We thank Dr. Tadashi Koyama for technical help. This work was supported in part by JSPS KAKENHI Grant Numbers 23249045, 15K09211, 15K19172; a grant to the Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan; a grant for research on allergic disease and immunology, Japan.

1. Preparations and Culture of Lung Epithelial Cells
<ol>
	<li>Culture A549 human lung epithelial cells (adherent cell line) in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 &#181;g/ml streptomycin.</li>
	<li>Remove and discard the cell culture media from culture dish, and wash once with 5 - 10 ml of phosphate buffered saline (PBS). After washing, immediately aspirate the PBS.</li>
	<li>Add 2 ml&#160;Trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05%) and incubate at 37 &#176;C and 5% CO2 for 3 min.</li>
	<li>Collect the detached cells in centrifuge tubes containing cell culture medium, centrifuge the tubes at RT for 4 min at 150 &#215; g.</li>
	<li>Resuspend the cells pellet in 2 ml of cell culture medium and remove a sample for cell counting. Count the number of the cells using a hemocytometer with Trypan blue stain to check cell viability.</li>
	<li>Seed A549 cells on 10 cm polystyrene plates at a density of 0.5 - 1.0 &#215; 106 cells/dish with 10 ml of medium for gel contraction assay. Seed the cells on 6 well polystyrene plates at a density of 0.5 - 1.0 &#215; 105 cells/well with 2 ml of medium for EMT confirmation. Incubate the cells at 37 &#176;C and 5% CO2 for 24 hr.</li>
</ol>
2. EMT Procedure
<ol>
	<li>Add 10 &#181;l of TGF-&#946;1 (5 &#181;g/ml) and 10 &#181;l of TNF-&#945; (10 &#181;g/ml) to the plate for gel contraction assay seeded in step 1.6. Add 2 &#181;l of TGF-&#946;1 (5 &#181;g/ml) and 2 &#181;l of TNF-&#945; (10 &#181;g/ml) to the plate for EMT confirmation seeded in step 1.6. Incubate at 37 &#176;C and 5% CO2 for 48 hr.</li>
</ol>
3. Confirmation of EMT Procedure by PCR and Western Blotting
<ol>
	<li>Confirm change in morphology (from cobble stone-like to spindle shape) of treated cells using phase contrast microscopy. 
	Note: Normal A549 cells have cobble stone-like and triangular shaped appearance that is a characteristic of epithelial cells, but after stimulation with TGF-&#946;1 and TNF-&#945;, the cells appear long and spindle shaped that is similar to mesenchymal cells14,15.</li>
	<li>Evaluate the expression of an epithelial marker, such as E-cadherin, and mesenchymal markers such as N-cadherin, vimentin, and &#945;-smooth muscle actin using PCR or Western blotting.
	<ol>
		<li>Extract total RNA from the cells using RNA extraction kit16 and synthesize cDNA using reverse transcriptase17 according to the manufacturer&#39;s protocol.</li>
		<li>Measure expression of mRNA levels using real time PCR system18 and monomeric cyanine dye PCR kit according to the manufacturers&#39; instructions. The specific primers for GAPDH (glyceraldehyde 3-phosphate dehydrogenase), ACTA2 (alpha-actin-2), CDH1 (cadherin-1; E-cadherin), and VIM (vimentin) are shown in Table 1.</li>
		<li>Lyse the cells using a lysis buffer (Table 2) solution containing 1% protease inhibitor cocktail. Measure all sample protein concentrations using a protein assay kit19,20, and apply the same amounts of protein to a polyacrylamide gel.
		<ol>
			<li>Perform SDS gel-electrophoresis and semi-dry transfer of the proteins to PVDF membrane21. Incubate the membrane with primary antibodies in blocking buffer for 1 - 2 hr. After incubation, wash twice the membrane with wash buffer and incubate the membrane 1 hr with second antibodies. 
			Note: See the Materials/Equipment Table for antibodies and dilutions used in these studies. See Table 2 for the components of blocking buffer.</li>
			<li>Wash the membrane with wash buffer twice. Take pictures of the membrane with the Western blotting detection kit22 using a cold CCD camera11,23.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Gel Contraction Assay for Evaluating EMT
<ol>
	<li>Aspirate the conditioned media from the cell culture vessel, and wash well with 5 - 10 ml of PBS to remove dead cells. After washing, immediately aspirate the PBS.</li>
	<li>Add 2 ml of 0.05% Trypsin/EDTA and incubate at 37 &#176;C and 5% CO2 for 3 min.</li>
	<li>Collect the detached cells in centrifuge tubes containing DMEM supplemented with trypsin inhibitor (1 mg/ml), centrifuge the tubes at RT for 4 min at 150 &#215;g.</li>
	<li>Mix type 1 collagen gel with distilled water, and 4&#215; concentrated DMEM to adjust the volumes to achieve a collagen concentration of 1.75 mg/ml, and 1&#215; DMEM concentration. Be sure to keep the gel medium on ice during this step. 
	Note: To make 6 ml of gel medium, mix well 3.5 ml of type 1 collagen gel (3 mg/ml), 1.5 ml of 4&#215; concentrated DMEM, and 1ml of distilled water.</li>
	<li>Resuspend the cell pellet in 500 &#181;l of PBS and remove a sample for cell counting. Count the number of the cells using a hemocytometer with trypan blue stain to check cell viability.</li>
	<li>Add the gel medium to adjust the volumes to achieve a cell density of 3.0 &#215; 105 cells/well (6.0 &#215; 105 cells/ml) and gently but quickly mix it by pipetting without gelation.</li>
	<li>Dispense 0.5 ml of the mixture into each well of a 24-well non-treated plate quickly and carefully to make neat cylindrical form. Be careful not to allow any air bubbles to contaminate the gels (Figure 1A). 
	Note: The gel medium containing the cells is viscous and can easily gel and form crescent shape in the well.</li>
	<li>Incubate the plate for 15 min in a cell incubator at 37 &#176;C, 5% CO2 and 95% humidity to gel completely.</li>
	<li>Detach gels from the plate without breaking by moving a sterilized spatula in a manner to draw a circumference in one direction. Using a spatula, gently transfer the gels to 60 mm tissue culture dishes containing 5 ml of DMEM/1% FBS with or without TGF-&#946;1 (5 ng/ml) and TNF-&#945; (10 ng/ml).</li>
	<li>Gently shake the dishes to ensure gels are floating on the medium. Incubate in a cell incubator at 37 &#176;C, 5% CO2 and 95% humidity.</li>
</ol>
5. Measurement of Gel Size
<ol>
	<li>Measure the collagen gel size after 0, 24, 48, and 72 hr using an image analysis system.
	<ol>
		<li>Turn on the gel documentation system (Figure 2A), and put dishes into the light shielding cabinet. Then take off the lid of dishes in the cabinet.</li>
		<li>Open the related gel analyzing software. Click the &#34;image acquisition button&#34; in the menu-bar to show the image of the gels in the cabinet. Then, click &#34;acquire&#34; in the menu bar to take pictures of the gels (Figure 2B).</li>
		<li>Click the &#34;detection button&#34; in the menu bar (Figure 2C) and adjust the measurement region (yellow circle in Figure 2C) by dragging the mouse. Then, click the &#34;auto-detect button&#34; in the menu bar (see button in Figure 2D). The software automatically detects the gels showing a heatmap (Figure 2D).</li>
		<li>Click &#34;OK&#34; to extract the outline of the gels by image processing and to calculate areas surrounded by the outline (Figure 2E). After the area is calculated, the calculated area is displayed in separate pop-up window. 
		Note: If the gels overlap each other during imaging, move gels gently using a sterile pipette tip.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

The protocol developed in this study comprises two steps. The first step is performed to induce EMT, while the second step is the gel contraction assay. Since it is important to confirm that cells underwent EMT, step 2 provides an excellent complement to the morphological and gene expression changes. Previous studies showed that EMT of A549 cells was induced by TGF-&#946;1 only24; however, as we have reported previously10, TNF-&#945; treatment enhances EMT and the acquisition of mesenchymal cell mar...

Fibrosis is involved in the pathogenesis of various chronic progressive diseases, such as interstitial pulmonary disease, cardiac fibrosis, liver cirrhosis, terminal renal failure, systemic sclerosis, and autoimmune disease1. Among interstitial lung diseases, idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease and shows poor prognosis. Pathological hallmark of IPF is the development of fibroblastic foci consisting of activated fibroblasts and myofibroblasts that are associated with the prognosis. The origins of such pulmonary fibroblasts are proposed to be derived from several mesenchymal cells, including originally resident pulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, epithelial-mesenchymal transition (EMT) has been proposed to be associated with the formation of mesenchymal cells2, and to contribute to the pathogenesis of fibrotic disorders.
It is thought that EMT plays important roles in the process of fetal development, wound healing, and progression of cancer, including tumor invasion and metastasis3. Following the process of EMT, epithelial cells obtain the ability of mesenchymal cells by loss of epithelial markers, such as E-cadherin, and by expression of mesenchymal markers, such as vimentin, and &#945;-smooth muscle actin (SMA)4,5. Previous studies showed the evidence that EMT process has been associated with the development of tissue fibrosis in the kidney6 and lung7. Additionally, chronic inflammation promotes fibrotic disease8; furthermore, such inflammatory cytokines as Tumor necrosis factor superfamily member 14 (TNFSF14; LIGHT), tumor necrosis factor (TNF)-&#945;, and interleukin-1&#946;, have been shown to enhance EMT9-12.
Collagen gel contraction assay, a collagen-based cell contraction assay in which fibroblasts are embedded in type I collagen gel three-dimensionally, is an ideal in vitro model for the evaluation of contractility. Contractility is one of the characteristic functions of fibroblasts and contributes to normal wound repair and fibrosis13. In this assay, it is thought that the attachment of fibroblasts to type I collagen through integrin-dependent mechanisms is supposed to produce mechanical tension under some conditions, and consequently lead to tissue contraction.
Here, the development of the gel contraction assay is reported to be adapted to evaluate the acquisition of contractile function in the cells that underwent EMT. This report demonstrates that this modified assay is suitable for evaluating contractility in mesenchymal cells that underwent EMT.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:167px;">For A549 medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:96px;">GIBCO</td>
			<td style="width:119px;">10437</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Transforming Growth Factor-&#946;1, Human, recombinant</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">209-16544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recombinant Human TNF-&#945;</td>
			<td style="width:96px;">R&#38;D systems</td>
			<td style="width:119px;">210-TA/CF</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">E-Cadherin (24E10) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#3195</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vimentin (D21H3) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#5741</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-&#945;-Tubulin antibody</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">T9026</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-Actin, &#945;-Smooth Muscle antibody&#160;</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">A5228</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-N-cadherin antibody</td>
			<td style="width:96px;">BD Transduction Laboratories</td>
			<td style="width:119px;">#610920</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA931-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA934-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">blocking reagent</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN418</td>
			<td>2% in TBS-T</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid</td>
			<td style="width:96px;">corning</td>
			<td style="width:119px;">#353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 mm cell culture dish</td>
			<td style="width:96px;">TPP</td>
			<td style="width:119px;">#93100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, powder</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">12100-046</td>
			<td>For 4&#215;DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">type 1 collagen gel</td>
			<td style="width:96px;">Nitta gelatin</td>
			<td style="width:119px;">Cellmatrix type I-A</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">24 well cell culture plate</td>
			<td style="width:96px;">AGC TECHNO GLASS</td>
			<td style="width:119px;">1820-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Documentation System&#160;</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">AE-6911FXN</td>
			<td>Gel imager</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">gel analyzing software</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">Densitograph, ver. 3.00</td>
			<td>analysing software bundled with AE-6911FXN</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 Well Plates, Non-Treated</td>
			<td style="width:96px;">IWAKI</td>
			<td style="width:119px;">1820-024</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNA extraction kit</td>
			<td style="width:96px;">Qiagen</td>
			<td style="width:119px;">74106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">reverse transcriptase</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">18080044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">real time PCR system</td>
			<td style="width:96px;">Stratagene</td>
			<td style="width:119px;">Mx-3000P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR green PCR kit</td>
			<td style="width:96px;">Qiagen</td>
			<td style="width:119px;">204145</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protease Inhibitor Cocktail (100X)</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">78429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVDF membrane</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">2392390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">protein assay kit</td>
			<td style="width:96px;">bio-rad</td>
			<td style="width:119px;">5000006JA&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyacrylamide gel</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">2331810</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">western blotting detection reagent</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN2232</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">cold CCD camera</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">Ez-Capture MG/ST</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin inhibitor</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">T9003-100MG</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Polyoxyethylene (20)Sorbitan Monolaurate</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">163-11512</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">polyoxyethylene (9) octyiphenyl ether</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">141-08321</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an in vitro assay to evaluate contractile function of mesenchymal cells that underwent epithelial-mesenchymal transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

During EMT, epithelial cells lose epithelial markers, like E-cadherin, and gain the expression of mesenchymal markers, such as vimentin and &#945;-smooth muscle actin4,5. Incubation of A549 human lung epithelial cells with TGF-&#946;1 and TNF-&#945; induces EMT. The appearance of normal A549 cells are cobble stone like shape and triangle shape that is a characteristic of epithelial cells (Figure 3A), but after stimulated with TGF-&#946;1 and TNF-&#945;, the app...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition at JoVE.com

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

development-an-vitro-assay-to-evaluate-contractile-function

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JoVE Journal

Developmental Biology

12.1K Views.  The University of Tokyo Hospital. Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Video: Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...