Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Summary

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

Abstract

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Introduction

Clonal studies have revealed heterogeneity in the long-term engraftment properties of adult HSCs, providing new insight into HSC subtypes and changes in HSC behavior during aging¹. However, similar studies of embryonic HSCs and their precursors have been more challenging. During early embryonic development, HSCs arise from a population of precursors known as hemogenic endothelium in a transient process referred to as the endothelial to hematopoietic transition². The first HSC, defined by their ability to provide robust, long-term multilineage engraftment following transplantation into conditioned adult recipients, are no....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Fred Hutchinson Cancer Research Center.

1. Preparation of AGM-EC Monolayers for Co-culture

1. 24 h prior to the AGM dissection and sort, make  AGM-EC culture media and filter sterilize.
2. Add 0.1% gelatin in water (100 µL/well) to each well of a 96-well plate and incubate at room temperature for 15 min. Aspirate the gelatin and leave the plate in a tissue culture hood with the lid removed until the wells are dry.
3. Dissociate the AGM-EC monolayers and plate to 96-well plates.

Results

Figure 1A shows a schematic of the experimental design. Once P-Sp/AGM tissues are dissected, pooled, and dissociated in collagenase, they are stained with antibodies to VE-Cadherin and EPCR for index sorting. Pre-HSC are enriched in cells sorted at VE-Cadherin⁺EPCR^high (Figure 1B). Other fluorochrome-conjugated antibodies can be included to retrospectively analyze additional phenotypic parameters, which are .......

Discussion

The study of HSC genesis during embryonic development necessitates means to detect HSC potential in hemogenic precursors yet lacking the competence to provide long-term multilineage hematopoietic reconstitution in transplanted adult recipients. In this protocol, we present a clonal assay of embryonic hemogenic precursors by stromal co-culture on vascular niche ECs from the AGM, which supports the maturation of precursors to HSC, with subsequent functional analysis in transplantation assays. Incorporation of index sorting.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AGM-EC culture media
Materials for culture of endothelial cells
TrypLE Express	Gibco	12605-028	Use to dissociate AGM-EC monolayers
Gelatin 0.1% in water	StemCell Technologies	7903	Use to adhere AGM-EC monolayers to plastic
96-well tissue culture plates	Corning	3599	Use to co-culture AGM-EC monolayers and index sorted clones
Dulbecco's Phosphate Buffered Saline (PBS)	Gibco	14190-144	Use for dissection and FACS staining
500 ml filter bottles	Fisher Scientific	9741202	Use to sterile filter Endothelial media
AGM-EC culture media (500 mls)
Iscove's Modified Dulbecco's Medium (IMDM)	Gibco	12440-053	400 mls
Hyclone Fetal Bovine Serum	Fisher Scientific	SH30088.03	100 mls
Penicillin Streptomycin	Gibco	15140-122	5 mls
Heparin	Sigma	H3149	50 mg dissolved in 10 mls media
L-glutamine (200 mM)	StemCell Technologies	7100	5 mls
Endothelial Mitogen (ECGS)	Alfa Aesar	J64516 (BT-203)	Add 10 mls media to dissolve and add
Materials for AGM index sort
Collagenase (0.25%)	StemCell Technologies	7902	Use for dissociation of embryonic tissues
3ml syringe	BD Biosciences	309657	Use to sterile filter antibody solutions for FACS
0.22 µM syringe-driven filter	Millipore	SLGP033RS	Use to sterile filter antibody solutions for FACS
5 ml polystyrene tube with cell-strainer cap	Corning	352235	Use to remove cell clumps prior to FACS
DAPI (prepared as 1 mg/ml stock in H2O)	Millipore	268298	Use to exclude dead cells from sort
anti-mouse CD16/CD32 (FcR block)	BD Biosciences	553141	Use to block non-specific staining to Fc receptors
Antibodies for AGM index sort
Anti-mouse CD144 PE-Cyanine7	eBioscience	25-1441-82	Staining
Rat IgG1 kappa Isotype Control, PE-Cyanine7	eBioscience	25-4301-81	Isotype control for CD144 PE-Cyanine7
Anti-mouse CD201 (EPCR) PerCP-eFluor710	eBioscience	46-2012-80	Staining
Rat IgG2b kappa Isotype Control, PerCP-eFlour710	eBioscience	46-4031-80	Isotype control for CD201 PerCP-eFluor710
Anti-mouse CD41 PE	BD Biosciences	558040	Staining
Rat IgG1 kappa Isotype Control, PE	BD Biosciences	553925	Isotype control for CD41 PE
Anti-mouse CD45 FITC	eBioscience	11-0451-85	Staining
Rat IgG2b kappa Isotype Control, FITC	eBioscience	11-4031-81	Isotype control for CD45 FITC
AGM-serum free media (10mls)
X-Vivo 20	Lonza	04-448Q	10 mls
recombinant murine stem cell factor (SCF)	Peprotech	250-03	10 ml (100 mg/ml stock) Final concentration 100 ng/ml
recombinant human FLT3 Ligand (FLT3L)	Peprotech	300-19	10 ml (100 mg/ml stock) Final concentration 100 ng/ml
recombinant human thrombopoietin (TPO)	Peprotech	300-18	2 ml (100 mg/ml stock) Final concentration 20 ng/ml
recombinant murine interleukin-3 (IL3)	Peprotech	213-13	2 ml (100 mg/ml stock) Final concentration 20 ng/ml
Materials for Staining co-cultured cells
96 well plate, V-bottom	Corning	3894	Use for FACS analysis
Antibodies for analysis of Index sorted clones co-cultured with endothelial cells
Anti-mouse CD45 PerCP-Cyanine5.5	eBioscience	45-0451-82	Staining
Rat IgG2b kappa Isotype Control, PE-Cyanine5.5	eBioscience	35-4031-80	Isotype control for CD45 PerCP-Cyanine5.5
Anti-mouse CD201 (EPCR) PE	eBioscience	12-2012-82	Staining
Rat IgG2b kappa Isotype Control, PE	eBioscience	12-4031-81	Isotype control for CD201 PE
Anti-mouse Ly-6A/E (Sca-1) APC	eBioscience	17-5981-83	Staining
Rat IgG2a kappa Isotype Control, APC	eBioscience	17-4321-81	Isotype control for Ly-6A/E APC
Anti-mouse F4/80 FITC	eBioscience	11-4801-81	Staining
Rat IgG2a kappa Isotype Control, FITC	eBioscience	11-4321-41	Isotype control for F4/80 FITC
Anti-mouse Ly-6G/C (Gr1) FITC	BD Biosciences	553127	Staining
Rat IgG2b kappa Isotype Control, FITC	BD Biosciences	556923	Isotype control for Ly-6G/C and CD3 FITC
Materials for tail vein injection for transplant
1/2 ml insulin syringes with 29G 1/2" needles	BD Biosciences	309306	Use for tail vein injection for transplantation
Antibodies for Peripheral Blood analysis following translant
Anti-mouse Ly-6G/C (Gr1) PerCP	Biolegend	108426	Staining
Rat IgG2b kappa Isotype Control, PerCP	Biolegend	400629	Isotype control for Ly-6G/C PerCP
Anti-mouse F4/80 PE	eBioscience	12-4801-82	Staining
Rat IgG2a kappa Isotype Control, PE	eBioscience	12-4321-80	Isotype control for F4/80 PE
Anti-mouse CD3 FITC	BD Biosciences	555274	Staining
Anti-mouse CD19 APC	BD Biosciences	550992	Staining
Rat IgG2a kappa Isotype Control, APC	BD Biosciences	553932	Isotype control for CD19 APC
Anti-mouse CD45.1 (A20) PE-Cyanine7	eBioscience	25-0453-82	Staining
Rat IgG2a kappa Isotype Control, PE-Cyanine7	eBioscience	25-4321-81	Isotype control for CD45.1 PE-Cyanine7
Anti-mouse CD45.2 (104) APC-eFluor780	eBioscience	47-0454-82	Staining
Rat IgG2a kappa Isotype Control, APC-eFluor780	eBioscience	47-4321-80	Isotype control for CD45.2 APC-eFluor780
Equipment
BD FACSAria II with DIVA software	BD Biosciences
BD FACSCanto II with plate reader	BD Biosciences
Haemocytometer	Fisher Scientific	S17040	For counting cells
Multi-channel pipette	Fisher Scientific	14-559-417	For dispensing cells
FACS analysis software	FlowJo/BD Biosciences		https://www.flowjo.com/solutions/flowjo