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Reversal of Diabetes by the Creation of Neo-Islet Tissues Into a Subcutaneous Site Using Islet Cell Sheets

Saito, Takahiro1,2; Ohashi, Kazuo2,3; Utoh, Rie2; Shimizu, Hirofumi1; Ise, Kazuya1; Suzuki, Hiroyuki1; Yamato, Masayuki2; Okano, Teruo2; Gotoh, Mitsukazu1

doi: 10.1097/TP.0b013e3182375835
Basic and Experimental Research

Background. There remains a paucity of therapeutic approaches to completely treat diabetes mellitus. This study was designed to develop a dispersed islet cell-based tissue engineering approach to engineer functional neo-islet tissues in the absence of traditional bioabsorbable scaffold matrices.

Methods. Specialized coated plastic dishes were prepared by covalently immobilizing a temperature-responsive polymer, poly(N-isopropylacrylamide), onto the plastic followed by coating with laminin-5. Dispersed rat islet cells were plated on the laminin-5-poly(N-isopropylacrylamide) dishes. After 2 days of culturing, islet cells were harvested as a uniformly connected tissue sheet by lowering the culture temperature from 37°C to 20°C for 30 min. Two harvested islet cell sheets were transplanted into the subcutaneous space of streptozotocin-induced diabetic severe combined immunodeficiency (SCID) mice to engineer neo-islet tissues in vivo. Therapeutic effects were investigated after the tissue engineering procedures.

Results. In all of the diabetic SCID mice transplanted with the islet sheets, serum hyperglycemia was successfully reverted to a steady normoglycemic level. The recipient SCID mice demonstrated positive for serum rat C-peptide and elevated serum insulin levels. Moreover, the islet cell sheet-transplanted SCID mice demonstrated rapid glucose clearance and return of serum glucose levels after intraperitoneal glucose tolerance test. Histological examination revealed that the transplanted islet cell sheets were structured as flat clusters of islet tissues in which an active vascular network manifested within and surrounding the newly formed tissues.

Conclusions. This study describes a new proof-of-concept therapeutic approach to engineer functional neo-islet tissues for the treatment of type 1 diabetes mellitus.

1 Department of Surgery, Fukushima Medical University, Fukushima, Japan.

2 Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Shinjuku, Tokyo, Japan.

This work was supported, in part, by Special Coordination Funds for Promoting Science and Technology (K.O., M.Y., and T.O.), Global Center of Excellence Program (K.O. and M.Y.), and Grant-in-Aid (No. 21300180 to K.O. and No. 20591227 to M.G.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

M.Y. serves as consultant and shareholder for CellSeed, Inc. (Japan). T.O. is an investor in CellSeed, Inc. (Japan) and an inventor/developer designated on the patent for the temperature-responsive culture surfaces (patent nos. JP1972502, US5284766, FR0382214, NL0382214, DE0382214, GB0382214, SE0382214, and CH0382214).

3 Address correspondence to: Kazuo Ohashi, M.D., Ph.D., Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.


T.S., K.O., R.U., H.Sh., K.I., and M.G. participated in research design; T.S., R.U., H.Su., K.I., and M.Y. participated in the performance of the research; T.S., R.U., and H.Sh. participated in data analysis.; K.O., K.I., T.O., and M.G. participated in interpreting the data; and T.S., K.O., R.U., and K.I. participated in writing the manuscript.

Received 1 February 2011. Revision requested 22 February 2011.

Accepted 12 September 2011.

Cell-based therapies using pancreatic islets have emerged as a promising new approach for the treatment of insulin-dependent diabetes mellitus. Currently, the preferred organ for the transplantation of islet cells is the liver. The majority of the clinical trials treating type 1 diabetes mellitus are designed for transplantation of islets into the liver through the portal vascular system (1, 2), but this approach is limited. There is a gradual loss of the transplanted islets rendering the majority of the recipients to switch back to insulin-dependent from their independent status (3). It has been suggested that portal infusion of islets is associated with a number of complications, which include instant blood-mediated inflammatory reactions (2, 4), complement cascade activation (5), and leukocyte infiltration (6). Ultimately, these immune-related problems result in graft failure, and the majority of the recipients switch back to insulin-dependent from their independent status (3).

To prolong the longevity of the transplanted islet cells, there are emerging new approaches designed to bioengineer functional islet systems at extra-hepatic sites, such as subcutaneous, subrenal, and abdominal spaces. Of these candidate sites, the subcutaneous site remains the most attractive, because the transplantation of islet cells and tissue systems to this particular area can be performed with minimal invasiveness (7–13). Dispersed islet cells infused into the subcutaneous space have been shown to survive, but only using approaches that either prevascularize the transplantation site (8, 14) or use a synthetic polymer scaffold to allow for cell attachment to the ectopic site (15–17). In the absence of these additional modifications, the success rate of the engraftment of the transplanted islet cells remains poor. Moreover, it is not clear whether these additional steps required to promote islet cell engraftment and survival are readily amenable to the clinical setting.

For these reasons, our laboratory developed a novel cell sheet technology in which individually dispersed cells are allowed to form a thin, contiguous monolayer. The cells comprised in this cell sheet format are able to communicate among themselves and act as an intrinsic biological system that can recognize and sense changes in physiological parameters after transplantation. In our previous investigations, cell sheets were engineered from a number of sources including oral mucosal cells (18), cardiomyocytes (19), liver cells (11), and recently islet cells (20). These newly engineered cell sheets have the advantage over individually dispersed cell clusters by allowing for multilayer approach leading to the creation of three-dimensional tissues (11, 19), which has been shown to prolong the viability and functionality of these cell sheets for therapeutic applications.

This study was designed to engineer functional islet tissues in a subcutaneous site using our novel cell sheet technology. Our results demonstrate that the islet cell sheets are capable of engraftment in the subcutaneous space and continually function as a newly bioengineered islet tissue to normal the glycemic index in the diabetic mice. In all, the islet tissue engineering approach described in this study is a unique and effective tissue engineering procedure as a potential treatment modality for diabetes mellitus and has many benefits, including its longevity of function, over previously published approaches using individually dispersed cell systems.

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Therapeutic Potential of Islet Tissue Engineering Using Islet Cell Sheet

As shown in Figure 1A, nonfasting blood glucose (NFBG) levels in all the recipient diabetic severe combined immunodeficiency (SCID) mice (n=7) returned to a state of normoglycemia within 1 week after the transplantation of islet cell sheets, whereas all the mice in the sham-operated control group (n=6) remained hyperglycemic. In some diabetic SCID mice, we performed the injection of dispersed islet cells (equivalent cell number of two islet cell sheets) into the subcutaneous site and found that there were minimal decreases in their NFBG levels, but all the mice showed persistent hyperglycemic status after the cell injection (Fig. 1A). In another set of experiments, stable and long-term (>110 days) therapeutic effects were confirmed (Fig. 1B). After diabetic mice achieved a state of normoglycemia, two mice were chosen for graft removal. Immediately after the graft removal, a steep rise in the NFBG levels of both mice was detected as it reattained a hyperglycemic state (Fig. 1B). The recipient SCID mice showed improved clinical conditions with a steady increase in body weight after the transplantation procedure (Fig. 1C).



Histological assessments of tissue samples were made using tissues harvested at days 4 (Fig. 2A) and 60 (Fig. 2B–F). The histology showed that clusters of islet tissues were formed at the transplantation site. The cells in the neo-islet tissues retained structural morphology characteristic of pancreas islets. Strong cytoplasmic expression of rat insulin and glucagon was observed following immunohistochemistry, which confirmed the islet-specific phenotypes of the neo-engineered islet tissues in vivo (Fig. 2B–D). We observed that numerous platelet-endothelial cell adhesion molecule (PECAM)-1-positive cells were found at close proximity to the insulin-positive grafts at day 4 (Fig. 2A). Furthermore, intense vascular networks composed of PECAM-1-positive vascular endothelial cells were formed within and surrounding the neo-engineered islet tissues at day 60 (Fig. 2E,F). These findings also demonstrated the ability to form blood vascular network at an early stage after cell sheet transplantation and also to synthesize and store insulin and glucagon. Species specificity of these histological staining was confirmed by negative immune complex signals detected in the normal mouse pancreas samples (data not shown).



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Functional Confirmation of the Engineered Neo-Islet Tissues

To further confirm the functionality of the engineered neo-islet tissues in vivo, intraperitoneal glucose tolerance tests (IPGTTs) were assessed at day 30. After administration of the glucose into the peritoneal space, the blood glucose levels of the control diabetic SCID mice were immediately elevated at over 500 mg/dL and remained above 350 mg/dL at the end of the experiment (150 min; Fig. 3). In contrast, the blood glucose levels of the recipient SCID mice and nondiabetic naive SCID mice showed temporal elevations at 15 and 30 min and thereafter showed sharp declines and returned to the normal levels (Fig. 3).



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Detection of Rat-Specific C-Peptide in the Recipient Mice

Because C-peptide is species-specific and is produced during the cleavage step from the catalysis of proinsulin, we measured the serum levels of rat-specific C-peptide. As shown in Figure 4A, significant amount of rat C-peptide was detected only in the islet cell sheet-transplanted SCID mice. Accordingly, significantly high insulin levels were detected in the blood samples of recipient SCID mice compared with those of sham-operated control diabetic mice. There was no statistical significance in the blood insulin levels between the graft recipient SCID mice versus normal SCID mice (Fig. 4B). These findings confirmed that the phenotypic correction of the diabetic status was due to the de novo production of rat insulin from the engineered neo-islet tissues.



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This study describes a novel tissue engineering approach that uses dispersed islet cells to form a contiguous monolayer sheet that can be readily used for transplantation into ectopic sites for the production of therapeutic proteins necessary for the treatment of diabetes mellitus. In recipient diabetic SCID mice receiving the transplanted islet cell sheets, normal levels of blood glucose were restored, and high serum levels of rat-specific C-peptide were detected. This strongly demonstrates that engineered neo-islet tissues were capable of producing and secreting insulin into the systemic circulation. The functionality of the engineered neo-islet tissues was further confirmed by the IPGTTs. The ability of the de novo engineered neo-islet tissues to sense and release insulin was likely attributed to the formation of a highly vascular network within and surrounding the transplanted tissues.

An important feature of our approach to engineer islet cell sheet in vitro is the use of a temperature-responsive poly(N-isopropylacrylamide) (PIPAAm)-grafted dishes (21, 22). A simple lowering of the incubator temperature allows for easy detachment of the cultured cell sheet without the use of harmful proteolytic enzymes. This enables us to harvest the cell sheets as a contiguous monolayer that retains its native intercellular communications and intracellular microstructures, which are essential for normal cellular function (11, 19, 21). These interwoven cells exhibited the formation and maintenance of desmosome structures during the 2-day culture period (20). In addition, numerous secretion granules were observed throughout the cytoplasm of the islet cells within the sheets (20). The detection of structural elements found in normal intact islet tissue is promising for the use of this approach in producing insulin in vivo.

It is interesting, however, the differences in the survivability between individually dispersed islet cells compared with our engineering cell sheet format. Although both the dispersed cells and our newly formed tissue sheets are capable of engrafting and functioning within the transplantation site, our data would suggest that the tissue sheet format can exhibit prolonged survivability compared with individually dispersed islet cells and/or clusters. One possible explanation is that islet cells are prone to progressing toward apoptotic cell death pathways once they are dispersed into single cells (19, 23–26), so it may be plausible that the islet cells within the cell sheet format may be protected from entering the cell death pathway and allow for the prolonged survival upon transplantation.

Another possibility may be the close proximity of the islet cells to the vascular network that is formed during the tissue engineering process. Researchers have reported that the diffusion-based oxygen supply is limited to 50 to 100 μm distance from the vascular channel (27). As islets are a clustered mass of cells made of approximately 3000 cells with a diameter of 50 to 400 μm, it is possible that the diffusion of oxygen is severely limited to only a small proportion of cells that are located at a proximal surface to the active vascular network. Considering the fact that the subcutaneous space is not actively vascularized, individual cells clustered in a ball may not be adequately perfused with nutrients. A number of researchers have designed vascularized platforms within the subcutaneous space in hopes of enhancing the islet survival time (28, 29). On the other hand, our monolayered cell sheet array may not be limited by the diffusion of gases for their survival in the absence of vascularized networks. It may also be possible that islet cell sheet has a high ability to recruit vascular endothelial cells, which results in the creation of an active vascular network. We speculate this latter possibility because of our findings that numerous PECAM-1-positive cells were detected around the neo-islet tissues at an early phase (day 4 after transplantation). It is, therefore, important to note that the present islet cell sheet-based approach does not require such preparation of vascularized platform but results in engineering functional islet tissues.

Another key benefit in the development of the islet cell sheet technology for transplantation into ectopic subcutaneous sites is its relatively minimal invasiveness to the patient and can be performed under local anesthesia. Moreover, accessibility to the transplanted site would be a simple procedure in cases where: (1) subsequent biopsies are needed to examine the engraft and differentiation of the islet cell sheets or (2) additional transplantation procedures can be performed to increase the therapeutic efficacy. However, to advance our current methodology toward the clinics, there are several remaining issues that need to be addressed. One of the major ones is the prevention of the transplanted cells from host immunologic allosensitization. Recently, cotransplantation of islets with Sertoli cells has shown to have immunoprotective effects on the islets from allogenic immune responses (30, 31). In response to this issue and these recent findings, we have attempted to engineer a temperature-responsive culture surface that would enable us to pattern a monolayer structure using multiple types of cells (32, 33). Using this type of culture system may facilitate the production of a monolayer islet cell sheet interwoven with Sertoli cells. Another important factor to consider is the size of the transplant area in the human patients. Considering the fact that there is a big difference in the body size between mice and human beings, a larger transplant area of neo-islet may be required to achieve therapeutic effects. One potential resolution for this issue would be to create more complex and multilayered stratified tissues within a confined space. Toward this goal, our group has attempted to create a stamp manipulator system that allows us to precisely stratify multiple cell sheets within a single confined site (32, 34). By integrating the cellular biology with the new tissue culture technologies, therapeutic targets may be treatable in the foreseeable future by using this multilayering approach.

As this methodology advances toward clinical consideration as a viable methodology to treat human disease, we have to be cognizant that the availability of islet cells will remain a limited resource. We found that a relatively small number of cells (∼3.3×106 islet cells) were needed to provide a state of persistent normoglycemia in mice, but further studies are needed to determine what the minimal number of cells are needed to maintain therapeutic efficacy. However, islet cell can be lost during the islet dispersion process and cell attachment process (35). For these reasons, further refinements in the cell culture methodologies are needed to optimize islet cell sheet engineering with the minimal number of cells isolated from donor samples.

In conclusion, we have experimentally succeeded in reverting the hyperglycemic state of a mouse model of diabetes mellitus by de novo engineering islet tissues in an ectopic subcutaneous space. In recent years, considerable efforts had been made in generating insulin-producing cells from other cellular sources, including embryonic stem cells (36, 37), or induced pluripotent stem cells (38). Because these cell-generation processes are normally conducted under the culture condition, our cell sheet engineering approach could contribute in advancing the regenerative medicine using these newly generated cells.

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Male Lewis rats (LEW/CrlCrlj, 8 to 12 weeks old; Charles River, Yokohama, Japan) were used as islet donors. Male SCID mice (C.B-17/lcr-scid/scidJcl, 7–10 weeks old; CLEA, Tokyo, Japan) were used as graft recipients. All animal studies were performed in accordance with the institutional guidelines.

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Islet Isolation and Single Cell Purification

Pancreatic islets were isolated from Lewis rats as described elsewhere (20, 39, 40). Islets were subsequently cultured in Roswell Park Memorial Institute 1640 medium (Sigma, St. Louis, MO). The next day, islets were dispersed using Trypsin–EDTA (Invitrogen, Carlsbad, CA) to obtain single cells (41).

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Islet Cell Culture on the Temperature-Responsive Culture Dish and Recovery of Islet Cell Sheet

Dispersed islet cells were cultured to engineer monolayered islet cell sheet as described previously (20). In brief, temperature-responsive culture dishes were created by covalently grafting PIPAAm by electron beam irradiation, and this surface was subsequently coated with rat laminin-5 (Millipore, Billerica, MA). A previous study confirmed that the grafted PIPAAm remained on the dish side during cell sheet harvesting process, and thus PIPAAm does not attach to the cell sheet side (42). Dispersed islet cells were plated at a density of 0.57×106 cells/cm2 on 35-mm dishes. When the cultured islet cells reached confluency at day 2, the cultured cells were detached from the PIPAAm dish as a uniformly connected tissue sheet by lowering the culture temperature to 20°C for 30 min.

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Induction of Diabetic Status and Neo-Islet Tissue Engineering Procedures

SCID mice were rendered diabetic by intraperitoneal injection of streptozotocin (Sigma; 0.22 mg/gram body weight). Only SCID mice that exhibited NFBG values more than 350 mg/dL for 2 consecutive days were categorized as diabetic mice. Islet cell sheets were recovered with the support membrane for transplantation into the subcutaneous site as previously described (11, 20). To transplant the islet cell sheets, an L-shaped skin incision in the left dorsal skin region was exposed. After a 5-min attachment period, the support membrane was carefully removed. An additional layer of islet cell sheet was then transplanted on top of the first sheet. Cell counting evaluation revealed that two layers of islet cell sheet were made up of 3.3±0.1×106 islet cells (n=5).

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Validation of Therapeutic Effects of Engineering Neo-Islet Tissues

Blood samples were periodically obtained by tail snipping to assess NFBG. At day 60, serum samples were obtained to measure rat-specific C-peptide levels and rat nonspecific insulin levels using enzyme-linked immunosorbent assay kits from Wako (Osaka, Japan) and Shibayagi (Gunma, Japan), respectively. For two recipient mice at either day 7 or 21, subcutaneous neo-islet tissues were excised by removing the portion of the surrounding abdominal wall and adjoining skin areas.

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Intraperitoneal Glucose Tolerance Tests

The functionality of the newly engineered neo-islet tissues was evaluated in vivo by performing IPGTTs at day 30. After 18 hr of fasting, the mice received intraperitoneal inoculation of a glucose solution (2 mg/g body weight).

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Histological and Immunohistochemical Analyses

At day 60, subcutaneous tissues were harvested and fixed in 10% buffered formalin. Specimens were paraffin-embedded and sectioned (5 μm thick) for hematoxylin-eosin staining and immunohistochemical staining. For immunohistochemical analyses, sections were incubated overnight at 4°C with either anti-rat insulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-rat glucagon (Progen, Heidelberg, Germany) followed by secondary antibody labeling. Visualization of the immune complexes was performed by incubating with 3,3′-diaminobenzidine. Sections were also used for immunofluorescence analysis of rat insulin and PECAM-1 by incubating with rabbit anti-rat insulin antibody (Santa Cruz Biotechnology) and goat anti-rat PECAM-1 antibody (Santa Cruz Biotechnology). Secondary antibody labeling was performed using Alexa-Fluor-594-conjugated anti-rabbit immunoglobulin (Invitrogen) and Alexa-Fluor-488-conjugated anti-goat immunoglobulin. The slides were mounted with mounting media containing 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).

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Statistical Analyses

All of the values calculated were provided as mean±standard error. The Student's t test was used for comparison between two groups. When the data set did not have equal variance, Mann-Whitney U test was used. When more than two groups were compared, an analysis of variance was performed followed by Games-Howell post hoc test. A probability value of P less than 0.05 was considered statistically significant.

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The authors thank Ms. K. Kanegae (Tokyo Women's Medical University), Ms. H. I and Ms. Y. Kikuta (Fukushima Medical University) for technical assistance, Mr. H. Watanabe (CellSeed) for providing the modified temperature-responsive culture surfaces, and Dr. F. Park (Medical College of Wisconsin) for his editorial assistance of this manuscript.

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Islet; Diabetes mellitus; Cell sheet engineering; Dispersed islet cells

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