Cell Transplantation

The islet tissue, called Brockmann bodies, in certain teleost fish is anatomically distinct from the pancreatic exocrine tissue and can be easily identified macroscopically. Expensive islet isolation procedures, such as required when procuring islet tissue from mammalian pancreases, are unnecessary. Tilapia islets transplanted into diabetic athymic nude mice will produce long-term normoglycemia and mammalian-like glucose tolerance profiles. Encapsulated tilapia islets function well after transplantation into euthymic recipients. Additionally, tilapia are potentially ideal xenogeneic donors because of markedly lower donor production costs, minimal islet procurement costs, and possibly decreased xenozoonotic potential relative to mammalian donors. Tilapia islets appear to be appropriately glucose responsive with high insulin output, can be cryopreserved, and are much more resistant to hypoxia than mammalian islets. Because tilapia and human insulin differ by 17 amino acids, we have cloned, sequenced, and modified the tilapia insulin gene by site-directed mutagenesis resulting in a tilapia insulin gene that codes for “humanized” insulin while still maintaining all of the tilapia regulatory (noncoding) sequences. We have now produced small numbers of founder transgenic tilapia with incorporation of our humanized tilapia insulin gene construct, and we have shown transmission and expression of the transgene in the beta cells and serum of their F1 offspring. Our ultimate goal is to achieve homologous recombination and to breed for homozygosity for the hybrid insulin gene. Subsequent generations of transgenic tilapia will undergo a program of genomic optimization selecting for growth, survival, and fecundity as well as stability of the transgene. Islets from the resulting transgenic fish, after extensive characterization, could be harvested, encapsulated, and then transplanted into diabetic patients.

In moderately diabetic rats (plasma glucose 20 – 30 mmol/L), where there is some residual pancreatic islet function, normoglycemia can be restored by transplantation of pancreatic islets into the liver via the portal vein. To examine whether normoglycemia can also be achieved in more severely diabetic animals (which more closely resemble human type I diabetes), we have compared the effect of transplanting 1000 islets intraportally in Lewis rats made moderately diabetic (55 mg/kg streptozotocin injected IP while nonfasting) or severely diabetic (65 mg/kg streptozotocin injected IP while fasting). In the moderately diabetic rats in which residual pancreatic insulin was 128 ± 40 mU insulin (2.0% of control), plasma glucose stabilized (32 ± 2.8 mmol/L at 1 week, 34 ± 2 mmol/L at 3 weeks) as did body weight (falling from 290 ± 5 to 265 ± 5 g at 1 week and 253 ± 6 g at 3 weeks). In contrast, in severely diabetic rats in which residual pancreatic insulin was only 13.5 ± 4.2 mU insulin (0.21% of control), there was a progressive rise in plasma glucose (30 ± 1.3 mmol/L at 1 week, 49 ± 4 mmol/L at 2 weeks, and 67 ± 7 mmol/L at 3 weeks) and a progressive fall in body weight (from 304 ± 10 to 260 ± 5 g by week 1 and to 209 ± 6 g by week 3). Following islet transplantation, nonfasting plasma glucose normalized in moderately diabetic rats (10.5 ± 0.6 vs. 9.1 ± 0.6 mmol/L in nondiabetic controls, NS) after 23 ± 5 days. In contrast, in the severely diabetic rats plasma glucose stabilized at 32 ± 5mmol/L (p < 0.05 compared to moderately diabetic group) but did not normalize. This difference was not attributable to different plasma glucose levels at the time of transplantation (35.1 ± 1.8 in moderately diabetic vs. 32.5 ± 2.5 mmol/L in severely diabetic rats). These observations demonstrate that residual native β-cells (equivalent to only 60 – 80 islets) contribute to the survival or function of intraportally transplanted islets.

The electrofusion-derived rat insulin-secreting cell line BRIN-BD11 was cultured in five different commercially available media to determine the optimum medium for the in vitro maintenance of such clonal cell lines. Cells were cultured in RPMI-1640, DMEM, McCOY'S, F-12K, or MEM culture medium supplemented with 10% (v/v) fetal bovine serum and antibiotics (100 U/ml penicillin and 0.1 g/L streptomycin). Insulin secretion studies performed after 10 days revealed RPMI-1640 to be the best performing medium in terms of insulin secretory responsiveness to a range of stimuli including glucose, l-alanine, l-arginine, carbachol, and glibenclamide. Insulin release was significantly decreased (p < 0.01 to p < 0.05) in all other media compared to RPMI-1640. Only the cells cultured in RPMI-1640 and DMEM showed a significant glucose-induced insulin secretory response (p < 0.01 and p < 0.05). McCOY'S gave the next best result followed by F-12K and MEM. After the 10-day culture period, the highest insulin content was found in cells cultured in RPMI-1640 and DMEM with significantly lower levels of insulin in cells cultured in McCOY'S, F-12K, and MEM (p < 0.01 to p < 0.001). RPMI-1640 was used for further studies to investigate the effects of 5.6 – 16.7 mmol/L glucose in culture on the secretory responsiveness of BRIN-BD11 cells. Significant responses to a number of nonglucidic secretagogues were seen following culture at 5.6 and 16.7 mmol/L glucose, although responsiveness was less than after culture with 11.1 mmol/L glucose. At 16.7 mmol/L glucose culture, glucose-stimulated insulin release was abolished.

Addition of rIL-6 to IL-6-dependent B9 cells starved for IL-6 for 16 – 20 h stimulated a vigorous proliferative response. Glucocorticoids (GCs), in a concentration-dependent manner, inhibited rIL-6-stimulated proliferation of B9 cells This inhibition was specific for the GCs, evident by the capacity of the GCs, dexamethasone, prednisolone, and hydrocortisone, but not non-GC steroids, to suppress rIL-6-dependent B9 cell proliferation. Furthermore, GC inhibition of IL-6-stimulated B9 cell proliferation was receptor mediated and was abrogated by the GC receptor antagonist, RU486. In addition to their reported effects on inhibition IL-6 expression, the results presented support the notion that GCs also acted distally by suppressing signal transduction through the IL-6 receptor.

Assays of C-peptide are used to monitor allogeneic islet graft function. However, it is not known whether xenogeneic C-peptide is metabolized and excreted in a fashion similar to endogenous and allogeneic C-peptide. In this study, injection of 10 times the physiological amount of porcine C-peptide into mice did not result in the excretion of the C-peptide in the urine. In contrast, when a physiological amount of porcine C-peptide was injected into athymic mice, urinary excretion of porcine C-peptide was readily detected. After injection of radioactively labeled porcine C-peptide into mice, the radioactive uptake in tissues belonging to the mononuclear phagocytic system was significantly increased in mice immunized towards the xenogeneic C-peptide. These results may reflect an immunological reactivity towards the C-peptide. Antibodies against porcine C-peptide could not be detected in the serum of any of the mice. However, porcine C-peptide was found to be glycosylated. Thus, a possible explanation to the lack of porcine C-peptide in the urine is that xenoreactive antibodies had bound to carbohydrate structures on the peptide and that the antibody – C-peptide complex had been cleared from the circulation by the mononuclear phagocytic system. Thus, the urinary excretion of xenogeneic C-peptide seems to be different from that of endogenous and allogeneic C-peptide. Consequently, determinations of donor-specific C-peptide may not properly reflect islet xenograft function. In fact, islet xenograft function may be underestimated.

In a model of transplantation rejection, we have tested whether a graft manipulated to secrete immunomodulators could protect itself from immune destruction. An insulinoma cell line having the NOD genotype but also expressing the neoantigen, SV40 T antigen, was transfected with CTLA4Ig or LFA3Ig to block signals in the co-stimulatory/adhesion pathways. This neoantigen is potent at inducing graft rejection. Secretion of CTLA4Ig and LFA3Ig by transfectants promoted survival of the insulinoma graft in young NOD mice. In immunodeficient mice, cell growth was similar for all transfectants. However, in immunocompetent NOD mice the survival/growth of test grafts was significantly better than that of the controls. Graft survival was enhanced additively, when the two test transfectants were cotransplanted. Endowing the graft the ability to secrete immunomodulators that block individual co-stimulatory/adhesion signals can contribute to transplantation success. Blockade of two signals (CD2 and CD28) in these pathways enhances this success.

Immunoisolation is the separation of transplanted cells from cells of the immune system using a semipermeable membrane. Using one such immunoisolation capsule – -the TheraCyte® device – -we have assessed the survival of encapsulated xenogeneic tissue in vivo as well as the contribution of CD4+ve T cells to encapsulated xenograft rejection. he foreign body reaction to the TheraCyte® capsule in vivo was assessed by transplanting empty capsules into normal mice. These capsules elicit a foreign body response by the host animal. Encapsulated CHO, NIT-1, and PK-15 cells were placed in culture and in immunodeficient mice to investigate their growth characteristics in the TheraCyte® device. These cell lines survive both in culture and in immunodeficient SCID mice. Xenogeneic PK cells were also transplanted into normal C57BL/6 mice. These cells do not survive in normal mice despite the absence of direct contact between infiltrating and encapsulated cells. In addition, the survival of encapsulated cells in mice treated with a single dose of anti-CD4 antibody was examined. This was assessed using two systems: 1) histological analysis of capsule sections; 2) a quantitative luciferase reporter system using PK cells transfected to express luciferase. In both cases, anti-CD4 antibody contributed to prolonged encapsulated xenogeneic cell survival. Encapsulated xenogeneic cells survive in immunodeficient mice but not normal mice. Treatment of normal mice with anti-CD4 antibody results in prolonged survival of xenogeneic cells that can be measured using a luciferase reporter system. These results highlight the contribution of CD4+ve T cells to encapsulated xenograft rejection.

We have shown previously that chitosan-polyvinyl pyrrolidone (PVP) hydrogels are blood compatible, islet compatible, and noncytotoxic to various cell types. Because of these potential applications of chitosan-PVP hydrogel, the present study was designed to investigate its effect on macrophage activation. Macrophages did not adhere to hydrogel in culture but maintained their viability and did not undergo apoptosis as confirmed by trypan blue staining and absence of DNA ladder. Hydrogel leach-out products did not exhibit cytotoxic effects on macrophage functionality at mitochondrial and lysosomal level as confirmed by tetrazolium reduction (MTT) and neutral red uptake (NRU) assay. On exposure to hydrogels, macrophages showed comparable expression of activation markers such as CD11b/CD18 (Mac-1), CD45, and CD14 to those cultured in the presence of PTFE, a known biocompatible control, indicating its nonactivating nature. Macrophage activation was also assessed by checking the level of messenger RNA of inflammatory cytokines such as IL-6 and TNF-α by reverse transcriptase polymerase chain reaction (RT-PCR), which did not show stastistically significant difference (p > 0.05) in the expression of these transcripts in both control and hydrogel-exposed macrophages. The nonimmunogenic nature of the hydrogel was further confirmed by the lack of induced proliferation of mouse splenic lymphocytes after exposure to hydrogel leach-outs. All these results point out that chitosan-PVP hydrogel did not activate macrophages and thus is immunocompatible. Our results indicate that this hydrogel could be a potential candidate for transplantation studies by virtue of its biocompatibility and imunocompatibility.

Biopsies removed from 57 patients considered for cartilage transplantation were grown at CTI Ltd. (47 biopsies) and at Tel Aviv University (10 biopsies). Tissue processing took place in dedicated laboratories. Explant cultures allowed cell number expansion. Fifty-four out of 57 biopsies grew cells. Fanning out of the cells began after 5 – 15 days in culture. Two passages later, cell numbers in the 107 range were achieved. Cells from all cultures expressed mRNA of aggrecan and link protein but not of alkaline phosphatase. Histochemical stains such as alcian blue pH 1 were negative in sparse monolayer cultures, but positive in pellet cultures. Immunohistochemistry demonstrated expression of collagen type I in monolayer cultures, switching to collagen type II in micromass cultures. Fibroblast growth factor receptor 3, a recently described characteristic receptor of precartilaginous cells, was expressed in monolayers and disappeared in micromass cultures. In conclusion, explants of articular chondrocytes cultured in vitro consistently yield monolayer cultures. The cells appear to revert to dedifferentiated chondrocytes, expressing a mesenchymal stem cell protein profile. Simultaneously, these cells regained their capacity to proliferate. Cultures held as micromass allowed reexpression of the differentiated phenotype traits.

Ex vivo gene therapy of Duchenne muscular dystrophy based on autologous transplantation of genetically modified myoblasts is limited by their premature senescence. MyoD-converted fibroblasts represent an alternative source of myogenic cells. In this study the forced MyoD-dependent conversion of murine NIH-3T3 fibroblasts into myoblasts under the control of an inducible promoter silent in the presence of tetracycline was evaluated. After tetracycline withdrawal this promoter drives the transcription of MyoD in the engineered fibroblasts, inducing their myogenesis and giving rise to β-galactosidase-positive cells. MyoD-expressing fibroblasts withdrew from the cell cycle, but were unable to fuse in vitro into multinucleated myotubes. Five days following implantation of engineered fibroblasts in muscles of C57BL/10J mice we observed a sevenfold increase of β-galactosidase-positive regenerating myofibers in animals not treated with antibiotic compared with treated animals. After 1 week the number of positive fibers decreased and several apoptotic myonuclei were detected. Three weeks following implantation of MyoD-converted fibroblasts in recipient mice, no positive “blue” fiber was observed. Our results suggest that transactivation by tetracycline of MyoD may drive an in vivo myogenic conversion of NIH-3T3 fibroblasts and that, in this experimental setting, apoptosis plays a relevant role in limiting the efficacy of engineered fibroblast transplantation. This work opens the question whether apoptotic phenomena also play a general role as limiting factors of cellmediated gene therapy of inherited muscle disorders.

Human neural progenitor cells, originally isolated from prenatal donor tissue at 17 weeks of development, were cultured as neurospheres and transplanted to the vitreous cavity of dystrophic Royal College of Surgeons rats with, or without, cyclosporin A immunosuppression. Donor cells were either unlabeled or prelabeled, the latter utilizing incubation with BrdU or adenoviral modification to express green fluorescent protein. Recipients of various ages were examined at 1, 2, and 4 weeks postgrafting. Transplanted human neural progenitor cells survived in the host vitreous for at least 4 weeks and maintained expression of green fluorescent protein for at least 2 weeks. After 2 weeks in vivo, grafted cells differentiated morphologically, coincident with expression of the neuronal marker MAP, indicating mature neuronal differentiation. The extensive intraretinal migration previously shown using rat progenitor cells in the Royal College of Surgeons rat model was not seen in this experiment, suggesting that high levels of neuronal migration may depend at least in part upon species-specific molecular cues. Human neural progenitor cells represent a renewable source of multipotent human cells capable of in vivo neuronal development and a potential means of delivering therapeutic factors intraocularly. Human neural progenitor cells therefore provide a useful tool for studies of neural development and differentiation in the dystrophic eye.