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>

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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><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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&amp;nbsp;</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>&lt;strong&gt;Materials&lt;/strong&gt;</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 &amp;mu;m cell strainer</td><td>Sysmex</td><td>04-004-2317</td><td></td></tr><tr><td>70 &amp;mu;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>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alt-R S.p. Cas9 Nuclease V3, 100 &amp;mu;g</td><td>IDT&amp;nbsp;</td><td>1081058</td><td></td></tr><tr><td>Animal free recombinant mouse stem cell factor&amp;nbsp;</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&amp;nbsp;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&amp;nbsp;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&amp;nbsp;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&amp;nbsp;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&amp;nbsp;APC/Cy7 (clone: 53-6.7)</td><td>Biolegend</td><td>100714</td><td></td></tr><tr><td>Anti-mouse Ly6G/Ly6C&amp;nbsp;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&amp;nbsp;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&amp;nbsp;</td><td>Corning</td><td>3330</td><td>negative surface charged</td></tr><tr><td>Custom synthetic sgRNA&amp;nbsp;</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&#039;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&amp;uuml;rks&amp;rsquo; 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...

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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 ...

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Research

JoVE Journal

Biology

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

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 ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Developmental Biology

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

Genetics

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Bioengineering

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Immunology and Infection

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Medicine

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

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

Summary

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Chapters in this video

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34⁺ Cells Using Valproic Acid

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications