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 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.
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'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'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'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 "memory" 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 "reprogramming" 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'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'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.
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
2. Coating Tissue Culture Plates
3. Making Enzyme Solution for Dissociation
4. Making Cell Culture Medium
5. Culturing and Irradiating 3T3 Cells as a Feeder Source
6. Obtaining, Preserving and Transporting Pediatric Esophageal Biopsies
7. Processing Patient Tissue for Downstream Culture and Analysis
8. Culturing and Expanding Cells
9. Freeze and Store Human Esophageal Epithelial Cells
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 "reprogrammed" 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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µm. Magnification is 200X. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 "guess and check" 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.
The authors have nothing to disclose.
We would like to acknowledge Connecticut Children’s Medical Center Strategic Research Funding for supporting this work.
Primocin | InVivogen | ant-pm2 | |
Isopentane | Sigma Aldrich | 277258-1L | |
Gelatin From Porcine Skin | Sigma Aldrich | G1890-100G | |
DMEM | Thermofisher Scientific | 11965092 | |
Cryomold | TissueTek | 4565 | |
Cryomatrix OCT | Thermofisher Scientific | 6769006 | |
15ml Conical Tubes | Denville Scientific | C1017-p | |
Complete Keratinocyte Serum Free Medium | Thermofisher Scientific | 10724011 | |
Penicillin Streptomycin | Thermofisher Scientific | 15140122 | |
Glutamax | Thermofisher Scientific | 35050061 | |
Insulin Solution | Sigma Aldrich | I9278-5ml | |
Human Epidermal Growth Factor (EGF) | Peprotech | AF-100-15 | |
ROCK Inhibitor (Y-27632) | Fisher Scientific | 125410 | |
F-12 Medium | Thermofisher Scientific | 11765054 | |
Fetal Bovine Serum | Denville Scientific | FB5001 | |
Dispase | Thermofisher Scientific | 17105041 | |
0.05% Trypsin-EDTA | Thermofisher Scientific | 25300062 | |
0.25% Trypsin-EDTA | Thermofisher Scientific | 25200072 | |
100mm Dishes | Denville Scientific | T1110-20 | |
150mm Dishes | Denville Scientific | T1115 | |
50ml Conicals | Denville Scientific | C1062-9 | |
Phosphate Buffered Saline Tablets | Fisher Scientific | BP2944-100 | |
5ml Pipettes | Fisher Scientific | 1367811D | |
10ml Pipettes | Fisher Scientific | 1367811E | |
25ml Pipettes | Fisher Scientific | 1367811 | |
9" Pasteur Pipettes | Fisher Scientific | 13-678-20D | |
NIH 3T3 Cells | ATCC | CRL1658 |