The authors do not have any acknowledgements.

All procedures described must be performed in accordance with the guidelines and regulations for the use of vertebrate animals, including prior approval by the local Institutional Animal Care and Use Committee.
1. Preparation of Dissecting Instruments and Culture Media
<ol>
	<li>Prewash all instruments to be used in dissection with 3% hydrogen peroxide and then autoclave at 121 &#176;C for 20 min. Filter BGJb media with antibiotic and antimycotic solution using a 0.22 &#181;m pore-sized filter with vacuum suction in a hood.</li>
</ol>
2. Preparation of Culture Equipment
<ol>
	<li>Cut polycarbonate membrane filters that will support the palatal organs in small triangles, spray with 70% alcohol and allow them to dry. Prepare Center-Well Organ Culture Dish (60x15 mm) by placing a clean and sterilized equilateral triangular-shaped wire grid (20 mm) over the organ culture plate. Then fill the well with 1 ml of BGJb media culture media. &#160;NOTE: The media should be sufficient to reach the level of the grid without submerging the palates. Place the membrane filters, shiny side down, on top of the grid (3-4 membranes per grid).Depending on the experiment, treatment (proteins, antibodies or inhibitors) can be added to the media</li>
</ol>
3. Mouse Embryo Collection and Preparation for Culture
<ol>
	<li>To obtain wild type mouse embryos, use timed 13.5 pregnant CD-1 mouse genetic background strain. Clean the dissection area with 70% ethanol. &#160; 
	NOTE: Other strains of mice can be used. We prefer CD-1 mice because of the bigger litter number.</li>
	<li>Euthanize pregnant mice at E13.5 dpc (days post coitus) with 5% Isoflurane followed by cervical dislocation to insure death using approved protocols.</li>
	<li>Spray 70% ethanol on the ventral abdominal surface to avoid having the abdominal hair stick to the instruments. Then open the abdominal cavity ventrally using a sharp-blunt operating scissors and micro-dissecting forceps to locate the uterus. Using the forceps, lift the entire uterus and separate it from the body by cutting at the uterine body and at the tips of the uterine horns with a light operating scissors.</li>
	<li>Place the uterus on a clean paper towel over ice (According to the protocol guide for care and use of laboratory animals). Cover the uterus with another clean paper towel to prevent contamination. Sterilize the instruments using 70% ethanol spray before further use. The uterus and the embryos can be transported to the laboratory on ice.</li>
	<li>In an open laminar flow hood, carefully make a small opening in the yolk sac using sharp-blunt operating scissors and micro-dissecting forceps. Make the opening wider and then gently push it to exteriorize the embryo from the yolk sac.</li>
	<li>Transfer the embryos immediately to a Petri dish with cold phosphate-buffered Saline 1X (PBS) pH 7.4. To assure the proper stage, examine the embryo under a dissecting microscope for the body, head size and limb morphology. 
	NOTE: Embryos that are not at the right stage of development are not used for the experiment.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53063/53063fig2.jpg" /> 
Figure 2. Limb Bud Morphology. The fore and hind limbs are checked for the degree of webbing indentation between the digits. At E13.5, all the digit condensations are prominent, with the webbing and distal indentations between digits appearing very distinct. The hind digits are behind by a half-day. 12 Upper row: forelimb, lower row: hind limb.
4. Palate Dissection
<ol>
	<li>Place one embryo at a time in a petri dish with PBS under the dissecting microscope. Using small dissecting scissors and tweezers separate the head from the body of the embryos.</li>
	<li>Carefully holding the mouse cranium above eye level with forceps (avoiding the maxillary process) make two incisions on both sides of the lip line. Then remove the mandible and tongue. Isolate the maxillary process region by making a cut at eye level and remove the brain, eyes, nasal septum and adjoining tissue.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/53063/53063fig3.jpg" /> 
Figure 3.Palate tissue collected from 13.5 embryos, when the palate shelves had elevated but not made contact. The palatal shelves were placed in organ culture on a filter at the air media interface for 72 hr in the static model. (A) Level of dissection on E13.5 embryo. (B)&#160;Blue stars demarcate areas to hold the tissue with forceps during dissection. (C)&#160;View of dissected palatal shelves placed on a trapezoid filter membrane. (D)&#160;Side view of culture dish after placement of the dissected palates and addition of culture media.
<ol start="3">
	<li>Place the palatal oral side up and remove the nasal septum and the tissue around it, keeping the primary palate attached to help orient it from anterior to posterior. Use a spoon spatula and very carefully transfer the palatal shelves (nasal side down) to the prepared filters on the grids in the culture dishes. &#160;NOTE: Adjust volume of media to allow proper air-media interface.</li>
	<li>Under the stereo microscope, dissect the primary palate and use tweezers to place the palatal shelves close to each other. The palatal shelves will not fuse if they are not placed in contact. Cut the triangular membrane filter where the anterior part of the palate is located. The trapezoid shape will aid in localizing the anterior portion of the palate.</li>
	<li>Culture the palatal shelves individually or with two or three in the same organ culture dish using BGJb culture media.</li>
</ol>
5. Culturing Mouse Palatal Shelves
<ol>
	<li>Incubate the tissues at 37 &#176;C in a humidified gas mixture (5% CO2 and 95% air) for 72 hr, and change the medium every 24 hr.</li>
</ol>

6. Palate Processing for Histological Analysis
<ol>
	<li>Fix the palates after 72 hr in culture with 4% formaldehyde/phosphate-buffered saline O/N at 4&#176;&#160;C. Then, gently with a transfer pipette wash the tissue in the culture dish with PBS. Wrap the palates in a piece of laboratory wipes and place in embedding cassettes. &#160; 
	NOTE: Wrapping the palates in laboratory wipes prevent their loss during the dehydration process.</li>
	<li>Dehydrate the tissue following 70%, 80% and up to 100% ethanol washes for 1 hr, followed by the regular paraffin embedding procedure. For embedding, place the anterior part of the palate facing the tray.</li>
	<li>Cut Serial 6 &#181;m sections in the coronal orientation from anterior to posterior. A complete section palate will yield 350-450 sections. Stain the sections with hematoxylin and eosin (H&#38;E).10 Score every 20th section using the previously described Mean Fusion Score (MFS) scale (Table 1). 8</li>
</ol>

<ol>
	<li>Nawshad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palatal+seam+disintegration+to+die+or+not+to+die+that+is+no+longer+the+question.">Palatal seam disintegration to die or not to die that is no longer the question.</a> Developmental dynamics an official publication of the American Association of Anatomists. 237, 2643-2656 (2008).</li><li>Strong, E. B., Buckmiller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+the+cleft+palate.">Management of the cleft palate.</a> Facial plastic surgery clinics of North America. 9, 15-25 (2001).</li><li>Witt, P. D., Marsh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+assessing+outcome+of+surgical+repair+of+cleft+lip+and+cleft+palate.">Advances in assessing outcome of surgical repair of cleft lip and cleft palate.</a> Plastic and reconstructive surgery. 100, 1907-1917 (1997).</li><li>4miloro, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> principles of oral and maxillofafcial surgery. 209, 231-249 (1984).</li><li>Mima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+epithelial+adhesion+molecule+CEACAM1+is+important+for+palate+formation.">Regulation of the epithelial adhesion molecule CEACAM1 is important for palate formation.</a> PloS one. 8, e61653 (2013).</li><li>Carette, M. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+medial+edge+epithelial+cells+during+palatal+fusion+in+vitro+an+analysis+by+DiI+labelling+and+confocal+microscopy.">The fate of medial edge epithelial cells during palatal fusion in vitro an analysis by DiI labelling and confocal microscopy.</a> Development (Cambridge, England). 114, 379-388 (1992).</li><li>Ferguson, M. W., Honig, L. S., Slavkin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+cultured+palatal+shelves+from+alligator+chick+and+mouse+embryos.">Differentiation of cultured palatal shelves from alligator chick and mouse embryos.</a> The Anatomical record. 209, 231-249 (1984).</li><li>San Miguel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin+reverse+signaling+controls+palate+fusion+via+a+PI3+kinase+dependent+mechanism.">Ephrin reverse signaling controls palate fusion via a PI3 kinase dependent mechanism.</a> Developmental dynamics an official publication of the American Association of Anatomists. 240, 357-364 (2011).</li><li>Kang, P., Svoboda, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI+3+kinase+activity+is+required+for+epithelial+mesenchymal+transformation+during+palate+fusion.">PI 3 kinase activity is required for epithelial mesenchymal transformation during palate fusion.</a> Developmental dynamics an official publication of the American Association of Anatomists. 225, 316-322 (2002).</li><li>RD, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histopathologic Technic and Practical Histochemistry. , (1965).</li><li>Kaufman, 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=.">.</a> The Atlas of Mouse Development. , (1992).</li><li>Taher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+analysis+of+murine+limb+development.">Global gene expression analysis of murine limb development.</a> PloS one. 6, e28358 (2011).</li></ol>

The authors do not have any competing financial interests.

The protocol in this article provides a method of dissecting palatal shelves from embryos at embryonic day 13.5. Palatal shelves from mouse embryos were cultured in a serum-free culture media in an atmosphere of 95% O2 5% CO2 at 37 &#176;C. Successful palatal dissection is critically dependent on multiple factors at each step during the procedure from the embryonic time of the euthanized mice to the completion of the culture. One of the most important factors influencing palatal fusion is the time t...

Orofacial clefts are the most predominant craniofacial birth defects. Also, taking in consideration all possible craniofacial defects, these are the second most common birth anomaly in newborns 2. Cleft palates occur at approximately 1 in every 700 births in the United States (US) every year, the incidence of cleft palate is equal to 475 children born with cleft palates per month or 15 children with clefts per day 3. Approximately 1% of kids born around the world each year exhibit some form of craniofacial dysmorphology.
Clefts of the palate and the lip need a very expensive and complicated procedure with lifelong implications for patients who have this anomaly. The estimated cost for each patient with oral cleft is approximately $100,0004. The treatment of a patient with cleft lip and palate requires a team of doctors including craniofacial surgeons, otolaryngologists, geneticists, anesthesiologists, speech-language pathologists, nutritionists, orthodontists, prosthodontists, psychologists, neurosurgeons, and ophthalmologists.
In palatogenesis, the secondary palate arises as paired outgrowths, which initially grow vertically and undergo palatal shelf elevation above the dorsum of the tongue. Following elevation, the paired palatal shelves grow towards the midline (at E14.5 &#8211;E15 in mice and week 9 in humans). The medial edge epithelium (MEE) that covers the shelf tip adheres forming the midline epithelial seam.
This is followed by epithelial to mesenchymal transition and/or apoptosis to allow mesenchymal confluence. Adhesion of opposing MEE is an essential event whose alteration causes cleft palate. However, only few studies investigated the initial adhesion of palatal shelves5. The hard palate forms by differentiation of mesenchymal cells into osteoblast. The abnormal development of the palate can produce cleft palates with or without involvement of the lip.
Palate organ culture techniques had been used widely for many labs over the past 30 years6,7.
In this protocol we describe in details a method of palatal dissection and static organ culture. The advantage of a motionless organ culture is that it allows palatal shelves to fuse. This technique had been used successfully in our laboratory for many fusion and signaling experiments8,9. However the scope of the technique is vast and can be used whenever static organ culture system is required, including the evaluation of responses to exogenous chemical agents, the effects of regulatory growth factors in different pathways and specific proteins.
<img alt="Figure 1" src="/files/ftp_upload/53063/53063fig1.jpg" /> 
Figure 1. Mouse Palatogenesis. Developmental stages of the mice palate. (B-F) Scanning electron micrographs (SEM) of the secondary palate at representative developmental times. Red arrows: show the initial part of palatal adhesion and fusion. Yellow arrows: point to the space between the primary and secondary palates that will disappear after fusion (reprinted from Kaufman11 with permission from PLOS one).

&#160;
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BGJb</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Lonza Inc.</td>
			<td>17-602E</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td>100x15mm</td>
		</tr>
		<tr>
			<td>Center-well organ culture dish</td>
			<td>Falcon&#160;</td>
			<td>#353037</td>
			<td>60x15 mm</td>
		</tr>
		<tr>
			<td>Wire triangular grid</td>
			<td>Custom made with stainless steel wire mesh to fit the organ culture dish well.&#160;&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate filter</td>
			<td>GE&#38; Water and Process Technologies</td>
			<td>K04BP04700</td>
			<td>Black 0.4 micron, 47 mm</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>ZEISS&#160; Stemi SR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture hood</td>
			<td>NUAIRE Laminar Flow</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissecting forceps</td>
			<td>Dumont Medical&#160;</td>
			<td></td>
			<td>#5</td>
		</tr>
		<tr>
			<td>Microdissecting scissors</td>
			<td>Kent Scientific Corporation</td>
			<td>INS14003-G</td>
			<td>Vannas Scissors, straight, 8cm long, 0.1mm tips, 5mm blades, German made</td>
		</tr>
		<tr>
			<td>Microdissecting scissors</td>
			<td>Kent Scientific Corporation</td>
			<td>INS500086</td>
			<td>Vannas Scissors, straight, 8.5cm long, 0.025mm x 0.015mm tips, 7mm blades</td>
		</tr>
		<tr>
			<td rowspan="2">Microdissecting scissors</td>
			<td rowspan="2">Kent Scientific Corporation</td>
			<td rowspan="2">INS14127-G</td>
			<td rowspan="2">Spring scissors curved curved, 10.5cm long, 8mm blades, German made</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Surgical currete (spoon spatula)</td>
			<td>Hu-friedy</td>
			<td></td>
			<td>CM 2/4</td>
		</tr>
		<tr>
			<td>Protector laboratory hood</td>
			<td>Labconco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Incubator</td>
			<td rowspan="3">Thermo Scientific</td>
			<td rowspan="3"></td>
			<td>Farma</td>
		</tr>
		<tr>
			<td>Series11 water Jacket</td>
		</tr>
		<tr>
			<td>Co2 incubator</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>GIBCO</td>
			<td>10010-023&#160;</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Fiber optic Light source</td>
			<td>Fiber-light&#160; Dolan-Jenner</td>
			<td>PL-750</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding cassette&#160;</td>
			<td>Statlab</td>
			<td>EC301</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Kim Wipes</td>
			<td rowspan="2">VWR</td>
			<td rowspan="2">470173-504</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

method of studying palatal fusion using static organ culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

The technique can be applied to different kinds of experiments.The following study was designed to test the teratogenic effect of nicotine in vitro on palatal fusion. Two strains of mice were utilized for this experiment, CD1 and C57.Palatal tissue was treated with 0.06 and 6 mM concentrations of nicotine hemisulfate. It was observed that palatal fusion patterns and timing were different in the two different strains. In CD1 mice, palatal shelves from the control group continued fusion in time dependent manner. H...

Watch this Scientific Journal Video about Method of Studying Palatal Fusion using Static Organ Culture at JoVE.com

Method of Studying Palatal Fusion using Static Organ Culture

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

method-of-studying-palatal-fusion-using-static-organ-culture

Research

JoVE Journal

Developmental Biology

8.7K Views.  Texas A&M University Baylor College of Dentistry. Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

Video: Method of Studying Palatal Fusion using Static Organ Culture

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

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 ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion is the merger of two or more cells to form a single cell. Here we provide a protocol for cell fusion of two different cell types. Fused hybrid cells are enriched by flow cytometry-based sorting, followed by fluorescence microscopy of hybrid cell structure and function. Fluorescently tagged proteins generated by genome editing are imaged inside fused cells, allowing cellular structures to be identified based on fluorescence emission and referenced back to the cell type of origin. This robust and general method can be applied to different cell types or organelles of interest, to understand cellular structure and function across a range of fundamental biological questions.

The purpose of this protocol is to fuse two different cell types to create hybrid cells. Fluorescence microscopy analysis of fused cells is used to track the cell of origin of cellular organelles. This assay can be used to explore how cellular structure and function respond to perturbation by cell fusion.

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion ...