We thank U. Ganapati for the establishment and testing of the mStrawberry OP9 cell line. Additionally, we thank H. Shafifor her assistance with the co-culture. H.P. is supported by a fellowship from the National Heart, Lung, and Blood Institute (F31HL087714) (H.P.). The content of this work is solely the responsibility of the author and does not necessarily represent the official views of the National Heart, Lung, And Blood Institute or the National Institutes of Health (NIH). The UCLA Flow Cytometry Core Facility is supported by the NIH (CA-16042 and AI-28697), the Jonsson Cancer Center, the UCLA AIDS Institute, and the UCLA School of Medicine.

1. Preparation of ES cells
<ol>
	<li>Culture ES cells according to standard protocol for your particular cell line. Make sure to prepare enough ES cells to plate onto of the mStrawberry OP9 cells for Day 0 (ES cells will be plated at 1x10^3 cells per cm2 at Day 0)</li>
</ol>
2. Preparation of OP9 cells
<ol>
	<li>Before growing mStrawberry OP9 cells, prepare the OP9 growth media 
	OP-9 Cell Culture Medium 
	<table border="1">
		<tbody>
			<tr>
				<td>&nbsp;</td>
				<td>Stock</td>
				<td>Final conc.</td>
				<td>Volume (mL)</td>
			</tr>
			<tr>
				<td>aMEM</td>
				<td>&nbsp;</td>
				<td>&nbsp;</td>
				<td>39.5</td>
			</tr>
			<tr>
				<td>FBS (OP-9 tested)</td>
				<td>100%</td>
				<td>20%</td>
				<td>10</td>
			</tr>
			<tr>
				<td>L-glutamine</td>
				<td>100X (200mM)</td>
				<td>2mM</td>
				<td>0.5</td>
			</tr>
			<tr>
				<td>&nbsp;</td>
				<td>&nbsp;</td>
				<td>&nbsp;</td>
				<td>50mL</td>
			</tr>
		</tbody>
	</table>
	 
	Store at 4&deg;C and use within one week of preparation</li>
	<li>In order to maintain mStrawberry OP9 cells, media must be changed every 2 days and cells should be subcultured prior to cells reaching confluency (subculture at 70-90% confluency). mStrawberry OP9 cell cultures are never grown beyond 90 % confluency as this will result in their differentiation into adipocytes. See Figure 3.
	<ol class="lalpha">
		<li>Subculture cells by washing cells 2X with PBS (or 1x with PBS + 1x 0.1% with Trypsin-EDTA)</li>
		<li>Add pre-warmed 0.1% Trypsin-EDTA at 37&deg;C for 5 minutes (or until cells detach)</li>
		<li>When the cells have detached, add OP-9 growth medium. Transfer cells to a 50mL conical tube.</li>
		<li>Pellet cells at 300 x g for 5 minutes at room temperature. Discard supernatant.</li>
		<li>Resuspend cells in fresh mStrawberry OP9 growth media</li>
		<li>Plate cells at a minimum density of 104cells/cm2</li>
		<li>Grow mStrawberry OP9 cells to confluency starting 3 days prior to day needed for co-culturing ES cells (Day 0, Day 5, or Day8/9 of the experiment)</li>
	</ol>
	</li>
</ol>
3. ES/mStrawberry OP9 Co-culture
<ol>
	<li>Prepare Co-culture differentiation media 
	<table border="1">
		<tbody>
			<tr>
				<td>&nbsp;</td>
				<td>Stock</td>
				<td>Final</td>
				<td>Volume (mL)</td>
			</tr>
			<tr>
				<td>aMEM</td>
				<td>&nbsp;</td>
				<td>&nbsp;</td>
				<td>39.5</td>
			</tr>
			<tr>
				<td>FBS</td>
				<td>100%</td>
				<td>20%</td>
				<td>10</td>
			</tr>
			<tr>
				<td>L-glutamine</td>
				<td>200mM</td>
				<td>2mM</td>
				<td>0.5</td>
			</tr>
			<tr>
				<td>&nbsp;</td>
				<td>&nbsp;</td>
				<td>&nbsp;</td>
				<td>50mL</td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>mStrawberry OP9 cells (Day &ndash;3)
	<ol class="lalpha">
		<li>ALWAYS Subculture OP9 cells on Day -3, so that you have confluent layers of mStrawberry OP9 cells for Day 0 of co-culture. See Figure 4b. Plate mStrawberry OP9 cells at 1x104/cm2 for confluency at Day 0. Be sure to subculture extra cells for Day 5 and Day 8/9 for the continuation of co-culture.</li>
	</ol>
	</li>
	<li class="jove_content">ES/mStrawberry OP9 co-culture Set-Up (Day 0)
	<ol class="lalpha">
		<li class="jove_content">Wash ES cells 2x with PBS</li>
		<li>Trypsinize ES cells with pre-warmed 0.25% Trypsin-EDTA, incubate for 5min @ 37&deg;C</li>
		<li>Add Co-culture differentiation media to cells</li>
		<li>Pellet ES cells at 400xg, 5-7min, RT. Discard supernatant</li>
		<li>Resuspend cells in Co-culture differentiation media</li>
		<li>Remove media from mStrawberry OP9 cells</li>
		<li>Add Co-culture differentiation media to mStrawberry OP9 flasks</li>
		<li>Plate 1x103 ES cells/cm2 to flask of mStrawberry OP9 cells. Incubate cells at 37&deg; C, 5% CO2 with humidity.</li>
	</ol>
	</li>
	<li>mStrawberry OP9 cells (Day 2)
	<ol class="lalpha">
		<li>Subculture mStrawberry OP9 so that you have confluent layers of mStrawberry OP9 cells for Day 5 of co-culture. Plate mStrawberry OP9 cells at 1x104/cm2 for confluency at Day 5 (as in step 3.2). Be sure to subculture extra cells for Day 8/9, if necessary (as in step 2.2).</li>
	</ol>
	</li>
	<li>ES/mStrawberry OP9 co-culture (Day 3)
	<ol class="lalpha">
		<li>Carefully remove medium from co-culture and replace with fresh Co-culture differentiation medium</li>
	</ol>
	</li>
	<li>
	 ES/mStrawberry OP9 co-culture (Day 5) 
	<ol class="lalpha">
		<li>Carefully wash co-cultures 2x with PBS</li>
		<li>Trypsinize cells with pre-warmed 0.25% Trypsin-EDTA</li>
		<li>Wash cells off flask with Co-culture differentiation media</li>
		<li>Pellet cells at 400xg, 7min, RT. Discard supernatant</li>
		<li>Add Co-culture differentiation media to cells. Use a 21-gauge bluntended needle to resuspend cells and to ensure for single cell suspension</li>
		<li>Count cells using a hemocytometer. Should have 100-150 fold increase in ES cell number from Day 0.</li>
		<li>Re-seed 6.48x104/cm2 &ndash; 7.76x104/cm2 co-culture cells on to a new layer of confluent, mStrawberry OP9 cells</li>
		<li>Day 5 ASSAYS -Remaining co-culture cells can be used for flow cytometry analysis, cytopreps(30K cells), ChIP assays, etc.</li>
	</ol>
	</li>
	<li>ES/mStrawberry OP9 co-culture (Day 8/9 and Day 12)
	<ol class="lalpha">
		<li>Remove differentiation media from flask and save. Wash monolayer 1x with PBS to remove hematopoietic cells in suspension. Do not toss out hematopoietic cells.</li>
		<li>Trypsinize adherent cells with pre-warmed 0.25% Trypsin-EDTA</li>
		<li>Wash cells off flask with ES differentiation media and add to hematopoietic cells found in suspension=</li>
		<li>Pellet cells at 400xg, 7min, RT. Discard supernatant</li>
		<li>Add Co-culture differentiation media or PBS to cells. Use a 21-gauge bluntended needle to resuspend cells and to ensure for single cell suspension Count cells using a hemocytometer.</li>
		<li>Co-culture can be continued for 14 days to evaluate more mature hematopoietic progenitors by re-seeding co-culture cells onto a new layer of confluent, mStrawberry OP9 cells at Day 8 or 9 and then again on Day 12. Re- seed optimized cell number based on required cell recovery for day of assay.</li>
	</ol>
	</li>
	<li>Evaluation of ES-derived progeny (Day 1-14). ES-derived progeny can be evaluated on any day of co-culture by flow sorting out mStrawberry negative cells (ES-derived progeny).
	<ol class="lalpha">
		<li>Remove differentiation media from flask and save. Wash monolayer 1x with PBS to remove hematopoietic cells in suspension. Do not toss out hematopoietic cells.</li>
		<li>Trypsinize adherent cells with pre-warmed 0.25% Trypsin-EDTA</li>
		<li>Wash cells off flask with Co-culture differentiation media and add to hematopoietic cells found in suspension</li>
		<li>Pellet cells at 400xg, 7min, RT. Discard supernatant</li>
		<li>Resuspend cells with 21-gauge bluntended needle and count</li>
		<li>ES-derived progeny (mStrawberry negative cells) can be flow sorted from mStrawberry positive OP9 cells. ES-derived progeny can then be characterized by flow cytometry analysis, cytopreps, ChIP assays, etc.</li>
	</ol>
	</li>
</ol>
4. Representative Results:
<img src="/files/ftp_upload/2559/2559fig1.jpg" alt="Figure 1" /> 
Figure 1. ES/mStrawberry OP9 Co-culture System. Our lab uses an in vitro ES cell co-culture system to model hematopoietic development. In this co-culture system, ES cells differentiate, into hemangioblasts at Day5, when placed on a confluent layer of mStrawberry OP9 cells. As the co-culture proceeds, hematopoietic precursors are present at Day 8, while terminally differentiated hematopoietic lineages appear by Day 14. Arrows indicate days of differentiation when passaging of ES-derived progeny onto new confluent layers of mStrawberry OP9 cells in necessary.
<img src="/files/ftp_upload/2559/2559fig2.jpg" alt="Figure 2" /> 
Figure 2. Proper ES cell Differentiation. ES cells are seeded onto a confluent layer of mStrawberry OP9 at Day 0. ES-derived progeny begin to become visible at Day 3 of differentiation, with ES-derived progeny looking like a cluster of cells with a defined border or beginning to form a whorl pattern. Hemangioblasts, which are the common mesodermal precursor for the endothelial and hematopoietic lineages, should appear as piled up whorls of cells at Day 5. For co-culture periods longer than five days, ES-derived progeny should appear as clusters of hematopoietic cells. Two to four celled clusters, at Day 6/7, should appear and grow into larger cell clusters by Day 8/9 of co-culture. Other ES derived progeny are also found tightly bound to the adherent stromal cell layer.
<img src="/files/ftp_upload/2559/2559fig3.jpg" alt="Figure 3" /> 
Figure 3. Unsuccessful ES cell differentiation. Results from this co-culture reflect improper ES cell differentiation. Note how the ES-derived progeny do not appear in a whorl pattern at Day 5. The absence of cells in a whorl pattern indicates improper hemangioblast formation. If hemangioblast do not develop properly, then continued co-culture will result in stunted hematopoietic lineage differentiation. Co-cultures with improper hemangioblast formation should be discarded.
<img src="/files/ftp_upload/2559/2559fig4.jpg" alt="Figure 4" /> 
Figure 4. mStrawberry OP9 cell cultures. (A) mStrawberry OP9 cell are subcultured at 70-90% confluency. (B) For proper differentiation, ES cells and ES-derived progeny are placed onto a overly confluent layer of mStrawberry OP9 cells.
<img src="/files/ftp_upload/2559/2559fig5.jpg" alt="Figure 5" /> 
Figure 5. Representative ES/mStrawberry OP9 Co-culture flow profile. All ES-derived progeny are mStrawberry negative cells and can be flow sorted from the mStrawberry OP9 cells. mStrawberry negative cell gate was determined using a traditional ES/OP9 co-culture.

<ol>
	<li>Baron, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origins+of+mammalian+hematopoiesis.">Embryonic origins of mammalian hematopoiesis.</a> Exp Hematol. 31, 1160-1160 (2003).</li><li>Baron, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+patterning+of+the+mouse+embryo:+implications+for+hematopoietic+commitment+and+differentiation.">Early patterning of the mouse embryo: implications for hematopoietic commitment and differentiation.</a> Exp Hematol. 33, 1015-1015 (2005).</li><li>Baron, M. H., Fraser, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specification+of+early+hematopoiesis+in+the+mammal.">The specification of early hematopoiesis in the mammal.</a> Curr Opin Hematol. 12, 317-317 (2005).</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> Exp Physiol. 85, 645-645 (2000).</li><li>Ganapati, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+notch+signaling+in+normal+and+neoplastic+hematopoiesis:+global+gene+expression+profiling+in+response+to+activated+notch+expression.">Modeling notch signaling in normal and neoplastic hematopoiesis: global gene expression profiling in response to activated notch expression.</a> Stem Cells. 25, 1872-1872 (2007).</li><li>Hogan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo : a laboratory manual. , (1994).</li><li>Keller, G., Kennedy, M., Papayannopoulou, T., Wiles, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+commitment+during+embryonic+stem+cell+differentiation+in+culture.">Hematopoietic commitment during embryonic stem cell differentiation in culture.</a> Mol Cell Biol. 13, 473-473 (1993).</li><li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Curr Opin Cell Biol. 7, (1995).</li><li>Nakano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphohematopoietic+development+from+embryonic+stem+cells+in+vitro.">Lymphohematopoietic development from embryonic stem cells in vitro.</a> Semin Immunol. 7, 862-862 (1995).</li><li>Nakano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+development+of+hematopoietic+system+from+mouse+embryonic+stem+cells:+a+new+approach+for+embryonic+hematopoiesis.">In vitro development of hematopoietic system from mouse embryonic stem cells: a new approach for embryonic hematopoiesis.</a> Int J Hematol. 65, 1-1 (1996).</li><li>Nakano, T., Kodama, H., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+lymphohematopoietic+cells+from+embryonic+stem+cells+in+culture.">Generation of lymphohematopoietic cells from embryonic stem cells in culture.</a> Science. 265, 1098-1098 (1994).</li><li>Turksen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Embryonic stem cells : methods and protocols. , (2002).</li></ol>

No conflicts of interest declared.

Proper differentiation of ES cells on confluent layer of mStrawberry OP9 cells results in the production of Flk1+ hemangioblasts at Day 5 of co-culture. Hemangioblasts should appear as piled up whorls of cells, as observed in Figure 1. For the remainder of the co-culture, visible ES-derived progeny should appear as clusters of hematopoietic cells (Figure 1). At Day6/7 two to four cell clusters should appear and grow into larger cell clusters (both adherent and in suspension) by Day 8/9 of co-culture. Other ES derived progeny are also found tightly bound within the adherent stromal cell layer.
Although not listed in the protocol, it is critical to pre-test the FBS used in the ES growth medium, the mStrawberry OP9 growth media and the ES/mStrawberry OP9 differentiation media. Properly tested serum should ensure proper growth of mStrawberry OP9 cells, as well as, proper differentiation of ES cells in co-culture until Day 5 of differentiation. As an alternative or in addition to mStrawberry OP9 cells, OP9 cells can be irradiated, at 8000 rads, prior to seeding at confluency (7.8x104 cells/cm2). Irradiation of OP9 cells allows for the elimination of repeated replating steps, as well as, allows for almost complete elimination of old, contaminating OP9 cells from downstream assays.
OP9 cells were engineered to express mStrawberry protein by transducing OP9 cells with 3ug/mL of a lentiviral vector expressing mStrawberry under the control of a CMV promoter (pRRLsin-CMV vector, UCLA vector core).
Standard ES/OP9 co-culture protocols entail the placement of ES cells onto a confluent layer OP9 cells, as well as, a day 5 replating step in order to reduce old, contaminating OP9 cells from cell passage. Additionally, only hematopoietic cells found in suspension are removed for evaluation of ES-derived progeny. However, with both a replating step and the harvesting of only suspension cells one potentially misses a substantial number of the developing ES-derived hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. In order to circumvent this issue our lab used a modified co-culture, through the use of mStrawberry-expressing OP9 cells. This assures that all ES progeny are transferred to the continued co-culture at day 5, and for the harvest of all ES-derived progeny for downstream assays. This modification provides a useful tools in the evaluation and characterization of hematopoietic progeny derived from both human and mouse ES lines.
This ES-mStrawberry OP9 co-culture model recapitulates the various stages primitive and definitive hematopoiesis. In studying the earliest stages of hematopoietic development, many people use the BL-CFC(Blast-like colony forming cell) assay, which assess the development of the hemangioblast from the ES cell. While the BL-CFC assay is a useful tool when investigating early hematopoietic development, the mStrawberry-ES co-culture systems exhibits increase utility as it allows for the assessment of hemangioblast, as well as, more adult hematopoietic lineages.
Previous work using traditional OP9/ES and EB differentiation protocols has shown that additional factors maybe necessary, such as overexpression of HoxB4, in order to derive long-term repopulating hematopoietic stem cells in vitro. Additional work is necessary to assess whether mStrawberry negative, sorted ES-derived populations contain true hematopoietic stem cells.

<table><tbody><tr><td colspan="4">OP-9 Cell Maintenance Medium Stock Components</td></tr><tr><td>aMEM</td><td>Invitrogen</td><td>12571-063</td><td></td></tr><tr><td>FBS</td><td>Omega Scientific</td><td>FB01</td><td>Pre-Tested</td></tr><tr><td>L-glutamine</td><td>Cellgro</td><td>25-005-C I</td><td>&lt;u&gt;200nM&lt;/u&gt;</td></tr><tr><td>Trypsin-EDTA</td><td>Stem Cell Technologies</td><td>07901</td><td></td></tr><tr><td colspan="4">ES/OP-9 Cell Differentiation Medium Stock Components</td></tr><tr><td>aMEM</td><td>Invitrogen</td><td>12571-063</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070</td><td>Pre-Tested</td></tr><tr><td>L-glutamine</td><td>Cellgro</td><td>25-005-CI</td><td>200mM</td></tr><tr><td>PBS (without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Stem Cell Technologies</td><td>07901</td><td></td></tr></tbody></table>

modified es / op9 co-culture protocol provides enhanced characterization of hematopoietic progeny

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.

Watch this Scientific Journal Video about Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny at JoVE.com

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

modified-es-op9-co-culture-protocol-provides-enhanced

Nanyang Technological University

Research

JoVE Journal

Biology

20.6K Views.  University of California, Los Angeles. mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

Video: Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

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 ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

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 ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Immunology and Infection

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line ...

Cancer Research

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

Utilizing Low Oxygen Tension to Reduce Hematopoietic Cells in Murine Bone Marrow Stromal Cell Cultures

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this reason, BMSCs, which support hematopoiesis and contribute to all the functions of the skeleton, continue to be widely used to study multipotent mesenchymal progenitors (MMPs) and to infer SSC function. Moreover, given the breadth of transgenic murine models used to study musculoskeletal diseases, the use of BMSCs also serves as a powerful tool to examine the molecular mechanisms regulating MMPs and SSCs. However, common isolation procedures for murine BMSCs result in over 50% of recovered cells being of hematopoietic origins, potentially hindering the interpretation of the data generated during these studies. Here, we describe a method using low oxygen tension or hypoxia for the selective elimination of CD45+ cells in BMSC cultures. Importantly, this method can be easily implemented to not only reduce hemopoietic contaminants but to also enhance the percentage of MMPs and putative SSCs in BMSC cultures.

A routine culture of bone marrow stromal cells (BMSCs) leads to the isolation of heterogeneous cell populations, with many cells being of hematopoietic origins. Here, we describe a method that utilizes low oxygen tension to greatly reduce hematopoietic contaminants in murine BMSC cultures.

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this ...

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

Summary

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Chapters in this video

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

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

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Utilizing Low Oxygen Tension to Reduce Hematopoietic Cells in Murine Bone Marrow Stromal Cell Cultures