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

Summary

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.

Abstract

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.

Introduction

Hematopoiesis is the process of making the multitude of mature blood cells required for an organism'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 HSPCs^3,4,5,6,7,8. 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 vivo^3,4,7,8,9. The zebrafish's quick generation time and development ex utero has led to its use in mutagenesis studies^{11,12,13,14,15} and drug screening^{16,17,18,19,20} 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 HSCs^21,22,23,24, as well as exploit the ability of HSCs to efflux dye^25,26. 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's expression. Studies have visualized and enumerated HSCs in the ventral wall of the dorsal aorta with microscopy utilizing this technique^3,4,8,9. Other laboratories have generated clonal strains of zebrafish^28,29 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 HSPCs^{31,32,33,34,35,36}. These assays show proliferation and differentiation of HSPCs in vitro^{31,32,33,34,35,36}, the rescue of hematopoietic defects³⁶, and an efficient method for discovering and testing cytokines^33,34,35. They have also been utilized to identify genes responsible for HSPC biology^31,32. 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.

Protocol

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

1. With 15 cm (w) x 15 cm (l) x 7 cm (h) plastic containers create 3 wash stations: 1) with 1 mL of 5% bleach in 1000 mL 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.
   NOTE: For each condition tested, at least 10 embryos will be needed. This washing procedure can accommodate up to 200 embryos.
2. With a transfer pipette, place embryos into a tea strainer and submerge eggs in wash station #1 for 5 min. Remove the tea strainer (with embryos inside) and place into wash station #2 for 5 min; repeat for wash station #3 for a final 5 min.
3. After the last wash, rinse embryos from tea strainer by turning it upside down over a clean 10 cm Petri dish and gently rinsing embryos off the tea strainer with sterile E3 and a transfer pipette.

2. Dechorionate Embryos

1. With a transfer pipette, remove and discard as much E3 as possible from the Petri dish and add 500 μL of dechorionation protease (10 mg/mL) to embryos. Incubate at room temperature for 5 min. Gently tap the side of the Petri dish to completely remove chorions.
2. With a serological pipette, add 20 mL of sterile E3 to dilute protease. Allow embryos to settle, and remove the E3 with a transfer pipette. Repeat this wash step 3 times to remove all traces of the dechorionation protease.

3. Preparing Embryo Samples

1. Using a P1000 pipette, place 10 embryos into a single sterile 1.5 mL microcentrifuge tube.
2. Remove E3 with a pipette and discard.
   Caution: Pipette with care as embryos can easily be displaced and discarded during the following wash steps.

4. Preparing Embryos for Dissociation

1. Transfer samples to laminar flow hood, and wash embryos by adding 1 mL sterile E3. Allow embryos to settle to bottom of tube and remove supernatant with a P1000 pipette. Repeat for a total of 3 washes.
2. After last wash, remove E3 and discard. Add 1 mL of 10 mM dithiothreitol (DTT) in E3 to remove the mucus coating (and any spores, yeast, or bacteria that may be trapped in it) surrounding the embryonic zebrafish. Lay microcentrifuge tube horizontally, and incubate at room temperature in laminar flow hood for 25 min.

5. Embryo Dissociation

1. Wash sample 3 times with 1 mL of sterile DPBS (with Ca²⁺ and Mg²⁺). After last wash add 500 μL of DPBS (with Ca²⁺ and Mg²⁺) and add 5 μL of 5 mg/mL (26 U/mL) dissociation protease.
   Caution: This enzyme needs Ca²⁺ and Mg²⁺, so ensure that the DPBS contains these ions.
2. Incubate samples at 37 °C on a horizontal orbital shaker at 180 rpm for 60 min. Place samples in the laminar flow hood and triturate embryos with a P1000 until samples are fully dissociated.
   Caution: Check embryos periodically to determine when they become dissociated. The embryo solution should have some amount of tissue present; it should not be completely homogenous. It is possible to over-digest, which will destroy the HSPCs (see Supplemental Figure 1).

6. Preparation of Dissociated Embryos

1. Pipette the 10 dissociated embryos onto the top reservoir of a 5 mL polystyrene round bottom tube with a 35 μm cell strainer cap.
2. With a pipette, rinse the Petri tube with sterile PBS (with no Ca²⁺ or Mg²⁺) and transfer solution to 5 mL polystyrene tube containing filtered cells from step 6.1. Repeat until 4 mL of liquid is present in the 5 mL polystyrene tube.
   NOTE: PBS, DPBS, or other isotonic buffered solutions may be used at this stage, as long as they do not contain Ca²⁺ or Mg²⁺
3. Centrifuge tubes at 4 °C and 300 x g for 5 min to pellet the homogenized cell sample. With a pipette, remove and discard supernatant from the round bottom tube. Take care to not disrupt the cells pelleted at the bottom of the tube. Resuspend the cells in 100 μL of PBS.

7. Preparation of Methylcellulose

1. Generate 1x complete methylcellulose (Table 1) stock solution and add 2.5 mL to sterile round-bottom 14 mL tubes with 3 mL syringes and 16 G needles. Use one tube for each condition tested.
2. Add cytokines, small molecules, or other agents to be investigated to each sample.
   1. For myeloid differentiation, add 1% Carp serum and 0.3 mg/mL recombinant zebrafish granulocyte colony stimulating factor (Gcsf)³⁴ to methylcellulose medium. See Svoboda et al.³⁷ for full description on how to generate carp serum and recombinant cytokines.
   2. For erythroid differentiation, add 1% Carp serum and 0.1 mg/mL recombinant zebrafish erythropoietin (Epo)³⁸ to methylcellulose medium.
   3. To examine multilineage progenitors, add Epo and Gcsf.
      Caution: Cytokines and additives should not total more than 10% of the total volume, as the medium will not be viscous enough to discern individual colonies.

8. Addition of Dissociated Embryos to Methylcellulose

1. Using a pipette, add 100 μL of the dissociated 10 embryos to the surface of the prepared methylcellulose with recombinant cytokines and carp serum. Cap lid and vortex gently to fully homogenize sample. Each tube should now contain the dissociated tissue of 10 embryos.
2. Using 3 mL syringes and 16 G needles, aliquot 1 mL of sample into 2 separate 35 mm Petri dishes. Make sure that the sample is fully dispersed throughout the Petri dish. Repeat for each sample.
3. Place 35 mm Petri dishes with cells in methylcellulose into a 15 cm Petri dish. To each 15 cm dish, add one 35 mm Petri plate with 5 mL of sterile water to humidify the samples. Incubate at 32 °C and 5% CO₂ for 7 - 10 days.

9. Visualization of HSPC-derived Colony Forming Units (CFUs)

1. After incubating 7 days, place samples onto an inverted microscope at 40 - 100X for visualization and enumeration. At this point, individual colonies can be isolated with a pipette and subjected to staining and/or gene analysis.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% Bovine Serum Albumin in Iscove's MDM	StemCell Technologies	9300
DPBS (10x) with Calcium2+ and Magnesium2+	Life Technologies	14080-055
HyClone PBS (1x)	GE Healthcare Life Sciences	sh30256.01
DMEM 500 mL	Corning CellGrow	10-017-CV
Ham's F12 500 mL	Corning CellGrow	10-080-CV
FBS 500 mL	Gemini Bio-Products	100-108
HEPES 100 mL (1 M)	Gibco life technologies	15-630-080
Penicillin/streptomycin (5000 U/mL and 5000 mg/mL) with L-Glutamine (200 mM)	Corning Mediatech	30-009-CI
Gentamycin Sulfate 10 mL (50 mg/mL)	Corning Mediatech	30-005-CR
1.5 mL MCF tube	FisherBrand	05-408-129
3 mL 23gx1 injection needle with Luer lock	BD Safety Glide	305905
5 mL polystyrene round bottom tube with cell strainer cap	Corning Falcon	352235
Methocel MC	Sigma-Aldrich	64630
14 mL Polystyrene round bottom tube	Corning Falcon	352057
10 mm polystyrene easygrip Petri dish	Corning Falcon	351008
Librease TM	Roche Sigma-Aldrich	5401119001	dissociation protease
Pronase	Roche Sigma-Aldrich	11459643001	dechorionation protease
15 cm Petri dish	Corning Falcon	351058
AB	Zebrafish International Resource Center (ZIRC)	ZL1	zebrafish strain used
Dithiolthreitol (DTT)	Sigma-Aldrich	646563