Name: retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation
Uploaded: 2014-10-20T15:00:00.000+00:00
Duration: 11 min 40 s
Description: Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.

1. Embryoid Body (EB) Formation
<ol>
	<li>Gelatin-adapt ESCs that have been maintained on mouse embryo fibroblasts (MEFs). Passage ESCs 3 times on 6 well tissue culture plate coated with 0.1% gelatin to remove MEFs.
	<ol>
		<li>Grow cells in ESC maintenance media with LIF to keep the cells undifferentiated (Table 1). Make sure cells never exceed 80% confluence. Use low passage (10 or less passages after removing from MEFs) cells for differentiation.</li>
	</ol>
	</li>
	<li>On the day before ESCs are cultured for EB differentiation, passage the cells so they will be approximately 50-70% confluent the next day. Continue culturing cells in ESC maintenance medium to retain cell pluripotency.</li>
	<li>On the day of the differentiation, aspirate ESC maintenance media and wash with 1 ml Phosphate Buffered Saline (PBS: 137 mM NaCl, 2.7 mM KCl, and 11.9 mM phosphate buffer, pH 7.4) per well of a 6-well plate.
	<ol>
		<li>Add 200 &#181;l of 0.25% trypsin/EDTA to each well and incubate at 37 &#176;C for 3 min.</li>
		<li>Inactivate the trypsin/EDTA with 800 &#181;l of differentiation media. Pipet up and down in a 2 ml serological pipet with a p200 tip to break-up the cells into a single cell suspension. Ensure that the p200 tip fits snugly on the end so media does not go up between the pipet and tip interface.</li>
		<li>Pipet up and down approximately 5-10 times. Take care not to create bubbles during the pipetting.</li>
	</ol>
	</li>
	<li>Transfer cells to a 15 ml conical tube. Bring up the volume to 10 ml with PBS. Centrifuge at 315 x g for 5 min at room temperature.</li>
	<li>Remove supernatant. Wash twice with 5 ml PBS to remove remaining media containing LIF. Centrifuge as in step 1.4 for each wash.
	<ol>
		<li>Resuspend the final cell pellet in 2 ml of differentiation media (Table 2).</li>
	</ol>
	</li>
	<li>Count cells with a hemocytometer or cell counter and plate 6,000-10,000 cells/ml in a 10 cm Petri plate (non-tissue culture treated).
	<ol>
		<li>Determine the exact cells/ml empirically for each ESC line. Use sterile Petri plates normally used for bacterial work or low adherence plates. 
		NOTE: For best differentiation results, the EBs should form spheres that remain in suspension without attaching to the plate. Test several brands of plates to find ones with the least adherence.</li>
		<li>Incubate the differentiating ESCs in a 37 &#176;C tissue culture incubator with 5% CO2.</li>
	</ol>
	</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col span="2" />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock</td>
			<td>Final Concentration</td>
			<td>Volume</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>1x</td>
			<td></td>
			<td>410 ml</td>
			<td>Sigma/Aldrich</td>
			<td>D5796</td>
		</tr>
		<tr>
			<td>FBS (ES Screened)</td>
			<td>100%</td>
			<td>15%</td>
			<td>75 ml</td>
			<td>Hyclone</td>
			<td>SH30070.03E</td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>11140</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>35050</td>
		</tr>
		<tr>
			<td>Penncillin/Streptomycin</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>15070</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>14.3 M</td>
			<td>114 &#181;M</td>
			<td>4 &#181;l</td>
			<td>Sigma/Aldrich</td>
			<td>M3148</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor (LIF)</td>
			<td>107 units/ml</td>
			<td>1,000 units/ml</td>
			<td>50 &#181;l</td>
			<td>Millipore</td>
			<td>ESG1107</td>
		</tr>
	</tbody>
</table>
Table 1: ESC Maintenance Media.
<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col span="2" />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock</td>
			<td>Final Concentration</td>
			<td>Volume</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>1x</td>
			<td></td>
			<td>410 ml</td>
			<td>Sigma/Aldrich</td>
			<td>D5796</td>
		</tr>
		<tr>
			<td>FBS (User Screened for optimal differentiation)</td>
			<td>100%</td>
			<td>15%</td>
			<td>75 ml</td>
			<td>Hyclone</td>
			<td>SH30070.03E</td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>11140</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>35050</td>
		</tr>
		<tr>
			<td>Penncillin/Streptomycin</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>15070</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>14.3 M</td>
			<td>114 &#181;M</td>
			<td>4 &#181;l</td>
			<td>Sigma/Aldrich</td>
			<td>M3148</td>
		</tr>
	</tbody>
</table>
Table 2: Differentiation Media.
2. Preparing EB Cells for Virus Infection
<ol>
	<li>At the desired stage of development (between day 2 and day 3), transfer the EBs from the 10 cm Petri dish to 15 ml conical tube. Wash the plate with 5 ml PBS and add the wash to the conical tube.</li>
	<li>Pellet the EBs at 315 x g at room temperature for 5 min. Wash the EB cells with 5 ml PBS.</li>
	<li>Add 1 ml of thawed cell detachment solution (Containing proteolytic and collagenolytic enzymes) and place the tubes in a 37 &#176;C water bath (or incubator) for 30 min with occasional agitation (from flicking or light vortexing).</li>
	<li>Add 1 ml of differentiation media. Pipet up and down with a 2 ml pipet with a p200 pipet tip. Place the tube in 37 &#176;C water bath again for 30-60 min.</li>
	<li>Spin down the cells at 315 x g for 5 min and wash the cells with PBS.</li>
</ol>
3. Virus Infection
<ol>
	<li>Resuspend the cells in 1.5 ml of differentiation media. Transfer the cell suspension to a 6-well non-tissue culture treated plate.</li>
	<li>Prepare the retrovirus by standard techniques ahead of time. Add 5 to 100 &#181;l of viral supernatant to cells depending on viral titer. Do not add polybrene to enhance infection as this inhibits the subsequent reformation of EBs.</li>
	<li>Spinoculate the cells with virus by centrifuging at 2,120 x g for 90 min at room temperature.</li>
</ol>
4. Differentiating Viral Infected EB Cells in Hanging Drops
<ol>
	<li>Add 2 ml of differentiation media to each well of infected EB cell suspension.</li>
	<li>Pipet 3.5 ml of infected EB cell suspension into a sterile reagent reservoir for multipipettors. Use the multipipettor to pipet 15-20 rows of eight 20 &#181;l drops onto the inverted lid of the 15 cm Petri plate.</li>
	<li>Add 10 ml of sterile PBS or water to the bottom half of the Petri plate. Then invert the lid with the drops and place onto the Petri plate. Ensure that the drops are hanging upside down from lid with PBS or water below to keep the chamber humidified.</li>
	<li>Place the dishes in the 37 &#176;C tissue culture incubator with 5.0% CO2.</li>
	<li>After 2 days, collect EBs formed in the hanging drops by inverting lid and washing with 4.5 ml of differentiation media.
	<ol>
		<li>Transfer media and EBs to a new 10 cm non-tissue culture Petri plate. Wash the lid once more with 3 ml of differentiation media and transfer to the 10 cm plate.</li>
	</ol>
	</li>
	<li>Harvest cells for analysis by flow cytometry after a total of 8 days culture starting from the initial transfer of cells to differentiation media (-LIF).</li>
</ol>
5. Preparing Cells for Flow Cytometer Analysis
<ol>
	<li>Transfer cells to a 15 ml conical tube with a 10 ml pipet. Wash the plate with 5 ml PBS and transfer to the same conical tube.</li>
	<li>Pellet cells at 315 x g. Wash pellets with 10 ml PBS.</li>
	<li>Add 1 ml of thawed cell detachment solution and place the tubes in a 37 &#176;C water bath for 30 min with occasional agitation. For detection of some cell surface antigens, trypsin may be used instead.</li>
	<li>Add 1 ml differentiation media and pipet up and down with a 2 ml pipet. Use a p200 tip to the end of the pipet to help break-up digested EB into single cell suspension.
	<ol>
		<li>Place the tube in a 37 &#176;C water bath again for 60 min in order for the integral membrane proteins to recycle to the cell surface.</li>
	</ol>
	</li>
	<li>Pellet cells at 315 x g for 5 min.</li>
	<li>Wash the cells with 0.1% Bovine Serum Albumin Fraction V (BSA) prepared in PBS (PBS/BSA) and incubate the cells with specific fluorescently tagged antibodies.</li>
</ol>
6. Flow Cytometry Analysis for Hematopoietic Progenitors
<ol>
	<li>Place 106 EB derived cells in a 5 ml 12 x 75 mm round bottom tube. For adjusting flow cytometer compensation settings, use compensation beads according to manufacturer instructions.</li>
	<li>Pellet cells at 315 x g. Pour off supernatant and resuspend pellet in residual PBS/BSA solution in the tube.</li>
	<li>For detecting ESC HPCs, incubate the cells with fluorescent labeled antibodies: anti-mouse CD41-PE (R-Phycoerytherin) and anti-mouse CD117 (cKit)-APC/CY7 (Allophycocyanin Cyanine 7).
	<ol>
		<li>Dilute antibodies 1:100 in PBS/BSA. Add 100 &#181;l of diluted antibody to each sample. Determine the correct dilution of antibody for each vendor, clone, and lot or antibody. Determine optimal dilution for labeling cells individually.</li>
	</ol>
	</li>
	<li>Incubate samples on ice in the dark for 30 min.</li>
	<li>Add 2 ml PBS/ BSA and pellet cells at 315 x g for 5 min.</li>
	<li>Aspirate supernatant and resuspend pellet in 300 &#181;l of PBS or another isotonic buffer.</li>
	<li>Filter resuspended cells through a cell strainer cap (0.35 &#181;M) to remove large cell aggregates.</li>
	<li>Analyze cells on a flow cytometer.</li>
</ol>

<ol>
	<li>Williams, R. L., 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=Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336, 684-687 (1988).</li><li>Desbaillets, I., Ziegler, U., Groscurth, P., Gassmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryoid+bodies:+an+in+vitro+model+of+mouse+embryogenesis.">Embryoid bodies: an in vitro model of mouse embryogenesis.</a> Experimental physiology. 85, 645-651 (2000).</li><li>Hopfl, G., Gassmann, M., Desbaillets, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+embryonic+stem+cells+into+embryoid+bodies.">Differentiating embryonic stem cells into embryoid bodies.</a> Methods Mol Biol. 254, 79-98 (2004).</li><li>Tian, X., Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+embryonic+stem+cells+towards+hematopoietic+cells:+progress+and+pitfalls.">Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.</a> Curr Opin Hematol. 15, 312-318 (2008).</li><li>Thomas, K. R., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+by+gene+targeting+in+mouse+embryo-derived+stem+cells.">Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.</a> Cell. 51, 503-512 (1987).</li><li>Mansour, S. L., Thomas, K. R., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+proto-oncogene+int-2+in+mouse+embryo-derived+stem+cells:+a+general+strategy+for+targeting+mutations+to+non-selectable+genes.">Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.</a> Nature. 336, 348-352 (1988).</li><li>Robertson, E., Bradley, A., Kuehn, M., Evans, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+genes+introduced+into+cultured+pluripotential+cells+by+retroviral+vector.">Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector.</a> Nature. 323, 445-448 (1986).</li><li>Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+expression+in+embryonic+stem+cells+and+hematopoietic+stem+cells.">Retroviral expression in embryonic stem cells and hematopoietic stem cells.</a> Mol Cell Biol. 20, 7419-7426 (2000).</li><li>Pfeifer, A., Ikawa, M., Dayn, Y., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+by+lentiviral+vectors:+lack+of+gene+silencing+in+mammalian+embryonic+stem+cells+and+preimplantation+embryos.">Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos.</a> Proc Natl Acad Sci U S A. 99, 2140-2145 (2002).</li><li>Smith-Arica, J. R., 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=Infection+efficiency+of+human+and+mouse+embryonic+stem+cells+using+adenoviral+and+adeno-associated+viral+vectors.">Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors.</a> Cloning and stem cells. 5, 51-62 (2003).</li><li>Lakshmipathy, U., 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=Efficient+transfection+of+embryonic+and+adult+stem+cells.">Efficient transfection of embryonic and adult stem cells.</a> Stem Cells. 22, 531-543 (2004).</li><li>Cook, B. D., Liu, S., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smad1+signaling+restricts+hematopoietic+potential+after+promoting+hemangioblast+commitment.">Smad1 signaling restricts hematopoietic potential after promoting hemangioblast commitment.</a> Blood. 117, 6489-6497 (2011).</li><li>Zafonte, B. 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=Smad1+expands+the+hemangioblast+population+within+a+limited+developmental+window.">Smad1 expands the hemangioblast population within a limited developmental window.</a> Blood. 109, 516-523 (2007).</li><li>Kyba, M., Perlingeiro, R. C., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HoxB4+confers+definitive+lymphoid-myeloid+engraftment+potential+on+embryonic+stem+cell+and+yolk+sac+hematopoietic+progenitors.">HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.</a> Cell. 109, 29-37 (2002).</li><li>Galvin, K. E., Travis, E. D., Yee, D., Magnuson, T., Vivian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+signaling+regulates+the+bone+morphogenic+protein+pluripotency+pathway+in+mouse+embryonic+stem+cells.">Nodal signaling regulates the bone morphogenic protein pluripotency pathway in mouse embryonic stem cells.</a> J Biol Chem. 285, 19747-19756 (2010).</li><li>Ismailoglu, I., Yeamans, G., Daley, G. Q., Perlingeiro, R. C., Kyba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodermal+patterning+activity+of+SCL.">Mesodermal patterning activity of SCL.</a> Exp Hematol. 36, 1593-1603 (2008).</li><li>Kyba, M., 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=Enhanced+hematopoietic+differentiation+of+embryonic+stem+cells+conditionally+expressing+Stat5.">Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5.</a> Proc Natl Acad Sci U S A. 100, 11904-11910 (2003).</li><li>Cao, M., 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=MiR-23a+regulates+TGF-beta-induced+epithelial-mesenchymal+transition+by+targeting+E-cadherin+in+lung+cancer+cells.">MiR-23a regulates TGF-beta-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells.</a> Int J Oncol. 41, 869-875 (2012).</li><li>Chan, M. 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=Molecular+basis+for+antagonism+between+PDGF+and+the+TGFbeta+family+of+signalling+pathways+by+control+of+miR-24+expression.">Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression.</a> EMBO J. 29, 559-573 (2010).</li><li>Huang, 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=Upregulation+of+miR-23a+approximately+27a+approximately+24+decreases+transforming+growth+factor-beta-induced+tumor-suppressive+activities+in+human+hepatocellular+carcinoma+cells.">Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells.</a> Int J Cancer. 123, (2008).</li><li>Min, 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=TGF-beta-associated+miR-27a+inhibits+dendritic+cell-mediated+differentiation+of+Th1+and+Th17+cells+by+TAB3,+p38+MAPK,+MAP2K4+and+MAP2K7.">TGF-beta-associated miR-27a inhibits dendritic cell-mediated differentiation of Th1 and Th17 cells by TAB3, p38 MAPK, MAP2K4 and MAP2K7.</a> Genes Immun. 13, 621-631 (2012).</li><li>Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D., Orkin, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD41+marks+the+initiation+of+definitive+hematopoiesis+in+the+mouse+embryo.">Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.</a> Blood. 101, 508-516 (2003).</li><li>Mitjavila-Garcia, M. 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=Expression+of+CD41+on+hematopoietic+progenitors+derived+from+embryonic+hematopoietic+cells.">Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells.</a> Development. 129, 2003-2013 (2002).</li><li>Warr, M. R., Pietras, E. M., Passegue, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+controlling+hematopoietic+stem+cell+functions+during+normal+hematopoiesis+and+hematological+malignancies.+Wiley+interdisciplinary+reviews.">Mechanisms controlling hematopoietic stem cell functions during normal hematopoiesis and hematological malignancies. Wiley interdisciplinary reviews.</a> Systems biology and medicine. 3, 681-701 (2011).</li><li>Dahl, R., Ramirez-Bergeron, D. L., Rao, S., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spi-B+can+functionally+replace+PU.1+in+myeloid+but+not+lymphoid+development.">Spi-B can functionally replace PU.1 in myeloid but not lymphoid development.</a> Embo J. 21, 2220-2230 (2002).</li><li>Dahl, R., 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=Regulation+of+macrophage+and+neutrophil+cell+fates+by+the+PU.1:C/EBPalpha+ratio+and+granulocyte+colony-stimulating+factor.">Regulation of macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor.</a> Nat Immunol. 4, 1029-1036 (2003).</li></ol>

The authors have no competing financial interests to disclose.

As discussed above, ESC clones that inducibly express a gene of interest can be generated using doxycycline systems, however, generation of these lines is time-consuming and labor intensive. Described in this protocol is a method to express a gene of interest in single cell suspension prepared from ESC derived EBs. These infected cells are then reformed into EBs by hanging drop to examine subsequent differentiation. In the example (Figure 3), it is shown that expression of the mirn23a cluster enhances hematopoietic development when expressed at day 3 of differentiation. At this point the development of nascent germ layer tissue has occurred including early mesoderm that gives rise to blood progenitors. At day 2 and 3 of RW4 ESC differentiation, expression of genes representing ectoderm (Pax6), endoderm (FoxA2), and mesoderm (T, Twist, and Tbx6) are detected by quantitative reverse transcriptase PCR (Q-RT-PCR, Data not shown). For mirn23a to enhance HPC production, it may need to be expressed in early mesoderm. This technique allows for the ability to determine if a transgene has different effects upon its temporal expression without expending a lot of effort to generate novel reagents. Upon observing phenotypes associated with distinct temporal expression, an investigator may want to now spend the effort to generate inducible lines where the expression of the transgene can be tightly controlled.
Other applications for this technique include performing rescue experiments in ESCs where both alleles of a gene of interest have been deleted (Double Knockout ESCs, DKOs). The wildtype gene or mutated versions of the deleted gene could be reintroduced into the differentiating EB cells. Additionally rescue of a phenotype by genes downstream of the deleted gene can be be assayed. This method can be used to examine the cell intrinsic nature of the observed phenotypes. For example in hematopoiesis, defects have been observed in genes that affect either the hematopoietic stem and progenitor cells themselves or sometimes the defect may affect cells in the microenvironment that are needed to support the development of stem and progenitor cells16,24. In this system, if the defect is intrinsic to hematopoietic progenitor cells, then reintroduction of the wildtype gene will only rescue hematopoietic development in the GFP+ (retroviral infected) cells. If the defect were non-cell autonomous, then retroviral infection would result in a rescue of hematopoietic development being observed in both GFP+ and GFP- populations.
In this protocol flow cytometry is used to identify hematopoietic progenitor cells by the cell surface expression of CD41 and CD117. Alternatively, after 8 days of differentiation infected GFP+ cells could be isolated by fluorescence activated cell sorting (FACs). The isolated cells could then be assayed for the presence of specific hematopoietic progenitors by hematopoietic colony assays in methylcellulose media as we have previously described25,26. Similarly, RNA can be extracted from sorted cells and analyzed for expression of lineage specific genes.
The advantage of the described technique is that it requires minimal effort to generate the needed reagents, and allows for temporal expression of a transgene in the developing EB. If a viral vector co-expressing the gene of interest with a fluorescent protein is already available the experiment can be performed immediately. There are a few limitations to be considered with choosing to use this protocol. The technique outlined here does require the ability to produce high-titer retrovirus to have a sufficient number of cells for analysis. Also note that use of the retrovirus can result in insertional mutagenesis that could yield a phenotype. However since this protocol is examining a pool of infected cells instead of a clonal population, it is less likely that a phenotype due to insertional mutagenesis will be observed over the short time of culture. The major limitation of the technique (compared to an inducible system such as at tet system) is that expression of the transgene cannot be silenced at a later time point, and there is no ability to control the levels of expression. ESCs that express a transgene under the control of doxycycline regulation is advantageous since it allows not only for turning on the gene in a temporal manner, but also allows for turning off the gene at a later timepoint. Varying the amount of an inducer such as doxycycline will also allow the user to fine-tune expression. Lastly the inducible systems allow for reproducible expression of the transgene in all cells, whereas in this retroviral protocol there is chimeric expression of the transgene. As discussed above though this chimeric expression could be valuable for some applications. The inducible systems clearly offer a lot of advantages. However, due to the ease and decreased expense of the technique described here, many investigators will find it useful for their studies of embryonic stem cell differentiation.

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)1. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)2. The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types3. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPCs)4. 
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13. 
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

<table><tbody><tr><td>DMEM</td><td>Sigma/Aldrich</td><td>D5796</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Sigma/Aldrich</td><td>M3148</td><td></td></tr><tr><td>FBS (ES Screened)</td><td>Hyclone</td><td>SH30070.03E</td><td></td></tr><tr><td>FBS (Defined)</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Life Technologies</td><td>11140</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>35050</td><td></td></tr><tr><td>Penn/Strep</td><td>Life Technologies</td><td>15070</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>25200</td><td></td></tr><tr><td>LIF ESGRO</td><td>Millipore</td><td>ESG1107</td><td></td></tr><tr><td>ACCUMAX</td><td>Millipore</td><td>SCR006</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>25200</td><td></td></tr><tr><td>Falcon Tissue Culture Plates 6-well</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>Falcon Non-treated Plates 6-well</td><td>Fisher Scientific</td><td>08-772-49</td><td></td></tr><tr><td>Falcon Petri dish 10 cm</td><td>Fisher Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>Falcon Petri dish 15 cm</td><td>Fisher Scientific</td><td>08-757-148</td><td></td></tr><tr><td>Falcon Tube 5 ml</td><td>Fisher Scientific</td><td>14-959-11A</td><td></td></tr><tr><td>Falcon Tube 5 ml with Cell Strainer Cap</td><td>Fisher Scientific</td><td>08-771-23</td><td></td></tr><tr><td>White sterile resevoirs</td><td>U.S.A. Scientific</td><td>111-0700</td><td></td></tr><tr><td>Rat anti-mouse CD41 PE-conjugated</td><td>Biolegend</td><td>133906</td><td></td></tr><tr><td>Rat anti-mouse CD117 APC/CY7-conjugated</td><td>Biolegend</td><td>105826</td><td></td></tr><tr><td>RW4 ESCs</td><td>ATCC</td><td>CRL-12418</td><td></td></tr></tbody></table>

retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

In these studies gelatinized RW4 (Derived from 129X1/SvJ mouse strain) ESCs were used. EBs were isolated at 3 days of differentiation. For best results the EBs should be spherical and non-adherent (Figure 1A, B). After spinoculation with virus co-expressing the fluorescent marker GFP along with a gene of interest, EBs were reformed by the hanging drop method. EBs were successfully reformed from cell suspensions prepared from 2.0, 2.5, and 3.0 day EBs. However, EBs could not be reformed from cell suspensions prepared from day 4.0 EBs. The chimerism of the EBs between infected and non-infected cells was observed by fluorescence microscopy at day 8 of differentiation (Figure 2A, B). The density of the EBs often makes the EBs appear to be 100% GFP positive. However, focusing on different focal planes can reveal the contribution of GFP positive and negative cells to the EBs (Figure 2B).
For analysis of HPC development, chimeric EBs were dissociated into single cell suspensions after 8 days of differentiation. Antagonizing the activity of miRNA(s) of the mirn23a cluster (miRs-23a, 24-2, and -27) results in an inability of ESCs to differentiate into blood progenitors (Manuscript in preparation). To determine if overexpression of the mirn23a cluster has the opposite effect, ESCs were infected at the onset of differentiation. However, this results in generation of decreased HPCs. Since the mirn23a cluster may be involved in TGF&#223;/BMP/Smad signaling18-21, the cluster may have distinct effects when expressed at different stages of EB development similar to the BMP4 activated Smad1 protein12,13. Using this protocol, EB single cell suspensions were infected after 3 days of differentiation. EBs were reformed by hanging drop, and subsequently analyzed at day 8 for HPC generation by assaying CD41 and CD117 cell surface expression by flow cytometry. CD41+ single positive cells are a pool of primitive and definitive HPCs, whereas CD41+CD117+ cells contain only definitive HPCs22,23. We observed a significant increase in the overall population of CD41+ HPCs in the MSCV-mirn23a infected cells compared to the MSCV control virus infected cells (Figure 3). The figure also demonstrates that a high contribution of infected cells to the reformed EBs can be achieved. It is important to infect EBs with both a control virus expressing the fluorescent protein alone, as well as infecting cells with the experimental virus. Infected populations (GFP+) should then be compared for differences in phenotype. Examining differences between uninfected cells versus infected cells may give erroneous results. Comparing GFP- (uninfected), and GFP+ (infected) populations there is a difference in the CD41 populations in both the MSCV and MSCV-mirn23a cultures (Figure 3). This increase may be due to retroviruses infecting proliferative cells.
<img alt="Figure 1" src="/files/ftp_upload/52022/52022fig1.jpg" /> 
Figure 1: Representative Day 3 Embryoid Bodies. RW4 ESCs were switched from ESC maintenance media into differentiation media and cultured in 10 cm plates. (A) 4X and (B) 10X images are shown of embryoid bodies that developed after 3 days of culture. In the 4X image the bar represents 1,000 &#181;m, whereas in the 10X image the bar represents 400 &#181;m.
<img alt="Figure 2" src="/files/ftp_upload/52022/52022fig2.jpg" /> 
Figure 2: Eight day Embryoid Bodies (EB) reformed from retroviral infected day 3 EB cells. (A) Left hand side images are bright field. (B) The three fluorescent columns of fluorescent images to the right are the same EB taken in different focal planes. In the 4X images the bar represents 1,000 &#181;m, whereas in the 10X images the bar represents 400 &#181;m.
<img alt="Figure 3" src="/files/ftp_upload/52022/52022fig3.jpg" /> 
Figure 3: Flow cytometry analysis of hematopoietic progenitor cells produced in chimeric EBs. Single cells suspensions were prepared from d3 EBs, and infected with the indicated retroviruses. EBs were reformed by hanging drop, and cultured an additional 8 days. Single cell suspensions were generated and incubated with fluorescently labeled antibodies to CD41, and CD117. Left hand histogram plots show the GFP- (non-infected cells) and GFP+ (infected cells) gates. The right hand panels show color dot plot of cells positive for CD41, and CD117 expression in the GFP- and GFP+ gates. Primitive hematopoietic progenitors are found in the CD41+ fractions, and definitive progenitors are present in both the CD41+CD117-, and CD41+CD117+ fractions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52022/52022fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

retroviral-infection-murine-embryonic-stem-cell-derived-embryoid-body

Nanyang Technological University

Research

JoVE Journal

Biology

8.4K Views.  Harper Cancer Research Institute. Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation. 

Video: Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

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 of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Developmental Biology

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

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

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

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

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Immunology and Infection

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 ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Medicine

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

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

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

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

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video