C-kit-derived CD11b+ cells are critical for cardiac allograft prolongation by autologous C-kit+ progenitor cells.

Autologous C-kit+ cells robustly prolong cardiac allografts. As C-kit+ cells can transdifferentiate to hematopoietic cells as well as non-hematopoietic cells, we aimed to clarify the class(es) of C-kit-derived cell(s) required for cardiac allograft prolongation. Autologous C-kit+ cells were administered post-cardiac transplantation and allografts were evaluated for C-kit+ inoculum-derived cells. Results suggested that alloimmunity was a major signal for trafficking of C-kit-derived cells to the allograft and demonstrated that C-kit+ inoculum-derived cells expressed CD11b early after transfer. Allograft survival studies with CD11b-DTR C-kit+ cells demonstrated a requirement for C-kit+-derived CD11b+ cells. Co-therapy studies demonstrated near complete abrogation of acute rejection with concomitant CTLA4-Ig therapy and no loss of prolongation in combination with Cyclosporine A. These results strongly implicate a C-kit-derived myeloid population as critical for allograft preservation and demonstrate the potential therapeutic application of autologous C-kit+ progenitor cells as calcineurin inhibitor-sparing agents and possibly as co-therapeutics for durable graft survival.

Characterization of rat spinal cord neurons cultured in defined media on microelectrode arrays.

Previous efforts to utilize mammalian spinal cord neurons as biosensor elements have relied on neuronal: glial co-cultures maintained in serum-containing media. We have examined the feasibility of culturing primary spinal cord neurons in serum-free medium, modified for neuronal longevity, on fabricated microelectrode arrays. Embryonic day 15 rat spinal cord cells were plated on trimethoxysilyl-propyldiethylenetriamine coated microelectrode arrays comprised of gold recording sites passivated with silicon nitride. Immunocytochemistry was performed to verify the presence of neurons and quantitatively assess astrocytes using antibodies against glial fibrillary acidic protein on the silicon nitride substrates. Modifications to culture media enabled viable neuronal culture to extend from approximately 14 days in vitro (DIV) to 40 DIV on the arrays containing only 1.1 +/- 0.5% (mean +/- SEM) astrocytes. Extracellular recording revealed tetrodotoxin-sensitive spontaneous electrical activity from the enriched neuronal culture. Threshold detection of extracellular potentials showed an increase in spike rate as a function of glutamate concentration with neurotoxicity at elevated levels. This approach suggests that functional measures related to biosensor applications, pharmacological screening, or the evaluation of neurological disease models can be implemented in a defined culture system.

Reversal of diabetes by transplantation of cryopreserved rat islets of Langerhans to the renal subcapsular space.

Intraportal transplantation of cryopreserved islets of Langerhans has been shown to reverse streptozotocin-induced diabetes in rats. This study demonstrates the feasibility of transplanting 3,000 cryopreserved islets beneath the kidney capsule of syngeneic streptozotocin-diabetic Wistar-Furth rats. The clinical indices of plasma glucose, urine volume, and urine glucose returned to baseline values following implantation of fresh and cryopreserved islets. The clinical indices post-transplant and the K values (decline in plasma glucose concentration, percent/min) during intravenous glucose tolerance tests were not significantly different in rats receiving freshly isolated or cryopreserved islet grafts. The rapid return to the diabetic state after nephrectomy proved conclusively that the reversal of diabetes was due to the grafted islets. This was confirmed by immunocytochemical localization of well confirmed by immunocytochemical localization of well granulated islets beneath the kidney capsule. We conclude that cryopreserved islets reverse streptozotocin-induced diabetes after renal subcapsular implantation and that the clinical response is similar to fresh isografts.

Prolongation of islet xenograft survival by cryopreservation.

Our attempt to reduce islet immunogenicity by slow cooling to -40 degrees C, storage at -196 degrees C, and rapid thawing is based on the differential susceptibility of various cell types to a freeze-thaw process. Five hundred rat islets (greater than or equal to 100 micron) were immediately implanted or cryopreserved and then implanted beneath the renal capsule of streptozocin-induced diabetic mice with or without an injection of anti-lymphocyte serum at the time of transplantation. Thirteen days after transplantation, all fresh xenografts had rejected, whereas 37.5% of cryopreserved grafts were still functioning. In immunosuppressed mice, 6.2% of fresh xenografts and 54.5% of cryopreserved grafts were functioning 19 days after transplantation. These results show that cryopreservation can extend xenograft survival.