This research is supported by The Netherlands Organization for Scientific Research (NWO) (VENI 016.146.079) and a LUMC Research fellowship both to AMS, and LUMC Bontius Stichting (MJG).

All experiments with human tissue specimens were approved by the ethics committee of the Leiden University Medical Center and conforms to the Declaration of Helsinki. All steps are performed with sterile equipment in a cell culture flow cabinet.
1. Preparations
<ol>
	<li>Prepare EPDC medium by mixing Dulbecco&#39;s modified Eagle&#39;s medium (DMEM low- glucose) and Medium 199 (M199) in a 1:1 ratio. Add 10% heat inactivated fetal bovine serum (FBS, heat inactivated for 25 min at 56 &#176;C) and supplement with 100 U/mL penicillin and 100 mg/mL streptomycin. Pre-warm the EPDC medium in a 37 &#176;C water bath.</li>
	<li>Pre-warm Trypsin 0.25%/EDTA (1:1) in a 37 &#176;C water bath.</li>
	<li>Coat wells with gelatin. Add 0.1% gelatin/PBS to each well and incubate the plates for at least 15 min at 37 &#176;C. Guidelines for the required cell culture plate are summarized in Table 1. Carefully remove all fluid before plating cells.</li>
	<li>Prepare a stock solution of 10 mM SB431542 (SB), diluted in DMSO (CAUTION). Make 50 &#181;L aliquots in conical bottom polypropylene centrifuge tubes, like Eppendorf tubes, and store them at -20 &#176;C. Note that SB aliquots can be thawed only once.</li>
</ol>
2. Retrieval and Storage of Adult and Fetal Heart Specimens
<ol>
	<li>Store human adult auricles directly upon removal during surgery in a 50-mL tube with 15 mL high-glucose DMEM containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 4 &#176;C. Samples are generally 3 - 5 cm in size and can be stored up to 48 h after dissection.</li>
	<li>Store fetal hearts obtained from elective abortion material in EPDC medium at 4 &#176;C up to 24 h after isolation. For this protocol, use samples with a gestational age between 12 - 22 weeks. Note that the whole heart can be used for isolation of the epicardium.</li>
</ol>
3. Isolation of the Epicardial Layer
<ol>
	<li>In the laminar flow cabinet, prepare a 100-mm cell-culture dish with PBS and place a separate droplet (~200 &#181;L) of PBS in the lid. Fill a 15-mL tube with 5 mL pre-warmed EPDC medium.</li>
	<li>Place the tissue in the cell-culture dish filled with PBS. Make sure that the tissue is moistened frequently during the procedure.</li>
	<li>Using a stereomicroscope, remove as much of the epicardial layer from the tissue sample as possible by peeling it off using forceps. 
	Note: The epicardium can be recognized as a very thin, transparent layer tightly adhered to the outside of the heart (Figure 1A). Try to avoid contamination with epicardial adipose tissue and blood vessels since this will hamper the isolation.</li>
	<li>Collect pieces of epicardial tissue in the droplet of PBS on the lid.</li>
	<li>Cut the epicardial tissue into small pieces (0.5 mm3) using a scalpel (Figure 1B), or using the sharp tips of small forceps.Note that the pieces should be able to pass a P1,000 pipet tip (step 3.6).</li>
	<li>Add 1 mL of trypsin to the epicardial tissue and collect the pieces and trypsin with a P1,000 pipet tip. Transfer the tissue into a 1.5-mL conical centrifuge tube (Figure 1C).</li>
	<li>Incubate the tube in a 37 &#176;C water bath for 10 min, while shaking at ~60 rpm.</li>
	<li>Remove the tube from the water bath, and clean the outside of the tube with 70% ethanol.</li>
	<li>Allow the tissue to sink to the bottom of the tube (Figure 1D) and carefully transfer the supernatant containing epicardial cells to the 15-mL tube containing 5 mL EPDC medium to inactivate the trypsin.</li>
	<li>Replenish the remaining tissue in the conical centrifuge tube with 1 mL trypsin, and mix by gently pipetting up and down.</li>
	<li>Repeat step 3.7 to 3.10 two times. At the final step, after a total of 30 min of trypsin incubation, transfer both the supernatant and the remaining epicardial tissue into the tube containing EPDC medium.</li>
	<li>When all cells are collected in EPDC medium, gently pass the suspension through a 10-mL syringe with a 19-gauge needle into a new 15-mL tube to mechanically dissociate the cells (Figure 1E). 
	Note: Make sure to homogenize the suspension by pipetting up and down before passing the solution through the needle to prevent the needle getting obstructed.</li>
	<li>To further dissociate the cells, repeat step 3.12 with a 21-gauge needle.</li>
	<li>Place a 100-&#181;m cell strainer on top of a 50-mL tube and transfer the medium containing the epicardial cells onto the strainer using a 10-mL pipette to remove all remaining clumps (Figure 1F).</li>
	<li>Wash the strainer to collect residual cells by pipetting 5 mL EPDC medium onto the strainer.</li>
	<li>Pipet the cell suspension from the 50-mL tube into a 15-mL tube. Since cells sink to the bottom of the tube, shake the solution gently before pipetting. 
	Note: While this step is not necessary, a smaller tube aids visualization of the pellet after centrifugation.</li>
	<li>Centrifuge at 200 x g for 5 min at room temperature (Figure 1G).</li>
	<li>Remove the supernatant and resuspend the cell pellet (Figure 1H) in the required volume of EPDC medium (Table 1). 
	Note: For fetal EPDCs, directly use EPDC medium containing 10 &#181;M of the ALK5 kinase inhibitor&#160;(EPDC+SB). Adult cells can be plated without SB during the first passage.</li>
	<li>Plate the cell suspension on the gelatin coated culture plates (Figure 1I). The size of the well depends on the size of the epicardial tissue sample (Table 1). Note that low confluency will induce the occurrence of EMT.</li>
	<li>Place the cells in the incubator for at least 48 h at 37 &#176;C, 5% CO2 to allow the cells to attach to the culture plate.</li>
</ol>
4. Culture of EPDCs
<ol>
	<li>Replenish the EPDC+SB medium at least every 3 days.</li>
	<li>Inspect the cells at least every 3 days using the microscope. When the cells reach confluency, i.e., when the culture plate is fully covered with cells, passage the cells in a 1:2 surface ratio. Note that reaching confluency can take ~5 - 10 days.
	<ol>
		<li>Aspirate the medium using a pipet or an aspiration system with, e.g., a glass pipet.</li>
		<li>Wash the cells carefully by adding PBS to the cells. Gently swirl the plate and aspirate the PBS.</li>
		<li>Add trypsin to the plate. Gently rotate the plate to cover all cells with trypsin and incubate the plate for 1 min at 37 &#176;C. 
		NOTE: Use as little trypsin as possible (indication: 200 &#181;L per&#160;well of a 6 well plate)</li>
		<li>Tap the plate to mechanically detach the cells from the bottom of the plate. Use the microscope to visually check if cells have detached. If not, incubate the cell culture plate for an extra minute.</li>
		<li>Add the required volume of EPDC+SB medium to the cells, resuspend by pipetting up and down, and transfer the cell suspension to a new gelatin coated culture plate. 
		NOTE: In general, cells can be kept in a cobblestone morphology up to passage 8.</li>
	</ol>
	</li>
</ol>
5. Induction of EMT in EPDCs
<ol>
	<li>To induce EMT, dissociate the cells with trypsin and transfer the cells to new gelatin coated wells in a 1:2 surface ratio in EPDC+SB medium, as described in 4, and incubate for at least 24 h at 37 &#176;C, 5% CO2.</li>
	<li>Check if confluency is 50 - 70% and if EPDCs have a cobblestone morphology. 
	NOTE: Confluency affects the ability of EPDCs to undergo EMT.</li>
	<li>Aspirate the medium from the cells and wash the cells carefully with PBS (see step 4.2.1 - 4.2.2).</li>
	<li>Stimulate cells with 1 ng/mL TGF&#946;3 in EPDC medium and place them in an incubator at 37 &#176;C, 5% CO2 for 5 days. Note that during stimulation, the EPDC medium containing TGF&#946;3 does not have to be replenished.</li>
	<li>Monitor the cells daily. After 5 days, cells that underwent EMT are recognized by a spindle-shaped morphology (Figure 2A).</li>
	<li>Culture spindle-shaped EPDCs according to the method described in 4 without addition of SB or TGF&#946; to the EPDC medium. Note that in general, spindle-shaped EPDCs can be cultured up to passage 20.</li>
	<li>Validate the occurrence of EMT with immunofluorescent staining using antibodies against the mesenchymal markers &#945;SMA or Vimentin or by phalloidin to detect the formation of F-actin stress fibers (Figure 2B), or using qRT-PCR for EMT-related genes (e.g., WT1, Periostin, Col1A1, &#945;SMA, N-Cadherin, MMP3, Snail, Slug) (Figure 2C and Table 2).</li>
</ol>

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H. P., Chang, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+Induction+of+Fetal+Cardiomyocyte+Proliferation+via+a+Retinoic+Acid-Inducible+Trophic+Factor.">Epicardial Induction of Fetal Cardiomyocyte Proliferation via a Retinoic Acid-Inducible Trophic Factor.</a> Developmental Biology. 250 (1), 198-207 (2002).</li><li>Pennisi, D. J., Ballard, V. L. T., Mikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium+is+required+for+the+full+rate+of+myocyte+proliferation+and+levels+of+expression+of+myocyte+mitogenic+factors+FGF2+and+its+receptor,+FGFR-1,+but+not+for+transmural+myocardial+patterning+in+the+embryonic+chick+heart.">Epicardium is required for the full rate of myocyte proliferation and levels of expression of myocyte mitogenic factors FGF2 and its receptor, FGFR-1, but not for transmural myocardial patterning in the embryonic chick heart.</a> Developmental Dynamics. 228 (2), 161-172 (2003).</li><li>Lavine, K. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epicardium+as+modulator+of+the+cardiac+autonomic+response+during+early+development.">The epicardium as modulator of the cardiac autonomic response during early development.</a> Journal of Molecular and Cellular Cardiology. 89, 251-259 (2015).</li><li>van Wijk, B., Gunst, Q. D., Moorman, A. F. M., van den Hoff, M. J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration+from+activated+epicardium.">Cardiac regeneration from activated epicardium.</a> PloS one. 7 (9), e44692 (2012).</li><li>Zhou, B., Honor, L. 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The authors have nothing to disclose.

Here we describe a detailed protocol to isolate and culture primary epicardial cells derived from human adult and fetal hearts. Extensive characterization of these cells has been previously published17. We have shown that both cell types can be maintained as epithelial cobblestone-like cells when cultured with the ALK5 kinase inhibitor SB431542. EMT is an integral part of epicardial activation in vivo during both development and the post-injury response. EMT can be studied using this meth...

The epicardium, a single-cell epithelial layer that envelopes the heart, is of vital importance for cardiac development and repair (reviewed in Smits et al.1). Developmentally, the epicardium arises from the proepicardial organ, a small structure located at the base of the developing heart. Around developmental day E9.5 in mouse, and 4 weeks post-conception in human, cells start to migrate from this cauliflower structure and cover the developing myocardium2. Once a single epithelial cell layer is formed, a portion of the epicardial cells undergoes&#160;epithelial to mesenchymal transition (EMT). During EMT, cells lose their epithelial characteristics, such as cell-cell adhesions, and obtain a mesenchymal phenotype which gives them the capacity to migrate into the developing myocardium. The formed epicardial derived cells (EPDCs) can differentiate into several cardiac cell types including fibroblasts, smooth muscle cells, and potentially cardiomyocytes and endothelial cells3, although differentiation of the latter two cell populations remains subject to debate (reviewed in Smits et al.4). Furthermore, the epicardium provides instructive paracrine signals to the myocardium to regulate its growth and vascularization5,6,7,8. Multiple studies have demonstrated that impaired epicardial formation leads to developmental defects in cardiac muscle9,10, vasculature11, and conduction system12, emphasizing the essential contribution of the epicardium to the formation of the heart.
Although in the adult heart the epicardium is present as a dormant layer, it becomes reactivated upon ischemia13. Epicardial reactivation post-injury recapitulates several of the processes described for cardiac development, including proliferation and EMT14, albeit less efficiently. Interestingly, although the exact mechanism is not fully understood, the epicardial contribution to repair can be improved by treatment with, e.g., Thymosin &#946;415 or modified VEGF-A mRNA16, resulting in ameliorated cardiac function after myocardial infarction. The epicardium is therefore considered an interesting cell source to enhance endogenous repair of the injured heart.
Mechanisms of cardiac development are often recapitulated during injury, although in a less efficient manner. In search of epicardial activators, it is paramount that we can determine and compare the full capacity of the fetal and adult epicardium. Moreover, from a therapeutic point of view, it is important that, in addition to animal experiments, we extend knowledge regarding the response of the human epicardium. Here, we describe a method to isolate and culture human adult and fetal epicardial derived cells (EPDCs) in an epithelial-cell-like morphology and to induce EMT. With this model, we aim to explore and compare adult and fetal epicardial cell behavior.
The main advantage of this protocol is the use of human epicardial material, which has not been thoroughly studied. Importantly, the described isolation and cell culture protocol provides a single uniform method to derive both fetal and adult cobble EPDCs, enabling a direct comparison between these two cell sources. Additionally, since the epicardium is isolated based on its location, it is ensured that the cells are actually epicardially derived17.
While human EPDC isolation methods have been established previously, these mostly rely on outgrowth protocols where pieces of cardiac or epicardial tissue are plated onto a cell culture dish18,19. This approach thereby selects specifically for cells that partially lose their epithelial phenotype in order to migrate, and that are more prone to undergo spontaneous EMT. In the current protocol, the epicardium is first processed into a single cell solution which allows the isolated EPDCs to maintain their epithelial state. This method therefore provides a solid in vitro model to study epicardial EMT.

<table border="0" cellpadding="0" cellspacing="0" width="798">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Dulbecco&#8217;s modified Eagle&#8217;s medium + GlutaMAX</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:119px;">21885-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Medium 199&#160;</td>
			<td>Gibco</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Fetal Bovine Serum&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Trypsin 0.25%</td>
			<td>Invitrogen</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Penicillin G sodium salt</td>
			<td style="width:133px;">Roth</td>
			<td style="width:119px;">HP48</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Streptomycin sulphate</td>
			<td style="width:133px;">Roth</td>
			<td>HP66</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Trypsin 1:250 from bovine pancreas</td>
			<td style="width:133px;">Serva</td>
			<td>37289</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">EDTA</td>
			<td style="width:133px;">Sigma</td>
			<td>E4884</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Gelatin from porcine skin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Culture plates 6 well</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Culture plates 12 well</td>
			<td style="width:133px;">Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Culture plates 24 well</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">SB 431542</td>
			<td style="width:133px;">Tocris</td>
			<td style="width:119px;">1614</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">102931</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">100-1000&#181;L Filtered Pipet Tips</td>
			<td style="width:133px;">Corning</td>
			<td style="width:119px;">4809</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">10-ml pipet</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td style="width:119px;">607180</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">5-ml pipet</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td style="width:119px;">606180</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Cell culture dish 100/20 mm</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td style="width:119px;">664160</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">PBS</td>
			<td>Gibco</td>
			<td style="width:119px;">10010056</td>
			<td>Or home-made and sterilized</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Eppendorf tubes 1.5 mL</td>
			<td style="width:133px;">Eppendorf</td>
			<td style="width:119px;">0030120086</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">15-ml centrifuge tubes</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">50-ml centrifuge tubes</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">10 mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>305959</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Needles 19 Gauge</td>
			<td>Becton Dickinson</td>
			<td>301700</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Needles 21 Gauge</td>
			<td>Becton Dickinson</td>
			<td>304432</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">EASYstrainer Cell Sieves, 100 &#181;m</td>
			<td>Greiner bio-one</td>
			<td>542000</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">TGF&#946;3&#160;</td>
			<td style="width:133px;">R&#38;D systems</td>
			<td style="width:119px;">243-B3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Monoclonal Anti-Actin, &#945;-Smooth Muscle</td>
			<td style="width:133px;">Sigma</td>
			<td>A2547&#160;</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Anti-Mouse Alexa Fluor 555</td>
			<td style="width:133px;">Invitrogen</td>
			<td>A31570</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:119px;">A12379</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Equipment</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Name</td>
			<td style="width:133px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Pipet P1,000</td>
			<td style="width:133px;">Gilson</td>
			<td style="width:119px;">F123602</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Pipet controller</td>
			<td style="width:133px;">Integra</td>
			<td style="width:119px;">155 015</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Stereomicroscope</td>
			<td style="width:133px;">Leica</td>
			<td style="width:119px;">M80</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Inverted Light Microscope</td>
			<td style="width:133px;">Olympus</td>
			<td style="width:119px;">CK2</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Centrifuge</td>
			<td style="width:133px;">Eppendorf</td>
			<td style="width:119px;">5702</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Waterbath</td>
			<td style="width:133px;">GFL</td>
			<td style="width:119px;">1083</td>
			<td></td>
		</tr>
	</tbody>
</table>

the isolation and culture of primary epicardial cells derived from human adult and fetal heart specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

Here, we outline a straightforward protocol to isolate EPDCs from human adult and fetal cardiac tissue (Figure 1). This protocol takes advantage of the easily accessible location of the epicardium on the outside of the heart (Figure 1A). Staining of the heart auricle after dissection demonstrates that the WT1+ epicardium is removed while the underlying subepicardial extracellular matrix and myocardial tissue remain intact (<stron...

Watch this Scientific Journal Video about The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens at JoVE.com

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

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Research

JoVE Journal

Developmental Biology

7.7K Views.  Leiden University Medical Center. The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

Video: The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...