Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

Summary

We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter system.

Abstract

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs), defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

Introduction

Hematopoietic stem cells (HSCs), which possess self-renewal capacity and multipotency, reside at the apex of the hematopoietic hierarchy^1,2. In 1988, Weissman and colleagues demonstrated for the first time that the isolation of mouse HSCs could be achieved using flow cytometry³. Subsequently, a fraction defined by a combination of cell surface markers, Lineage⁻c-Kit⁺Sca-1⁺CD150⁺CD34^−/loFlk2⁻, was reported to contain all HSCs in mice^4,5....

Protocol

All the animal experiments described were approved by the RIKEN Center for Biosystems Dynamics Research.

1. Preconditioning of the recipient mice

1. Prepare male C57BL/6 congenic mice aged 8-10 weeks old as recipient mice. The number of recipient mice depends on the experimental protocol. We typically prepare 10-20 mice for each condition.
   1. Feed the mice with sterilized water supplemented with enrofloxacin (170 mg/L). As irradiated recipient mice are highly.......

Discussion

Traditionally, cell surface marker-defined HSCs have been prepared to study the functions of HSCs, such as self-renewal capacity and multi-potency^19,20,21. However, the immunophenotypically defined (Lineage⁻c-Kit⁺Sca-1⁺CD150⁺CD34^−/loFlk2⁻) HSC fraction contains two discrete HSC populations: LT-HSCs and ST-HSCs⁹<.......

Materials

Name	Company	Catalog Number	Comments
0.2 mL Strip of 8 Tubes, Dome Cap	SSIbio	3230-00
0.5M EDTA pH 8.0	Iinvtrogen	AM9260G
100 µm Cell Strainer	Falcon	352360
30G insulin syringe	BD	326668
40 µm Cell Strainer	Falcon	352340
5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap	FALCON	352235
7-AAD Viability Staining Solution	BioLegend	420404
96 well U-Bottom	FALCON	351177
Anti-APC-MicroBeads	Milteny biotec	130-090-855
Aspirator with trap flask	Biosan	FTA-1
B220-Alexa Fluor 700 (RA3-6B2)	BioLegend	103232
B220-Biotin (RA3-6B2)	BioLegend	103204
B220-BV786 (RA3-6B2)	BD Biosciences	563894
B6.CD45.1 congenic mice	Sankyo Labo Service	N/A
Baytril 10%	BAYER	341106546
BD FACS Aria II special order system	BD	N/A
Brilliant stain buffer	BD	566349
CD11b-Alexa Fluor 700 (M1/70)	BioLegend	101222
CD11b-Biotin (M1/70)	BioLegend	101204
CD11b-BUV395 (M1/70)	BD Biosciences	563553
CD11b-BV711 (M1/70)	BD Biosciences	563168
CD127-Alexa Fluor 700 (A7R34)	Invitrogen	56-1271-82
CD150-BV421 (TC15-12F12.2)	BioLegend	115943
CD16/CD32-Alexa Fluor 700 (93)	Invitrogen	56-0161-82
CD34-Alexa Fluor 647 (RAM34)	BD Biosciences	560230
CD34-FITC (RAM34)	Invitrogen	11034185
CD3-Alexa Fluor 700 (17A2)	BioLegend	100216
CD3ε -Biotin (145-2C11)	BioLegend	100304
CD3ε -BV421 (145-2C11)	BioLegend	100341
CD45.1/CD45.2 congenic mice	N/A	N/A	Bred in our Laboratory
CD45.1-FITC (A20)	BD Biosciences	553775
CD45.2-PE (104)	BD Biosciences	560695
CD4-Alexa Fluor 700 (GK1.5)	BioLegend	100430
CD4-Biotin (GK1.5)	BioLegend	100404
CD8a-Alexa Fluor 700 (53-6.7)	BioLegend	100730
CD8a-Biotin (53-6.7)	BioLegend	100704
Centrifuge Tube 15ml	NICHIRYO	00-ETS-CT-15
Centrifuge Tube 50ml	NICHIRYO	00-ETS-CT-50
c-Kit-APC-eFluor780 (2B8)	Invitrogen	47117182
D-PBS (-) without Ca and Mg, liquid	Nacalai	14249-24
Fetal Bovine Serum	Thermo Fisher	10270106
Flk2-PerCP-eFluor710 (A2F10)	eBioscience	46135182
FlowJo version 10	BD Biosciences	https://www.flowjo.com/solutions/flowjo
Gmmacell 40 Exactor	Best theratronics	N/A
Gr-1-Alexa Fluor 700 (RB6-8C5)	BioLegend	108422
Gr-1-Biotin (RB6-8C5)	BioLegend	108404
Hoxb5-tri-mCherry mice (C57BL/6J background)	N/A	N/A	Bred in our Laboratory
IgG from rat serum, technical grade, >=80% (SDS-PAGE), buffered aqueous solution	Sigma-Aldrich	I8015-100MG
isoflurane	Pfizer	4987-114-13340-3
Kimwipes S200	NIPPON PAPER CRECIA	6-6689-01
LS Columns	Milteny biotec	130-042-401
Lysis buffer	BD	555899
MACS  MultiStand	Milteny biotec	130-042-303
Microplate for Tissue Culture (For Adhesion Cell) 6Well	IWAKI	3810-006
MidiMACS Separator	Milteny biotec	130-042-302
Mouse Pie Cages	Natsume Seisakusho	KN-331
Multipurpose refrigerated Centrifuge	TOMY	EX-125
NARCOBIT-E (II)	Natsume Seisakusho	KN-1071-I
NK-1.1-PerCP-Cy5.5 (PK136)	BioLegend	108728
Penicillin-Streptomycin Mixed Solution	nacalai	26253-84
Porcelain Mortar φ120mm with Pestle	Asone	6-549-03
Protein LoBind Tube 1.5 mL	Eppendorf	22431081
Sca-I-BUV395 (D7)	BD Biosciences	563990
Stainless steel scalpel blade	FastGene	FG-B2010
Streptavidin-BUV737	BD Biosciences	612775
SYTOX-red	Invitrogen	S34859
Tailveiner Restrainer for Mice standard	Braintree	TV-150 STD
TCRb-BV421 (H57-597)	BioLegend	109230
Ter-119-Alexa Fluor 700 (TER-119)	BioLegend	116220
Ter-119-Biotin (TER-119)	BioLegend	116204
Terumo 5ml Concentric Luer-Slip Syringe	TERUMO	SS-05LZ
Terumo Hypodermic Needle 23G x 1	TERUMO	NN-2325-R