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.
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).
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).
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.
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.
MATERIALS AND METHODS
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.
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).
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.
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).
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.
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).
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).
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.
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.
1. Merani S, Toso C, Emamaullee J, et al. Optimal implantation site for pancreatic islet
transplantation. Br J Surg
2008; 95: 1449.
2. Bennet W, Groth CG, Larsson R, et al. Isolated human islets trigger an instant blood mediated inflammatory reaction: Implications for intraportal islet
transplantation as a treatment for patients with type 1 diabetes. Ups J Med Sci
2000; 105: 125.
3. Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet
transplantation. N Engl J Med
2006; 355: 1318.
4. van der Windt DJ, Bottino R, Casu A, et al. Rapid loss of intraportally transplanted islets: An overview of pathophysiology and preventive strategies. Xenotransplantation
2007; 14: 228.
5. Balamurugan AN, Bottino R, Giannoukakis N, et al. Prospective and challenges of islet
transplantation for the therapy of autoimmune diabetes. Pancreas
2006; 32: 231.
6. Roep BO, Stobbe I, Duinkerken G, et al. Auto- and alloimmune reactivity to human islet
allografts transplanted into type 1 diabetic patients. Diabetes
1999; 48: 484.
7. Fox IJ, Schafer DF, Yannam GR. Finding a home for cell transplants: Location, location, location. Am J Transplant
2006; 6: 5.
8. Pileggi A, Molano RD, Ricordi C, et al. Reversal of diabetes by pancreatic islet
transplantation into a subcutaneous, neovascularized device. Transplantation
2006; 81: 1318.
9. Kawakami Y, Iwata H, Gu Y, et al. Modified subcutaneous tissue with neovascularization is useful as the site for pancreatic islet
transplantation. Cell Transplant
2000; 9: 729.
10. Hussey AJ, Winardi M, Wilson J, et al. Pancreatic islet
transplantation using vascularised chambers containing nerve growth factor ameliorates hyperglycaemia in diabetic mice. Cells Tissues Organs
2010; 191: 382.
11. Ohashi K, Yokoyama T, Yamato M, et al. Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat Med
2007; 13: 880.
12. Yokoyama T, Ohashi K, Kuge H, et al. In vivo engineering of metabolically active hepatic tissues in a neovascularized subcutaneous cavity. Am J Transplant
2006; 6: 50.
13. Ohashi K, Waugh JM, Dake MD, et al. Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases. Hepatology
2005; 41: 132.
14. Kawakami Y, Iwata H, Gu YJ, et al. Successful subcutaneous pancreatic islet
transplantation using an angiogenic growth factor-releasing device. Pancreas
2001; 23: 375.
15. Dufour JM, Rajotte RV, Zimmerman M, et al. Development of an ectopic site for islet
transplantation, using biodegradable scaffolds. Tissue Eng
2005; 11: 1323.
16. Blomeier H, Zhang X, Rives C, et al. Polymer scaffolds as synthetic microenvironments for extrahepatic islet
2006; 82: 452.
17. Vériter S, Mergen J, Goebbels RM, et al. In vivo selection of biocompatible alginates for islet
encapsulation and subcutaneous transplantation. Tissue Eng Part A
2010; 16: 1503.
18. Ohki T, Yamato M, Murakami D, et al. Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model. Gut
2006; 55: 1704.
19. Shimizu T, Sekine H, Isoi Y, et al. Long-term survival and growth of pulsatile myocardial tissue grafts engineered by the layering of cardiomyocyte sheets. Tissue Eng
2006; 12: 499.
20. Shimizu H, Ohashi K, Utoh R, et al. Bioengineering of a functional sheet of islet
cells for the treatment of diabetes mellitus
2009; 30: 5943.
21. Yang J, Yamato M, Sekine H, et al. Tissue engineering using laminar cellular assemblies. Adv Mater
2009; 21: 1.
22. Yang J, Yamato M, Shimizu T, et al. Reconstruction of functional tissues with cell sheet engineering
2007; 28: 5033.
23. Grossmann J. Molecular mechanisms of “detachment-induced apoptosis—Anoikis.” Apoptosis
2002; 7: 247.
24. Thomas F, Wu J, Contreras JL, et al. A tripartite anoikis-like mechanism causes early isolated islet
2001; 130: 333.
25. Thomas FT, Contreras JL, Bilbao G, et al. Anoikis, extracellular matrix, and apoptosis factors in isolated cell transplantation. Surgery
1999; 126: 299.
26. Wang RN, Rosenberg L. Maintenance of beta-cell function and survival following islet
isolation requires re-establishment of the islet
-matrix relationship. J Endocrinol
1999; 163: 181.
27. Tsai AG, Friesenecker B, Mazzoni MC, et al. Microvascular and tissue oxygen gradients in the rat mesentery. Proc Natl Acad Sci U S A
1998; 95: 6590.
28. Balamurugan AN, Gu Y, Tabata Y, et al. Bioartificial pancreas transplantation at prevascularized intermuscular space: Effect of angiogenesis induction on islet
2003; 26: 279.
29. Fumimoto Y, Matsuyama A, Komoda H, et al. Creation of a rich subcutaneous vascular network with implanted adipose tissue-derived stromal cells and adipose tissue enhances subcutaneous grafting of islets in diabetic mice. Tissue Eng Part C Methods
2009; 15: 437.
30. Dufour JM, Rajotte RV, Kin T, et al. Immunoprotection of rat islet
xenografts by cotransplantation with sertoli cells and a single injection of antilymphocyte serum. Transplantation
2003; 75: 1594.
31. Kin T, Rajotte RV, Dufour JM, et al. Development of an immunoprivileged site to prolong islet
allograft survival. Cell Transplant
2002; 11: 547.
32. Elloumi Hannachi I, Itoga K, Kumashiro Y, et al. Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing. Biomaterials
2009; 30: 5427.
33. Itoga K, Yamato M, Kobayashi J, et al. Cell micropatterning using photopolymerization with a liquid crystal device commercial projector. Biomaterials
2004; 25: 2047.
34. Tsuda Y, Shimizu T, Yamato M, et al. Cellular control of tissue architectures using a three-dimensional tissue fabrication technique. Biomaterials
2007; 28: 4939.
35. Callewaert H, Gysemans C, Cardozo AK, et al. Cell loss during pseudoislet formation hampers profound improvements in islet
lentiviral transduction efficacy for transplantation purposes. Cell Transplant
2007; 16: 527.
36. Assady S, Maor G, Amit M, et al. Insulin production by human embryonic stem cells. Diabetes
2001; 50: 1691.
37. Shi Y. Generation of functional insulin-producing cells from human embryonic stem cells in vitro. Methods Mol Biol
2010; 636: 79.
38. Tateishi K, He J, Taranova O, et al. Generation of insulin-secreting islet
-like clusters from human skin fibroblasts. J Biol Chem
2008; 283: 31601.
39. Carter JD, Dula SB, Corbin KL, et al. A practical guide to rodent islet
isolation and assessment. Biol Proced Online
2009; 11: 3.
40. Sutton R, Peters M, McShane P, et al. Isolation of rat pancreatic islets by ductal injection of collagenase. Transplantation
1986; 42: 689.
41. Peakman M, McNab GL, Heaton ND, et al. Development of techniques for obtaining monodispersed human islet
1994; 57: 384.
42. Akiyama Y, Kushida A, Yamato M, et al. Surface characterization of poly(N-isopropylacrylamide) grafted tissue culture polystyrene by electron beam irradiation, using atomic force microscopy, and X-ray photoelectron spectroscopy. J Nanosci Nanotechnol
2007; 7: 796.