Name: conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation
Uploaded: 2017-03-22T13:00:00.000+00:00
Duration: 10 min 15 s
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. Coating Tissue Culture Plates
<ol>
	<li>Approximately 2 h prior to cell isolation, add enough 0.1 % gelatin to cover a tissue culture dish (100 mm) and incubate at 37 &#176;C in a cell culture incubator. 
	NOTE: These plates can also be made ahead of time and kept in the incubator before use.</li>
</ol>
3. Making Enzyme Solution for Dissociation
<ol>
	<li>Approximately 30 min prior to cell isolation, weigh out 10 units of dispase on a balance based on the units per milligram concentration on the vial. Place the powder in a 15 mL conical tube with 10 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) and place in a 37 &#176;C water bath.</li>
</ol>
4. Making Cell Culture Medium
<ol>
	<li>To make 50 mL of conditional reprogramming medium, mix the following ingredients into a sterile conical tube: 35 mL of F12 (Ham&#39;s Medium), 11.5 mL of DMEM, 0.5 mL of L-glutamine, 0.5 mL of 100x penicillin/streptomycin, 2.5 mL of fetal bovine serum (FBS), 250 &#956;g of human insulin, 500 ng of human epidermal growth factor (EGF).</li>
	<li>To make 500 mL of 3T3 medium, mix 50 mL of FBS, 5 mL of 100x penicillin/streptomycin and 5 mL of L-glutamine with 500 mL of DMEM.</li>
	<li>To make 500 mL of keratinocyte serum-free medium, thaw EGF and bovine pituitary extract (BPE) supplements (provided with the media). Add the supplements to the 500 mL bottle of base medium and add 5 mL of 100x penicillin/streptomycin</li>
</ol>
5. Culturing and Irradiating 3T3 Cells as a Feeder Source
<ol>
	<li>Prepare 3T3 medium, comprising DMEM, 10% FBS, 1x penicillin/streptomycin and 2 mM L-glutamine.</li>
	<li>Culture 3T3 cells in tissue-treated T-75 flasks at least 3 days before the arrival of patient samples.
	<ol>
		<li>Just prior to the arrival of patient samples, trypsinize 3T3 cells using 5 mL of 0.25% Trypsin-EDTA per flask and incubate at 37 &#176;C for 5 min or until most cells are floating</li>
	</ol>
	</li>
	<li>Add 5 mL of 3T3 medium to stop the reaction. Aspirate the cell suspension and transfer to a 15 mL conical tube. Spin for 5 min at 300 x g.</li>
	<li>Aspirate the medium and resuspend the cells in a defined volume of Trypan blue for cell counting. Combine 10 &#181;L of cells with 10 &#181;L of Trypan blue to exclude dead cells. Load 10 &#181;L of this solution on a hemocytometer for cell counting. 
	NOTE: For this protocol, seeding density for irradiated 3T3 cells is 10,900 cells per cm2, which is equal to 600,000 cells per 100 mm plate.</li>
	<li>Aliquot the number of cells necessary to plate a 100 mm plate per patient sample and resuspend in 25 mL of 3T3 medium in a 50 mL conical tube and irradiate with 3000 rads.</li>
	<li>Following irradiation, spin cells down for 5 min at 300 x g and resuspend in conditional reprogramming medium at 5 mL per 100 mm plate.</li>
	<li>Set the tube aside until the plating of patient cells takes place later in the protocol (see step 7.6).</li>
</ol>
6. Obtaining, Preserving and Transporting Pediatric Esophageal Biopsies
<ol>
	<li>Add 5 mL of complete keratinocyte serum-free medium with 100 &#181;g/mL&#160; primocin to 15 mL conical tubes and bring it to the medical facility on ice. 
	NOTE: These tubes of medium can be stored at 4 &#176;C for up to 2 months.</li>
	<li>Obtain approximately 8 esophageal biopsies at the time of endoscopy and separate as follows:
	<ol>
		<li>Place six biopsies in the 15 mL conical containing complete keratinocyte serum free medium with 100 &#181;g/mL primocin and place on wet ice.</li>
		<li>Place one biopsy in 5 mL of buffered formalin and place on wet ice.</li>
		<li>Place one biopsy in a small biopsy cryomold with optimal cutting temperature compound.
		<ol>
			<li>Immediately drop this mold into a beaker of isopentane placed in liquid nitrogen. Once frozen, place the cryomold in a specimen bag and store on dry ice in a polystyrene foam container.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Place the tubes containing biopsies in medium or formalin inside a sealable bag and then into a shipping box containing wet ice. Seal and label the box with a biohazard label and transport it to the laboratory. 
	NOTE: The container that contains the dry ice and frozen biopsy is also sealed and labeled with a biohazard label.</li>
</ol>
7. Processing Patient Tissue for Downstream Culture and Analysis
<ol>
	<li>Upon arrival at the laboratory, transfer the frozen biopsy on dry ice to -80 &#176;C freezer for downstream sectioning or RNA extraction. Place the sample in formalin. Store in a 4 &#176;C refrigerator and allow to fix overnight.</li>
	<li>Spin the remaining biopsies down at 300 x g, and aspirate the medium. Add 10 mL of dispase solution to the conical tube, seal and allow it to incubate at 37 &#176;C in a water bath for 15 min.</li>
	<li>Following the incubation, spin the biopsies down at 300 x g. Aspirate the medium and resuspend biopsies in 5 mL of 0.05% Trypsin-EDTA.</li>
	<li>Transfer the 5 mL of trypsin-EDTA containing the biopsies to a 100 mm sterile plate and mince the biopsies using 2 sterilized razor blades as fine as possible.</li>
	<li>Transfer the 5 mL of trypsin-EDTA containing minced biopsies to a new 15 mL conical. Rinse the plate twice with an additional 5 mL of 0.05% Trypsin-EDTA and add to the conical tube. Incubate the tube for 10 min in a 37 &#176;C water bath.</li>
	<li>Following the incubation, spin the biopsies down at 300 x g (1.4 RPM) for 5 min and aspirate the medium. Add 5 mL of conditional reprogramming medium to the conical tube as well as the 5 mL of conditional reprogramming medium containing the irradiated feeder cells (see step 5.7).</li>
	<li>Add ROCK inhibitor (1 &#956;L/mL) to the 10 mL of cell suspension and plate after aspirating the gelatin from the tissue culture dish. Incubate at 37 &#176;C.</li>
</ol>
8. Culturing and Expanding Cells
<ol>
	<li>After approximately 5 days of culture, aspirate the initial medium containing the minced-biopsy pieces and rinse the dish 2-3 times with 5 mL sterile PBS. Add new medium to the plate including ROCK inhibitor at 10 &#956;M.</li>
	<li>Upon reaching 70% confluency, expand the cells. Add 5 mL of 0.05% Trypsin-EDTA to the plate and incubate in a 37 &#176;C incubator no more than 2.5 min to remove the feeder cells. Following that incubation, aspirate the trypsin-EDTA and rinse the plate with 5 mL of PBS.</li>
	<li>Aspirate the PBS and add 5 mL of 0.25% Trypsin-EDTA to the plate and incubate for 5-10 min or until the cells are loose from the plate. Add equal amounts of medium to the plate to stop the reaction and transfer all of the fluid to a 50 mL conical tube and spin down at 300 x g for 5 min.</li>
	<li>Resuspend cells in a defined volume of conditional reprogramming medium and mix 10 &#956;L of the suspension with 10 &#956;L of trypan blue. Count on a hemocytometer to determine total cell number.</li>
	<li>To expand cells, add 1 million human esophageal cells to a 150 mm plate along with 1.2 million irradiated 3T3 cells in a total volume of 15 mL. Add fresh ROCK inhibitor at 10&#956;M and add the cell suspension to a gelatin-coated 150 mm dish.</li>
</ol>
9. Freeze and Store Human Esophageal Epithelial Cells
<ol>
	<li>To make freezing medium, add 10% DMSO to conditional reprogramming medium. After trypsinizing and counting, aliquot the cells to freeze into a new 50 mL conical tube and spin down at 300 x g for 5 min.</li>
	<li>Aspirate the medium and add freezing medium at a volume of 2 mL per cryovial. Once the cell suspension is homogenous, aliquot 106 cells in pre-labeled cyrovials. Place in a freezing container at -80 &#176;C for at least 24 h prior to moving the vials to long-term liquid nitrogen storage.</li>
</ol>

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The authors declare they have no competing financial interests.

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 reprogramming, it is reasonable to expect differences in growth kinetics, phenotype and ability to expand to multiple passages. Typically, 4 esophageal biopsies are obtained from each patient for cell isolation. Once dissociated, we net approximately 300,000-600,000 cells at day 0. This expands to over 10 million cells by passage 3 on average. Morphology of colonies that form should look similar to cobblestone morphology described for epithelial cell lines25 (Figure 2). A sample of cells in culture should be analyzed via qRT-PCR, flow cytometry and immunofluorescence for epithelial markers to ensure the populations expanding are indeed epithelial in origin (Figure 3). Should the cells not express epithelial markers (E-Cadherin, EpCAM, TJP1), the cell line should be terminated and discarded. It is expected that greater than 90% of the cells present are EpCAM positive if they are epithelial in origin. In order to evaluate if the cultures are changing phenotype or losing epithelial expression, immunofluorescence or flow cytometry should be utilized to evaluate epithelial markers (E-cadherin), progenitor expression (P63), and epithelial tight junction proteins (TJP1). As the cells are passaged it is expected that phenotype should not change, proliferation should not decrease and these cells should display epithelial expression as well as progenitor expression (Figure 4). Furthermore, normal esophageal tissue that is dissociated and plated in conditional reprogramming culture appears to have a population doubling time of approximately 36-40 h (Figure 5). Cells that are found not to double for more than 5 days are likely to be senescent and should be discarded.
It has been described that cells isolated from patients with inflammatory processes present, such as EoE, possess epithelial cells that have a decreased proliferative rate, decreased E-cadherin expression (Figure 3, Patients 26 and 28) and a high rate of apoptosis26. Therefore, it is not unreasonable to have difficulty culturing cells from a severely ill patient using this method. It is possible that cells will only withstand a few passages or even just initial passage. This should still provide the investigator with a greater number of cells than originally isolated. However, this number may not be adequate for all proposed studies. Since these cells are being used to ultimately study esophageal disease or construct an esophageal replacement, it is important to remember these cells are only responsible for mucosal regeneration. Other cell types such as fibroblasts, neurons, smooth muscle cells and blood vessels are also just as important to the study of esophageal disease and for tissue engineering applications.
This technique offers a patient-specific cell source that is an invaluable tool for studying disease pathogenesis as well as serving as a potential reservoir of patient-specific cells for esophageal tissue engineering applications. Cells utilized for seeding biomaterials for surgical implantation must be free of genetic abnormalities, viral sequences and not form teratomas. The use of stem cells (multipotent or pluripotent) generates too many unwanted cell types following differentiation, some of which can form teratomas if they did not undergo differentiation. Moreover, this cell source phenotype is consistent and does not require purification or removal of unwanted cells. The ideal cell source will be cells that are found in the same anatomical location that is being engineered. In this approach these esophageal epithelial cells are being used to repopulate the mucosa of an esophageal scaffold. Additional cell types are needed for esophageal regeneration; however, the focus of this technique was to ensure the mucosa was reseeded and not incomplete as this can lead to leakage, stricture and perforation.
In addition to utilizing these cells for engineering new surgical options for the esophagus, these cells can also be used to study esophageal disease processes as well as screen for new therapeutic approaches to treat these conditions. Eosinophilic esophagitis (EoE) is described as an allergic disease of the esophagus, with the exact mechanism and pathogenesis not yet fully described. Current treatment regiments include elimination diets, oral steroids and multiple endoscopies. No assay exists to evaluate what triggers the inflammatory process for each patient, which leads doctors to use a &#34;guess and check&#34; approach which is long and often frustrating for the patient. Since this method of patient-specific cell expansion does not utilize lentiviral transduction or genetic modification, the possibility of permanent changes to the genotype of the cells from the reprogramming process is minimal. This means the cells theoretically remain unchanged from the in vivo environment. The ability to test patient cells in vitro to identify the triggering agent would be novel and could ultimately lead to development of new diagnostics or therapeutics for these pediatric patients.

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.

<table><tbody><tr><td>Primocin</td><td>InVivogen</td><td>ant-pm2</td><td></td></tr><tr><td>Isopentane</td><td>Sigma Aldrich</td><td>277258-1L</td><td></td></tr><tr><td>Gelatin From Porcine Skin</td><td>Sigma Aldrich</td><td>G1890-100G</td><td></td></tr><tr><td>DMEM</td><td>Thermofisher Scientific</td><td>11965092</td><td></td></tr><tr><td>Cryomold</td><td>TissueTek</td><td>4565</td><td></td></tr><tr><td>Cryomatrix OCT</td><td>Thermofisher Scientific</td><td>6769006</td><td></td></tr><tr><td>15 mL Conical Tubes</td><td>Denville Scientific</td><td>C1017-p</td><td></td></tr><tr><td>Complete Keratinocyte Serum Free Medium</td><td>Thermofisher Scientific</td><td>10724011</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Thermofisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Glutamax</td><td>Thermofisher Scientific</td><td>35050061</td><td></td></tr><tr><td>Insulin Solution</td><td>Sigma Aldrich</td><td>I9278-5ml</td><td></td></tr><tr><td>Human Epidermal Growth Factor (EGF)</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>ROCK Inhibitor (Y-27632)</td><td>Fisher Scientific</td><td>125410</td><td></td></tr><tr><td>F-12 Medium&amp;nbsp;</td><td>Thermofisher Scientific</td><td>11765054</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Denville Scientific</td><td>FB5001</td><td></td></tr><tr><td>Dispase</td><td>Thermofisher Scientific</td><td>17105041</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25300062</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25200072</td><td></td></tr><tr><td>100 mm Dishes</td><td>Denville Scientific</td><td>T1110-20</td><td></td></tr><tr><td>150 mm Dishes</td><td>Denville Scientific</td><td>T1115</td><td></td></tr><tr><td>50 mL Conicals</td><td>Denville Scientific</td><td>&amp;nbsp; C1062-9&amp;nbsp;</td><td></td></tr><tr><td>Phosphate Buffered Saline Tablets</td><td>Fisher Scientific</td><td>BP2944-100</td><td></td></tr><tr><td>5 mL Pipettes</td><td>Fisher Scientific</td><td>1367811D</td><td></td></tr><tr><td>10 mL Pipettes</td><td>Fisher Scientific</td><td>1367811E</td><td></td></tr><tr><td>25 mL Pipettes</td><td>Fisher Scientific</td><td>1367811</td><td></td></tr><tr><td>9&quot; Pasteur Pipettes</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>NIH 3T3 Cells</td><td>ATCC</td><td>CRL1658</td><td></td></tr></tbody></table>

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). To ensure that fibroblasts are removed from the plate prior to removing the epithelial cells, 0.05% trypsin-EDTA is used for up to 2.5 min to remove the feeders. The absence of feeders should look similar to Figure 2D. The adherent epithelial cells are then trypsinized with 0.25% Trypsin-EDTA and replated on new irradiated feeder cells (Figure 2E).
Phenotypic characterization of these cells via qRT-PCR demonstrates upregulation of P63, a marker of progenitor cells at the end of passage 1, while still maintaining E-cadherin and TJP1 expression as compared to the initial biopsy sample. This indicates that cells have been &#34;reprogrammed&#34; to a more progenitor/stem-like state that are more proliferative compared to non-reprogrammed cells. When these cells are taken from the conditional reprogramming conditions and plated on normal tissue culture plates, the expression of P63 decreases and TJP1 and E-Cadherin (CDH1) increases demonstrating a shift in phenotype from progenitor cell to a more differentiated epithelial cell (Figure 3A). These cells were also analyzed at passage 1 for epithelial markers via flow cytometry and greater than 90% of the cells present from three patients were positive for EpCAM (Figure 3B). Cells were also analyzed via immunofluorescence at passages 1 and 3 to ensure the phenotype over those 3 passages was not changing. The staining demonstrates the persistence of epithelial marker E-cadherin (Figure 4A-D), progenitor marker P63 (Figure 4E-H), proliferation marker KI-67 (Figure 4I-L) and epithelial tight junction protein TJP1 (Figure 4M-P).
Lastly, a growth curve was plotted using a cell line from a non-diseased patient to evaluate the doubling time of these cells in conditional reprogramming culture. Cells were plated at 100,000 cells at day 0 and were found to be just slightly over 500,000 cells at day 4 (Figure 5). The doubling time was calculated to be approximately 36-40 h. It is expected that diseased cells will have a slower doubling time, however, every patient should be assessed separately to account for individual differences.
<img alt="Figure 1" src="/files/ftp_upload/55243/55243fig1.jpg" /> 
Figure 1: Overview of Isolation and Culturing Human Esophageal Epithelial Cells from Pediatric Biopsies. Biopsies are obtained from pediatric patients after informed consent and transported in keratinocyte serum-free medium to the lab. These biopsies are then treated with 1 U/ mL of dispase, followed by mincing the tissue with sterile razor blades and finally treating with 0.05% Trypsin-EDTA. The cell suspension is then added to irradiated feeder cells in conditional reprogramming medium. <a href="http://cloudflare.jove.com/files/ftp_upload/55243/55243fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55243/55243fig2.jpg" /> 
Figure 2: Culturing and Expanding Human Esophageal Epithelial Cells. After 5 days in conditional reprogramming medium, esophageal epithelial cells begin to form small cobblestone colonies (A), which eventually converge to form larger colonies (B). Once the tissue culture dish reaches 70% confluence (C), the plates are treated for a very brief time with 0.05% Trypsin-EDTA to remove the feeder cells, while still maintaining the adherence of the epithelial cells (D). The epithelial cells are then removed with 0.25% Trypsin-EDTA and replated with new feeder cells. Just 24 h after splitting these cells they will form new colonies and continue to proliferate (E). Magnification is 100X. <a href="http://cloudflare.jove.com/files/ftp_upload/55243/55243fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55243/55243fig3.jpg" /> 
Figure 3: Flow Cytometry and qRT-PCR Analysis of Cells Expanded in Culture. (A) Representative gene expression of cells in culture at passage 1 of conditional reprogramming demonstrated a 6 fold increase in P63 expression, a marker of progenitor cells as well as a decrease in epithelial markers CDH1 and TJP1 as compared to the biopsy gene expression. This indicates the cell population is in a more progenitor-like state and therefore is more highly proliferative. Gene expression 24 h post-reprogramming (after the removal of feeder cells and ROCK Inhibitor) demonstrates increased CDH1 and TJP1 expression but a decrease in P63 expression. (B) Flow cytometry analysis of cells from multiple patients obtained at passage 1 demonstrate greater than 90% of the cells expressing EpCam. Patient 27 represents non-EoE patient cells, while Patients 26 and 28 are EoE patient cells. <a href="http://cloudflare.jove.com/files/ftp_upload/55243/55243fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55243/55243fig4.jpg" /> 
Figure 4: Immunofluorescence Staining of Cells at Passages 1 and 3. Cells grown in conditional reprogramming medium with irradiated feeder cells express E-Cadherin (A-D) as well as progenitor marker P63 (E-H). These cells are still proliferative at passage 3 (I-L). Lastly, these cells express tight junction protein 1 (TJP1), which is also consistent with an epithelial phenotype (M-P). Nuclei are counterstained with DAPI (Blue) and antibodies are detected using an Alexa Fluor 546 Antibody (Orange). 2nd Antibody (Ab) control represents non-specific binding of the secondary antibody to the tissue (Q, R). Scale bars = 50 &#181;m. Magnification is 200X. <a href="http://cloudflare.jove.com/files/ftp_upload/55243/55243fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55243/55243fig5.jpg" /> 
Figure 5: Growth Curve of Cells in Conditional Reprogramming. Cells in conditional reprogramming medium from a non-diseased patient were seeded at a defined density and replicates of three were counted each day for 4 days. The growth curve was generated using these cell counts and doubling time was calculated based on the numbers obtained at Day 4 and Day 0. The doubling time of these cells is approximately 36-40 h. Error bars indicate standard error. <a href="http://cloudflare.jove.com/files/ftp_upload/55243/55243fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

conditional-reprogramming-pediatric-human-esophageal-epithelial-cells

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.9K Views.  UConn Health. 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.

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

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Bioengineering

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Medicine

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Neuroscience

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

Human Esophageal Organoids and Air-Liquid Interface Culture Protocols

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

Immunology and Infection

Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescence-activated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively high-throughput method for identifying and understanding esophageal stem cell niche components in vitro.

Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell ...