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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 hierarchy1,2. 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, Lineagec-Kit+Sca-1+CD150+CD34/loFlk2, was reported to contain all HSCs in mice4,5....

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.......

Representative Results

Previously, self-renewal capacity has been measured using competitive transplantation assays, in which donor HSCs are thought to retain their self-renewal capacity only if multi-lineage donor cells in the recipient peripheral blood are observed17. In addition, several reports define LT-HSCs as cells that continue to produce peripheral blood cells several months after the second bone marrow transplantation10,18. Therefore, in order to compa.......

Discussion

Traditionally, cell surface marker-defined HSCs have been prepared to study the functions of HSCs, such as self-renewal capacity and multi-potency19,20,21. However, the immunophenotypically defined (Lineagec-Kit+Sca-1+CD150+CD34/loFlk2) HSC fraction contains two discrete HSC populations: LT-HSCs and ST-HSCs9<.......

Acknowledgements

We gratefully acknowledge Hiroshi Kiyonari for the animal care and for providing recipient mice at RIKEN BDR, as well as Hitomi Oga, Kayoko Nagasaka, and Masaki Miyahashi for laboratory management at Kobe University. The authors also greatly appreciate the ongoing support for this work. Masanori Miyanishi was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP17K07407 and JP20H03268, The Mochida Memorial Foundation for Medical and Pharmaceutical Research, The Life Science Foundation of Japan, The Takeda Science Foundation, The Astellas Foundation for Research on Metabolic Disorders, and AMED-PRIME, AMED under Grant Number JP18gm....

Materials

NameCompanyCatalog NumberComments
0.2 mL Strip of 8 Tubes, Dome CapSSIbio3230-00
0.5M EDTA pH 8.0IinvtrogenAM9260G
100 µm Cell StrainerFalcon352360
30G insulin syringeBD326668
40 µm Cell StrainerFalcon352340
5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap CapFALCON352235
7-AAD Viability Staining SolutionBioLegend420404
96 well U-BottomFALCON351177
Anti-APC-MicroBeadsMilteny biotec130-090-855
Aspirator with trap flaskBiosanFTA-1
B220-Alexa Fluor 700 (RA3-6B2)BioLegend103232
B220-Biotin (RA3-6B2)BioLegend103204
B220-BV786 (RA3-6B2)BD Biosciences563894
B6.CD45.1 congenic mice Sankyo Labo ServiceN/A
Baytril 10%BAYER341106546
BD FACS Aria II special order system BDN/A
Brilliant stain bufferBD566349
CD11b-Alexa Fluor 700 (M1/70)BioLegend101222
CD11b-Biotin (M1/70)BioLegend101204
CD11b-BUV395 (M1/70)BD Biosciences563553
CD11b-BV711 (M1/70)BD Biosciences563168
CD127-Alexa Fluor 700 (A7R34)Invitrogen56-1271-82
CD150-BV421 (TC15-12F12.2)BioLegend115943
CD16/CD32-Alexa Fluor 700 (93)Invitrogen56-0161-82
CD34-Alexa Fluor 647 (RAM34)BD Biosciences560230
CD34-FITC (RAM34)Invitrogen11034185
CD3-Alexa Fluor 700 (17A2)BioLegend100216
CD3ε -Biotin (145-2C11)BioLegend100304
CD3ε -BV421 (145-2C11)BioLegend100341
CD45.1/CD45.2 congenic miceN/AN/ABred in our Laboratory
CD45.1-FITC (A20)BD Biosciences553775
CD45.2-PE (104)BD Biosciences560695
CD4-Alexa Fluor 700 (GK1.5)BioLegend100430
CD4-Biotin (GK1.5)BioLegend100404
CD8a-Alexa Fluor 700 (53-6.7)BioLegend100730
CD8a-Biotin (53-6.7)BioLegend100704
Centrifuge Tube 15mlNICHIRYO00-ETS-CT-15
Centrifuge Tube 50mlNICHIRYO00-ETS-CT-50
c-Kit-APC-eFluor780 (2B8)Invitrogen47117182
D-PBS (-) without Ca and Mg, liquid Nacalai14249-24
Fetal Bovine SerumThermo Fisher10270106
Flk2-PerCP-eFluor710 (A2F10)eBioscience46135182
FlowJo version 10BD Biosciences https://www.flowjo.com/solutions/flowjo
Gmmacell 40 ExactorBest theratronicsN/A
Gr-1-Alexa Fluor 700 (RB6-8C5)BioLegend108422
Gr-1-Biotin (RB6-8C5)BioLegend108404
Hoxb5-tri-mCherry mice (C57BL/6J background) N/AN/ABred in our Laboratory
IgG from rat serum, technical grade, >=80% (SDS-PAGE), buffered aqueous solutionSigma-AldrichI8015-100MG
isofluranePfizer4987-114-13340-3 
Kimwipes S200NIPPON PAPER CRECIA 6-6689-01
LS ColumnsMilteny biotec130-042-401
Lysis buffer BD555899
MACS  MultiStandMilteny biotec130-042-303
Microplate for Tissue Culture (For Adhesion Cell) 6WellIWAKI3810-006
MidiMACS SeparatorMilteny biotec130-042-302
Mouse Pie CagesNatsume SeisakushoKN-331
Multipurpose refrigerated CentrifugeTOMYEX-125
NARCOBIT-E (II)Natsume SeisakushoKN-1071-I
NK-1.1-PerCP-Cy5.5 (PK136)BioLegend108728
Penicillin-Streptomycin Mixed Solutionnacalai26253-84
Porcelain Mortar φ120mm with PestleAsone6-549-03
Protein LoBind Tube 1.5 mL Eppendorf22431081
Sca-I-BUV395 (D7)BD Biosciences563990
Stainless steel scalpel bladeFastGeneFG-B2010
Streptavidin-BUV737BD Biosciences612775
SYTOX-redInvitrogenS34859
Tailveiner Restrainer for Mice standardBraintreeTV-150 STD
TCRb-BV421 (H57-597)BioLegend109230
Ter-119-Alexa Fluor 700 (TER-119)BioLegend116220
Ter-119-Biotin (TER-119)BioLegend116204
Terumo 5ml Concentric Luer-Slip SyringeTERUMOSS-05LZ
Terumo Hypodermic Needle 23G x 1TERUMONN-2325-R

References

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Hematopoietic Stem CellsHSCLong term HSCShort term HSCHoxb5Hoxb5 reporter MiceBone MarrowCell IsolationCell StainingFlow CytometryCell GatingLineage negativeC kitSca 1Flk2CD150CD34

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