:GFP3 double-positive animals, allow for live, real-time visualization of HSC emergence and expansion in vivo3,4,7,8,9. The zebrafish&#39;s quick generation time and development ex utero has led to its use in mutagenesis studies11,12,13,14,15 and drug screening16,17,18,19,20 for compounds that hold therapeutic promise for human blood disorders. Overall, conservation of the hematopoietic system, the presence and easy development of transgenic lines, and quick regeneration time has made the zebrafish an inexpensive, quick, flexible, and ideal model for hematopoietic studies.
Numerous methods of isolating and testing HSCs have been developed in mammalian hematopoietic systems. Investigators can utilize a combination of cell surface receptors to mark HSCs21,22,23,24, as well as exploit the ability of HSCs to efflux dye25,26. After they are labeled, fluorescence-activated cell sorting (FACS) allows their physical separation. Proving that a cell is an HSC requires irradiating a host animal to destroy endogenous HSPCs, transplanting putative HSCs, and observing long-term, multi-lineage reconstitution of all mature blood cell types. These assays work well in mice, as there are numerous cell-surface antibodies against hematopoietic cells and inbred mouse strains that facilitate immune matching for transplantation. However, few zebrafish hematopoietic cell-surface antibodies have been generated27, hindering the identification and isolation of HSCs. The most common way to mark and isolate zebrafish HSCs is with transgenic animals, whereby a cell-specific promoter sequence is driving a fluorescent protein&#39;s expression. Studies have visualized and enumerated HSCs in the ventral wall of the dorsal aorta with microscopy utilizing this technique3,4,8,9. Other laboratories have generated clonal strains of zebrafish28,29 and have performed successful transplants in MHC-matched animals30. However, these techniques are cost prohibitive to many laboratories, are technically difficult, and are time consuming. To address these issues, laboratories have generated several in vitro assays to test for the presence, the proliferation rates, and the differentiation capacity of HSPCs31,32,33,34,35,36. These assays show proliferation and differentiation of HSPCs in vitro31,32,33,34,35,36, the rescue of hematopoietic defects36, and an efficient method for discovering and testing cytokines33,34,35. They have also been utilized to identify genes responsible for HSPC biology31,32. In this study, we take these assays a step further, allowing the quantitation of HSPCs in a developing zebrafish embryo. These assays can also be utilized to quantitate the number of HSPCs in mutant animals and animals treated with hematopoietic-disruptive drugs. In essence, these assays are fast, present few technical challenges, and are inexpensive ways to quantitate HSPC numbers, examine their proliferation, and investigate blocks in differentiation.

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			<td height="40" style="height:40px;width:341px;">Penicillin/streptomycin (5000 U/mL 
			and 5000 mg/mL) with L-Glutamine (200 mM)</td>
			<td style="width:115px;">Corning Mediatech</td>
			<td style="width:119px;">30-009-CI</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Gentamycin Sulfate 10 mL (50 mg/mL)</td>
			<td style="width:115px;">Corning Mediatech</td>
			<td style="width:119px;">30-005-CR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:341px;">1.5 mL MCF tube</td>
			<td style="width:115px;">FisherBrand</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">3 mL 23gx1 injection needle with Luer lock</td>
			<td style="width:115px;">BD Safety Glide</td>
			<td style="width:119px;">305905</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">5 mL polystyrene round bottom tube with cell strainer cap</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">352235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:341px;">Methocel MC</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">64630</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">14 mL Polystyrene round bottom tube</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">352057</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">10 mm polystyrene easygrip Petri dish</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">351008</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Librease TM</td>
			<td style="width:115px;">Roche Sigma-Aldrich</td>
			<td>5401119001</td>
			<td>dissociation protease</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Pronase</td>
			<td style="width:115px;">Roche Sigma-Aldrich</td>
			<td style="width:119px;">11459643001</td>
			<td>dechorionation protease</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">15 cm Petri dish</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:341px;">AB</td>
			<td style="width:115px;">Zebrafish International Resource Center (ZIRC)</td>
			<td style="width:119px;">ZL1</td>
			<td>zebrafish strain used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:341px;">Dithiolthreitol (DTT)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td>646563</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

To assess HSPC numbers in embryonic zebrafish, 48 hpf embryos were digested, plated in methylcellulose with exogenous hematopoietic-supportive growth factors, and incubated for 7 days (Figure 1A). After 7 days, colony forming units (CFUs) were enumerated (Figure 1B) and imaged (Figure 1C). By controlling the different cytokines added, one can control...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

The overall goal of this assay is to enumerate and examine the differentiation of hematopoietic progenitor cells in the developing zebrafish embryo. This method can help answer some really important questions in the hematopoietic field, such as, how many stem and progenitor cells are actually in a developing zebrafish embryo. The main advantage of this technique, is that it's quick, it's relatively inexpensive and it's easy to perform.Though this method can provide insight into the number of progenitors present in the developing zebrafish, it also allows researchers to measure the functional effect that a drug or genetic mutation has on blood progenitor formation. We had the idea for this method, when we were knocking down genes that we hypothesized were crucial for blood development. We predicted that we would be able to measure the difference between normally developing embryos and those that were morphants with this assay.To bleach zebrafish embryos, use plastic containers to create three wash stations, with one containing one milliliter of five percent bleach per one liter of E3 and two stations containing sterile E3.With a transfer pipette, place embryos into a tea strainer and submerge them in wash station number one for five minutes. Then transfer the tea strainer with the embryos into wash station number two for five minutes. Repeat for wash station number three for a final five minutes.After the last wash, remove the embryos from the tea strainer by turning it upside down over a clean 10 centimeter petri dish and use a transfer pipette and sterile E3 to gently rinse the embryos off the strainer. To dechorionate the embryos, use a transfer pipette to remove and discard as much E3 as possible from the petri dish. Then add 500 microliters of dechorionation prodeas to the embryos and incubate them at room temperature for five minutes.Next, gently tap the side of the petri dish to completely remove the chorions. Then, with a serological pipette, add 20 milliliters of sterile E3 to dilute the prodeas. Allow embryos to settle and use a pipette to remove the E3.Repeat this wash step three times to remove all traces of the dechorionation prodeas.To prepare embryo samples, use a pipette to place 10 embryos into a single sterile 1.5 milliliter microcentrifuge tube. Then, carefully pipette out the E3 and discard it. Transfer samples to a laminar flow hood, and wash the embryos by adding one milliliter of sterile E3, allowing embryos to settle to the bottom of the tube.Then, use a P1000 pipette to remove the supernatant. Repeat for a total of three washes. Following the last wash, remove the E3 and discard it.Then add one milliliter of 10 millimolar DTT in E3 to remove the mucus coating and any spores, yeasts or bacteria that may be trapped in it surrounding the embryos. Now, lay the microcentrifuge tube horizontally and incubate it at room temperature for 25 minutes. To dissociate the embryos, use one milliliter of sterile DPBS to wash the sample three times.After the last wash, add 500 microliters of DPBS with calcium and magnesium and five microliters of five milligram per milliliter dissociation prodeas. Incubate the samples at 37 degrees Celsius on a horizontal orbital shaker at 180 rpm for 60 minutes. Then, place the samples and the laminar flow hood and use a P1000 to triturate the embryos until the samples are fully dissociated.Use caution and check embryos periodically to determine when they are dissociated. You want some tissue to be present. It is possible to over digest the embryos which will destroy the progenitor cells.Pipette the 10 dissociated embryos onto the top reservoir of a five milliliter poly styrene round bottom tube with a 35 micrometer cell strainer cap. With a pipette and sterile PBS, rinse the embryo tube and transfer the solution to the five milliliter poly styrene tube containing filtered cells. Repeat the rinse until four milliliters of liquid is present in the tube.Centrifuge the tubes at 300 times the gravity and four degrees Celsius for five minutes to palette the homogenized cell sample. With a pipette, remove and discard the supernatant from the round bottom tube, taking care to not disrupt the cells pelleted at the bottom. Then use 100 microliters of PBS to re-suspend the cells.Prepare one X complete methylcellulose stock solution and use three milliliter syringes and 16 gage needles to add 2.5 milliliters of sterile round bottom 14 milliliter tubes. Use one tube for each condition tested. For myeloid differentiation, add one percent carp serum and 0.3 milligram per milliliter of recombinant zebrafish granulocyte colony-stimulating factor or GCSF to methyl cellulose medium.For erythroid differentiation, add one percent carp serum and 0.1 milligram per milliliter of recombinant zebrafish erythropoietin or EPO to methyl cellulose medium. To examine multi-lineage progenitors, add EPO and GCSF. Using a pipette, add 100 microliters of the 10 dissociated embryos to the surface of the prepared methyl cellulose with the recombinant cytokines in carp serum.Cap the lid and gently vortex to fully homogenize the sample. Each tube should now contain the dissociated tissue of 10 embryos. Using three milliliter syringes and 16 gage needles, aliquot one milliliter of sample into two separate 35 millimeter petri dishes.Make sure that the sample is fully dispersed throughout the petri dish. Repeat for each sample. Place the 35 millimeter petri dishes with cells and methyl cellulose into a 15 centimeter petri dish.To each large plate, add one 35 millimeter dish with five milliliters of sterile water to humidify the samples. Incubate the dishes at 32 degrees Celsius and five percent carbon dioxide for seven to 10 days. Finally, visualize the colony forming units according to the text protocol.After seven days in culture with hematopoietic supportive factors, progenitors from 48 HPF embryonic zebrafish were counted and imaged. Importantly, the quantity of CF used directly correlates with the number of embryos plated. Plating either four or eight embryos per milliliter, which correlates to starting with 10 or 20 embryos, at the beginning of the experiment, yields approximately 1200 or 2400 CFUs respectively.Once mastered, this technique can be done in two and a half hours if performed properly. While attempting this procedure, it's important to remember not to over digest the embryos as this will destroy the progenitor cells. With this procedure, you can compare the number of progenitors present at different time points.You can compare mutant animals with wild type animals, as well as test the effect of drugs on blood development. After watching this video, you should have a good understanding of how to perform a methyl cellulose assay to enumerate zebrafish hematopoietic progenitors. You can alter this procedure to fit your specific experimental needs, such as testing specific cytokines.Don't forget, that working with vertebrate blood and needles can be extremely hazardous and care should always be taken when performing this assay.

Research

JoVE Journal

Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

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Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

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Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

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Induction of Hypoxia in Living Frog and Zebrafish Embryos

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Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

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Single-cell Photoconversion in Living Intact Zebrafish

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The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...