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Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures
* These authors contributed equally
In This Article
Summary
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.
Abstract
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.
Introduction
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 ....
Protocol
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
- PVA stock solution
- Take 50 mL of tissue culture quality water in a small glass bottle (suitable for autoclaving). Warm.......
Representative Results
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.......
Discussion
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
Acknowledgements
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.
....Materials
| Name | Company | Catalog Number | Comments |
| 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 |
References
- Laurenti, E., Göttgens, B. From haematopoietic stem cells to complex differentiation landscapes. Nature. 553 (7689), 418-426 (2018).
- Eaves, C. J. Hematopoietic stem cells: concepts, definitions, and the new rea....
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