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.
Self-renewing multipotent 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.
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 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.
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
2. HSC bone marrow extraction and c-Kit+ enrichment
3. Initiating cell cultures with c-Kit-enriched HSPCs
4. Initiating cell cultures with FACS purified HSCs
5. Performing media changes
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.
7. Transducing cultured HSPCs with lentiviral vector
NOTE: Initiate cultures with sufficient numbers of cells, to perform this at the desired experimental time point.
8. Flow cytometric analysis of HSPC cultures
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– fraction to be ~10% (Figure 2). These results are similar for cell cultures initiated from c-Kit enriched bone marrow or FACS purified HSCs. Following transduction with a GFP-expressing lentiviral vector (at 20 transduction units/cell), we expect ~30% GFP+ cells (Figure 3).
When confluent, we expect the cultures to be at ~2 million cells/mL. Within the culture, we expect to see mainly small round cells, although it is normal to see a small frequency of larger round (megakaryocyte-like) cells. Within the cell cultures initiated from c-Kit-enriched bone marrow, some initial cell death is expected14. Approximately 50% cell death is expected within the first 24-48 h, before the first medium changes are performed. Initial dead cell debris in these cultures likely comes from cell death within the cultures rather than bone debris (from the crushed bones), since the c-Kit-enrichment step should deplete such bone debris. Cell numbers, however, return to 80%-100% of the seeded cell numbers after 1 week (see Table 5 for expected cell density values). If poor results are seen, we recommend troubleshooting the protocol (Table 6). In this case, it can be most simple to batch-test reagents using the c-Kit-enriched bone marrow protocol (Section 3), as this avoids complications associated with FACS-sorting. Note that the representative results are based on the use of reagents and equipment detailed in the Table of Materials; similar results may be achieved using reagents from different vendors, however, validation (and titration) of new reagents is likely to be necessary.
Figure 1: Gating strategy for FACS purification of HSCs from c-Kit-enriched bone marrow. Sequential gating used to identify CD150+CD34–c-Kit+Sca1+Lineage– cells from c-Kit-enriched bone marrow. Abbreviations: FACS = fluorescence-activated cell sorting; HSCs = hematopoietic stem cells; SSC-A = side scatter-peak area; FSC-A = forward scatter-peak area; FSC-W = forward scatter-peak width; FSC-H = forward scatter-peak height; SSC-H = side scatter-peak height; PI = propidium iodide; PE = phycoerythrin; APC = allophycocyanin; FITC = fluorescein isothiocyanate. Please click here to view a larger version of this figure.
Figure 2: Gating strategy for flow cytometric analysis of cultured HSPCs. Sequential gating used to identify CD201+CD150+c-Kit+Sca1+Lineage– cells from HSPC cultures. Abbreviations: HSPCs = hematopoietic stem and progenitor cells; SSC-A = side scatter-peak area; FSC-A = forward scatter-peak area; FSC-W = forward scatter-peak width; FSC-H = forward scatter-peak height; SSC-H = side scatter-peak height; PI = propidium iodide; PE = phycoerythrin; APC = allophycocyanin. Please click here to view a larger version of this figure.
Figure 3: Representative results from lentiviral vector transduction. Representative GFP expression following cultured HSPC transduction with a GFP-expressing lentiviral vector (20 transduction units/cell) at 48 h after transduction. Abbreviations: GFP = green fluorescent protein; HSPCs = hematopoietic stem and progenitor cells; FSC-H = forward scatter-peak height. Please click here to view a larger version of this figure.
Reagent | Final concentration in F-12 | Volume (μL) |
Ham’s F-12 medium | N/A | 958 |
100x Penicillin-Streptomycin-Glutamine | 1x | 10 |
1 M HEPES | 10 mM | 10 |
100 mg/mL PVA stock | 1 mg/mL | 10 |
100x ITSX | 1x | 10 |
100 μg/mL TPO stock | 100 ng/mL | 1 |
10 μg/mL SCF stock | 10 ng/mL | 1 |
Table 1: HSC media composition. Media reagent volumes for 1 mL of complete medium. Abbreviations: PVA = polyvinyl alcohol; SCF = stem cell factor; TPO = thrombopoietin; ITSX = insulin-transferrin-selenium-ethanolamine.
Antibody | Concentration (µg/mL) | Volume (µL) |
Ly6G/Ly6C – biotin | 0.5 | 100 |
Ter119 – biotin | 0.5 | 100 |
CD4 – biotin | 0.5 | 25 |
CD8a – biotin | 0.5 | 25 |
CD45R – biotin | 0.5 | 50 |
CD127 – biotin | 0.5 | 50 |
Sterile PBS | N/A | 350 |
Table 2: Biotinylated antibody cocktail. Antibody volumes for the stock of the biotin antibody cocktail.
Antibody | Concentration (mg/mL) | Volume (µL) |
CD34 FITC | 0.5 | 4 |
c-Kit APC | 0.2 | 1 |
Sca1 PE | 0.2 | 1 |
Streptavidin APC/Cy7 | 0.2 | 1 |
CD150 PE/Cy7 | 0.2 | 1 |
CD48 BV421 | 0.2 | 1 |
Sterile PBS | N/A | 291 |
Table 3: Fresh HSC antibody cocktail. Antibody volumes for the fresh HSC antibody cocktail. Abbreviations: PE = phycoerythrin; APC = allophycocyanin; FITC = fluorescein isothiocyanate.
Antibody | Concentration (mg/mL) | Volume (µL) |
c-Kit BV421 | 0.2 | 12.5 |
Sca1 PE | 0.2 | 12.5 |
CD150 PE/Cy7 | 0.2 | 12.5 |
CD4 APC/Cy7 | 0.2 | 5 |
CD8 APC/Cy7 | 0.2 | 5 |
Ter119 APC/Cy7 | 0.2 | 5 |
CD127 APC/Cy7 | 0.2 | 5 |
CD45R APC/Cy7 | 0.2 | 5 |
Ly6C/Ly6G APC/Cy7 | 0.2 | 5 |
CD201 APC | 0.2 | 5 |
Sterile PBS | N/A | 27.5 |
Table 4: Cultured HSC antibody cocktail (100x). Antibody volumes for the 100x stock of the cultured HSC antibody cocktail. Abbreviations: PE = phycoerythrin; APC = allophycocyanin; FITC = fluorescein isothiocyanate.
Culture | Cell density at 24-48 h | % KSL at 24-48 h | Cell density at 1 week | % KSL at 1 week |
Fresh c-Kit+ bone marrow plating | 0.5×106 mL-1 | 25-35% | 1-2×106 mL-1 | 50-60% |
HSPC electroporation | 0.5×106 mL-1 | 25-35% | 0.5-1.5×106 mL-1 | 25-35% |
HSPC lentivirus | 1.5×106 mL-1 | 20-25% | 1-2×106 mL-1 | 45-55% |
Table 5: Expected results. Expected cell densities and frequencies of c-Kit+Sca1+Lineage– cells following (1) seeding of c-Kit+ bone marrow, (2) electroporation, and (3) lentiviral vector transduction based on seeding at 1 × 106 cells mL-1. Abbreviations: KSL = c-Kit+Sca1+Lineage–; HSPC = hematopoietic stem and progenitor cell.
Issue | Likely cause/s | Solution/s | |||
Flow rate of LS column is unusually slow. | LS column is clogged. | Remove the column from the magnet. Elute the cell suspension using the plunger and repeat with another 5 mL of PBS. Repeat the column enrichment with a fresh column and fresh filter. | |||
Large amount of cell death within the first 7 days of culture | Using expired reagents or reagents in incorrect ratios. | Ensure regents are at proper concentration and stored correctly. | |||
Inaccurate incubator temperature. | Regular servicing of incubators and ensuring that the incubator is kept closed as much as possible. | ||||
Culture collapse after 7 days of culture | Wrong cell type sorted | Seek advice from HSC FACS experts on sorting protocols. Validate reagents with c-Kit-enriched bone marrow. | |||
Incomplete media changes being performed | Perform more complete media changes. | ||||
Excessive (e.g., >50%) overall cell death after transduction | Lentiviral vector toxicity. | Reduce amount of virus particles used. | |||
Shorten incubation times to 5 h. | |||||
Excessive (e.g., >50%) overall cell death after transfection | Cells left in P3 solution too long | Recover cells in media as soon as possible, electroporate only 2 samples at a time and be gentle when pipetting (and use wide-bore pipette tips). | |||
Pipetting too vigorously kill cells | |||||
Low transfection efficiency | Degradation of RNPs and/or Cas9 enzyme. | Incorrect storage, store Cas9 at -20 °C, and RNA at -80 °C (reconstituted with RNase-free water). | |||
Low transduction efficiency | Low lentiviral vector activity | Titrate lentiviral vector for specific cell population, avoid freeze/thawing lentiviral vector |
Table 6: Common troubleshooting issues. Summary of common issues and suggested troubleshooting.
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 demonstrated14,15. The in vivo validation of these HSC and c-Kit+ HSPC cultures can be found in these publications, while readers can refer to other published protocols for in vivo transplantation protocols21. We do not see major differences in lineage chimerism between fresh and cultured HSPCs following transplantation, however, the reconstitution potency of individual HSCs after ex vivo expansion is yet to be determined in detail. The large numbers of HSCs generated by this approach opens up new ways to interrogate HSCs using molecular or biochemical assays that require large cell numbers. Additionally, being able to generate genetically modified HSCs within these culture systems should allow us to further probe the mechanisms regulating HSC activity and the hematopoietic system. For example, these systems are amenable to performing genetic screens16.
Mechanistically, we do not yet fully understand why PVA and other polymers can replace serum albumin and support efficient HSC expansion; we believe PVA at least partially replaces serum albumin through stabilizing cytokines within the media9. Additionally, the lack of poorly defined bioactive contaminants found in serum albumin products appears to reduce HSC differentiation. The use of synthetic polymers should also help to reduce batch-to-batch variability and confounding effects associated with these bioactive contaminants when studying HSCs ex vivo22. There is also still more to be learnt regarding why certain polymers provide better support for HSCs ex vivo.
While already powerful, further optimization and characterization of these culture protocols should be possible. In particular, it would be useful to improve the purity of HSCs within these cultures. Additional markers distinguishing the long-term HSC compartment from these cultures would also help track and isolate these cell types. It is also of interest to see whether this system can be eventually used to quantify HSC activity ex vivo, without the need for in vivo transplantation assays. We also do not yet understand for how long HSCs can be expanded ex vivo, although it is certainly longer than 6-8 weeks if maintained properly8. Finally, while these cultures provide a useful model to study mouse HSCs, it is also important to develop equivalent culture systems for human HSCs to provide a more tractable system to study human HSC biology and hematopoiesis, and eventually, to generate HSCs for clinical stem cell transplantation therapies.
The authors have nothing to disclose.
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.
Equipment | |||
Dissection kit | Fisher Scientific | 12764416 | |
Hemocytometer | Appleton Woods Ltd | HC002 | |
P3 Primary Cell 4D-Nucleofector X Kit | Lonza | V4XP-3024 | |
Pestle and mortar | Scientific Laboratory Supplies Limited | X18000 | |
QuadroMACS separator | Miltenyi Biotec | 130-090-976 | |
Materials | |||
5 mL syringe | VWR International Ltd | 720-2519 | |
19 G needle | VWR International Ltd | 613-5394 | |
50 μm cell strainer | Sysmex | 04-004-2317 | |
70 μm cell strainer | Corning | 431751 | |
Kimtech wipes | VWR International Ltd | 115-2075 | |
LS MACS column | Miltenyi Biotec | 130-042-401 | |
Reagents | |||
Alt-R S.p. Cas9 Nuclease V3, 100 μg | IDT | 1081058 | |
Animal free recombinant mouse stem cell factor | Peprotech | AF-250-03 | |
Animal free recombinant mouse thrombopoietin | Peprotech | AF-315-14 | |
Anti-mouse CD117 APC (clone: 2B8) | ThermoFisher | 17-1171-83 | |
Anti-mouse CD117 BV421 (clone: 2B8) | Biolegend | 105828 | |
Anti-mouse CD127 APC/Cy7 (clone: A7R34) | Biolegend | 135040 | |
Anti-mouse CD127 biotin (clone: A7R34) | Biolegend | 135006 | |
Anti-mouse CD150 PE/Cy7 (clone: TC15-12F12.2) | Biolegend | 115914 | |
Anti-mouse CD201 APC (clone: eBio1560) | ThermoFisher | 17-2012-82 | |
Anti-mouse CD34 FITC (clone: RAM34) | ThermoFisher | 11-0341-85 | |
Anti-mouse CD4 APC/Cy7 (clone: RM4-5) | Biolegend | 100526 | |
Anti-mouse CD4 biotin (clone: RM4-5) | Biolegend | 100508 | |
Anti-mouse CD45R APC/Cy7 (clone: RA3-6B2) | Biolegend | 103224 | |
Anti-mouse CD45R biotin (clone: RA3-6B2) | Biolegend | 103204 | |
Anti-mouse CD48 BV421 (clone: HM48-1) | Biolegend | 103428 | |
Anti-mouse CD8 biotin (clone: 53-6.7) | Biolegend | 100704 | |
Anti-mouse CD8a APC/Cy7 (clone: 53-6.7) | Biolegend | 100714 | |
Anti-mouse Ly6G/Ly6C APC/Cy7 (clone: RB6-8C5) | Biolegend | 108424 | |
Anti-mouse Ly6G/Ly6C biotin (clone: RB6-8C5) | Biolegend | 108404 | |
Anti-mouse Sca1 PE (clone: D7) | Biolegend | 108108 | |
Anti-mouse Ter119 APC/Cy7 (clone: TER-119) | Biolegend | 116223 | |
Anti-mouse Ter119 biotin (clone: TER-119) | Biolegend | 116204 | |
CellBIND plates, 24-well | Corning | 3337 | negative surface charged |
CellBIND plates, 96-well | Corning | 3330 | negative surface charged |
Custom synthetic sgRNA | Synthego, Sigma Aldrich, IDT | Custom order | |
Fetal bovine serum | Merck Life Science UK Limited | F7524-50ML | |
Fibronectin Coated plates, 96-well | BD Biosciences | 354409 | |
Ham's F-12 Nutrient Mix | Gibco | 11765054 | |
Insulin-Transferrin-Selenium-X (100x) | Gibco | 51500.056 | |
Phosphate buffered saline | Alfa Aesar | J61196.AP | |
Polyvinyl alcohol | Sigma Aldrich | P8136 | |
Propidium Iodide | Enzo Life Sciences (UK) Ltd | EXB-0018 | |
Streptavidin APC/Cy7 | Biolegend | 405208 | |
Türks’ solution | Sigma Aldrich | 109277 | |
Virkon | Mettler-Toledo Ltd | 95015662 |