Induced pluripotent stem (iPS) cells have captured the world’s attention and directed an unprecedented focus on regenerative medicine. The potential of iPS cells to aid in the development of new treatments for various diseases is exciting, and researchers are only beginning to discover their potential benefits for humans. iPS cells are more effective if they are interconnected with tissues; however, new technologies are needed to create and transplant these tissues. This study introduces a new connection between endoscopy and regenerative medicine in gastroenterology through specifically addressing how cell sheet technology can be a viable method of tissue creation and transplantation.
Key points
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Research on the use of regenerative medicine in the gastrointestinal field is gaining momentum around the world.
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In this first-ever clinical study of its type in the gastrointestinal field, a new treatment was developed that involved the transfer of tissue-engineered oral mucosal epithelial cell sheets to post–endoscopic submucosal dissection ulcerations.
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This new treatment showed promising results, indicating that it can potentially prevent post–endoscopic submucosal dissection esophageal stricture.
Video of the cell sheet transplantation technique accompanies this article
Introduction
Induced pluripotent stem (iPS) cells, which led to the 2012 Nobel Prize in Physiology or Medicine for Dr Shinya Yamanaka, have captured the world’s attention and directed an unprecedented focus on regenerative medicine. The potential of iPS cells to aid in the development of new treatments for various diseases is very exciting, and researchers are only beginning to discover their potential benefits for humans. iPS cells are more effective if they are interconnected with tissues; however, new technologies are needed to create and transplant these tissues. In the field of regenerative medicine, clinical research involving cell sheet technology has already been conducted. This study introduces a new connection between endoscopy and regenerative medicine in gastroenterology through specifically addressing how cell sheet technology can be a viable method of tissue creation and transplantation.
Introduction
Induced pluripotent stem (iPS) cells, which led to the 2012 Nobel Prize in Physiology or Medicine for Dr Shinya Yamanaka, have captured the world’s attention and directed an unprecedented focus on regenerative medicine. The potential of iPS cells to aid in the development of new treatments for various diseases is very exciting, and researchers are only beginning to discover their potential benefits for humans. iPS cells are more effective if they are interconnected with tissues; however, new technologies are needed to create and transplant these tissues. In the field of regenerative medicine, clinical research involving cell sheet technology has already been conducted. This study introduces a new connection between endoscopy and regenerative medicine in gastroenterology through specifically addressing how cell sheet technology can be a viable method of tissue creation and transplantation.
Post–endoscopic submucosal dissection esophageal stricture
Esophageal endoscopic submucosal dissection (ESD) has recently become a standard endoscopic treatment technique, similar to gastric ESD. However, after a large esophageal ESD, esophageal stricture often arises because of large artificial ulceration. A severe esophageal stricture occurring after ESD is difficult to treat and can require frequent endoscopic balloon dilations. Limitations regarding not only the depth but also the size of the tumor exist in the guidelines of the Japan Esophageal Society on the application of endoscopic resection. Ono and colleagues reported that 90% of patients with lesions more than three-fourths the circumference of the esophagus experienced stricture after ESD. Recently, reports on steroid use for esophageal stricture have been increasing ; however, this has been accompanied by many problems, such as delayed wound healing, immune suppression, optical damage, psychiatric disturbances, diabetes, peptic ulcerations, osteoporosis, and susceptibility to tuberculosis infection. The authors developed a procedure involving the endoscopic transplantation of cultured autologous oral mucosal epithelial cell sheets to an ulcer after endoscopic resection in a canine model to assess the degree to which it could prevent post-ESD esophageal stricture.
Regenerative medicine in endoscopy
The number of reports on regenerative medicine in endoscopy and gastroenterology is increasing. Isolated oral mucosal epithelial cells have been injected into the submucosal tissue after esophageal endoscopic mucosal resection (EMR) in a porcine model. Epithelialization of the ulceration was confirmed 2 weeks after the injection of epithelial cells. Autologous adipose tissue–derived stromal cells that were not cultured were endoscopically injected into submucosal tissue after esophageal EMR in a canine model. The epithelialization of ulcerations was determined to be promoted by autologous adipose tissue–derived stromal cells. The tubular scaffold of the decellularized porcine urinary bladder has been shown to attach, as if through biologic glue, to the surface of a circumferential ulceration after multiple EMRs in a canine model. Furthermore, the subsequent placement of a scaffold made of a decellularized xenogeneic extracellular matrix (ECM) promoted a constructive remodeling response and minimized stricture.
Cell sheet technology
A temperature-responsive polymer [poly( N -isopropylacrylamide)] covalently bound to the surface of a temperature-responsive culture dish has been used as a cell sheet. The culture dish was coated with the temperature-responsive polymer that had a hydrophobic surface at 37°C (normal culture conditions), allowing cells to attach to the surface of the dish ( Fig. 1 A). When the temperature was reduced to 32°C, the surface of the temperature-responsive culture dish became hydrophilic, and the cells were released from the dish. The adhesion molecules that maintain contact between cells can be destroyed through the conventional method of freeing cells from the dish using proteases. In contrast, all of the cells can be removed as a continuous sheet along with the extracellular matrix deposited at the base of the culture dish using a temperature-responsive dish (see Fig. 1 B). Because adhesion molecules are retained on the surface of the cells, the cell sheet can be transplanted onto various tissues without sutures by attaching it for a short period. The number of cells used in single-cell injections is low, whereas the scaffolding technique has no cell content, relying on confluent cells for healing to occur. Cell sheets contain high cell density and can also be used to transplant stem cells.
Treatment using autologous oral mucosal epithelial cell sheets for the prevention of stricture
The authors developed a regenerative medical treatment using tissue-engineered epithelial cell sheets. Epithelial cells isolated from the patient’s own oral mucosa were seeded c
c Oral mucosal epithelial cells were collected by remove of epithelial layers after treatment with 1000 U/ml dispase (Godo Shusei, Tokyo, Japan) at 37°C for 2 hours. Epithelial layers were carefully removed from the substantia propria by surgical forceps and treated with 0.25% trypsin/0.1% ethylenediamine tetraacetic acid (EDTA) (GIBCO-Invitrogen, Carlsbad, CA, USA) for 20 minutes at 37°C. Isolated epithelial cells were suspended in a keratinocyte culture medium (KCM) composed of a basal mixture of 3 parts Dulbecco’s modified Eagle’s medium (DMEM) and 1 part nutrient mixture F-12 Ham (Sigma), and supplemented with 2 nmol/L triiodothyronine (Wako Pure Chemicals, Osaka, Japan), 5 μg/mL insulin (Eli Lilly, Indianapolis, IN, USA), 10 ng/mL epidermal growth factor (Higeta Shoyu, Chiba, Japan), 0.4 μg/mL hydrocortisone (Kowa Pharmaceutical, Tokyo, Japan), 1 nM cholera toxin (List Biological Laboratories, Campbell, CA), 0.25 μg/mL amphotericin B (Bristol-Myers Squibb, New York, NY, USA), 40 μg/mL gentamicin (Schering-Plough, Kenilworth, NJ, USA), and 5% autologous human serum.
onto temperature-responsive culture inserts and cultured dd Suspended epithelial cells were seeded onto temperature-responsive polymer (N-isopropylacrylamide) grafted cell culture inserts (UpCell Insert; CellSeed, Tokyo, Japan) were prepared with the use of commercial cell culture inserts (Falcon; Becton Dickinson, Franklin Lakes, NJ, USA) at a density of 4 to 8×104 cells/cm 2 , and cultured for 16 days at 37°C in a humidified atmosphere containing 5% CO 2 .
for 16 days at 36°C ( Fig. 2 [1–2]). Then, the autologous cell sheets ( Figs. 3 and 4 ) were then transplanted with endoscopic forceps onto the bed of the esophageal ulcer after ESD (see Fig. 2 [3–4]; Fig. 5 A). The authors reported 9 cases that underwent oral mucosal epithelial cell sheet transplantation. Cell sheet transplantation effectively prevented post-ESD esophageal stricture.
