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>

<ol>
	<li>Weissman, I. L., Shizuru, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+of+the+identification+and+isolation+of+hematopoietic+stem+cells,+and+their+capability+to+induce+donor-specific+transplantation+tolerance+and+treat+autoimmune+diseases.">The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases.</a> Blood. 112 (9), 3543-3553 (2008).</li><li>Majeti, R., Park, C. Y., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+hierarchy+of+multipotent+hematopoietic+progenitors+in+human+cord+blood.">Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood.</a> Cell Stem Cell. 1 (6), 635-645 (2007).</li><li>Spangrude, G. J., Heimfeld, S., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+mouse+hematopoietic+stem+cells.">Purification and characterization of mouse hematopoietic stem cells.</a> Science. 241 (4861), 58-62 (1988).</li><li>Ogawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+function+of+c-kit+in+hemopoietic+progenitor+cells.">Expression and function of c-kit in hemopoietic progenitor cells.</a> Journal of Experimental Medicine. 174 (1), 63-71 (1991).</li><li>Ikuta, K., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+hematopoietic+stem+cells+express+mouse+c-kit+but+do+not+depend+on+steel+factor+for+their+generation.">Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.</a> Proceedings of the National Academy of Sciences of the United States of America. 89 (4), 1502-1506 (1992).</li><li>Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+lymphohematopoietic+reconstitution+by+a+single+CD34-low/negative+hematopoietic+stem+cell.">Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.</a> Science. 273 (5272), 242-245 (1996).</li><li>Christensen, J. L., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flk-2+is+a+marker+in+hematopoietic+stem+cell+differentiation:+A+simple+method+to+isolate+long-term+stem+cells.">Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (25), 14541-14546 (2001).</li><li>Kiel, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121 (7), 1109-1121 (2005).</li><li>Morrison, S. J., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-term+repopulating+subset+of+hematopoietic+stem+cells+is+deterministic+and+isolatable+by+phenotype.">The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.</a> Immunity. 1 (8), 661-673 (1994).</li><li>Spangrude, G. J., Brooks, D. M., Tumas, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+repopulation+of+irradiated+mice+with+limiting+numbers+of+purified+hematopoietic+stem+cells:+In+vivo+expansion+of+stem+cell+phenotype+but+not+function.">Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: In vivo expansion of stem cell phenotype but not function.</a> Blood. 85 (4), 1006-1016 (1995).</li><li>Dykstra, B., Olthof, S., Schreuder, J., Ritsema, M., de Haan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+reveals+multiple+functional+defects+of+aged+murine+hematopoietic+stem+cells.">Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells.</a> Journal of Experimental Medicine. 208 (13), 2691-2703 (2011).</li><li>Grover, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+reveals+molecular+and+functional+platelet+bias+of+aged+haematopoietic+stem+cells.">Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells.</a> Nature Communications. 7, 11075 (2016).</li><li>Kataoka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evi1+is+essential+for+hematopoietic+stem+cell+self-renewal,+and+its+expression+marks+hematopoietic+cells+with+long-term+multilineage+repopulating+activity.">Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity.</a> Journal of Experimental Medicine. 208 (12), 2403-2416 (2011).</li><li>Gazit, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgd5+identifies+hematopoietic+stem+cells+in+the+murine+bone+marrow.">Fgd5 identifies hematopoietic stem cells in the murine bone marrow.</a> Journal of Experimental Medicine. 211 (7), 1315-1331 (2014).</li><li>Acar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+imaging+of+bone+marrow+shows+non-dividing+stem+cells+are+mainly+perisinusoidal.">Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal.</a> Nature. 526 (7571), 126-130 (2015).</li><li>Chen, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hoxb5+marks+long-term+haematopoietic+stem+cells+and+reveals+a+homogenous+perivascular+niche.">Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche.</a> Nature. 530 (7589), 223-227 (2016).</li><li>Ema, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+self-renewal+capacity+in+single+hematopoietic+stem+cells+from+normal+and+Lnk-deficient+mice.">Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice.</a> Developmental Cell. 8 (6), 907-914 (2005).</li><li>Morita, Y., Ema, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+and+hierarchy+within+the+most+primitive+hematopoietic+stem+cell+compartment.">Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.</a> Journal of Experimental Medicine. 207 (6), 1173-1182 (2010).</li><li>Yamamoto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+unveils+self-renewing+lineage-restricted+progenitors+generated+directly+from+hematopoietic+stem+cells.">Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.</a> Cell. 154 (5), 1112-1126 (2013).</li><li>Fathman, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+CD11A+on+hematopoietic+stem+cells+denotes+the+loss+of+long-term+reconstitution+potential.">Upregulation of CD11A on hematopoietic stem cells denotes the loss of long-term reconstitution potential.</a> Stem Cell Reports. 3 (5), 707-715 (2014).</li><li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Haas, S., Trumpp, A., Milsom, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+hematopoietic+stem+cell+heterogeneity.">Causes and consequences of hematopoietic stem cell heterogeneity.</a> Cell Stem Cell. 22 (5), 627-638 (2018).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+heterogeneity:+Subtypes,+not+unpredictable+behavior.">Hematopoietic stem cell heterogeneity: Subtypes, not unpredictable behavior.</a> Cell Stem Cell. 6 (3), 203-207 (2010).</li><li>Muller-Sieburg, C. E., Sieburg, H. B., Bernitz, J. M., Cattarossi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+heterogeneity:+Implications+for+aging+and+regenerative+medicine.">Stem cell heterogeneity: Implications for aging and regenerative medicine.</a> Blood. 119 (17), 3900-3907 (2012).</li><li>Duran-Struuck, R., Dysko, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+bone+marrow+transplantation+(BMT):+Providing+optimal+veterinary+and+husbandry+care+to+irradiated+mice+in+BMT+studies.">Principles of bone marrow transplantation (BMT): Providing optimal veterinary and husbandry care to irradiated mice in BMT studies.</a> Journal of the American Association for Laboratory Animal Science. 48 (1), 11-22 (2009).</li><li>Nishi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+minimum+requirements+for+successful+haematopoietic+stem+cell+transplantation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hoxb5+defines+the+heterogeneity+of+self-renewal+capacity+in+the+hematopoietic+stem+cell+compartment.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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 &#181;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&#160;</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&#160;</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&#949; -Biotin (145-2C11)</td>
			<td>BioLegend</td>
			<td>100304</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3&#949; -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&#160;</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&#160;</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>Hoxb5-tri-mCherry mice (C57BL/6J background)&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Bred in our Laboratory</td>
		</tr>
		<tr>
			<td>IgG from rat serum, technical grade, &#62;=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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes S200</td>
			<td>NIPPON PAPER CRECIA&#160;</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&#160;</td>
			<td>BD</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS&#160; 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 &#966;120mm with Pestle</td>
			<td>Asone</td>
			<td>6-549-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 1.5 mL&#160;</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 compa...

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

Research

JoVE Journal

Biology

1.5K Views.  Kobe University Graduate School of Medicine. 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.

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

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.

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

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 ...

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 ...

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 ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...