Topographical arrangement of α- and ß-cells within neo-islet tissues engineered by islet cell sheet transplantation in mice.

BACKGROUND: We established a procedure to engineer therapeutic neo-islets in subcutaneous spaces in mice by transplanting contiguous layers of islet cell sheets. In this study, we investigated the cellular arrangements of α and ß within these engineered neo-islets in vivo as a function of time after sheet transplantation. METHODS AND RESULTS: Temperature-responsive culture dishes optimized for dispersed islet cell culture were prepared by covalently immobilizing a temperature-responsive polymer poly(N-isopropylacrylamide) (PIPAAm) on plastic dishes followed by laminin-5 coating. Dispersed islet cells obtained from Lewis rats were plated onto the PIPAAm dishes. After reaching confluence at day 2, islet cells were harvested as uniformly spread islet cell sheets by lowering the culture temperature from 37°C to 20°C for 20 minutes. Islet sheet transplantation was performed to creat neo-islet tissues in the subcutaneous spaces of SCID mice with streptozotocin-induced diabetes. This neo-islet engineering approach successfully lowered mouse blood glucose levels, achieving euglycemia at day 5 and thereafter. Histologic analyses of samples obtained at day 4 revealed that neo-islet tissues in the subcutaneous spaces showed heterogeneous cellular alignment of α and ß cells. In contrast, analyses of samples at days 14 and 60 revealed α and ß cells predominantly located at the peripheral and central parts of the engineered tissues, respectively. CONCLUSIONS: Reassembly of α and ß cells occurred in neo-islet tissues engineered by sheet transplantation. The unique cellular arrangements in neo-islet tissues, which were similar to those in naïve pancreatic islets, may contribute to their longevity and long-term function.

Production of islet cell sheets using cryopreserved islet cells.

BACKGROUND: To establish novel islet-based therapies, our group has recently developed technologies to create a contiguous, monolayered sheet made from freshly dispersed islet cells. Islet cell sheets generated from freshly isolated cells are easily transplantable for engraftment into subcutaneous sites in rodents. The use of a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), grafted culture dishes with laminin-5 coating is an important feature of this process. To expand the utility of this protocol, the present study was performed to assess whether sheets generated using cryopreserved islet cells maintained viability and normal cellular phenotypes. METHODS: Dispersed islet cells obtained from Lewis rats were, cryopreserved using University of Wisconsin solution and 10% dimethyl sulfoxide. Specially coated plastic dishes were prepared by covalently immobilizing PIPAAm onto the culture plastic, followed by a coating of rat laminin-5. After 1 month of cryopreservation, the thawed cells were plated onto the PIPAAm-coated dishes. RESULTS: Viability of the thawed islet cells as assessed by trypan blue exclusion test was 86% ± 5%. Thawed dispersed islet cells favorably attached to PIPAAm dishes could be harvested as a contiguous cell sheet using a simple change in culture temperature conditions. Electron microscopy showed the harvested islet cell sheet to retain cell-cell connections and numerous secretion granules. CONCLUSIONS: The present data indicated that dispersed islet cells, which were appropriately frozen and thawed, represent another viable cells source to create functional islet sheets for tissue engineering and potential clinical applications.

Developmentally and regionally regulated participation of epidermal cells in the formation of collagen lamella of anuran tadpole skin.

We investigated the cellular mechanism of formation of subepidermal thick bundles of collagen (collagen lamella) during larval development of the bullfrog, Rana catesbeiana, using cDNA of alpha1(I) collagen as a probe. The originally bilayered larval epidermis contains basal skein cells and apical cells, and the collagen lamella is directly attached to the basement membrane. The basal skein cells above the collagen lamella and fibroblasts beneath it intensively expressed the alpha1(I) gene. As the skin developed, suprabasal skein cells ceased expression of the gene. Concomitantly, the fibroblasts started to outwardly migrate, penetrated into the lamella and formed connective tissue between the epidermis and the lamella. These fibroblasts intensively expressed the gene. As the connective tissue developed, the basal skein cells ceased to express the gene and were replaced by larval basal cells that did not express the gene. These dynamic changes took place first in a lateral region of the body skin and proceeded to all other regions except the tail. Isolated cultured skein cells expressed the gene and extracellularly deposited its protein as the type I collagen fibrils. Thus, it is concluded that anuran larval epidermal cells can autonomously and intrinsically synthesize type I collagen.

Cell-type specific and thyroid hormone-dependent expression of genes of alpha1(I) and alpha2(I) collagen in intestine during amphibian metamorphosis.

Both the epithelium and the mesenchyme of the larval small intestine of anurans undergoes metamorphic conversion into the adult counterparts. The conversion of the mesenchyme has been poorly understood especially at the molecular level, whereas the changes of the epithelium have been extensively studied. The present study investigated the metamorphic changes of the mesenchyme of tadpoles of bullfrog, Rana catesbeiana, focusing on the expression of genes of type I collagen. By using the cDNA clones coding for a 1(I) and a 2(I) collagen as probes, expression of each collagen gene was examined. These genes were drastically up-regulated at the climax period of spontaneous metamorphosis, which was precociously mimicked by treating tadpoles with thyroid hormone. The increased expression of these genes at the climax stage was well correlated with the conversion of the thin larval mesenchyme to more thick and dense adult connective tissues of the intestine. In situ hybridization identified the fibroblasts that were actively expressing the collagen genes and, therefore, were thought to be responsible for the remodeling. These results strongly suggest that the expression of type I collagen genes is regulated during the intestinal remodeling in a cell-type specific and thyroid hormone-dependent manner.