Name: conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation
Uploaded: 2017-03-22T13:00:00
Description: Watch this Scientific Journal Video about Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation at JoVE.com

We would like to acknowledge Connecticut Children's Medical Center Strategic Research Funding for supporting this work.

Esophageal biopsies were obtained after informed consent was obtained from the parents/guardians of the pediatric patients and in accordance with institutional review board (IRB#13-094).
1. Sterilizing Instruments and Gelatin Solution
<ol>
	<li>Autoclave forceps, razor blades and scissors prior to handling tissue to prevent contamination.</li>
	<li>To make 200 mL of 0.1% gelatin solution, combine 200 mL of distilled water with 0.2 g of gelatin. Autoclave and cool prior to use.</li>
</ol>
2....

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Esophagus+tissue+engineering:+hybrid+approach+with+esophageal+epithelium+and+unidirectional+smooth+muscle+tissue+component+generation+in+vitro.">Esophagus tissue engineering: hybrid approach with esophageal epithelium and unidirectional smooth muscle tissue component generation in vitro.</a> J Gastrointest Surg. 13 (6), 1037-1043 (2009).</li><li>Macheiner, T., Kuess, A., Dye, J., Saxena, A. 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G., Gaines, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+in+regenerative+medicine:+an+argument+for+continued+research+on+human+embryonic+stem+cells.">Induced pluripotent stem cells in regenerative medicine: an argument for continued research on human embryonic stem cells.</a> Regen Med. 4 (5), 759-769 (2009).</li><li>Ghaedi, M., 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=Human+iPS+cell-derived+alveolar+epithelium+repopulates+lung+extracellular+matrix.">Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix.</a> J Clin Invest. 123 (11), 4950-4962 (2013).</li><li>Huang, S. X., 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=Efficient+generation+of+lung+and+airway+epithelial+cells+from+human+pluripotent+stem+cells.">Efficient generation of lung and airway epithelial cells from human pluripotent stem cells.</a> Nat Biotechnol. , (2013).</li><li>Ogaki, S., Morooka, M., Otera, K., Kume, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cost-effective+system+for+differentiation+of+intestinal+epithelium+from+human+induced+pluripotent+stem+cells.">A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells.</a> Sci Rep. 5, 17297 (2015).</li><li>Ehrhardt, C., Kim, K. J., Lehr, C. 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A., 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=Conditionally+reprogrammed+cells+represent+a+stem-like+state+of+adult+epithelial+cells.">Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells.</a> Proc Natl Acad Sci U S A. 109 (49), 20035-20040 (2012).</li><li>Davis, A. A., 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=A+human+retinal+pigment+epithelial+cell+line+that+retains+epithelial+characteristics+after+prolonged+culture.">A human retinal pigment epithelial cell line that retains epithelial characteristics after prolonged culture.</a> Invest Ophthalmol Vis Sci. 36 (5), 955-964 (1995).</li><li>Carro, O. M., Evans, S. A., Leone, C. 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The most important steps in order to isolate and expand esophageal epithelial cells from patient biopsies are: 1) adequately dissociating biopsy tissue with minimal cell death; 2) ensure ROCK inhibitor is added to the cell culture medium at every medium change; 3) Do not use more feeder cells than recommended; 4) maintain a clean aseptic culture; and 5) passage cells just prior to reaching confluence.
Due to the patient-related differences in biopsy samples obtained for conditional reprogrammi...

Esophageal tissue engineering and eosinophilic esophagitis (EoE) have been the focus of research in many laboratories over the last decade. Congenital defects, such as esophageal atresia, are seen in approximately 1 in 4,000 live births, which results in the incomplete development of the esophagus leading to the inability to eat1. The incidence and prevalence of EoE have been on the rise ever since the identification of the disease entity in 1993. The incidence of EoE varied from 0.7 to 10/100,000 per person-year and the prevalence ranged from 0.2 to 43/100,0002. A new attractive surgical approach to treating long gap esophageal atresia consists in generating tissue constructs for implantation utilizing the patient&#39;s own cells. These cells in conjunction with synthetic scaffolding will generate an autologous construct that does not require immune suppression. Some groups have already begun to investigate the use of stem-like cells for esophageal tissue engineering 3 as well as the use of native esophageal epithelial cells to repopulate the mucosa4-7. Diseases that are present in the esophagus of pediatric patients are often hard to diagnose or study without intervention. Furthermore, utilizing animal models or in vitro immortalized cell line models for pediatric diseases like EoE do not encompass the exact disease pathogenesis or patient specific differences8. Therefore, the ability to study a patient&#39;s disease process in vitro in order to identify specific disease triggering antigens, evaluate underlying mechanisms and investigate drug treatments would be novel and provide clinicians with information that can aid in patient treatment.
There have been many autologous or patient-specific cell types that have been proposed for use in tissue engineering and studying human disease pathogenesis. However, some of these cell types are limited in their capability to generate enough cells of a specific phenotype to seed a large scaffold or perform high throughput in vitro studies. The use of pluripotent or multipotent stem cells has been the topic of much research discussion, however, limitations and shortcomings for using these cells have been well described9. The use of human embryonic stem cells is highly debated and presents many ethical issues. Most importantly, these cells form teratomas, which are similar to a tumor, if they are not differentiated from their pluripotent state prior to delivering them into a living host10. Furthermore, the use of embryonic stem cells would not be patient-specific and could elicit an allogenic response and the need for immune suppression10. Induced pluripotent stem cells (iPSCs) are pluripotent cells that can be derived from a patient&#39;s own cells. Somatic cells, such as skin cells, can be induced to a pluripotent state using a variety of integrative and non-integrative techniques. These cells then serve as a patient-specific cell sources for tissue engineering or disease investigation. The integration of unwanted genetic material into these cells is a concern many have described and even if sequences are completely removed iPSCs appear to conserve an epigenetic &#34;memory&#34; towards the cell type from which they were derived11. These cells also will form teratomas in vivo if not differentiated prior to transplantation11. Many differentiation protocols have been investigated focusing on epithelial lineages12,13,14, however, it is very important to note that the cell types resulting at the end of differentiation are not homogenous and only possess a fraction of the cell type of interest. This results in low yield and the need to purify the desired cell type. Although iPSCs are a potential patient-specific cell source, the process to obtain a cell type of interest for either tissue engineering or disease investigation is very inefficient.
Human epithelial cells have been successfully isolated from a variety of both diseased and non-diseased tissues in the human body including: lung15, breast16, small intestine17, colon18, bladder19 and esophagus20. It is important to note that human primary cells have a finite number of passages in which the phenotype is maintained21,22. Unfortunately, this means that the number of cells needed for disease investigation or for seeding an engineered scaffold for implantation may not be achieved. Therefore, new techniques are needed to expand patient cells while still maintaining an epithelial phenotype. Conditional reprogramming of normal and cancerous epithelial cells utilizing feeder cells and ROCK inhibitor was described in 2012 by Liu et al.23. This technique was utilized to expand cancerous epithelial cells obtained from biopsies of prostate and breast cancer using irradiated feeder cells, ROCK inhibitor and conditional reprogramming medium. The goal was to generate enough cells for in vitro assays such as drug screening. This technique is capable of expanding epithelial cells indefinitely by &#34;reprogramming&#34; these cells to a stem or progenitor-like state, which is highly proliferative. It has been demonstrated that these cells are non-tumorigenic and do not possess the capability to form teratomas23,24. Furthermore, no chromosomal abnormalities or genetic manipulations were present after passaging these cells in culture using this technique23,24. Most importantly, these cells are only able to differentiate into the native cell type of interest. Therefore, this technique offers a large reservoir of patient-specific epithelial cells for disease investigation or tissue engineering without the need for immortalization.
Obtaining epithelial tissue from a specific organ in order to study disease processes is often limited and not always possible due to patient risk. For those patients suffering from esophageal disease or defects, endoscopic biopsy retrieval is a minimally invasive approach for obtaining epithelial tissue that can be dissociated and conditionally reprogrammed to provide an indefinite cell source that is specific to the mucosa of that patient&#39;s esophagus. This then allows for in vitro studies of the epithelial cells to evaluate disease processes and screen for potential therapeutics. One disease process that could greatly benefit from this approach is Eosinophilic Esophagitis, which has been described as allergic disease of the esophagus8. Allergy tests as well as therapeutic approaches could be evaluated in vitro using the patient&#39;s own epithelial cells and this data can then be passed onto the treating physician to develop individualized treatment plans. The technique of conditional reprogramming in conjunction with obtaining endoscopic biopsies from pediatric patients offers the ability to expand normal esophageal epithelial cells indefinitely from any patient. This cell source could therefore be teamed together with natural or synthetic scaffolding to provide a patient-specific surgical option for defects, disease or trauma. Having an indefinite cell number would help engineer esophageal constructs that possess a completely reseeded lumen with esophageal epithelial cells in order to help facilitate regeneration of the remaining cell types.

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			<td height="21" style="height:21px;width:385px;">Primocin</td>
			<td style="width:172px;">InVivogen</td>
			<td style="width:119px;">ant-pm2</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:385px;">Isopentane</td>
			<td style="width:172px;">Sigma Aldrich</td>
			<td style="width:119px;">277258-1L</td>
			<td></td>
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			<td height="21" style="height:21px;width:385px;">Gelatin From Porcine Skin</td>
			<td style="width:172px;">Sigma Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td></td>
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			<td height="21" style="height:21px;width:385px;">DMEM</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">11965092</td>
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			<td height="21" style="height:21px;width:385px;">Cryomold</td>
			<td style="width:172px;">TissueTek</td>
			<td style="width:119px;">4565</td>
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			<td height="21" style="height:21px;width:385px;">Cryomatrix OCT</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">6769006</td>
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			<td height="21" style="height:21px;width:385px;">15ml Conical Tubes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">C1017-p</td>
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			<td height="21" style="height:21px;width:385px;">Complete Keratinocyte Serum Free Medium</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">10724011</td>
			<td></td>
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			<td height="21" style="height:21px;width:385px;">Penicillin Streptomycin</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">15140122</td>
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			<td height="21" style="height:21px;width:385px;">Glutamax</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">35050061</td>
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			<td height="21" style="height:21px;width:385px;">Insulin Solution</td>
			<td style="width:172px;">Sigma Aldrich</td>
			<td style="width:119px;">I9278-5ml</td>
			<td></td>
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			<td height="21" style="height:21px;width:385px;">Human Epidermal Growth Factor (EGF)</td>
			<td style="width:172px;">Peprotech</td>
			<td style="width:119px;">AF-100-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">ROCK Inhibitor (Y-27632)</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">125410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">F-12 Medium&#160;</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">11765054</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Fetal Bovine Serum</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">FB5001</td>
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			<td height="21" style="height:21px;width:385px;">Dispase</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">17105041</td>
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			<td height="21" style="height:21px;width:385px;">0.05% Trypsin-EDTA</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">25300062</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">0.25% Trypsin-EDTA</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">25200072</td>
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			<td height="21" style="height:21px;width:385px;">100mm Dishes</td>
			<td style="width:172px;">Denville Scientific</td>
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			<td height="21" style="height:21px;width:385px;">150mm Dishes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td>T1115</td>
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			<td height="21" style="height:21px;width:385px;">50ml Conicals</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">&#160; C1062-9&#160;</td>
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			<td height="21" style="height:21px;width:385px;">Phosphate Buffered Saline Tablets</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">BP2944-100</td>
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			<td height="21" style="height:21px;width:385px;">5ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">10ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811E</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">25ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811</td>
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			<td height="21" style="height:21px;width:385px;">9&#34; Pasteur Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">NIH 3T3 Cells</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CRL1658</td>
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conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

A summary of the key steps in isolating esophageal epithelial cells from patient biopsies is summarized in Figure 1. Colonies of epithelial cells will form in approximately 4-5 days and will be surrounded by fibroblast feeder cells (Figure 2A). As these colonies expand they will merge with other colonies to form larger colonies (Figure 2B). Once the cultures have become 70% confluent, they need to be expanded (Figure 2C)....

Watch this Scientific Journal Video about Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation at JoVE.com

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

The overall goal of this procedure is to expand patient-derived esophageal epithelial cells from endoscopic biopsies to study esophageal diseases, or to create a 3D esophageal scaffold for downstream modeling or esophageal tissue replacement. This method can be used to better understand the role of esophageal epithelial cells in esophageal disease. They can also be used to see 3D scaffolds for modeling or implantation.The advantage to this technique is it allows us to generate patient specific epithelial cells from the very small piece of tissue without the need for any genetic or permanent modifications. The implications of this technique extend toward understanding the pathogenesis of eosinophilic esophagitis, as well as developing treatments for esophageal atresia and eosinophilic esophagitis. Generally, individuals new to this technique will struggle with the ability to completely remove the feeder cells prior to harvesting human esophageal epithelial cells in order to maintain purity.Demonstrating this technique will be Christopher Foster, a technician from our laboratory. At least three days before the patient samples are obtained, plate the 3T3 cells in 3T3 culture medium in tissue-treated T75 flasks. On the day of the experiment, just prior to obtaining the samples, detach the 3T3 cells with 5 milliliters of 0.25%Trypsin-EDTA per flask for about five minutes at 37 degrees Celsius.When most of the cells are floating, add 5 milliliters of 3T3 medium to stop the reaction, and transfer the cell suspension into a 15 milliliter conical tube for centrifugation. Re-suspend the pellet in medium for counting. Then, transfer the appropriate number of cells for seeding into a 15 milliliter conical tube.Next, add 25 milliliters of fresh 3T3 medium to the cells for irradiation at 3000 rads. Then, collect the cells by centrifugation, and re-suspend the pellet in conditional programming medium at five milliliters per 100 millimeter plate. After obtaining approximately six esophageal biopsies at the time of endoscopy, place six biopsies in a 15 milliliter conical tube containing complete serum-free keratinocyte medium, supplemented with Primocin on wet ice.Place one biopsy in five milliliters of buffered Formalin on wet ice, and place one biopsy in a small biopsy cryomold containing embedding medium. Immediately immerse the mold in a beaker of ice or pentane inside a container of liquid nitrogen. Once the tissue is frozen, place the cryomold in a specimen bag on dry ice, in a polystyrene foam container.Then, place the tubes of biopsies inside a sealable bag inside a shipping box with the biohazard label containing wet ice, and seal and label both containers with a biohazard sticker for transport to the laboratory. Upon entering the lab, immediately transfer the frozen biopsy to a minus 80 degree Celsius storage for downstream sectioning or RNA extraction. Place the sample in Formalin in a four degree Celsius refrigerator for overnight fixation and place the remaining biopsies in the centrifuge.Replace the medium in each tube with 10 milliliters of dispase solution, and seal the tubes for a 15 minute incubation at 37 degree Celsius in a water bath. At the end of the incubation, spin down the biopsies again, and re-suspend the samples in five milliliters of 0.25%Trypsin-EDTA. Transfer the biopsies to a single 100 milliliter sterile plate, and use two sterilized razors to mince the tissues as finely as possible.When all of the tissue has been cut, transfer the tissue fragments to a new 15 milliliter tube, and rinse the plate two times with five milliliters of 0.05%Trypsin-EDTA. Pool the washes in the tube, and return the samples to a 37 degree Celsius water bath. After 10 minutes, centrifuge the digested sample pieces and the feeder cells, and re-suspend the pellets in five milliliters of conditional reprogramming medium, each.Then, combine the feeder and esophageal biopsy samples into one tube, and add rho kinase, or ROCK inhibitor, to the cells for their co-culture on a gelatin-coated tissue culture dish. After approximately five days, remove the medium and minced biopsy pieces by aspiration, and rinse the dish two to three times with five milliliters of sterile PBS. After the last wash, add 10 milliliters of fresh medium supplemented with ROCK inhibitor, to the plate, and return the co-culture to the incubator until the cells reach 70%confluency.To expand the cells, remove the feeders with five milliliters of 0.05%Trypsin-EDTA at 37 degree Celsius for no more than two and a half minutes. Rinse the plate with five milliliters of fresh PBS to remove the floating feeder cells, and add five milliliters of 0.25%Trypsin-EDTA for a five to 10 minute incubation until the biopsy sample cells are loosened from the bottom of the plate. Then, stop the reaction with 10 milliliters of fresh medium, and transfer the cells into a 50 milliliter conical tube for centrifugation.Re-suspend the pellet in fresh conditional reprogramming medium for counting, and, now, account one times 10 to the sixth human esophageal cells into a new conical tube. Add 1.2 times 10 to the sixth irradiated 3T3 cells to the esophageal cells, and bring the total volume in the tube to 15 milliliters with fresh conditional reprogramming medium. Then, add fresh ROCK inhibitor to the cells, and plate the co-culture onto a gelatin-coated 150 millimeter dish.To freeze the esophageal cells for minus 80 degree Celsius storage, remove the feeders and detach the esophageal cells as just demonstrated. Next, transfer the cells into a new 50 milliliter conical tube for centrifugation, and re-suspend the pellet at a one times 10 to the sixth cells per two milliliter of freezing medium concentration. Then, transfer two milliliters of cells into each pre-labeled cryovial, and store the samples at minus 80 degree Celsius for at least 24 hours before moving them to long-term liquid nitrogen storage.Epithelial cell colonies will form in approximately four to five days, and will be surrounded by fibroblast feeder cells. As these colonies expand, they will merge with other colonies to form larger colonies. Once the culture has become 70%confluent, they need to be expanded.After the feeder cells have been removed, the adherent epithelial cells can be trypsonized and re-plated onto new irradiated feeder cells. Phenotypic characterizati6on of these cells via qRT-PCR reveals an up-regulation of P63, a progenitor cell marker at the end of passage one, compared to the initial biospy sample, indicating that cells have been reprogrammed to a more progenitor stem-like state. When these cells are transferred to normal tissue culture plates, the P63 expression decreases and tight junction protein one, or TJP1, and E-cadherin expressions increase, demonstrating a phenotype shift from progenitor cell to a more differentiated epithelial cell.Flow cytometric analysis of the cells after passage one indicates that greater than 90%of the cells are also positive for EpCAM. Immunofluorescence staining demonstrates that the cells retain their epithelial marker, progenitor marker, proliferation marker, and epithelial tight junction protein expression. The doubling time of esophageal cells from a non-diseased patient has been calculated to be approximately 36 to 40 hours, although it is likely that the doubling time of diseased cells will be slower.Once mastered, this technique should be completed in approximately one hour. While attempting this procedure, be sure to use sterile technique to prevent contamination, and be sure to work expeditiously to maintain cell viability. Following this procedure, other assays, such as transwell culture, can be performed in order to evaluate which antigens are causing esophageal inflammation, or pathogenesis.After development, this assay will pave the way for researchers to identify triggering antigens causing eosinophilic esophagitis, as well as new treatment options for treating these patients. After watching this video, you should have a good understanding of how to isolate and expand esophageal epithelial cells from endoscopic biopsies for use in studying esophageal diseases, or generating 3D models. Don't forget that working with sharp instruments and biological tissues can be hazardous.Be sure to dispose of sharp instruments properly, and use good biosafety practices.

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Research

JoVE Journal

Developmental Biology

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 ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

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 ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...