We thank the WIMM Flow Cytometry Core for flow cytometry access, and the WIMM Virus Screening Core for lentiviral vector generation. This work was funded by the Kay Kendall Leukaemia Fund and the UK Medical Research Council.

All animal procedures, including breeding and euthanasia, must be performed within institutional and national guidelines. The experiments detailed below were approved by the UK Home Office. See the Table of Materials for a list of all materials, reagents, and equipment used in this protocol.
1. Preparing stock solutions
<ol>
	<li>PVA stock solution
	<ol>
		<li>Take 50 mL of tissue culture quality water in a small glass bottle (suitable for autoclaving). Warm the water to near boiling in a microwave.</li>
		<li>Weigh out 5 g of PVA powder and add to the water. 
		NOTE: It is recommended to only dissolve 1-5 g of PVA. Dissolving larger amounts may result in incomplete reconstitution.</li>
		<li>Close the lid tightly and shake to mix. Then, loosen the lid. 
		NOTE: Be careful when re-opening, as the pressure may change due to the changing water temperature.</li>
		<li>Autoclave and allow to cool. Close the lid tightly and shake to mix. Make aliquots in sterile tubes and store for up to 3 months at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Dissolve lyophilized cytokines in F-12 medium containing 1 mg/mL PVA. Reconstitute the stem cell factor to 10 &#181;g/mL and thrombopoietin to 100 &#181;g/mL to generate 1:1,000 stocks. Make aliquots in sterile tubes and store long-term at -80 &#176;C. Alternatively, store the aliquots at 4 &#176;C for up to 1 week. 
	NOTE: Avoid the reconstitution of cytokines in bovine serum albumin due to the negative impact on mouse HSC expansion.</li>
</ol>
2. HSC bone marrow extraction and c-Kit+ enrichment
<ol>
	<li>Dissect the femurs, tibias, pelvises, and spine from freshly euthanized 8-12-week-old C57BL/6 mice (via CO2 asphyxiation and/or cervical dislocation) and place the bones in PBS on ice. 
	NOTE: Ensure the surfaces and tools are sterilized with 70% ethanol.</li>
	<li>Clean the bones using lint-free delicate task wipes, removing muscle and spinal cord, and add to a mortar with ~3 mL of PBS. 
	NOTE: Ensure the pestle and mortar are sterilized with 70% ethanol and then washed out once with PBS.</li>
	<li>Crush the bones with the pestle without grinding to minimize shearing forces. Break up large bone marrow fragments released into the PBS using a 19 G needle attached to a 5 mL syringe. Transfer the cell suspension through a 70 &#181;m filter into a 50 mL conical tube.</li>
	<li>Once the suspension has been transferred, repeat with fresh PBS until the bones are bleached and no marrow is visible. Aim for an end volume of ~30 mL per mouse and ~50 mL for two mice.</li>
	<li>Mix the bone marrow cells, collect 10 &#181;L of cells, and count using T&#252;rks&#39; solution at a dilution of 1:10-1:20 with a hemocytometer. 
	NOTE: One mouse is expected to yield 2-5 &#215; 108 whole bone marrow cells.</li>
	<li>Spin at 450 &#215; g for 5 min at 4 &#176;C, discard the supernatant, and resuspend the pellet in cold PBS (350 &#181;L for one mouse or 500 &#181;L for two mice).</li>
	<li>Add 0.2 &#181;L of allophycocyanin (APC) anti-c-Kit antibody per 10 million cells and incubate for 30 min in the dark at 4 &#176;C.</li>
	<li>Add 5 mL of cold PBS to the incubated cells to wash off excess antibody and filter through a 50 &#181;m filter into a fresh 15 mL conical tube.</li>
	<li>Wash the original tube with 7 mL of cold PBS and transfer through the filter. If filter clogging occurs, scratch the filter surface with a P1000 tip.</li>
	<li>Spin at 450 &#215; g for 5 min at 4 &#176;C, discard the supernatant, and resuspend the pellet in cold PBS (350 &#181;L for one mouse or 500 &#181;L for two mice).</li>
	<li>Add 0.2 &#181;L of anti-APC microbeads per 10 million cells and incubate for 15 min in the dark at 4 &#176;C. Add 12 mL of sterile PBS to wash off excess microbeads.</li>
	<li>Spin down the cells at 450 &#215; g for 5 min at 4 &#176;C and resuspend in 2 mL of cold PBS. While the cells are spinning in this step, proceed to step 2.13.</li>
	<li>Prepare for column enrichment by placing a magnetic filtration column into the magnet of a magnetic column separator, with a 50 &#181;m filter on top and a 15 mL conical tube below.</li>
	<li>Run 3 mL of sterile PBS through the 50 &#181;m filter and filtration column. Once the PBS has run through, pass the cell suspension through the column, followed by three washes of 3 mL of cold PBS each time. For each wash, wait for the column to stop dripping before adding the next wash.</li>
	<li>Remove the column from the magnet and place it on top of a fresh 15 mL tube. Add 5 mL of cold PBS, fit the column plunger onto the column, and elute the cells by pushing the plunger.</li>
	<li>Mix the c-Kit-enriched cells, collect 10 &#181;L of cells, and count using T&#252;rks&#39; solution at a dilution of 1:2 with a hemocytometer. The typical yield from one mouse is 2-5 &#215; 106 c-Kit+ cells. 
	NOTE: At this point, the cells can be directly seeded into HSC media or prepared for HSC fluorescence-activated cell sorting (FACS) purification.</li>
</ol>
3. Initiating cell cultures with c-Kit-enriched HSPCs
<ol>
	<li>Prepare fresh medium (Table 1) for the number of cells/wells required. Seed 0.5-1 million cells per mL for c-Kit-enriched HSPCs.</li>
	<li>Spin down the c-Kit-enriched cells and resuspend in HSC medium at the desired cell density.</li>
	<li>Transfer the cells to fibronectin-coated or negative surface charged plates, at 200 &#181;L per 96-well plate or 1 mL per 24-well plate.</li>
	<li>Place the cells in a tissue culture incubator set to 37 &#176;C and 5% CO2.</li>
</ol>
4. Initiating cell cultures with FACS purified HSCs
<ol>
	<li>Prepare an appropriate volume of the biotinylated lineage antibody stain: 3 &#181;L of master mix (Table 2) per 10 million cells, diluted 1:100 in PBS.</li>
	<li>Spin down the c-Kit-enriched cells and resuspend in the lineage antibody stain for 30 min at 4 &#176;C.</li>
	<li>Wash with 10 mL of sterile PBS and spin down at 450 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Prepare an appropriate volume of the fresh HSC antibody stain (Table 3): 300 &#181;L per 10 million cells. Alongside this sample staining, prepare appropriate staining control samples for compensation and gating. 
	NOTE: Work in a tissue culture hood with the light off when using dye-conjugated antibodies.</li>
	<li>Resuspend the cells in the HSC antibody stain and incubate at 4 &#176;C for 90 min. Mix the cells every 20-30 min by tapping to prevent cell pelleting.</li>
	<li>Wash with 10 mL of sterile PBS and spin down at 450 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant, flick the pellet to disrupt, and resuspend in sterile PBS with 0.5 &#181;g/mL propidium iodide (PI).</li>
	<li>Prepare fresh medium (Table 1) for the number of wells required, and plate into fibronectin or negative surface charged plates (200 &#181;L per 96-well plate or 1 mL per 24-well plate).</li>
	<li>Prepare the FACS machine for sorting and sort CD150+CD34-c-Kit+Sca1+Lineage- HSCs directly into media-containing wells (see Figure 1 for the standard FACS gating strategy used here). Sort up to 200 cells per 96-well plate well or up to 1,000 cells per 24-well plate well. 
	NOTE: FACS machines should be operated by a trained scientist. It is recommended that users contact their local FACS facility to discuss this sorting strategy if they are not experienced in FACS isolation of mouse HSCs.</li>
</ol>
5. Performing media changes
<ol>
	<li>For cell cultures initiated from c-Kit-enriched cells, begin media changes after 2 days. For cell cultures initiated from FACS-isolated HSCs, begin media changes after 5 days.</li>
	<li>Prepare sufficient fresh prewarmed (~37 &#176;C) HSC medium (Table 1) for all wells.</li>
	<li>Gently remove the plate from the tissue culture incubator. 
	NOTE: As HSPCs on negative surface charged plates are more easily disturbed than those on fibronectin, extra care should be taken when changing the medium on negative surface charged plates to avoid disturbing the cell cultures.</li>
	<li>Using a pipette or vacuum pump, slowly remove ~90%-95% of the medium from the well meniscus. 
	NOTE: Avoid drawing up the medium from the base of the well, otherwise many cells will be removed.</li>
	<li>Add 200 &#181;L (for 96-well plates; 1 mL for 24-well plates) of fresh medium to the well.</li>
	<li>Return the plate to the tissue culture incubator.</li>
	<li>Repeat steps 5.1-5.6 every 2-3 days until the experimental end point.</li>
	<li>For cell cultures initiated with c-Kit-enriched HSPCs (section 3), split the cultures at a ratio of 1:2-1:3 after ~3 weeks. For cell cultures initiated with FACS purified HSCs (section 4), split the cultures at a ratio of 1:2-1:3 after ~3 weeks and when the cultures are &#62;90% confluent. 
	&#8203;NOTE: The exact timeline will depend on the experimental interests. These cultures have been characterized for 4-8 weeks8, but additional culture lengths may be possible.</li>
</ol>
6. Electroporating cultured HSPCs
NOTE: This protocol is for electroporation of Cas9/sgRNA ribonucleoprotein (RNP), but could be adapted for electroporation of mRNA or other recombinant proteins. Initiate cultures with sufficient numbers of cells in order to perform this at the desired experimental time point.
<ol>
	<li>Perform a medium change 1 day before electroporation, as described in section 5. 
	NOTE: A minimum of an overnight culture before electroporation is recommended. However, cells are typically cultured for 1-3 weeks before transduction.</li>
	<li>On the day of electroporation, set up the nucleofector. Turn on the machine. On the touchscreen, select the X module and then the cuvette size used.</li>
	<li>Prepare sufficient P3 solution (according to the manufacturer&#39;s instructions) for the scale of the electroporation (100 &#181;L per cuvette or 20 &#181;L per microcuvette) and allow to equilibrate to room temperature.</li>
	<li>Prepare sufficient fresh medium (Table 1) for the number of wells being plated and add 500 &#181;L of medium for a 24-well plate well or 100 &#181;L of media to a 96-well plate well.</li>
	<li>Thaw out sgRNA (prediluted to 2 &#181;g/mL in RNase-free water) on ice and mix 16 &#181;g of sgRNA with 30 &#181;g of Cas9 enzyme (at 10 &#181;g/mL) in a sterile PCR tube. Include an extra PCR tube containing only Cas9 protein as a control. Mix by flicking the tube, then briefly spin down. Incubate at 25 &#176;C for 10 min in a thermocycler to complex the RNP, and then keep the tubes on ice. 
	NOTE: This can be scaled down fivefold if performing electroporation in microcuvettes.</li>
	<li>Mix and transfer the HSPCs for electroporation into a tube; collect 10 &#181;L of the cells and count using T&#252;rks&#39; solution at a dilution of 1:2 with a hemocytometer. 
	NOTE: It is recommended to electroporate 1-5 million cells per 100 &#181;L cuvette (scaled down fivefold for the microcuvette).</li>
	<li>Spin down the appropriate number cells in a 1.5 mL tube at 450 &#215; g for 5 min at 4 &#176;C. Aspirate as much of the supernatant as possible and resuspend with 100 &#181;L of nucleofection buffer.</li>
	<li>Transfer the cell suspension immediately into the PCR tube containing the complexed RNP and pipette up and down slowly to gently mix and transfer to a 100 &#181;L electroporation cuvette. Eject the mixture into the cuvette slowly and in one fluid motion to avoid the formation of air bubbles in the cuvette.</li>
	<li>On the electroporator, select the position of the wells being electroporated. Using the touchscreen, select the cell type program CD34+, human, or type in the pulse code EO100. Press the OK button.</li>
	<li>Transfer the cuvettes to the electroporator. Press the Start button on the touchscreen to initiate electroporation. Directly after electroporation, add 500 &#181;L of the culture medium to the cuvette (100 &#181;L if using a microcuvette).</li>
	<li>Gently transfer the cells to the prepared plate and return to the tissue culture incubator.</li>
	<li>For procedures employing an AAV6 donor template, add the vector immediately after the cells have been transferred to a fibronectin-coated plate at a concentration of 5,000 vectors/cell.</li>
	<li>After 6-18 h, prepare fresh medium and perform a medium change as described in section 5. For this medium change, remove only 80%-90% of the medium. 
	NOTE: Avoid drawing up the medium from the base of the well.</li>
	<li>Analyze the cells by flow cytometry after 2 or more days (see Section 8 for details).</li>
	<li>Continue to perform medium changes every 2 days, as described in section 5, until the experimental end point is reached. 
	&#8203;NOTE: Editing rates of CRISPR/Cas9 using this method rely highly on the targeting efficiency of the designed sgRNA. Editing rates up to 95% have been observed with an efficient guide15. Guidelines for sgRNA design have been previously detailed elsewhere19,20.</li>
</ol>
7. Transducing cultured HSPCs with lentiviral vector
NOTE: Initiate cultures with sufficient numbers of cells, to perform this at the desired experimental time point.
<ol>
	<li>Generate and titer lentiviral vector (depending on the experimental goals). Thaw lentiviral vector on ice.</li>
	<li>Perform a medium change (as in section 5); then, mix and transfer the HSPCs for transduction into a tube. Collect 10 &#181;L of the cells and count using T&#252;rks&#39; solution at a dilution of 1:2 with a hemocytometer. 
	NOTE: Cells can be transduced immediately following plating. However, cells are typically cultured for 1-3 weeks before transduction.</li>
	<li>Replate the required dose of cells for transduction (typically 100,000 cells per 96-well plate). Separately, plate un-transduced negative control cells. 
	NOTE: If using negative surface charged plates, transfer the cells to fibronectin-coated plates for lentiviral vector transduction.</li>
	<li>Add lentiviral vector to each well of cells: add 20 transduction units per cell to achieve ~30% transduction efficiency. However, determine the lentiviral vector dose empirically, depending on the experimental requirements. 
	NOTE: Ensure lentiviral vector is disposed of according to institutional guidelines.</li>
	<li>Return the cells to the tissue culture incubator for 6 h. After, perform a medium change as described in section 5. 
	NOTE: The supernatant contains live virus. Dispose of according to institutional guidelines.</li>
	<li>Analyze the cells by flow cytometry (e.g., for GFP expression) after 2 days or more. See section 8 for details.</li>
	<li>Continue to perform medium changes every 2 days, as described in section 5, until the experimental end point is reached.</li>
</ol>
8. Flow cytometric analysis of HSPC cultures
<ol>
	<li>Prepare a concentrated cultured ex vivo HSC antibody mix in PBS containing 2% FBS (Table 4). Store the mixture at 4 &#176;C in the dark for up to 1 month. 
	NOTE: Work in a tissue culture hood with the lights off when using dye-conjugated antibodies.</li>
	<li>Add 2 &#181;L of concentrated antibody mix to 50 &#181;L of cells. Incubate for 30 min at 4 &#176;C in the dark. Alongside this sample staining, prepare appropriate staining control samples for compensation and gating.</li>
	<li>Add 200-1,000 &#181;L of PBS containing 2% FBS and centrifuge at 450 &#215; g for 5 min at 4 &#176;C. Remove as much supernatant as possible and resuspend in 100-500 &#181;L of PBS containing 2% FBS and 0.5 &#181;g/mL PI.</li>
	<li>Set up the flow cytometer and record at least 10,000 live cells per samples. 
	NOTE: Flow cytometers should be operated by a trained scientist. Users must contact their local FACS facility to discuss this analysis if they are not experienced in flow cytometry.</li>
	<li>Export the data in FCS format and analyze the data using appropriate flow cytometry analysis software. See Figure 2 for the standard gating strategy used here.</li>
</ol>

<ol>
	<li>Laurenti, E., G&#246;ttgens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+haematopoietic+stem+cells+to+complex+differentiation+landscapes.">From haematopoietic stem cells to complex differentiation landscapes.</a> Nature. 553 (7689), 418-426 (2018).</li><li>Eaves, C. J. <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+cells:+concepts,+definitions,+and+the+new+reality.">Hematopoietic stem cells: concepts, definitions, and the new reality.</a> Blood. 125 (17), 2605-2613 (2015).</li><li>Wilkinson, A. C., Igarashi, K. J., 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=Haematopoietic+stem+cell+self-renewal+in+vivo+and+ex+vivo.">Haematopoietic stem cell self-renewal in vivo and ex vivo.</a> Nature Reviews Genetics. 21 (9), 541-554 (2020).</li><li>Crane, G. M., Jeffery, E., 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=Adult+haematopoietic+stem+cell+niches.">Adult haematopoietic stem cell niches.</a> Nature Reviews Immunology. 17 (9), 573-590 (2017).</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>Granot, N., Storb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+hematopoietic+cell+transplantation:+challenges+and+progress.">History of hematopoietic cell transplantation: challenges and progress.</a> Haematologica. 105 (12), 2716-2729 (2020).</li><li>Wilkinson, A. C., 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=Stabilizing+hematopoietic+stem+cells+in+vitro.">Stabilizing hematopoietic stem cells in vitro.</a> Current Opinion in Genetics &amp; Development. 64, 1-5 (2020).</li><li>Wilkinson, A. C., 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=Long-term+ex+vivo+haematopoietic-stem-cell+expansion+allows+nonconditioned+transplantation.">Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.</a> Nature. 571 (7763), 117-121 (2019).</li><li>Nishimura, 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=Use+of+polyvinyl+alcohol+for+chimeric+antigen+receptor+T-cell+expansion.">Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion.</a> Experimental Hematology. 80, 16-20 (2019).</li><li>Becker, H. 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=A+single+cell+cloning+platform+for+gene+edited+functional+murine+hematopoietic+stem+cells.">A single cell cloning platform for gene edited functional murine hematopoietic stem cells.</a> bioRxiv. , (2022).</li><li>Kobayashi, 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=Environmental+optimization+enables+maintenance+of+quiescent+hematopoietic+stem+cells+ex+vivo.">Environmental optimization enables maintenance of quiescent hematopoietic stem cells ex vivo.</a> Cell Reports. 28 (1), 145-158 (2019).</li><li>Kobayashi, H., Takubo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+method+to+maintain+quiescent+human+hematopoietic+stem+cells.">A culture method to maintain quiescent human hematopoietic stem cells.</a> Journal of Visualized Experiments. (171), e61938 (2021).</li><li>Aal Wilson, ., 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=Hematopoietic+stem+cells+reversibly+switch+from+dormancy+to+self-renewal+during+homeostasis+and+repair.">Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.</a> Cell. 135 (6), 1118-1129 (2008).</li><li>Ochi, K., Morita, M., Wilkinson, A. C., Iwama, A., Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-conditioned+bone+marrow+chimeric+mouse+generation+using+culture-based+enrichment+of+hematopoietic+stem+and+progenitor+cells.">Non-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells.</a> Nature Communications. 12 (1), 3568 (2021).</li><li>Wilkinson, A. C., 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=Cas9-AAV6+gene+correction+of+beta-globin+in+autologous+HSCs+improves+sickle+cell+disease+erythropoiesis+in+mice.">Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice.</a> Nature Communications. 12 (1), 686 (2021).</li><li>Haney, M. S., 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=Large-scale+in+vivo+CRISPR+screens+identify+SAGA+complex+members+as+a+key+regulators+of+HSC+lineage+commitment+and+aging.">Large-scale in vivo CRISPR screens identify SAGA complex members as a key regulators of HSC lineage commitment and aging.</a> bioRxiv. , (2022).</li><li>Che, J. L. C., 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+and+characterization+of+in+vitro+expanded+hematopoietic+stem+cells.">Identification and characterization of in vitro expanded hematopoietic stem cells.</a> EMBO Reports. 23 (10), e55502 (2022).</li><li>Zhang, Q., Konturek-Ciesla, A., Yuan, O., Bryder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+potential+of+murine+hematopoietic+stem+cells:+a+rare+property+only+partially+predicted+by+phenotype.">Ex vivo expansion potential of murine hematopoietic stem cells: a rare property only partially predicted by phenotype.</a> bioRxiv. , (2022).</li><li>Schindele, P., Wolter, F., Puchta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+guide+RNA+design+guidelines+for+efficient+genome+editing.">CRISPR guide RNA design guidelines for efficient genome editing.</a> Methods in Molecular Biology. 2166, 331-342 (2020).</li><li>Hanna, R. E., Doench, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+analysis+of+CRISPR-Cas+experiments.">Design and analysis of CRISPR-Cas experiments.</a> Nat Biotechnol. 38 (7), 813-823 (2020).</li><li>Wilkinson, A. C., Ishida, R., Nakauchi, H., Yamazaki, S. <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+ex+vivo+expansion+of+mouse+hematopoietic+stem+cells.">Long-term ex vivo expansion of mouse hematopoietic stem cells.</a> Nature Protocols. 15 (2), 628-648 (2020).</li><li>Ieyasu, 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=An+all-recombinant+protein-based+culture+system+specifically+identifies+hematopoietic+stem+cell+maintenance+factors.">An all-recombinant protein-based culture system specifically identifies hematopoietic stem cell maintenance factors.</a> Stem Cell Reports. 8 (3), 500-508 (2017).</li></ol>

The authors have no conflict of interests.

We hope that this protocol provides a useful approach to investigate HSC biology, hematopoiesis, and hematology more generally. Since the initial development of the PVA-based culture method for FACS-purified HSCs8, the method has been extended. For example, the method has been shown to work with c-Kit enriched with bone marrow and with negative surface charged plates14. Its compatibility with transduction and electroporation has also been demonstrated14</s...

The hematopoietic system supports a range of essential processes in mammals, from oxygen supply to fighting pathogens, through specialized blood and immune cell types. Continuous blood production (hematopoiesis) is required to support blood system homeostasis, which is sustained by hematopoietic stem and progenitor cells (HSPCs)1. The most primitive hematopoietic cell is the hematopoietic stem cell (HSC), which has unique capacities for self-renewal and multilineage differentiation2,3. This is a rare cell population, mainly found in the adult bone marrow4, where they occur at a frequency of just approximately one every 30,000 cells. HSCs are thought to support life-long hematopoiesis and help to re-establish hematopoiesis following hematological stress. These capacities also allow HSCs to stably reconstitute the entire hematopoietic system following transplantation into an irradiated recipient5. This represents the functional definition of an HSC and also forms the scientific basis for HSC transplantation therapy, a curative treatment for a range of blood and immune diseases6. For these reasons, HSCs are a major focus of experimental hematology.
Despite a large focus of research, it has remained challenging to stably expand HSCs ex vivo7. We recently developed the first long-term ex vivo expansion culture system for mouse HSCs8. The approach can expand transplantable HSCs by 234-899-fold over a 4 week culture. In comparison to alternative approaches, the major change in the protocol was the removal of serum albumin and its replacement with a synthetic polymer. Polyvinyl alcohol (PVA) was identified as an optimal polymer for the mouse HSC cultures8, which has now also been used to culture other hematopoietic cell types9. However, another polymer called Soluplus (a polyvinyl caprolactam-acetate-polyethylene glycol graft copolymer) has also recently been identified, which appears to improve clonal HSC expansion10. Prior to the use of polymers, serum albumin in the form of fetal bovine serum, bovine serum albumin fraction V, or recombinant serum albumin were used, but these had limited support for HSC expansion and only supported short-term (~1 week) ex vivo culture7. However, it should be noted that HSC culture protocols that retain HSCs in a quiescent state can support a longer ex vivo culture time11,12.
In comparison with other culture methods, a major advantage of PVA-based cultures is the number of cells that can be generated and the length of time the protocol can be used to track HSCs ex vivo. This overcomes several barriers in the field of experimental hematology, such as the low numbers of HSCs isolatable per mouse (only a few thousand) and the difficulty to track HSCs over time in vivo. However, it is important to remember that these cultures stimulate HSC proliferation, while the in vivo HSC pool is predominantly quiescent at a steady state13. Additionally, although the cultures are selective for HSCs, additional cell types do accumulate with the cultures over time, and transplantable HSCs only represent approximately one in 34 cells after 1 month. Myeloid hematopoietic progenitor cells appear to be the major contaminating cell type in these HSC cultures8. Nevertheless, we can use these cultures to enrich for HSCs from heterogeneous cell populations (e.g., c-Kit+ bone marrow HSPCs14). It also supports transduction or electroporation of HSCs for genetic manipulation14,15,16. To help identify HSCs from the heterogeneous cultured HSPC population, CD201 (EPCR) has recently been identified as a useful ex vivo HSC marker10,17,18, with transplantable&#160;HSCs restricted to the CD201+CD150+c-Kit+Sca1+Lineage- fraction.
This protocol describes methods to initiate, maintain, and assess PVA-based mouse HSC expansion cultures, as well as protocols for genetic manipulation within these cultures using electroporation or lentiviral vector transduction. These methods are expected to be useful for a range of experimental hematologists.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection kit</td>
			<td>Fisher Scientific</td>
			<td>12764416</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Appleton Woods Ltd</td>
			<td>HC002</td>
			<td></td>
		</tr>
		<tr>
			<td>P3 Primary Cell 4D-Nucleofector X Kit</td>
			<td>Lonza&#160;</td>
			<td>V4XP-3024</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle and mortar</td>
			<td>Scientific Laboratory Supplies Limited</td>
			<td>X18000</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>VWR International Ltd</td>
			<td>720-2519</td>
			<td></td>
		</tr>
		<tr>
			<td>19 G needle</td>
			<td>VWR International Ltd</td>
			<td>613-5394</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#956;m cell strainer</td>
			<td>Sysmex</td>
			<td>04-004-2317</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Corning</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech wipes</td>
			<td>VWR International Ltd</td>
			<td>115-2075</td>
			<td></td>
		</tr>
		<tr>
			<td>LS MACS column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease V3, 100 &#956;g</td>
			<td>IDT&#160;</td>
			<td>1081058</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal free recombinant mouse stem cell factor&#160;</td>
			<td>Peprotech</td>
			<td>AF-250-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal free recombinant mouse thrombopoietin</td>
			<td>Peprotech</td>
			<td>AF-315-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD117 APC (clone: 2B8)</td>
			<td>ThermoFisher</td>
			<td>17-1171-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD117 BV421 (clone: 2B8)</td>
			<td>Biolegend</td>
			<td>105828</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD127&#160;APC/Cy7 (clone: A7R34)</td>
			<td>Biolegend</td>
			<td>135040</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD127 biotin (clone: A7R34)</td>
			<td>Biolegend</td>
			<td>135006</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD150 PE/Cy7 (clone: TC15-12F12.2)</td>
			<td>Biolegend</td>
			<td>115914</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD201&#160;APC (clone: eBio1560)</td>
			<td>ThermoFisher</td>
			<td>17-2012-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD34 FITC (clone: RAM34)</td>
			<td>ThermoFisher</td>
			<td>11-0341-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD4&#160;APC/Cy7 (clone: RM4-5)</td>
			<td>Biolegend</td>
			<td>100526</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD4 biotin (clone: RM4-5)</td>
			<td>Biolegend</td>
			<td>100508</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45R&#160;APC/Cy7 (clone: RA3-6B2)</td>
			<td>Biolegend</td>
			<td>103224</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45R biotin (clone: RA3-6B2)</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD48 BV421 (clone: HM48-1)</td>
			<td>Biolegend</td>
			<td>103428</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD8 biotin (clone: 53-6.7)</td>
			<td>Biolegend</td>
			<td>100704</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a&#160;APC/Cy7 (clone: 53-6.7)</td>
			<td>Biolegend</td>
			<td>100714</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ly6G/Ly6C&#160;APC/Cy7 (clone: RB6-8C5)</td>
			<td>Biolegend</td>
			<td>108424</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ly6G/Ly6C biotin (clone: RB6-8C5)</td>
			<td>Biolegend</td>
			<td>108404</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Sca1 PE (clone: D7)</td>
			<td>Biolegend</td>
			<td>108108</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119&#160;APC/Cy7 (clone: TER-119)</td>
			<td>Biolegend</td>
			<td>116223</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119 biotin (clone: TER-119)</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>CellBIND plates, 24-well</td>
			<td>Corning</td>
			<td>3337</td>
			<td>negative surface charged</td>
		</tr>
		<tr>
			<td>CellBIND plates, 96-well&#160;</td>
			<td>Corning</td>
			<td>3330</td>
			<td>negative surface charged</td>
		</tr>
		<tr>
			<td>Custom synthetic sgRNA&#160;</td>
			<td>Synthego, Sigma Aldrich, IDT</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Merck Life Science UK Limited</td>
			<td>F7524-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Coated plates, 96-well</td>
			<td>BD Biosciences</td>
			<td>354409</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix</td>
			<td>Gibco</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-X (100x)</td>
			<td>Gibco</td>
			<td>51500.056</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Alfa Aesar</td>
			<td>J61196.AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl alcohol</td>
			<td>Sigma Aldrich</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Enzo Life Sciences (UK) Ltd</td>
			<td>EXB-0018</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin APC/Cy7</td>
			<td>Biolegend</td>
			<td>405208</td>
			<td></td>
		</tr>
		<tr>
			<td>T&#252;rks&#8217; solution</td>
			<td>Sigma Aldrich</td>
			<td>109277</td>
			<td></td>
		</tr>
		<tr>
			<td>Virkon</td>
			<td>Mettler-Toledo Ltd</td>
			<td>95015662</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo expansion and genetic manipulation of mouse hematopoietic stem cells in polyvinyl alcohol-based cultures

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout life and reconstitute the entire blood system following transplantation. HSCs are used clinically in stem cell transplantation therapies, which represent curative treatment for a range of blood diseases. There is substantial interest in both understanding the mechanisms that regulate HSC activity and hematopoiesis, and developing new HSC-based therapies. However, the stable culture and expansion of HSCs ex vivo has been a major barrier in studying these stem cells in a tractable ex vivo system. We recently developed a polyvinyl alcohol-based culture system that can support the long-term and large-scale expansion of transplantable mouse HSCs and methods to genetically edit them. This protocol describes methods to culture and genetically manipulate mouse HSCs via electroporation and lentiviral transduction. This protocol is expected to be useful to a wide range of experimental hematologists interested in HSC biology and hematopoiesis.

For the FACS purification of HSCs, we expect that within the c-Kit-enriched bone marrow, ~0.2% of the cells are the CD150+CD34-c-Kit+Sca1+Lineage- population for young (8-12-week-old) C57BL/6 mice (Figure 1). However, it is likely that transgenic mice or mice of different ages display differing HSC frequencies. After 4 weeks of culture, we expect the CD201+CD150+c-Kit+Sca1+Lineage- f...

Watch this Scientific Journal Video about Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures at JoVE.com

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Presented here is a protocol to initiate, maintain, and analyze mouse hematopoietic stem cell cultures using ex vivo polyvinyl alcohol-based expansion, as well as methods to genetically manipulate them by lentiviral transduction and electroporation.

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout ...

ex-vivo-expansion-genetic-manipulation-mouse-hematopoietic-stem-cells

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2.5K Views.  University of Oxford. Presented here is a protocol to initiate, maintain, and analyze mouse hematopoietic stem cell cultures using ex vivo polyvinyl alcohol-based expansion, as well as methods to genetically manipulate them by lentiviral transduction and electroporation.

Video: Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

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 ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...