Name: Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells (Video) | JoVE Journal
Uploaded: 2023-05-19T13:00:00.000+00:00
Duration: 6 min 41 s
Description: Learn to isolate long-term and short-term hematopoietic stem cells using a Hoxb5 reporter system and flow cytometry. This method reveals self-renewal capacity differences and stem cell heterogeneity mechanisms.

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5 minutes

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 JP18gm6110020. Taro Sakamaki is supported by JSPS KAKENHI Grant Numbers JP21K20669 and JP22K16334 and was supported by the JSPS Core-to-Core Program and RIKEN Junior Research Associate Program. Katsuyuki Nishi was supported by JSPS Grant Number KAKENHI JP18J13408.

All the animal experiments described were approved by the RIKEN Center for Biosystems Dynamics Research.
1. Preconditioning of the recipient mice
<ol>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Total body irradiation
	<ol>
		<li>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&#39;s irradiator and mouse strain.</li>
		<li>Return them to their cages after the total body irradiation.</li>
	</ol>
	</li>
</ol>
2. Collection of the donor bone marrow cells
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;g/mL streptomycin).</li>
	<li>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 &#181;m cell strainer into a 50 mL tube.</li>
	<li>Repeat step 2.5 until the solution becomes clear. Usually, three times is enough.</li>
</ol>
3. Separation of the c-kit+ cells by magnetic sorting
<ol>
	<li>Antibody staining for magnetic sorting
	<ol>
		<li>Centrifuge the samples at 400 x g and 4 &#176;C for 5 min. Aspirate the supernatant, and resuspend the pellet in 1 mL of cell staining buffer. Add 10 &#181;L 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.</li>
		<li>Add the c-Kit antibody (clone 2B8) at a concentration of 4 &#181;g/mL, and mix with a P1,000 pipette. Incubate on ice for 15 min.</li>
		<li>Add 5 mL of cell staining buffer, and mix well. Centrifuge the samples at 400 x g, 4 &#176;C, 5 min. Aspirate the supernatant, and resuspend the pellet in 500 &#181;L of cell staining buffer.</li>
		<li>Add 35 &#181;L of anti-APC microbeads to enrich the c-Kit+ cells, and mix with a P1,000 pipette. Incubate on ice for 15 min.</li>
		<li>Add 4-5 mL of cell-staining buffer. Centrifuge the samples at 400 x g and 4 &#176;C for 5 min. Aspirate the supernatant, and resuspend the pellet in 1 mL of cell staining buffer.</li>
	</ol>
	</li>
	<li>Sorting the c-Kit+ cells magnetically
	<ol>
		<li>Follow the manufacturer&#39;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 &#181;m cell strainer and load the sample onto the magnetic sorting column.</li>
		<li>Wash by adding 3 mL of cell staining buffer three times. Add the cell staining buffer only when the column reservoir is empty.</li>
		<li>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. 
		&#8203;NOTE: In case of accidental sample loss, we keep the flow-through until the end of the experiment.</li>
	</ol>
	</li>
</ol>
4. Hematopoietic stem cell staining
<ol>
	<li>Centrifuge the sample prepared in step 3.2.3 at 400 x g and 4 &#176;C for 5 min. Aspirate the supernatant.</li>
	<li>Add the CD34 antibody (clone RAM34) at a concentration of 50 &#181;g/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.</li>
	<li>Prepare the antibody master mix according to Table 1. Add 100 &#181;L 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.</li>
	<li>Add 4-5 mL of cell staining buffer. Centrifuge the sample at 400 x g and 4 &#176;C for 5 min. Aspirate the supernatant. Add streptavidin-BUV737 at a concentration of 3 &#181;g/mL to the pellet, and incubate on ice for 30 min.</li>
	<li>Add 4-5 mL of cell staining buffer. Centrifuge the sample at 400 x g and 4 &#176;C for 5 min. Aspirate the supernatant, and resuspend the pellet in 400 &#181;L of cell staining buffer. Keep the sample on ice.</li>
</ol>
5. Supporting cell preparation
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;g/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.</li>
	<li>Filter the cell suspension through a 40 &#181;m 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.</li>
	<li>Transfer 200 &#181;L 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.</li>
</ol>
6. Hoxb5pos or Hoxb5neg pHSC sorting
<ol>
	<li>Gating setup
	<ol>
		<li>Transfer 400 &#181;L of the sample prepared in step 4.5 to a round-bottom polystyrene test tube with a 35 &#181;m cell strainer snap cap. Prepare dead cell staining reagent, and add it to the sample before the analysis according to the manufacturer&#39;s instructions.</li>
		<li>Turn on a flowcytometer, and start the analysis software according to the manufacturer&#39;s instructions. Then, press Load, and acquire the data. 
		NOTE: A flow cytometer equipped with five lasers and a 70 &#181;m nozzle is recommended to enhance the purity of the sorted cells.</li>
		<li>After excluding doublets, dead cells, and lineage-positive cells, gate the Lineage&#8722;c-Kit+Sca-1+ fraction. Next, gate out the Flk2+ fraction. Then, gate Hoxb5pos or Hoxb5neg pHSCs in the CD150+CD34&#8722;/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&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/lowFlk2&#8722; fraction.</li>
	</ol>
	</li>
	<li>Hoxb5pos or Hoxb5neg pHSC sorting
	<ol>
		<li>Prepare a 1.5 mL low protein binding tube with 600 &#181;L of Ca2+- and Mg2+ -free PBS supplemented with 10% heat-inactivated FBS.</li>
		<li>Set the 1.5 mL low protein binding tube on a sort collection device, and sort the Hoxb5pos or Hoxb5neg 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.</li>
		<li>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.</li>
		<li>Sort the Hoxb5pos or Hoxb5neg pHSCs into a 96-well plate with the supporting cells using the gating strategy set in step 6.1.3. Sort 10 Hoxb5pos or Hoxb5neg 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.</li>
		<li>Typically, 500-1,000 Hoxb5pos pHSCs and 1,500-4,000 Hoxb5neg 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).</li>
	</ol>
	</li>
</ol>
7. Transplantation
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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. 
	&#8203;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.</li>
	<li>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 &#8804;200 &#181;L.</li>
	<li>Observe until the mice are conscious and moving about in a clean cage. Return them to their cages after confirming their recovery.</li>
</ol>
8. Peripheral blood analysis
<ol>
	<li>Collect 50 &#181;L of peripheral blood from the tail vein, and resuspend it with 100 &#181;L 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 &#176;C for 5 min, and discard the supernatant.</li>
	<li>Add 200 &#181;L of red blood cell lysis buffer, and incubate on ice for 3 min. Centrifuge the samples at 400 x g and 4 &#176;C for 5 min, and discard the supernatant. Repeat one more time.</li>
	<li>Add 200 &#181;L of cell staining buffer. Centrifuge the samples at 400 x g and 4 &#176;C for 5 min, and discard the supernatant.</li>
	<li>Prepare antibody master mix according to Table 2. Add 50 &#181;L of antibody master mix, and incubate on ice for 30 min.</li>
	<li>Add 150 &#181;L of cell staining buffer. Centrifuge the samples at 400 x g and 4 &#176;C for 5 min, and discard the supernatant.</li>
	<li>Add 200 &#181;L of cell staining buffer. Centrifuge the samples at 400 x g and 4 &#176;C for 5 min, and discard the supernatant. Resuspend in 200 &#181;L of cell staining buffer, and add dead cell staining reagent before the analysis according to the manufacturer&#39;s instructions.</li>
	<li>Analyze peripheral blood chimerism using a flow cytometer as described previously16.&#160;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.</li>
</ol>

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<li>Nishi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+the+minimum+requirements+for+successful+haematopoietic+stem+cell+transplantation%5BTitle%5D)+AND+%22British+Journal+of+Haematology%22%5BJournal%5D)">Identification of the minimum requirements for successful haematopoietic stem cell transplantation.</a> British Journal of Haematology. 196 (3), 711-723 (2022).</li>
<li>Sakamaki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hoxb5+defines+the+heterogeneity+of+self-renewal+capacity+in+the+hematopoietic+stem+cell+compartment%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Hoxb5 defines the heterogeneity of self-renewal capacity in the hematopoietic stem cell compartment.</a> Biochemical and Biophysical Research Communications. 539, 34-41 (2021).</li>
</ol>

The authors declare no conflicts of interest associated with this study.

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 (Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722;) HSC fraction contains two discrete HSC populations: LT-HSCs and ST-HSCs9<...

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, Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722;, was reported to contain all HSCs in mice4,5,6,7,8.
Immunophenotypically defined (Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722;) 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 capacity9,10 and multipotency11,12. 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 capacity9,10.
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-HSCs13,14,15; 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 Hoxb5pos cells. A competitive transplantation assay using Hoxb5pos pHSCs and Hoxb5neg pHSCs revealed that only Hoxb5pos pHSCs possess long-term self-renewal capacity, while Hoxb5neg 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 Hoxb5pos/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.

<table><tbody><tr><td>0.2 mL Strip of 8 Tubes, Dome Cap</td><td>SSIbio</td><td>3230-00</td><td></td></tr><tr><td>0.5M EDTA pH 8.0</td><td>Iinvtrogen</td><td>AM9260G</td><td></td></tr><tr><td>100 &amp;micro;m Cell Strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>30G insulin syringe</td><td>BD</td><td>326668</td><td></td></tr><tr><td>40 &amp;micro;m Cell Strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td><td>FALCON</td><td>352235</td><td></td></tr><tr><td>7-AAD Viability Staining Solution</td><td>BioLegend</td><td>420404</td><td></td></tr><tr><td>96 well U-Bottom</td><td>FALCON</td><td>351177</td><td></td></tr><tr><td>Anti-APC-MicroBeads</td><td>Milteny biotec</td><td>130-090-855</td><td></td></tr><tr><td>Aspirator with trap flask</td><td>Biosan</td><td>FTA-1</td><td></td></tr><tr><td>B220-Alexa Fluor 700 (RA3-6B2)</td><td>BioLegend</td><td>103232</td><td></td></tr><tr><td>B220-Biotin (RA3-6B2)</td><td>BioLegend</td><td>103204</td><td></td></tr><tr><td>B220-BV786 (RA3-6B2)</td><td>BD Biosciences</td><td>563894</td><td></td></tr><tr><td>B6.CD45.1 congenic mice&amp;nbsp;</td><td>Sankyo Labo Service</td><td>N/A</td><td></td></tr><tr><td>Baytril 10%</td><td>BAYER</td><td>341106546</td><td></td></tr><tr><td>BD FACS Aria II special order system&amp;nbsp;</td><td>BD</td><td>N/A</td><td></td></tr><tr><td>Brilliant stain buffer</td><td>BD</td><td>566349</td><td></td></tr><tr><td>CD11b-Alexa Fluor 700 (M1/70)</td><td>BioLegend</td><td>101222</td><td></td></tr><tr><td>CD11b-Biotin (M1/70)</td><td>BioLegend</td><td>101204</td><td></td></tr><tr><td>CD11b-BUV395 (M1/70)</td><td>BD Biosciences</td><td>563553</td><td></td></tr><tr><td>CD11b-BV711 (M1/70)</td><td>BD Biosciences</td><td>563168</td><td></td></tr><tr><td>CD127-Alexa Fluor 700 (A7R34)</td><td>Invitrogen</td><td>56-1271-82</td><td></td></tr><tr><td>CD150-BV421 (TC15-12F12.2)</td><td>BioLegend</td><td>115943</td><td></td></tr><tr><td>CD16/CD32-Alexa Fluor 700 (93)</td><td>Invitrogen</td><td>56-0161-82</td><td></td></tr><tr><td>CD34-Alexa Fluor 647 (RAM34)</td><td>BD Biosciences</td><td>560230</td><td></td></tr><tr><td>CD34-FITC (RAM34)</td><td>Invitrogen</td><td>11034185</td><td></td></tr><tr><td>CD3-Alexa Fluor 700 (17A2)</td><td>BioLegend</td><td>100216</td><td></td></tr><tr><td>CD3&amp;epsilon; -Biotin (145-2C11)</td><td>BioLegend</td><td>100304</td><td></td></tr><tr><td>CD3&amp;epsilon; -BV421 (145-2C11)</td><td>BioLegend</td><td>100341</td><td></td></tr><tr><td>CD45.1/CD45.2 congenic mice</td><td>N/A</td><td>N/A</td><td>Bred in our Laboratory</td></tr><tr><td>CD45.1-FITC (A20)</td><td>BD Biosciences</td><td>553775</td><td></td></tr><tr><td>CD45.2-PE (104)</td><td>BD Biosciences</td><td>560695</td><td></td></tr><tr><td>CD4-Alexa Fluor 700 (GK1.5)</td><td>BioLegend</td><td>100430</td><td></td></tr><tr><td>CD4-Biotin (GK1.5)</td><td>BioLegend</td><td>100404</td><td></td></tr><tr><td>CD8a-Alexa Fluor 700 (53-6.7)</td><td>BioLegend</td><td>100730</td><td></td></tr><tr><td>CD8a-Biotin (53-6.7)</td><td>BioLegend</td><td>100704</td><td></td></tr><tr><td>Centrifuge Tube 15ml</td><td>NICHIRYO</td><td>00-ETS-CT-15</td><td></td></tr><tr><td>Centrifuge Tube 50ml</td><td>NICHIRYO</td><td>00-ETS-CT-50</td><td></td></tr><tr><td>c-Kit-APC-eFluor780 (2B8)</td><td>Invitrogen</td><td>47117182</td><td></td></tr><tr><td>D-PBS (-) without Ca and Mg, liquid&amp;nbsp;</td><td>Nacalai</td><td>14249-24</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher</td><td>10270106</td><td></td></tr><tr><td>Flk2-PerCP-eFluor710 (A2F10)</td><td>eBioscience</td><td>46135182</td><td></td></tr><tr><td>FlowJo version 10</td><td>BD Biosciences&amp;nbsp;</td><td>https://www.flowjo.com/solutions/flowjo</td><td></td></tr><tr><td>Gmmacell 40 Exactor</td><td>Best theratronics</td><td>N/A</td><td></td></tr><tr><td>Gr-1-Alexa Fluor 700 (RB6-8C5)</td><td>BioLegend</td><td>108422</td><td></td></tr><tr><td>Gr-1-Biotin (RB6-8C5)</td><td>BioLegend</td><td>108404</td><td></td></tr><tr><td>&lt;em&gt;Hoxb5&lt;/em&gt;-tri-mCherry mice (C57BL/6J background)&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>Bred in our Laboratory</td></tr><tr><td>IgG from rat serum, technical grade, &gt;=80% (SDS-PAGE), buffered aqueous solution</td><td>Sigma-Aldrich</td><td>I8015-100MG</td><td></td></tr><tr><td>isoflurane</td><td>Pfizer</td><td>4987-114-13340-3&amp;nbsp;</td><td></td></tr><tr><td>Kimwipes S200</td><td>NIPPON PAPER CRECIA&amp;nbsp;</td><td>6-6689-01</td><td></td></tr><tr><td>LS Columns</td><td>Milteny biotec</td><td>130-042-401</td><td></td></tr><tr><td>Lysis buffer&amp;nbsp;</td><td>BD</td><td>555899</td><td></td></tr><tr><td>MACS&amp;nbsp; MultiStand</td><td>Milteny biotec</td><td>130-042-303</td><td></td></tr><tr><td>Microplate for Tissue Culture (For Adhesion Cell) 6Well</td><td>IWAKI</td><td>3810-006</td><td></td></tr><tr><td>MidiMACS Separator</td><td>Milteny biotec</td><td>130-042-302</td><td></td></tr><tr><td>Mouse Pie Cages</td><td>Natsume Seisakusho</td><td>KN-331</td><td></td></tr><tr><td>Multipurpose refrigerated Centrifuge</td><td>TOMY</td><td>EX-125</td><td></td></tr><tr><td>NARCOBIT-E (II)</td><td>Natsume Seisakusho</td><td>KN-1071-I</td><td></td></tr><tr><td>NK-1.1-PerCP-Cy5.5 (PK136)</td><td>BioLegend</td><td>108728</td><td></td></tr><tr><td>Penicillin-Streptomycin Mixed Solution</td><td>nacalai</td><td>26253-84</td><td></td></tr><tr><td>Porcelain Mortar &amp;phi;120mm with Pestle</td><td>Asone</td><td>6-549-03</td><td></td></tr><tr><td>Protein LoBind Tube 1.5 mL&amp;nbsp;</td><td>Eppendorf</td><td>22431081</td><td></td></tr><tr><td>Sca-I-BUV395 (D7)</td><td>BD Biosciences</td><td>563990</td><td></td></tr><tr><td>Stainless steel scalpel blade</td><td>FastGene</td><td>FG-B2010</td><td></td></tr><tr><td>Streptavidin-BUV737</td><td>BD Biosciences</td><td>612775</td><td></td></tr><tr><td>SYTOX-red</td><td>Invitrogen</td><td>S34859</td><td></td></tr><tr><td>Tailveiner Restrainer for Mice standard</td><td>Braintree</td><td>TV-150 STD</td><td></td></tr><tr><td>TCRb-BV421 (H57-597)</td><td>BioLegend</td><td>109230</td><td></td></tr><tr><td>Ter-119-Alexa Fluor 700 (TER-119)</td><td>BioLegend</td><td>116220</td><td></td></tr><tr><td>Ter-119-Biotin (TER-119)</td><td>BioLegend</td><td>116204</td><td></td></tr><tr><td>Terumo 5ml Concentric Luer-Slip Syringe</td><td>TERUMO</td><td>SS-05LZ</td><td></td></tr><tr><td>Terumo Hypodermic Needle 23G x 1</td><td>TERUMO</td><td>NN-2325-R</td><td></td></tr></tbody></table>

isolation method for long-term and short-term hematopoietic stem cells

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

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),&#160;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.

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...

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.

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells ...

isolation-method-for-long-term-and-short-term-hematopoietic-stem-cells

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Biology

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Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

This video demonstrates a technique for generating human immune system (HIS) mice. CD34+ hematopoietic stem cells (HSCs) are isolated from human cord blood buffy coat cells using antibody-coated magnetic beads. The CD34+ HSCs are expanded in a growth medium enriched with cytokines and cryopreserved. Finally, the thawed HSCs are injected into an immunodeficient mouse pup to establish the HIS mouse model.

Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

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A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Stromal Cell Isolation From Hematopoietic Organs

We describe a protocol for extracting high-quality nuclei from cryopreserved induced pluripotent stem cell-derived stromal/endothelial and blood cell types to support single-nucleus next-generation sequencing analyses. Producing high-quality, intact nuclei is imperative for multiomics experiments but can be a barrier to entry in the field for some laboratories.

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Learn to isolate long-term and short-term hematopoietic stem cells using a Hoxb5 reporter system and flow cytometry. This method reveals self-renewal capacity differences and stem cell heterogeneity mechanisms.

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

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