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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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-HSCs9,10. Therefore, the specific analysis of bonafide HSCs, LT-HSCs, has not yet been achieved. Accordingly, an isolation method for LT-HSCs using the Hoxb5 reporter system will significantly benefit the search for the molecular mechanisms of self-renewal capacity.
Here, we will discuss critical steps in this protocol. First, step 1 to step 7 need to be completed without interruption. These steps usually take 9-12 h, and it is important to keep the samples at 4 &#176;C throughout these procedures, as much as possible, in order to maintain sample viability. Next, approximately 1 x 108 bone marrow cells are harvested from a mouse. Thus, we need to use a sufficient volume of antibodies in order to reproduce the staining performance. In addition, the antibody for the CD34 antigen (clone; RAM34) requires 90 min for sufficient staining, while 30 min is enough for other antibodies. Second, irradiation usually causes pancytopenia in the recipient mice. If recipient-derived neutrophils persist in many recipient mice, this indicates that the radiation dose was insufficient. In such a case, optimization of the radiation dose is recommended. Third, if most of the mice die soon after the transplantation, there are two possible explanations: an inadequate number of supporting cells or unsuccessful retro-orbital injection.
For decades, it has been controversial whether the bonafide HSC fraction is homogeneous or heterogeneous22,23,24. In this study, the recipient mice that received the Hoxb5pos pHSC transplantation presented different donor chimeras and differentiation patterns (Figure 4A), indicating that this fraction could be heterogeneous. However, these fluctuations could be caused both by the use of unpurified bone marrow cells as the supporting cells and the different radio-sensitivities of individual mice25.
In summary, we have demonstrated a step-by-step protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. To date, the detection of LT-HSCs has depended on the competitive transplantation assay, which requires more than 8 months. In contrast, the Hoxb5 reporter system enables us to identify both LT-HSCs and ST-HSCs prospectively and use them for various functional analyses. Figure 4 and Figure 5 also show that the Hoxb5 expression level seems to be correlated with the degree of donor chimerism in the second recipient mice. Additionally, taking advantage of the Hoxb5 reporter system, we previously revealed that LT-HSCs and ST-HSCs work in a complementary fashion for continuous hematopoietic reconstitution after hematopoietic stem cell transplantation26. Moreover, we demonstrated that exogenous Hoxb5 expression could partially reverse the cell fate of ST-HSCs to that of LT-HSCs, indicating that the presence or absence of Hoxb5 explains the heterogeneity of self-renewal ability in the cell surface marker-defined HSC fraction27.
In addition to these findings, the prospective isolation of LT-HSCs allows us to analyze LT-HSCs under various physiological conditions, such as aging, inflammation, and so on. These analyses will greatly facilitate the understanding of the functions of LT-HSCs.

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

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 compare their self-renewal abilities, 10 Hoxb5pos or Hoxb5neg pHSCs isolated from Hoxb5 reporter mice were transplanted into lethally irradiated primary recipient mice with 2 x 105 whole bone marrow cells. Then, 16 weeks after the primary transplantation, 1 x 107 bone marrow cells isolated from the primary recipient mice were transplanted into lethally irradiated secondary recipient mice to assess the long-term self-renewal capacity (Figure 1). Figure 2 shows representative flow cytometry plots of the bone marrow analysis of the Hoxb5-tri-mCherry mice. Approximately 20%-25% of the cells in the pHSC fraction defined by Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722; were Hoxb5pos pHSCs, which account for only 0.001%-0.00125% of mouse bone marrow. Figure 3 displays representative flow cytometry plots of the peripheral blood analysis in the recipient mice. The CD45.2 donor mice (Hoxb5-tri-mCherry mice), CD45.1/CD45.2 supporting cells, and CD45.1 recipient mice were prepared, respectively, to separately analyze the donor, supporting, and recipient cells.
Figure 4&#160;shows peripheral blood analyses in the recipient mice at 4 weeks, 8 weeks, 12 weeks, and 16 weeks after transplantation to confirm donor chimerism. These analyses revealed that although Hoxb5pos and Hoxb5neg pHSCs present similar donor chimerism 4 weeks after transplantation, continuous hematopoiesis was observed only in the Hoxb5pos pHSC recipients (Figure 4A,B). On the other hand, Hoxb5neg HSCs started losing the ability to produce hematopoietic cells 8 weeks after transplantation (Figure 4A,B). In the secondary transplantation analysis, only the Hoxb5pos pHSC recipients presented robust hematopoiesis (Figure 5A,B). In contrast, donor cells were hardly observed in the Hoxb5neg pHSC recipient mice, suggesting that Hoxb5neg pHSCs lose their self-renewal ability within 16 weeks after transplantation in primary recipient mice. These data demonstrate that Hoxb5 expression can be used as a specific marker for LT-HSCs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64488/64488fig1v2.jpg" /> 
Figure 1: Experimental schematic for long-term hematopoietic reconstitution assays. The recipient mice were lethally irradiated and competitively transplanted with 10 HSCs and 2 x 105 whole bone marrow cells (supporting cells). For secondary transplants, 1 x 107 whole bone marrow cells were transferred from the primary recipient mice. Abbreviations: PB = peripheral blood; WBM = whole bone marrow. This figure has been modified from Chen et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64488/64488fig1largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64488/64488fig02.jpg" /> 
Figure 2: Gating strategy for sorting Hoxb5pos and Hoxb5neg pHSCs. Representative flow cytometry gating to isolate LKS, Flk2&#8722;, pHSC, Hoxb5pos, and Hoxb5neg pHSCs after the exclusion of doublets and dead cells. The values indicate the percentage of each fraction &#177; s.d. (n = 3). The lineages include B220, CD3&#949;, CD4, CD8a, Gr-1, and Ter-119. This figure has been modified from Chen et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64488/64488fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64488/64488fig3v2.jpg" /> 
Figure 3: Representative FACS plots of peripheral blood in a recipient mouse. Gating scheme to identify peripheral blood cells (NK cell, granulocyte, monocyte, T cell, and B cell) in a recipient mouse after the exclusion of doublets and dead cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64488/64488fig3largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64488/64488fig4v2.jpg" /> 
Figure 4: Chimerism in recipient mice after primary transplantation. (A)&#160;Percentage chimerism at 4 weeks, 8 weeks, 12 weeks, and 16 weeks in primary recipients receiving 10 Hoxb5neg (n = 9), Hoxb5lo (n = 13), or Hoxb5hi (n = 18) pHSCs. Each column represents an individual mouse. The Hoxb5hi fraction was defined as the top 5% of Hoxb5 expression and others as the Hoxb5lo fraction. (B) The average donor lineage contribution in 10 cell primary transplants. The error bars denote the s.d.&#160;This figure has been modified from Chen et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64488/64488fig4largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64488/64488fig5v2.jpg" /> 
Figure 5: Chimerism in the recipient mice after the secondary transplantation. (A)&#160;Percentage chimerism at 4 weeks, 8 weeks, 12 weeks, and 16 weeks following the whole bone marrow secondary transplantation. (B)&#160;Individual donor chimerism by lineage in whole bone marrow secondary recipients. Each line represents an individual mouse. This figure has been modified from Chen et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64488/64488fig5largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Clone</td>
			<td>Concentration</td>
			<td>Fluorochromes</td>
		</tr>
		<tr>
			<td>Flk-2</td>
			<td>A2-F10</td>
			<td>4 &#956;g/mL</td>
			<td>PerCP/eFlour710</td>
		</tr>
		<tr>
			<td>CD150</td>
			<td>TC15-12F12.2</td>
			<td>4 &#956;g/mL</td>
			<td>BV421</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>M1/70</td>
			<td>4 &#956;g/mL</td>
			<td>BV711</td>
		</tr>
		<tr>
			<td>Sca-1</td>
			<td>D7</td>
			<td>4 &#956;g/mL</td>
			<td>BUV395</td>
		</tr>
		<tr>
			<td>CD16/32</td>
			<td>93</td>
			<td>4 &#956;g/mL</td>
			<td>A-700</td>
		</tr>
		<tr>
			<td>CD127</td>
			<td>A7R34</td>
			<td>4 &#956;g/mL</td>
			<td>A-700</td>
		</tr>
		<tr>
			<td>CD3&#949;</td>
			<td>145-2C11</td>
			<td>10 &#956;g/mL</td>
			<td>Biotin</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>GK1.5</td>
			<td>10 &#956;g/mL</td>
			<td>Biotin</td>
		</tr>
		<tr>
			<td>CD8a</td>
			<td>53-6.7</td>
			<td>10 &#956;g/mL</td>
			<td>Biotin</td>
		</tr>
		<tr>
			<td>Gr-1</td>
			<td>RB6-8C5</td>
			<td>10 &#956;g/mL</td>
			<td>Biotin</td>
		</tr>
		<tr>
			<td>B220</td>
			<td>RA3-6B2</td>
			<td>10 &#956;g/mL</td>
			<td>Biotin</td>
		</tr>
		<tr>
			<td>Ter119</td>
			<td>TER119</td>
			<td>10 &#956;g/mL</td>
			<td>Biotin</td>
		</tr>
	</tbody>
</table>
Table 1: Antibody master mix for hematopoietic stem cell staining.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Clone</td>
			<td>Concentration</td>
			<td>Fluorochromes</td>
		</tr>
		<tr>
			<td>CD45.1</td>
			<td>A20</td>
			<td>1 &#956;g/mL</td>
			<td>FITC</td>
		</tr>
		<tr>
			<td>CD45.2</td>
			<td>104</td>
			<td>1 &#956;g/mL</td>
			<td>PE</td>
		</tr>
		<tr>
			<td>Gr-1</td>
			<td>RB6-8C5</td>
			<td>2.5 &#956;g/mL</td>
			<td>A700</td>
		</tr>
		<tr>
			<td>NK1.1</td>
			<td>PK136</td>
			<td>1 &#956;g/mL</td>
			<td>PerCP-Cyanine5.5</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>M1/70</td>
			<td>1 &#956;g/mL</td>
			<td>BUV395</td>
		</tr>
		<tr>
			<td>CD3&#949;</td>
			<td>145-2C11</td>
			<td>1 &#956;g/mL</td>
			<td>BV421</td>
		</tr>
		<tr>
			<td>TCR&#946;</td>
			<td>H57-597</td>
			<td>1 &#956;g/mL</td>
			<td>BV421</td>
		</tr>
		<tr>
			<td>B220</td>
			<td>RA3-6B2</td>
			<td>1 &#956;g/mL</td>
			<td>BV786</td>
		</tr>
	</tbody>
</table>
Table 2: Antibody master mix for peripheral blood cell staining.

Watch this Scientific Journal Video about Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells at JoVE.com

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

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

Nanyang Technological University

Research

JoVE Journal

Biology

1.7K Views.  Kobe University Graduate School of Medicine. De hematopoëtische stamcel- of HSC-isolatiemethode op lange en korte termijn zal onderzoekers helpen de celvernieuwingsmechanismen en biologische basis van heterogeniteit in het HSC-compartiment beter te begrijpen. Een transcriptiefactor, Hoxb5, die een exclusieve marker van HSC's op lange termijn kan zijn, werd geïdentificeerd. Op basis hiervan werd een Hoxb5-reporter muizenlijn opgezet en werden de lange termijn en de korte termijn HSC's met succes geïsoleerd.Om te beginnen met he...

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

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

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

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Immunology and Infection

High-Quality Nuclei from iPSC-Derived Hematopoietic Cells for Multiomics Studies

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Author Spotlight: Advancing In Vitro Blood Cell Production with Single-Cell Multiomics and Functional Genomics

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.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational ...

Developmental Biology

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

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

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

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.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

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.

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

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

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

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.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Bioengineering

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

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.

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Isolation and Magnetic Separation of Murine CD34+ Stem Cells from the Vascular Wall

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

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34+ Stem Cells Using Magnetic Bead Screening-Coupled Flow Cytometry

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.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

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

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