Novel FACS strategy for identification of early hematopoietic progenitors including BFU-e, CFU-e and erythroid-biased MPPs

doi:10.1038/protex.2018.031

Method Article

Novel FACS strategy for identification of early hematopoietic progenitors including BFU-e, CFU-e and erythroid-biased MPPs

https://doi.org/10.1038/protex.2018.031

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We used single-cell RNA-seq and fate potential assays to develop a novel FACS strategy that identifies erythroid-biased multipotent progenitors, and erythroid committed progenitors, directly in freshly isolated mouse bone marrow. It also identifies subsets enriched for progenitors of the basophil/ mast cell lineage or the megakaryocytic lineage. It divides Kit+CD55+ hematopoietic progenitors in mouse bone marrow into five populations, as follows: P1, highly purified CFU-e progenitors, also called Committed Erythroid Progenitors (CEPs); P2, highly enriched BFU-e progenitors, also called Early Erythroid Progenitors (EEPs); P2 also contains some CFU-e, and uncommitted progenitors with erythroid/megakaryocytic/basophil potential (EBMegPs); P3, enriched for basophil/mast cell progenitors; P4, enriched for megakaryocytic progenitors; and P5, containing EBMegPs and erythroid/ megakaryocytc/ basophil-biased MPPs.

Cell biology

Developmental biology

Biological techniques

erythropoiesis

hematopoiesis

erythroid progenitors

CFU-e

BFU-e

MPPs

basophils

mast cells

megakaryocytes

EBMegPs

Flow cytometry

Although CFU-e and BFU-e progenitors were defined functionally over four decades ago, their complete and pure prospective isolation from murine bone-marrow has not been possible. Our recent single-cell RNA-seq study developed a FACS strategy that achieves their isolation, as well as the isolation of a number of other early hematopoietic progenitors.

In this protocol, we first enrich Kit+ cells from total mouse bone marrow, using magnetic beads. This population is then labeled with antibodies against CD55, CD150, CD105, CD41, CD49f and CD71. Kit+CD55- cells are lympho/ myeloid progenitors, whereas Kit+CD55+ cells contain erythroid/ megakaryocytic/ basophil progenitors. We subdivide Kit+CD55+ cells into 5 subsets, P1 to P5. P5 contains uncommitted progenitors biased towards the erythroid/ megakaryocytic/ basophil lineages, including MPPs. P4, P3, and P1/P2 contain megakaryocytic, basophil/ mast cell, and erythroid progenitors, respectively. Specifically, P2 contains largely BFU-e cells, whereas P1 contains CFU-e, and no other progenitors. Of note, P1 can be further sub-divided into earlier CFU-e, forming larger colonies, and later CFU-e, forming smaller colonies, by selecting for P1-CD71^medium and P1-CD71^high gates, respectively.

Reagents

Staining buffer (1X PBS; 0.2% BSA; 0.08% Glucose)

Easy Sep buffer (1X PBS; 2% FBS; 1mM EDTA)

Mouse Streptavidin RapidSpheres Isolation Kit (STEMCELL technologies, Cat No. 19860A)

Rat Serum (STEMCELL technologies, Cat No. 13551, or equivalent)

0.5 M EDTA, pH 8.0 (Invitrogen by Life technologies, Cat No. 15575-038, or equivalent)

Biotin anti-TER119 (BD Biosciences, Clone TER119, Cat No. 553672)

Biotin anti-CD11b (BD Biosciences, clone M1/70, Cat No. 557395)

Biotin anti-Ly-6G and Ly-6C (BD Biosciences, clone RB6-8C5, Cat No. 553125)

Biotin anti-CD4 (BD Biosciences, clone RM4-5, Cat No. 553045)

Biotin anti-CD8a (Ly-2) (BD Biosciences, clone 53-6.7, Cat No. 553029)

Biotin anti-CD19 (BD Biosciences, clone 1D3, Cat No. 553784)

Streptavidin, Alexa Fluor™ 488 conjugate (Invitrogen, Cat No. S11223)

CD117- APC Cy7 (Biolegend, clone 2B8, Cat No. 105826)

TER119-BUV395 (BD Biosciences, clone TER-119, Cat No. 563827)

CD71- PE Cy7 (Biolegend, clone RI7217, Cat No. 113812)

CD55-AF647 (Biolegend, clone RIKO-3, Cat No. 131806)

CD105-PE (Biolegend, clone MJ7/18, Cat No. 120408)

CD150-BV650 (Biolgened, clone TC15-12F12.2, Cat No. 115931)

CD41-BV605 (Biolegend, clone MWReg30, Cat No. 133921)

CD49f – BV421 (Biolegend, clone GoH3, Cat No. 313624)

DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) (invitrogen, Cat No. D1306)

Dissection equipment, 70% ethanol in water spray, Mortar and Pestle, 2ml syringes, 26-gauge needles, 70 μm cell strainer, microcentrifuge, “The Big Easy” magnet (Cat No. 18001) from STEMCELL technologies (or equivalent)

1. Preparation of cell suspensions from mouse bone marrow:

• Obtain femora and tibiae from 10 mice and remove the muscles

• Cut the tip of the bones and flush the shaft with 2 to 3 ml of staining buffer or medium using a 2 ml syringe with a 26-gauge needle.

• Crush the bones after flushing, using mortar & pestle to obtain any remaining cells

• Pass cells through 70 μm cell strainer to remove cell clumps. Moisten filter with buffer or medium before use.

• Pellet cells by centrifugation at 1400 rpm for 5 min.

• Resuspend the cells in staining buffer and count. (approximately 2 x 10⁹ cells are obtained from 10 wild type mice.)

2. EasySep mouse Streptavidin RapidSpheres negative selection for Lin+ cells:

• Mouse Streptavidin RapidSpheres Isolation Kit (Cat No. 19860A) and “The Big Easy” magnet (Cat No. 18001) from STEMCELL technologies are used for this selection.

• For Negative selection for Easy Sep sort, cells are resuspended at 2 x 10⁸ cells/ ml in Easy Sep buffer.

• For tibia/femur of 10 mice, cells are resuspended in total volume of 10ml EasySep buffer, containing Rat Serum (1% final concentration) to block the Fc receptors, and a cocktail of lineage-specific antibodies, each at a final concentration of 0.005 mg/ml: of biotin lanti-TER119, anti-CD11b (Clone M1/70), anti-Ly-6G and Ly-6C (Clone RB6-8C5), anti-CD4 (Clone RM4-5), anti-CD8a (Ly-2) (Clone 53-6.7), anti-CD19 (Clone 1D3). Mix and incubate for 30 min on ice.

• Vortex RapidSpheres and add to sample at 100μl/ml (1ml /10ml). Mix well and incubate on ice for 15 min.

• Divide 10 ml total volume of sample into two 5 ml samples (Use 15 ml conical Falcon tubes).

• Bring each sample to a 10 ml total volume with Easysep buffer, and place the tube in The Big Easy magnet for 10 minutes. Pour the supernatant into a new empty 15 ml Falcon tube. Repeat the magnet wash for higher purity.

• Pellet lineage- depleted cells by centrifugation at 1400 rpm for 5 min.

3. Staining for populations P1 to P5:

• Wash the lineage-depleted cells with cold staining buffer and pellet by centrifugation at 1400 rpm for 5 min.

• Stain for 1 hour on ice in 1ml of staining buffer containing 1% Rat Serum and the following antibodies:

• 0.01 mg/mL Streptavidin Alexa Fluor 488

• 0.001 mg/mL CD117- APC Cy7

• 0.002 mg/mL TER119-BUV395

• 0.0001 mg/mL CD71- PE Cy7

• 0.01 mg/mL CD55-AF647

• 0.004 mg/mL CD105-PE

• 0.004 mg/mL CD150-BV650

• 0.002 mg/mL CD41-BV605

0.0005 mg/mL CD49f – BV421

• Wash cells with cold staining buffer and resuspend in 1ml staining buffer containing DAPI and 5 mM EDTA.

• Use cells for FACS analysis or for Cell sorting

• Any sorting needs to be performed at low pressure/wide nozzle conditions. E.g. 100 μM/14 psi or 130 μM/12 psi on a BD FACSAria II, following the gating strategy illustrated in Figure 1

• We include single-color controls and ‘fluorescence minus one’ controls in order adjust voltages, set compensation for spectral overlap, and place gates correctly

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Novel FACS strategy for identification of early hematopoietic progenitors including BFU-e, CFU-e and erythroid-biased MPPs

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Novel FACS strategy for identification of early hematopoietic progenitors including BFU-e, CFU-e and erythroid-biased MPPs

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Version 1

Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Troubleshooting

Additional Declarations

Associated Publications

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Version 1

Privacy Policy

Terms of Service

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