Name: Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny (Video) | JoVE Journal
Uploaded: 2011-06-07T13:00:00.000+00:00
Duration: 8 min 36 s
Description: Learn a modified ES/OP9 co-culture protocol to differentiate murine embryonic stem cells into hematopoietic lineages using mStrawberry OP9 stromal cells. This method enables complete characterization of all ES-derived progeny for studying hematopoietic development and defects.

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5 minutes

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> </td>
				<td>Stock</td>
				<td>Final conc.</td>
				<td>Volume (mL)</td>
			</tr>
			<tr>
				<td>aMEM</td>
				<td> </td>
				<td> </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> </td>
				<td> </td>
				<td> </td>
				<td>50mL</td>
			</tr>
		</tbody>
	</table>
	 
	Store at 4°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°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> </td>
				<td>Stock</td>
				<td>Final</td>
				<td>Volume (mL)</td>
			</tr>
			<tr>
				<td>aMEM</td>
				<td> </td>
				<td> </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> </td>
				<td> </td>
				<td> </td>
				<td>50mL</td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>mStrawberry OP9 cells (Day –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°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° 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 – 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="Murine ES/mStrawberry OP9 co-culture timeline; cell harvest, passage days; research diagram." data-altupdatedat="1788235205000" data-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="Hematopoietic differentiation stages, microscope images; cell development, hemangioblast stage, precursors." data-altupdatedat="1788235205000" data-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="Hemangioblast formation, Day 5, microscope image, improper development pattern, cell study analysis." data-altupdatedat="1788235205000" data-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="mStrawberry OP9 cultures, microscope image, non-confluent vs confluent cell growth comparison." data-altupdatedat="1788235205000" data-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="Flow cytometry analysis, scatter plot diagram, mStrawberry+ cell sorting, SSC-A vs FSC-A data." data-altupdatedat="1788235205000" data-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/pubmed?term=((Embryonic+origins+of+mammalian+hematopoiesis%5BTitle%5D)+AND+%22Exp+Hematol%22%5BJournal%5D)">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/pubmed?term=((Early+patterning+of+the+mouse+embryo%3A+implications+for+hematopoietic+commitment+and+differentiation%5BTitle%5D)+AND+%22Exp+Hematol%22%5BJournal%5D)">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/pubmed?term=((The+specification+of+early+hematopoiesis+in+the+mammal%5BTitle%5D)+AND+%22Curr+Opin+Hematol%22%5BJournal%5D)">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/pubmed?term=((Embryoid+bodies%3A+an+in+vitro+model+of+mouse+embryogenesis%5BTitle%5D)+AND+%22Exp+Physiol%22%5BJournal%5D)">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/pubmed?term=((Modeling+notch+signaling+in+normal+and+neoplastic+hematopoiesis%3A+global+gene+expression+profiling+in+response+to+activated+notch+expression%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHogan%2C+B+%22Manipulating+the+mouse+embryo+%3A+a+laboratory+manual%22">Manipulating the mouse embryo : a laboratory manual</a>. , 2nd ed, Cold Spring Harbor Laboratory Press. Plainview, N.Y. (1994).</li>
<li>Keller, G., Kennedy, M., Papayannopoulou, T., Wiles, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hematopoietic+commitment+during+embryonic+stem+cell+differentiation+in+culture%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((In+vitro+differentiation+of+embryonic+stem+cells%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Lymphohematopoietic+development+from+embryonic+stem+cells+in+vitro%5BTitle%5D)+AND+%22Semin+Immunol%22%5BJournal%5D)">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/pubmed?term=((In+vitro+development+of+hematopoietic+system+from+mouse+embryonic+stem+cells%3A+a+new+approach+for+embryonic+hematopoiesis%5BTitle%5D)+AND+%22Int+J+Hematol%22%5BJournal%5D)">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>
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<li>Turksen, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aTurksen%2C+K+%22Embryonic+stem+cells+%3A+methods+and+protocols%22">Embryonic stem cells : methods and protocols</a>. , Humana Press. Totowa, N.J. (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

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.

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 ...

The overall goal of the following experiment is to differentiate murine embryonic stem cells into various primitive and definitive hematopoietic lineages. And to characterize all ES progeny, murine hematopoietic cell differentiation is achieved by placing ES cells onto a confluent fluorescently labeled M strawberry OP nine stromal cell layer. Each day of differentiation or co-culture represents the various stages of hematopoietic development at the appropriate harvest time.Differentiated ES cell progeny can be separated from the M strawberry OP nine stromal cell line by cell sorting. This ES differentiation protocol recapitulates primitive and definitive hematopoiesis as observed in vivo and can be used to characterize hematopoietic defects associated with knockout ES cell lines without the use of embryos. Hello, welcome to the lab of Dr.Judith Gassen at UCLA's Johnson Comprehensive Cancer Center.My name is he Alicia Poz. The main advantage of the technique I'm going to show you today over existing ESO nine co-culture methods is that this procedure allows for the passage of all day five and day eight cells, as well as the complete separation of all ES derived progeny. From the nine stroma M strawberry OP nine cells are grown in OP nine cell culture medium, which must be changed every two days.The OP nine cells should be subculture three days prior to when they're needed for co culturing. It is important to note that during maintenance, the OP nine cells should never be maintained at a density above 90%Co fluency first wash the cells two times with PBS, then add PREWARM 0.1%trypsin EDTA for five minutes when the cells have detached. ADD OP nine cell culture medium and transfer the cells to a 50 milliliter conical tube.Tube pellet cells at 300 G for five minutes at room temperature after centrifugation, discard the supernatant and resuspend cells in fresh M strawberry OP nine cell culture medium plate cells at a minimum density of 10 to the fourth cells centimeter squared on the day the co-culture experiment begins. The OP nine cells that were subculture three days ago should be confluent. The ES cells should be cultured according to the standard protocol for your particular cell line.Wash ES cells two times with PBS trypsin eyes with 0.25%tryin EDTA and incubate for five minutes at 37 degrees Celsius. Next, add co-culture differentiation media to the ES cells and transfer them to 15 milliliter conical tubes. Pellet the ES cells at 400 G for five to seven minutes at room temperature.After removing the supernatant Resus suspend ES cells in co-culture differentiation media. Use a 21 gauge blunt end needle to obtain a single cell suspension. Count cells using trian blue and a hemo cytometer.Now retrieve the flask of M strawberry. OP nine cells from the incubator. Remove media from the OP nine cells and add co-culture differentiation media to the flask plate.One times 10 to the third ES cells per centimeter square to each flask of M strawberry OP nine cells incubate cells at 37 degrees Celsius, 5%carbon dioxide with humidity. This is day zero and the co-culture should look like this. Three days after the start of the ES M strawberry OP nine CO-culture.Mostly large flat clusters of ES derived progeny with defined borders are observed and some whirls of cells will have begun to form. Carefully remove medium from the co-culture and replace with fresh differentiation Medium five days after the start of the co-culture. Proper differentiation of ES cells on a confluent layer of M strawberry OP nine cells should result in heman eblasts, which appear as piled up worlds of cells.The most critical aspect of this procedure is making sure you get proper differentiation on day five of co-culture. At this point, you could see the heman eblast colonies not only microscopically but macroscopically as well. Carefully wash the co-culture two times with PBS and trypsin ice cells with 0.25%tripsin EDTA, wash cells off flask with ES differentiation media and transfer to a tube pellet cells at 400 G for seven minutes at room temperature.After centrifugation, remove the supernatant and add new media. Then use a 21 gauge blunt ended needle to resuspend cells and to ensure a single cell suspension, count the cells using a hemo cytometer, there should be a 100 to 150 fold increase in ES cell number from day zero reseed co-culture cells onto a new layer of confluent M strawberry OP nine cells on day eight or nine and day 12, count the cells again. Remove the media with the hematopoietic cells and reserve.Wash the monolayer one time with PBS To remove hematopoietic cells in suspension and reserve, add 0.25%tripsin EDTA to flask after trypsin adherence cells wash cells off the flask with ES differentiation media and add to the hematopoietic cells found in suspension palate cells at 400 G for seven minutes at room temperature. Finally resuspend the cells using a 21 gauge blunt end needle and count the cells. The cells are now ready to be replated or used in downstream assays.The ES M strawberry OP nine co-culture system can be used to model hematopoietic development outlined. Here is an overview of the co-culture time course days when differentiated ES progeny need to be transferred to new M strawberry OP nine cells are indicated. Proper differentiation of ES cells on a confluent layer of M strawberry OP nine cells will result in the production of flick one positive.He angio blasts at day five of co-culture Heman osteoblasts are the common mesodermal precursor for the endothelial and hematopoietic lineages and should appear as piled up worlds of cells as shown in this image. As the co-culture proceeds two to four celled clusters should appear at day six or seven and grow into larger cell clusters. By day eight or nine hematopoietic precursors are present by day six while terminally differentiated hematopoietic lineages appear by day 14.In contrast, a co-culture with improper ESL differentiation will result in ES derived progeny that do not appear in a world pattern at day five. The absence of cells in a world pattern indicates improper GIO blasts formation. A co-culture with improper eblast formation will ultimately result in stunted hematopoietic lineage differentiation and should be discarded with this modified ES OP nine Co-culture protocol.ES derived progeny are m strawberry negative and can be flow sorted from M strawberry positive. OP nine cells on any day of co-culture for evaluation. Results from a representative experiment are shown here.Previously published ES OP nine co-culture protocols have focused solely on the passage and evaluation of the non-adherent floating cells. In this modified protocol, all ES derived cells, both adherent and non-adherent cells are passaged and can be evaluated. This leads to increased yields of hematopoietic cells, allows for the identification of rare populations of cells, and provides for complete characterization of all ES progeny, both hematopoietic and non hematopoietic.

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Research

JoVE Journal

Biology

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.

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

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

This video describes the technique for long term 2D co-culture of leukemia cells with bone marrow stromal cells to study their interaction with the bone marrow microenvironment. The 2D coculture model utilizes the acute lymphoblastic leukemia cells and the bone marrow stromal or osteoblasts that influence tumor cell phenotype. This study will support investigation of the mechanisms that underlie bone marrow supported survival of leukemic cells during chemotherapy exposure.

2D Co-culture of Leukemic Cells with BMSCs: A Method to Obtain Subpopulations of Leukemic 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.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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.

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

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.

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

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

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.

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

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Freezing Human ES Cells

Learn a modified ES/OP9 co-culture protocol to differentiate murine embryonic stem cells into hematopoietic lineages using mStrawberry OP9 stromal cells. This method enables complete characterization of all ES-derived progeny for studying hematopoietic development and defects.

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

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