Name: ex vivo expansion and genetic manipulation of mouse hematopoietic stem cells in polyvinyl alcohol-based cultures
Uploaded: 2023-02-10T13:45:00
Description: 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

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...

<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>

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 ...

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Biology

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

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 ...

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

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 ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

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 ...

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 ...

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 ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

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

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) ...

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

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 ...

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

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 ...