Name: isolation method for long-term and short-term hematopoietic stem cells
Uploaded: 2023-05-19T13:00:00
Description: Watch this Scientific Journal Video about Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells at JoVE.com

We gratefully acknowledge Hiroshi Kiyonari for the animal care and for providing recipient mice at RIKEN BDR, as well as Hitomi Oga, Kayoko Nagasaka, and Masaki Miyahashi for laboratory management at Kobe University. The authors also greatly appreciate the ongoing support for this work. Masanori Miyanishi was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP17K07407 and JP20H03268, The Mochida Memorial Foundation for Medical and Pharmaceutical Research, The Life Science Foundation of Japan, The Takeda Science Foundation, The Astellas Foundation for Research on Metabolic Disorders, and AMED-PRIME, AMED under Grant Number JP18gm6110020. Taro Sakamaki is supported by JSPS KAKENHI Grant Numbers JP21K20669 and JP22K16334 and was supported by the JSPS Core-to-Core Program and RIKEN Junior Research Associate Program. Katsuyuki Nishi was supported by JSPS Grant Number KAKENHI JP18J13408.

All the animal experiments described were approved by the RIKEN Center for Biosystems Dynamics Research.
1. Preconditioning of the recipient mice
<ol>
	<li>Prepare male C57BL/6 congenic mice aged 8-10 weeks old as recipient mice. The number of recipient mice depends on the experimental protocol. We typically prepare 10-20 mice for each condition.
	<ol>
		<li>Feed the mice with sterilized water supplemented with enrofloxacin (170 mg/L). As irradiated recipient mice are highly...

<ol>
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W., 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=Upregulation+of+CD11A+on+hematopoietic+stem+cells+denotes+the+loss+of+long-term+reconstitution+potential.">Upregulation of CD11A on hematopoietic stem cells denotes the loss of long-term reconstitution potential.</a> Stem Cell Reports. 3 (5), 707-715 (2014).</li><li>Oguro, H., Ding, L., 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=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Haas, S., Trumpp, A., Milsom, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+hematopoietic+stem+cell+heterogeneity.">Causes and consequences of hematopoietic stem cell heterogeneity.</a> Cell Stem Cell. 22 (5), 627-638 (2018).</li><li>Schroeder, T. <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+cell+heterogeneity:+Subtypes,+not+unpredictable+behavior.">Hematopoietic stem cell heterogeneity: Subtypes, not unpredictable behavior.</a> Cell Stem Cell. 6 (3), 203-207 (2010).</li><li>Muller-Sieburg, C. E., Sieburg, H. B., Bernitz, J. 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Traditionally, cell surface marker-defined HSCs have been prepared to study the functions of HSCs, such as self-renewal capacity and multi-potency19,20,21. However, the immunophenotypically defined (Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722;) HSC fraction contains two discrete HSC populations: LT-HSCs and ST-HSCs9<...

Hematopoietic stem cells (HSCs), which possess self-renewal capacity and multipotency, reside at the apex of the hematopoietic hierarchy1,2. In 1988, Weissman and colleagues demonstrated for the first time that the isolation of mouse HSCs could be achieved using flow cytometry3. Subsequently, a fraction defined by a combination of cell surface markers, Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722;, was reported to contain all HSCs in mice4,5,6,7,8.
Immunophenotypically defined (Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722;) HSCs (hereafter, pHSCs) were previously considered functionally homogeneous. However, recent single-cell analyses have revealed that pHSCs still exhibit heterogeneity with respect to their self-renewal capacity9,10 and multipotency11,12. Specifically, two populations seem to exist in the pHSC fraction with regard to their self-renewal capacity: long-term hematopoietic stem cells (LT-HSCs), which have continuous self-renewal capacity, and short-term hematopoietic stem cells (ST-HSCs), which have transient self-renewal capacity9,10.
To date, the molecular mechanisms of self-renewal capacity that distinguish LT-HSCs and ST-HSCs remain poorly understood. It is crucial to isolate both cell populations based on their self-renewal capacities and to discover underlying molecular mechanisms. Several reporter systems have also been introduced to purify LT-HSCs13,14,15; however, the LT-HSC purity defined by each reporter system is variable, and exclusive LT-HSC purification has not been achieved to date.
Therefore, developing an isolation system for LT-HSCs and ST-HSCs will accelerate research regarding self-renewal capacity in the pHSC fraction. In the isolation of LT-HSCs and ST-HSCs, a study using multi-step, unbiased screening identified a single gene, Hoxb5, that is heterogeneously expressed in the pHSC fraction16. Additionally, bone marrow analysis of the Hoxb5 reporter mice revealed that approximately 20%-25% of the pHSC fraction consists of Hoxb5pos cells. A competitive transplantation assay using Hoxb5pos pHSCs and Hoxb5neg pHSCs revealed that only Hoxb5pos pHSCs possess long-term self-renewal capacity, while Hoxb5neg pHSCs lose their self-renewal capacity within a short period, indicating that Hoxb5 identifies LT-HSCs in the pHSC fraction16.
Here, we demonstrate a step-by-step protocol to isolate LT-HSCs and ST-HSCs using the Hoxb5 reporter system. In addition, we present a competitive transplantation assay to assess the self-renewal capacity of Hoxb5pos/neg pHSCs (Figure 1). This Hoxb5 reporter system allows us to prospectively isolate LT-HSCs and ST-HSCs and contributes to the understanding of LT-HSC-specific characteristics.

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			<td>0.2 mL Strip of 8 Tubes, Dome Cap</td>
			<td>SSIbio</td>
			<td>3230-00</td>
			<td></td>
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		<tr>
			<td>0.5M EDTA pH 8.0</td>
			<td>Iinvtrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>30G insulin syringe</td>
			<td>BD</td>
			<td>326668</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>FALCON</td>
			<td>352235</td>
			<td></td>
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			<td>7-AAD Viability Staining Solution</td>
			<td>BioLegend</td>
			<td>420404</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well U-Bottom</td>
			<td>FALCON</td>
			<td>351177</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-APC-MicroBeads</td>
			<td>Milteny biotec</td>
			<td>130-090-855</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator with trap flask</td>
			<td>Biosan</td>
			<td>FTA-1</td>
			<td></td>
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		<tr>
			<td>B220-Alexa Fluor 700 (RA3-6B2)</td>
			<td>BioLegend</td>
			<td>103232</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-Biotin (RA3-6B2)</td>
			<td>BioLegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-BV786 (RA3-6B2)</td>
			<td>BD Biosciences</td>
			<td>563894</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.CD45.1 congenic mice&#160;</td>
			<td>Sankyo Labo Service</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril 10%</td>
			<td>BAYER</td>
			<td>341106546</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS Aria II special order system&#160;</td>
			<td>BD</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant stain buffer</td>
			<td>BD</td>
			<td>566349</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-Alexa Fluor 700 (M1/70)</td>
			<td>BioLegend</td>
			<td>101222</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-Biotin (M1/70)</td>
			<td>BioLegend</td>
			<td>101204</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-BUV395 (M1/70)</td>
			<td>BD Biosciences</td>
			<td>563553</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-BV711 (M1/70)</td>
			<td>BD Biosciences</td>
			<td>563168</td>
			<td></td>
		</tr>
		<tr>
			<td>CD127-Alexa Fluor 700 (A7R34)</td>
			<td>Invitrogen</td>
			<td>56-1271-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD150-BV421 (TC15-12F12.2)</td>
			<td>BioLegend</td>
			<td>115943</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16/CD32-Alexa Fluor 700 (93)</td>
			<td>Invitrogen</td>
			<td>56-0161-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34-Alexa Fluor 647 (RAM34)</td>
			<td>BD Biosciences</td>
			<td>560230</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34-FITC (RAM34)</td>
			<td>Invitrogen</td>
			<td>11034185</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-Alexa Fluor 700 (17A2)</td>
			<td>BioLegend</td>
			<td>100216</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3&#949; -Biotin (145-2C11)</td>
			<td>BioLegend</td>
			<td>100304</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3&#949; -BV421 (145-2C11)</td>
			<td>BioLegend</td>
			<td>100341</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.1/CD45.2 congenic mice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Bred in our Laboratory</td>
		</tr>
		<tr>
			<td>CD45.1-FITC (A20)</td>
			<td>BD Biosciences</td>
			<td>553775</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.2-PE (104)</td>
			<td>BD Biosciences</td>
			<td>560695</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-Alexa Fluor 700 (GK1.5)</td>
			<td>BioLegend</td>
			<td>100430</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-Biotin (GK1.5)</td>
			<td>BioLegend</td>
			<td>100404</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8a-Alexa Fluor 700 (53-6.7)</td>
			<td>BioLegend</td>
			<td>100730</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8a-Biotin (53-6.7)</td>
			<td>BioLegend</td>
			<td>100704</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube 15ml</td>
			<td>NICHIRYO</td>
			<td>00-ETS-CT-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube 50ml</td>
			<td>NICHIRYO</td>
			<td>00-ETS-CT-50</td>
			<td></td>
		</tr>
		<tr>
			<td>c-Kit-APC-eFluor780 (2B8)</td>
			<td>Invitrogen</td>
			<td>47117182</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS (-) without Ca and Mg, liquid&#160;</td>
			<td>Nacalai</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Flk2-PerCP-eFluor710 (A2F10)</td>
			<td>eBioscience</td>
			<td>46135182</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo version 10</td>
			<td>BD Biosciences&#160;</td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
			<td></td>
		</tr>
		<tr>
			<td>Gmmacell 40 Exactor</td>
			<td>Best theratronics</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gr-1-Alexa Fluor 700 (RB6-8C5)</td>
			<td>BioLegend</td>
			<td>108422</td>
			<td></td>
		</tr>
		<tr>
			<td>Gr-1-Biotin (RB6-8C5)</td>
			<td>BioLegend</td>
			<td>108404</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoxb5-tri-mCherry mice (C57BL/6J background)&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Bred in our Laboratory</td>
		</tr>
		<tr>
			<td>IgG from rat serum, technical grade, &#62;=80% (SDS-PAGE), buffered aqueous solution</td>
			<td>Sigma-Aldrich</td>
			<td>I8015-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Pfizer</td>
			<td>4987-114-13340-3&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes S200</td>
			<td>NIPPON PAPER CRECIA&#160;</td>
			<td>6-6689-01</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Milteny biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis buffer&#160;</td>
			<td>BD</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS&#160; MultiStand</td>
			<td>Milteny biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate for Tissue Culture (For Adhesion Cell) 6Well</td>
			<td>IWAKI</td>
			<td>3810-006</td>
			<td></td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Milteny biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Pie Cages</td>
			<td>Natsume Seisakusho</td>
			<td>KN-331</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose refrigerated Centrifuge</td>
			<td>TOMY</td>
			<td>EX-125</td>
			<td></td>
		</tr>
		<tr>
			<td>NARCOBIT-E (II)</td>
			<td>Natsume Seisakusho</td>
			<td>KN-1071-I</td>
			<td></td>
		</tr>
		<tr>
			<td>NK-1.1-PerCP-Cy5.5 (PK136)</td>
			<td>BioLegend</td>
			<td>108728</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution</td>
			<td>nacalai</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcelain Mortar &#966;120mm with Pestle</td>
			<td>Asone</td>
			<td>6-549-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 1.5 mL&#160;</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>Sca-I-BUV395 (D7)</td>
			<td>BD Biosciences</td>
			<td>563990</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel scalpel blade</td>
			<td>FastGene</td>
			<td>FG-B2010</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-BUV737</td>
			<td>BD Biosciences</td>
			<td>612775</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX-red</td>
			<td>Invitrogen</td>
			<td>S34859</td>
			<td></td>
		</tr>
		<tr>
			<td>Tailveiner Restrainer for Mice standard</td>
			<td>Braintree</td>
			<td>TV-150 STD</td>
			<td></td>
		</tr>
		<tr>
			<td>TCRb-BV421 (H57-597)</td>
			<td>BioLegend</td>
			<td>109230</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter-119-Alexa Fluor 700 (TER-119)</td>
			<td>BioLegend</td>
			<td>116220</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter-119-Biotin (TER-119)</td>
			<td>BioLegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>Terumo 5ml Concentric Luer-Slip Syringe</td>
			<td>TERUMO</td>
			<td>SS-05LZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Terumo Hypodermic Needle 23G x 1</td>
			<td>TERUMO</td>
			<td>NN-2325-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation method for long-term and short-term hematopoietic stem cells

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs),&#160;defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

Previously, self-renewal capacity has been measured using competitive transplantation assays, in which donor HSCs are thought to retain their self-renewal capacity only if multi-lineage donor cells in the recipient peripheral blood are observed17. In addition, several reports define LT-HSCs as cells that continue to produce peripheral blood cells several months after the second bone marrow transplantation10,18. Therefore, in order to compa...

Watch this Scientific Journal Video about Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells at JoVE.com

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter system.

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells ...

The long-term and short-term hematopoietic stem cell or HSC isolation method will help researchers better understand the cell renewal mechanisms and biological basis of heterogeneity in HSC compartment. A transcription factor, Hoxb5, which may be an exclusive marker of long-term HSCs, was identified. Based on this, a Hoxb5-reporter mice line was established and the long-term and the short-term HSCs were successfully isolated.To begin the collection of the donor bone marrow cells, sterilize a mortar and pestle with 70%ethanol and let them dry completely. Equilibrate those with cell-staining buffer comprised of calcium and magnesium-free PBS-containing supplements. Put the bones isolated from the donor male Hoxb5-tri-mCherry mouse in the mortar and add three milliliters of the cell-staining buffer.Brush the bones open with the pestle, then disaggregate the cell clumps by gentle pipetting and transfer the cell suspension through a 100-micron cell strainer into a 50-milliliter tube. Repeat the process with fresh cell-staining buffer until the solution becomes clear. Usually, repeating three times is enough to yield a clear solution.After isolating the femurs and tibiae from the CD45.1.2 congenic mouse, cut both ends of the bones with sharp sterile scissors. Using a 23-gauge needle fitted with a five-milliliter syringe filled with ice-cold cell suspension buffer, flush the bone marrow out into a sterile cell culture dish containing cell suspension buffer. Disaggregate the cell clumps by gentle pipetting, then filter the cell suspension through a 40-micron cell strainer using a P1000 pipette.For the gating setup, prepare the sample following the instructions provided in the text, and transfer 400 microliters of it to a round bottom polystyrene test tube with a 35-micron cell strainer snap cap. Prepare and add dead cell-straining reagent to the sample before the analysis, following the manufacturer's instructions. Then, turn on the flow cytometer and launch the analysis software following the manufacturer's instructions.Then, press Load to begin acquiring the data. After excluding doublets, dead cells, and lineage-positive cells, gate the lineage-negative, the c-kit-positive, Sca-1-positive fraction. Next, gate out the Flk2-positive fraction.Then gate Hoxb5-positive or Hoxb5-negative pHSCs in the CD150-positive, CD34-negative, or low fraction. Next, prepare a 1.5-millimeter low-protein binding tube with 600 microliters of calcium and magnesium-free PBS, supplemented with 10%heat-inactivated FBS. To perform the Hoxb5-positive or Hoxb5-negative pHSC sorting, set the prepared 1.5-milliliter low-protein binding tube on a sort collection device and sort the Hoxb5-positive or Hoxb5-negative pHSCs into the tube using the previously mentioned gating strategy.In the first sorting, use the yield from the sorting precision mode. Set the 96-well plate with the previously prepared supporting cells on the automated cell deposition unit stage. Then, set the 1.5-milliliter low-protein binding tube to the loading port of the flow cytometer.Sort the Hoxb5-positive or Hoxb5-negative pHSCs into the 96-well plate with the supporting cells using the previously mentioned gating strategy. Sort 10 Hoxb5-positive or Hoxb5-negative pHSCs to test their self-renewal capacities. The flow cytometry plots of the bone marrow analysis of the donor Hoxb5-tri-mCherry mice revealed approximately 20 to 25%of the cells in the pHSC fraction defined by lineage-negative, c-kit-positive, Sca-1-positive, CD150-positive, CD34-negative, or low, Flk2-negative were Hoxb5-positive pHSCs.Shown here are the representative flow cytometry plots of the peripheral blood analysis of the recipient mice. Further, the peripheral blood analysis of the recipient mice after primary transplantation revealed that although Hoxb5-positive and Hoxb5-negative pHSCs presented similar donor chimerism four weeks after transplantation, continuous hematopoiesis was observed in only the Hoxb5-positive pHSC recipients. The Hoxb5-negative HSCs started losing the ability to produce hematopoietic cells eight weeks after transplantation.In the secondary transplantation analysis, only the Hoxb5-positive pHSC recipients presented robust hematopoiesis. In contrast, donor cells were hardly observed in the Hoxb5-negative pHSC recipient mice, suggesting Hoxb5-negative pHSCs lost self-renewal ability within 16 weeks of primary transplantation. This protocol usually takes nine to 12 hours, so it is important to keep the sample at four degrees Celsius throughout the procedures as much as possible to maintain cell viability.

isolation-method-for-long-term-and-short-term-hematopoietic-stem-cells

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Long-term Imaging Mammalian Cells using Wide-Field Microscopy

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 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 ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

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

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

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

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...