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.

OP-9 Cell Maintenance Medium Stock Components
<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</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>200nM</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Stem Cell Technologies</td>
			<td>07901</td>
			<td>
			</td>
		</tr>
	</tbody>
</table>
ES/OP-9 Cell Differentiation Medium Stock Components
<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</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 Ca2+ and Mg2+)</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 ...

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

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

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

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...