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Leading regenerative medicine research in Japan (2) -All 3 episodes-

Dr. Teruo Okano

Photo:Dr. Teruo Okano

Dr. Teruo Okano

Using cell sheets to make replacement parts for organs

Interviewer :
What can you make using cell sheets?

Okano :
Cell sheets secrete on their surface facing the culture surface adhesion proteins including fibronectin and laminin-5, that bind with the culture surface. This "sticky" surface makes the cell sheets capable of functioning as a sort of adhesive tape. In the past, we had to use DISPASE(a protease) to detach the cells from the surface, which meant the adhesion proteins would be decomposed and made the sheets difficult to manipulate. But using our proprietary cell sheets, we can make epithelial sheets of all types.
Between 2001 and 2003, we worked with Tohoku University Professor Koji Nishida, then an ophthalmolgist at the Osaka University School of Medicine, to develop sheets of corneal epithelium for use in regenerative transplantation. By culturing a 2 mm square of oral mucosal epithelial cells of the patient, we could make a sheet that maintains its own adhesive proteins and can be detached from the culture surface by temperature control, which could then be used in suture-free autologous transplantation, in which the transplant adheres securely to the cornea within about 10 minutes. This cell sheet transplantation has already been used successfully in 30 patients.

Interviewer :
Isn't it difficult to conduct clinical trials?

Okano :
Yes, it takes an enormous amount of data to conduct a full-fledged clinical trial of safety and efficacy in Japan.

Cell sheets in corneal transplantation attract worldwide attention

Chart:Cell sheets in corneal transplantation attract worldwide attention

Okano :
When I presented this work at an international scientific meeting, I met Professor Odile Damour from France who was very interested in our work and since then has been strongly supportive of it. She read our New England Journal of Medicine paper, and proposed to join our clinical trial for this treatment to be conducted in France by our Japanese venture business CellSeed Inc. Then we started jointly the clinical trial.
This involved testing the process of collecting a 2 mm square of oral mucosal epithelial cells from the lining of the patient's mouth, culturing it to make a cell sheet and using it in corneal transplantation. We treated 26 cases between September 2007 and July 2009, and are now in the middle of a one-year follow-up. If all goes well, we plan to seek approval in June next year or later. If granted, it will allow us to begin marketing the treatment in Europe. We have also had successes treating cornea damage that conventional transplants had not been able to help in Japan. One of our collaborators in France has encouragingly said that this is a treasure for humanity.

Cell sheets also used in regenerative approaches to cardiomyopathy and esophageal cancer.

Photo:Dr. Teruo Okano

Dr. Teruo Okano

Interviewer :
I understand that your group has had some successes in using cell sheets to treat cardiomyopathy.

Okano :
We have been working with Dr. Yoshiki Sawa, a cardiologist at Osaka University. We have received approval from the TWMU Institutional Review Board to use our cell sheets in patients within the university, but in order for us to use them in other university hospitals, we would need to conduct a full-fledged clinical trial. So we have been training physicians in Dr. Sawa's group in how to grow their own cell sheets, and after a long collaboration, have begun to do some clinical studies at Osaka University.
Dr. Sawa's group accepted a patient with dilated cardiomyopathy who had been waiting for a year and a half using a left ventricular pump while waiting for a transplant. It is extremely difficult to find donors in Japan, meaning that patients have to wait for long periods.
In our procedure, myoblasts (muscle progenitor cells) were collected from the patient's leg muscles, grown in cell sheets, and re-transplanted into the heart of the patient. These cells secrete constantly angiogenesis induction factors, and after three months the patient was off heart support, and in seven months was discharged from the hospital.
Resection of esophageal cancers usually involves opening the neck, chest and abdomen. We have used endoscopy to introduce cell sheets developed from oral mucosal cells following resection of tumors of the esophageal epithelium, which was less invasive and helped reduce post-operative stenosis, allowing the patient to return after only a few days of hospitalization.
You can see how we have developed leading applications for cell sheets in the treatment of conditions of the cornea, heart and esophagus. These sheets secrete growth factors such as VEGF and HGF and cytokines inducing angiogenesis, allowing their engraftment into the transplant site. We are able to engraft 100% of the transplanted cells into the target site by attaching cell sheets to it. This is not possible if we use cells separated from each other by treatment with a protease, where they are not able to communicate with surrounding tissues in this manner, which impairs engraftment. This is one of the features of our technology, and one of the true dreams of regenerative medicine

Interviewer :
How is it that cells derived from an organ are able to become a part of another organ after transplanted?

Okano :
In the cornea, for example, when we transplant a sheet of cells derived from oral mucosa, it forms a communicative environment with surrounding tissues, in which it "differentiates" into corneal epithelium, or forms a transparent epithelial tissue. Similarly in the heart, we see that attached cell sheets begin to form gap junctions with heart muscles within about 30 minutes of transplantation. Heart muscle cells express connexin 43, and its hexamers (connexon hexamers) form the junctions, allowing the attached cells to give "pulses" in synchrony with electrical signals.

Cell sheets at millimeter thickness

Okano :
If you stack multiple layers of cell sheets to form a tissue, they will necrose if they do not receive sufficient oxygen and nutrients. If the tissue is less thick than 100 to 200 μm, oxygen and glucose can be supplied by simple diffusion. But if it is thicker, capillaries are needed to carry such resources. When designing a tissue with multiple layers of cell sheets of approximately 100 μm each, we have found that by adding 5-10% of vascular endothelial cells to the culture, we can achieve formation of a capillary network like structure. When we transplant this 100μm thick construct into host subcutaneous tissues, within 10 hours we see the vascular network of the donor begins to form connections with its host counterparts, enabling blood flow and stable integration. By repeating the process every 10 hours, we are able to build tissues with thicknesses on the millimeter scale.


Interviewed by Miwako Honma (supervisor of iPS Trend website), Katsuaki Sato (Japan Science Technology Agency)
Published on 2 November, 2009

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Dr. Teruo Okano

Professor, Tokyo Women's University,
Director, Institute of Advanced Biomedical Engineering and Science,
Professor, University of Utah,
Member, Science Council of Japan

After receiving his Ph. D. in polymer chemistry from Waseda University in 1979, Dr. Okano served as an assistant and lecturer at Tokyo Women's Medical University (TWMU) before taking an assistant professorship at the University of Utah. He returned to TWMU as an assistant professor, and was appointed to professorship at both universities in 1994. He was appointed director of the TWMU medical engineering facility in 1999, and has served as the head of the Institute of Advanced Biomedical Engineering and Science at the same university since 2001.
His research interests include biomaterials, artificial organs, drug delivery systems, and regenerative medical engineering. He is particularly focused on how regenerative functions can be achieved by fine-tuning polymer structure. He developed the concept of cell sheet engineering, which first entered the clinic with bioengineered corneal epithelium. He hopes to extend to regenerative applications in the cardiovascular system, liver, and bladder as well. He is the director of the Japanese Society for Biomaterials, and a member of the board of the Japan Society of Drug Delivery System, the Japanese Society for Tissue Engineering, and the Japanese Society of Inflammation and Regeneration.

Awards and Honors
1990, 1995, 1996 Outstanding Paper Award from the American Controlled Release Society
1992 Japanese Society for Biomaterials Prize
1997 Clemson Award for Basic Research
1998 Society of Polymer Science Japan Prize
2000 Founders Award, Controlled Release Society
2000 Fellow, Biomaterials Science and Engineering
2005 Leona Esaki Prize
2006 Nagai Innovation Award, Controlled Release Society
2009 Medal with Purple Ribbon for scholarly or artistic achievement

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