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 m....

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 tr....

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