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^4,5,6,7,8.

Immunophenotypically defined (Lineage⁻c-Kit⁺Sca-1⁺CD150⁺CD34^−/loFlk2⁻) HSCs (hereafter, pHSCs) were previously considered functionally homogeneous. However, recent single-cell analyses have revealed that pHSCs still exhibit heterogeneity with respect to their self-renewal capacity^9,10 and multipotency^11,12. Specifically, two populations seem to exist in the pHSC fraction with regard to their self-renewal capacity: long-term hematopoietic stem cells (LT-HSCs), which have continuous self-renewal capacity, and short-term hematopoietic stem cells (ST-HSCs), which have transient self-renewal capacity^9,10.

To date, the molecular mechanisms of self-renewal capacity that distinguish LT-HSCs and ST-HSCs remain poorly understood. It is crucial to isolate both cell populations based on their self-renewal capacities and to discover underlying molecular mechanisms. Several reporter systems have also been introduced to purify LT-HSCs^13,14,15; however, the LT-HSC purity defined by each reporter system is variable, and exclusive LT-HSC purification has not been achieved to date.

Therefore, developing an isolation system for LT-HSCs and ST-HSCs will accelerate research regarding self-renewal capacity in the pHSC fraction. In the isolation of LT-HSCs and ST-HSCs, a study using multi-step, unbiased screening identified a single gene, Hoxb5, that is heterogeneously expressed in the pHSC fraction¹⁶. Additionally, bone marrow analysis of the Hoxb5 reporter mice revealed that approximately 20%-25% of the pHSC fraction consists of Hoxb5^pos cells. A competitive transplantation assay using Hoxb5^pos pHSCs and Hoxb5^neg pHSCs revealed that only Hoxb5^pos pHSCs possess long-term self-renewal capacity, while Hoxb5^neg pHSCs lose their self-renewal capacity within a short period, indicating that Hoxb5 identifies LT-HSCs in the pHSC fraction¹⁶.

Here, we demonstrate a step-by-step protocol to isolate LT-HSCs and ST-HSCs using the Hoxb5 reporter system. In addition, we present a competitive transplantation assay to assess the self-renewal capacity of Hoxb5^pos/neg pHSCs (Figure 1). This Hoxb5 reporter system allows us to prospectively isolate LT-HSCs and ST-HSCs and contributes to the understanding of LT-HSC-specific characteristics.

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 susceptible to infection, keep the cages as clean as possible.
      NOTE: Supplementation with antibiotics starts 24 h prior to the irradiation and continues for 3 weeks after transplantation to avoid infections.
2. Total body irradiation
   1. Total body irradiation destroys the recipient bone marrow cells to ensure engraftment of the donor cells. Transfer the recipient mice to an irradiation cage. Lethally irradiate the recipient mice with a single dose of 8.7 Gy at 12-16 h before transplantation.
      NOTE: The radiation dose and time may vary depending on the equipment. The lethal radiation dose should be confirmed with the researcher's irradiator and mouse strain.
   2. Return them to their cages after the total body irradiation.

2. Collection of the donor bone marrow cells

1. Prepare a male Hoxb5-tri-mCherry mouse aged 12 weeks old, and euthanize the mouse by CO₂ exposure followed by cervical dislocation or using methods approved by the local animal ethics committee.
   NOTE: The reagent volume per mouse is described in the following steps.
2. Under sterile conditions, remove the skin, and expose the bones (femur, tibia, pelvis, humerus). Cut the major muscles, and take the bones (femur, tibia, pelvis, humerus) from the mouse. Place them in sterile cell culture dishes with Ca²⁺- and Mg²⁺-free, ice-cold PBS.
3. Trim the muscles and fibrous tissues from the bones using tweezers, small scissors, and wipes to prevent contamination. Be careful not to break the bones during this step. Discard any broken bones in order to maintain sterility.
4. Sterilize a mortar and pestle with 70% ethanol (EtOH), and let them dry completely. Equilibrate with cell-staining buffer (Ca²⁺- and Mg²⁺-free PBS supplemented with 2% heat-inactivated FBS, 2 mM EDTA, 100 U/mL penicillin, and 100 µg/mL streptomycin).
5. Put the bones in the mortar, and add 3 mL of cell-staining buffer. Crush the bones open with the pestle. Disaggregate the cell clumps by gentle pipetting, and transfer the cell suspension through a 100 µm cell strainer into a 50 mL tube.
6. Repeat step 2.5 until the solution becomes clear. Usually, three times is enough.

3. Separation of the c-kit⁺ cells by magnetic sorting

1. Antibody staining for magnetic sorting
   1. Centrifuge the samples at 400 x g and 4 °C for 5 min. Aspirate the supernatant, and resuspend the pellet in 1 mL of cell staining buffer. Add 10 µL of rat-IgG (5 mg/mL) to reduce non-specific antibody binding, and gently pipet up and down using a P1,000 pipette. Incubate on ice for 15 min.
   2. Add the c-Kit antibody (clone 2B8) at a concentration of 4 µg/mL, and mix with a P1,000 pipette. Incubate on ice for 15 min.
   3. Add 5 mL of cell staining buffer, and mix well. Centrifuge the samples at 400 x g, 4 °C, 5 min. Aspirate the supernatant, and resuspend the pellet in 500 µL of cell staining buffer.
   4. Add 35 µL of anti-APC microbeads to enrich the c-Kit⁺ cells, and mix with a P1,000 pipette. Incubate on ice for 15 min.
   5. Add 4-5 mL of cell-staining buffer. Centrifuge the samples at 400 x g and 4 °C for 5 min. Aspirate the supernatant, and resuspend the pellet in 1 mL of cell staining buffer.
2. Sorting the c-Kit⁺ cells magnetically
   1. Follow the manufacturer's instructions to sort the cells. In brief, prime a magnetic sorting column with 3 mL of cell staining buffer. Filter the sample (1 mL) through a 40 µm cell strainer and load the sample onto the magnetic sorting column.
   2. Wash by adding 3 mL of cell staining buffer three times. Add the cell staining buffer only when the column reservoir is empty.
   3. Put the magnetic sorting column on top of an ice-cold 15 mL tube and add 5 mL of cell staining buffer. Flush out the cells by firmly pushing the plunger into the column. Keep the flow-through on ice.
      ​NOTE: In case of accidental sample loss, we keep the flow-through until the end of the experiment.

4. Hematopoietic stem cell staining

1. Centrifuge the sample prepared in step 3.2.3 at 400 x g and 4 °C for 5 min. Aspirate the supernatant.
2. Add the CD34 antibody (clone RAM34) at a concentration of 50 µg/mL to the pellet, and incubate on ice for 60 min.
   NOTE: Preparation of the supporting cells during the first hour of incubation with CD34 is recommended to shorten the processing time.
3. Prepare the antibody master mix according to Table 1. Add 100 µL of the master mix to the sample, and incubate on ice for 30 min. The antibody for the CD34 antigen (clone; RAM34) requires 90 min for sufficient staining.
4. Add 4-5 mL of cell staining buffer. Centrifuge the sample at 400 x g and 4 °C for 5 min. Aspirate the supernatant. Add streptavidin-BUV737 at a concentration of 3 µg/mL to the pellet, and incubate on ice for 30 min.
5. Add 4-5 mL of cell staining buffer. Centrifuge the sample at 400 x g and 4 °C for 5 min. Aspirate the supernatant, and resuspend the pellet in 400 µL of cell staining buffer. Keep the sample on ice.

5. Supporting cell preparation

1. Prepare a CD45.1⁺ CD45.2⁺ congenic mouse aged 12 weeks old; ideally, this should be the same age as the donor mouse. Euthanize the mouse by CO₂ exposure followed by cervical dislocation or using methods approved by the local animal ethics committee.
   NOTE: In the provided example, CD45.1⁺ CD45.2⁺ congenic mice were bred in-house by crossing B6.CD45.1 congenic mice with C57BL/6J mice¹⁶.
2. Under sterile conditions, take both femurs and tibiae, and place them in sterile cell culture dishes with Ca²⁺- and Mg²⁺-free, ice-cold PBS. Trim the muscles and fibrous tissues from the bones using tweezers and small scissors.
3. Cut both ends of the bones with sharp, sterile scissors. Use a 23 G needle and a 5 mL syringe filled with ice-cold cell-suspension buffer (Ca²⁺- and Mg²⁺-free PBS supplemented with 2% heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin) to flush the bone marrow out into a sterile cell culture dish with cell suspension buffer. Disaggregate the cell clumps by gentle pipetting.
4. Filter the cell suspension through a 40 µm cell strainer using a P1,000 pipette. Count the cell number of the cell suspension using a hemocytometer and prepare a bone marrow cell suspension containing 1 x 10⁶ cells/mL.
5. Transfer 200 µL of the bone marrow cell suspension (2 x 10⁵ cells) to a 96-well, round-bottom plate. Keep on ice until use.
   NOTE: Round-bottom plates are recommended for easy cell collection.

6. Hoxb5^pos or Hoxb5^neg pHSC sorting

1. Gating setup
   1. Transfer 400 µL of the sample prepared in step 4.5 to a round-bottom polystyrene test tube with a 35 µm cell strainer snap cap. Prepare dead cell staining reagent, and add it to the sample before the analysis according to the manufacturer's instructions.
   2. Turn on a flowcytometer, and start the analysis software according to the manufacturer's instructions. Then, press Load, and acquire the data.
      NOTE: A flow cytometer equipped with five lasers and a 70 µm nozzle is recommended to enhance the purity of the sorted cells.
   3. After excluding doublets, dead cells, and lineage-positive cells, gate the Lineage⁻c-Kit⁺Sca-1⁺ fraction. Next, gate out the Flk2⁺ fraction. Then, gate Hoxb5^pos or Hoxb5^neg pHSCs in the CD150⁺CD34^−/low fraction (Figure 2). Perform compensation for the spectral overlap in the first experiment.
      NOTE: Hoxb5 positive cells are expected to represent 20%-25% of the Lineage⁻c-Kit⁺Sca-1⁺CD150⁺CD34^−/lowFlk2⁻ fraction.
2. Hoxb5^pos or Hoxb5^neg pHSC sorting
   1. Prepare a 1.5 mL low protein binding tube with 600 µL of Ca²⁺- and Mg²⁺ -free PBS supplemented with 10% heat-inactivated FBS.
   2. Set the 1.5 mL low protein binding tube on a sort collection device, and sort the Hoxb5^pos or Hoxb5^neg pHSCs into the 1.5 mL tube using the gating strategy set in step 6.1.3. In the first sorting, use the yield from the sorting precision mode.
   3. Set the 96-well plate with the supporting cells prepared in step 5.5 on the automated cell deposition unit (ACDU) stage. Set the 1.5 mL low protein binding tube prepared in step 6.2.2 to the loading port of a flow cytometer.
   4. Sort the Hoxb5^pos or Hoxb5^neg pHSCs into a 96-well plate with the supporting cells using the gating strategy set in step 6.1.3. Sort 10 Hoxb5^pos or Hoxb5^neg pHSCs to test their self-renewal capacities.
      NOTE: Double-sorting is recommended to enhance the purity. In the second sorting, use purity as the recommended sorting precision mode.
   5. Typically, 500-1,000 Hoxb5^pos pHSCs and 1,500-4,000 Hoxb5^neg pHSCs are harvested after double-sorting. Proceed with the transplantation procedures as soon as possible after the HSC sorting to enhance the cell viability (ideally within 1-2 h).

7. Transplantation

1. Place the HSC-sorted, 96-well plate prepared in step 6.2.4 onto ice. Handle the HSC-sorted, 96-well plate in sterile conditions, ideally in a cell hood.
2. Anesthetize a recipient mouse with 2% isoflurane in a gas anesthesia induction chamber. Once the animal is fully anesthetized, remove it, and place it on its side. To ensure sufficient anesthesia, confirm no movement in response to a noxious stimulus.
3. After the confirmation of proper anesthesia, perform a retro-orbital injection as soon as possible to prevent the mouse from regaining consciousness. The retro-orbital injection takes less than 30 s.
   ​NOTE: Since the injection time is short, we finish the procedure without applying ophthalmic ointment in the eyes. However, if the procedure takes longer, we recommend the use of ophthalmic ointment.
4. Gently pipette the cells in the HSC-sorted 96-well plate to mix them. Collect the donor cells in the sorting plate using a 30 G insulin syringe, and inject them into the retro-orbital venous plexus of the recipient mice. The recommended injectable volume is ≤200 µL.
5. Observe until the mice are conscious and moving about in a clean cage. Return them to their cages after confirming their recovery.

8. Peripheral blood analysis

1. Collect 50 µL of peripheral blood from the tail vein, and resuspend it with 100 µL of Ca²⁺- and Mg²⁺-free PBS with 2 mM EDTA. Transfer all the samples to a 96-well plate. Centrifuge at 400 x g and 4 °C for 5 min, and discard the supernatant.
2. Add 200 µL of red blood cell lysis buffer, and incubate on ice for 3 min. Centrifuge the samples at 400 x g and 4 °C for 5 min, and discard the supernatant. Repeat one more time.
3. Add 200 µL of cell staining buffer. Centrifuge the samples at 400 x g and 4 °C for 5 min, and discard the supernatant.
4. Prepare antibody master mix according to Table 2. Add 50 µL of antibody master mix, and incubate on ice for 30 min.
5. Add 150 µL of cell staining buffer. Centrifuge the samples at 400 x g and 4 °C for 5 min, and discard the supernatant.
6. Add 200 µL of cell staining buffer. Centrifuge the samples at 400 x g and 4 °C for 5 min, and discard the supernatant. Resuspend in 200 µL of cell staining buffer, and add dead cell staining reagent before the analysis according to the manufacturer's instructions.
7. Analyze peripheral blood chimerism using a flow cytometer as described previously¹⁶. Collect blood at 4 weeks, 8 weeks, 12 weeks, and 16 weeks after transplantation to follow multi-lineage reconstitution. Representative flow cytometry plots are provided in Figure 3.

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 transplantation^10,18. 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-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