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The revascularization of islets of Langerhans transplanted in heterotopic sites like the liver by portal vein embolization or the renal subcapsular space is a major process necessary for the viability of grafted cells. This process has been extensively studied by different techniques and the results have shown that islet revascularization is an early phenomenon that takes place soon after transplantation. In this report we have analyzed by a double indirect immunofluorescence technique, the revascularization process of purified endocrine islet beta-cells transplanted in the renal subcapsular space of syngeneic rats. Lewis rats were grafted with islets cultured for 24 h, with a suspension of purified beta-cells cultured for 24 h, and with a suspension of purified beta plus nonbeta-cells cultured for 24 h. Rats were killed at different days after implantation and the kidney bearing the grafts were snap frozen and immunohistochemically stained with a rabbit anti factor VIII antiserum (which labels endothelial cells). Immunocytochemical analysis revealed that cultured islets completed revascularization by days 3-5 after transplantation, as shown by the detection of capillary endothelial cells within and surrounding the islets. Within purified endocrine beta-cell grafts, the presence of numerous endothelial cells was not observed until days 10-14, indicating that revascularization of beta-cells with host vessels is not such an early phenomenon as it takes place in whole isolated islets. Conversely, the addition of a population of endocrine nonbeta-cells to the purified islet cell grafts, partially accelerated the revascularization of pure beta-cell grafts, which showed the presence of abundant capillary endothelial cells already at day 7 after transplantation, indicating that some other unidentified factors besides the absence of endothelial cells may explain the retardation of beta-cell grafts revascularization.

The role of the thymus in the ongoing acquisition of tolerance to self antigens has made it an attractive site for islet transplantation. Several studies have reported survival of rodent islet allografts in the thymus without requiring the long-term use of immunosuppressive agents; however, the degree of glucose homeostasis in the intrathymic islet transplant recipients has not been examined. We transplanted 500, 1000, or 2000 syngeneic islets into the thymus of streptozotocin-induced diabetic Wistar-Furth rats, and compared the metabolic response of these recipients with animals receiving 2000 syngeneic islets under the kidney capsule. Three of four recipients which received 2000 islets under the kidney capsule achieved normoglycemia (< or =8.4 mmol/L) within 1 wk and all animals became normoglycemic within 2 wk posttransplantation. In contrast, intrathymic implantation of 2000 islets induced normoglycemia in only one of six recipients during the same time interval, and when this number was reduced to 1000 or 500 islets, none of the recipients (n = 6) normalized within 1 wk posttransplantation. Animals that received an intrathymic transplant were glucose intolerant compared to normal controls and animals with subcapsular islet transplant. Removal of the graft-bearing organs resulted in hyperglycemia in all cases, and examination of the grafts revealed the presence of numerous well-granulated insulin-containing cells in both sites. The cellular insulin content of the subcapsular grafts (67.4 +/- 12.1 microg; n = 4) was significantly higher (p < or =0.05) than what was extracted from intrathymic grafts (9.5 +/- 1.2 microg from 1000 islets; n = 3 and 20.0 +/- 4.6 microg from 2000 islets; n = 3). We conclude that 2000 syngeneic islets implanted either in the thymus or beneath the kidney capsule can normalize hyperglycemia in streptozotocin-diabetic rats; however, normal glucose tolerance was not established in intrathymic islet recipients, suggesting that a higher number of islets may be necessary to achieve normal glucose homeostasis.

The ability to replace damaged myocardial tissue with new striated muscle would constitute a major advance in the treatment of diseases that irreversibly injure cardiac muscle cells. The creation of focal grafts of skeletal muscle has been reported following the intramural injection of skeletal myoblasts into both normal and injured myocardium. The goals of this study were to determine whether skeletal myoblast-derived cells can be engrafted into the murine heart following arterial delivery. The murine heart was seeded with genetically labeled C2C12 myoblasts introduced into the arterial circulation of the heart via a transventricular injection. A transventricular injection provided access to the coronary and systemic circulations. Implanted cells were characterized using histochemical staining for beta-galactosidase, immunofluorescent staining for muscle-specific antigens, and electron microscopy. Initially the injected cells were observed entrapped in myocardial capillaries. One week after injection myoblasts were present in the myocardial interstitium and were largely absent from the myocardial capillary bed. Implanted cells underwent myogenic development, characterized by the expression of a fast-twitch skeletal muscle sarcoendoplasmic reticulum calcium ATPase (SERCA1) and formation of myofilaments. Four months following injection myoblast-derived cells began to express a slow-twitch/cardiac protein, phospholamban, that is normally not expressed by C2C12 cells in vitro. Most surprisingly, regions of close apposition between LacZ labeled cells and native cardiomyocytes contained structures that resembled desmosomes, fascia adherens junctions, and gap junctions. The cardiac gap junction protein, connexin43, was localized to some of the interfaces between implanted cells and cardiomyocytes. Collectively, these findings suggest that arterially delivered myoblasts can be engrafted into the heart, and that prolonged residence in the myocardium may alter the phenotype of these skeletal muscle-derived cells. Further studies are necessary to determine whether arterial delivery of skeletal myoblasts can be developed as treatment for myocardial dysfunction.

The testis has been suggested as an immune privileged site for islet transplantation. The present study evaluated this hypothesis by transplanting islets from Wistar Furth rats into (a) the testes; (b) the subcapsular space of the kidneys; or (c) the cryptorchid abdominal testes of streptozotocin-induced diabetic Swiss ND4 mice. Transplantation of 800 rat islets into the cryptorchid testes normalized blood glucose for 9.3 +/- 1.4 (Mean +/- SD) days, not significantly different from that of the scrotal testis site (12.4 +/- 1.3), or when the subcapsular space of the kidneys was used (11.5 +/- 1.2). When mouse islets were isotransplanted into the cryptorchid testes of diabetic mice, normoglycemia was maintained for the entire 3 month study period. Histologic examination of the islet xenograft-bearing cryptorchid testes at day 7 post transplantation and 2 days after returning to hyperglycemia revealed lymphocyte infiltration surrounding and inside the graft. No lymphocyte infiltration was seen in the isograft bearing-testes at 3 mo after transplantation. Cyclosporine A (CsA, 15 mg/kg/day) administration to the islet xenograft recipient slightly prolonged the normoglycemic period to 13.7 +/- 1.8 days (p < 0.01). Increasing CsA dose to 25 mg/kg induced a 66% (4/6) mortality, and did not further prolong the normoglycemic period. Using a lower number of rat islets (200 or 400 islets), prolonged graft survival was achieved in some (4 out of 20) animals when the cryptorchid testis was used. In contrast, transplantation of 400 rat islets into the subcapsular space of the kidneys was not associated with prolonged graft survival.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of membrane antigens on rat hepatocytes transplanted syngeneically and heterotopically was analyzed immunohistochemically using monoclonal antibodies against rat hepatocytes. Isolated adult and fetal hepatocytes were able to survive in the spleen, salivary gland, thymus, or subcapsular region of the kidney for various periods after transplantation. Fairly clear expression of HAM2, 4, and 8 antigens was observed on hepatocytes transplanted into syngeneic spleen, suggesting that the cells might be functionally equivalent to hepatocytes in situ. HAM4 antigen was localized specifically on the newly formed bile-canalicular faces of hepatocytes. The expression of HAM2 (MHC class I) antigen on the transplanted hepatocytes appeared much stronger on the side facing lymphoid tissues, than on the other faces, suggesting that some immunological reactions may take place between hepatocytes and lymphoid tissue. HAM8 antigen, which is localized on gap junctions between neighboring hepatocytes in rat liver, was also recognized between transplanted hepatocytes. In salivary glands where hepatocytes were transplanted, bile-canaliculus-like structures were observed not only between neighboring hepatocytes but also between hepatocytes and salivary acinar cells, suggesting good interaction between the two different epithelial cell types. Hepatocytes transplanted into thymus appeared viable, but most showed fatty degeneration. Some healthy hepatocytes survived in the interlobular connective tissue and the thymic cortical tissue. When fetal hepatocytes were transplanted heterotopically, they formed a mass consisting of hepatocytes and bile duct-like structures 7 wk after transplantation. The inoculated hepatocytes possessed HAM4 antigen, which was not recognized on fetal hepatocytes at day 14 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardium lacks the ability to regenerate following injury. This is in contrast to skeletal muscle (SKM), in which capacity for tissue repair is attributed to the presence of satellite cells. It was hypothesized that SKM satellite cells multiplied in vitro could be used to repair injured heart muscle. Fourteen dogs underwent explantation of the anterior tibialis muscle. Satellite cells were multiplied in vitro and their nuclei were labeled with tritiated thymidine 24 h prior to implantation. The same dogs were then subjected successfully to a myocardial injury by the application of a cryoprobe. The cells were suspended in serum-free growth medium and autotransplanted within the damaged muscle. Medium without cells was injected into an adjacent site to serve as a control. Endpoints comprised histology using standard stains as well as Masson trichrome (specific for connective tissue), and radioautography. In five dogs, satellite cell isolation, culture, and implantation were technically satisfactory. In three implanted dogs, specimens were taken within 6-8 wk. There were persistence of the implantation channels in the experimental sites when compared to the controls. Macroscopically, muscle tissue completely surrounded by scar tissue could be seen. Masson trichrome staining showed homogeneous scar in the control site, but not in the test site where a patch of muscle fibres containing intercalated discs (characteristic of myocardial tissue) was observed. In two other dogs, specimens were taken at 14 wk postimplantation. Muscle tissue could not be found. These preliminary results could be consistent with the hypothesis that SKM satellite cells can form neo-myocardium within an appropriate environment. Our specimens failed to demonstrate the presence of myocyte nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)