Impact of ex vivo administration of mesenchymal stem cells on the function of kidney grafts from cardiac death donors in rat.

BACKGROUND: Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC administration in marginal donor grafts may help avoid the ischemia-reperfusion injury associated with solid organ transplants. Given the reports of side effects after intravenous MSC administration, local MSC administration to the target organ might be a better approach. We administered adipose tissue-derived MSCs (AT-MSCs) ex vivo to donor rat kidneys obtained after cardiac death (CD). METHODS: Using male Lewis rats (8-10 weeks), and a marginal transplant model of 1hr CD plus 1hr sub-normothermic ET-Kyoto solution preservation were conducted. AT-MSCs obtained from double-reporter (luciferase-LacZ) transgenic Lewis rats were injected either systemically (1.0 × 10(6) cells/0.5 mL) to bilaterally nephrectomized recipient rats that had received a marginal kidney graft (n = 6), or locally via the renal artery (500 µL ET-Kyoto solution containing the same number of AT-MSCs) to marginal kidney grafts, which were then preserved (1 hour; 22°C) before being transplanted into bilaterally nephrectomized recipient rats (n = 8). Serum was collected to assess the therapeutic effects of AT-MSC administration, and the recipients of rats surviving to Day 14 were separately evaluated histopathologically. Follow-up was by in vivo imaging and histological LacZ staining, and tumor formation was evaluated in MSC-injected rats at 3 months. RESULTS: Systemic injection of MSC did not improve recipient survival. In vivo imaging showed MSCs trapped in the lung that later became undetectable. Ex vivo injection of MSCs did show a benefit without adverse effects. At Day 14 after RTx, 75% of the rats in the AT-MSC-injected group (MSC[+]) had survived, whereas 50% of the rats in the AT-MSC-non-injected group (MSC[-]) had died. Renal function in the MSC(+) group was improved compared with that in the MSC(-) group at Day 4. LacZ staining revealed AT-MSCs attached to the renal tubules at 24 hours after RTx that later became undetectable. Histopathologic examination showed little difference in fibrosis between the groups at Day 14. No teratomas or other abnormalities were seen at 3 months.

Canine mesenchymal chondrosarcoma of the ribs.

The tumor of the thoracic cavity, which arose from the ribs, was diagnosed as mesenchymal chondrosarcoma. No distant metastasis was observed. Histologically, the tumor was characterized by the nests of well-defined cartilaginous tissue within a proliferation of primitive mesenchymal cells. Additionally, the deformed blood vessels compressed by the proliferating mesenchymal cells exhibited clear stag-horn appearance. Immunohistochemically, most neoplastic cells that formed multifocal cartilaginous islands were positive for S-100 protein, while the surrounding mesenchymal cells were negative. This is the first report of canine mesenchymal chondrosarcoma of the ribs.

Effects of cryopreservation of DNA synthesis in the inner cell mass of in vitro matured/in vitro fertilized bovine embryos frozen in various cryoprotectants.

The rate of DNA synthesis in the inner cell mass (ICM) of frozen-thawed bovine embryos was examined. Bovine blastocysts derived from in vitro matured/in vitro fertilized oocytes were frozen with 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.4 M glycerol plus 0.25 M sucrose (GL). Viable embryos, after thawing and culture beyond the blastocysts stage, were examined by immunocytochemical staining for detection of DNA synthesis by ICM cells. The numbers of bromodeoxyuridine-immunoreactive ICM cells of frozen-thawed embryos prepared with EG (10.7) and GL (11.5) were significantly lower than those of unfrozen embryos (17.3). The results suggest that the rates of proliferation of ICM cells of frozen-thawed bovine embryos tend to be lower than those of unfrozen embryos irrespective of the cryoprotectant used.

Cryopreservation of isolated rat islets of Langerhans in the presence of ethylene glycol or dimethyl sulfoxide: evaluation of toxicity and the dynamic pattern of subsequent insulin release in vitro.

The toxic effect of ethylene glycol (EG) on the pattern of dynamic insulin release from rat pancreatic islets with or without freezing was investigated in comparison with that of dimethyl sulfoxide (Me2SO). Sixty islets were perifused (1 ml/min) consecutively with D-glucose (1.67 mM for 30 min followed by 16.7 mM for 60 min and 1.67 mM for 60 min) after exposure to 2.0 M EG or Me2SO for 1 h at either 22 or 0 degrees C. During the second period of perifusion, the insulin output from islets exposed to Me2SO or EG at 22 degrees C decreased to 53 and 51% of that from nontreated control islets, respectively. On the other hand, the islets exposed to EG at 0 degrees C exhibited 86% of the control insulin output under the same perifusion conditions, and this appeared to be higher than that of islets exposed to Me2SO (60%) at 0 degrees C. Frozen islets, after exposure to 2.0 M EG or Me2SO for 1 h at 0 degrees C, responded positively to 16.7 mM D-glucose, and the typical biphasic pattern of insulin secretion was observed. The insulin output from these islets during the second period of perifusion was not comparable to that from unfrozen control islets. In particular, the mean insulin output of EG-cryopreserved islets during the second period accounted for 99% of that from unfrozen control islets. The present findings suggest the possible use of EG as an alternative cryoprotectant to Me2SO.

Differential freezing tolerance of rat pancreatic islets depending on their size variation.

We have investigated the freezing tolerance of rat pancreatic islets. Freshly isolated rat pancreatic islets were divided into three groups based on their longest diameter (small; 100 - 200 microns, medium; 201 - 300 microns, large; > 300 microns). They were then cryopreserved at a slow cooling rate (-0.3 degrees C/min) in the presence of dimethyl sulfoxide (Me2SO) or ethylene glycol (EG). After storage at -196 degrees C for 1 - 4 weeks, they were thawed and their ability to secrete insulin in response to fluctuations in glucose concentration was examined during three consecutive static incubations in vitro (1st; 2.8 mM, 2nd; 16.7 mM, 3rd; 2.8 mM). Morphological examination of the beta-granule population was determined by image analysis, and correlation with islets size was analyzed. The amount of insulin released from large-sized islets was significantly suppressed in EG (p < 0.05) and Me2SO (p < 0.01) groups compared to unfrozen islets. However, the mean volume of the large-sized islets isolated from one rat accounted for 43.0% of the total volume. On the other hand, the amount of insulin released from small- and medium-sized islets did not differ from those of unfrozen islets, and their mean volumes were 13.2 and 43.8% respectively. The percentage of cells with beta-granules was significantly correlated with size in both EG (r = -0.52) and Me2SO (r = -0.35) groups, but no significant correlation was observed in the unfrozen islets groups. These findings suggest that large-sized islets are more susceptible to freezing injury than small- or medium-sized islets. Moreover, the volume distribution of isolated islets indicated that it may be important to retain the ability of insulin secretion from the large-sized islets.

Intrauterine transplantation of isogenic pancreatic islets in experimental diabetic rats.

The effect of intrauterine transplantation (IU group) as a potential immunologically privileged site on the diabetic state of the recipient was compared with that of conventional intraperitoneal transplantation (IP group) using Fisher 344 rats. Islets were isolated from the pancreata of normal rats and transplanted into the uterus and peritoneal cavity of the isogenic rats with experimental diabetes, which were treated with estradiol benzoate and progesterone. Although all the rats in both groups became normoglycemic within 4 days after transplantation, all of those in the IU group relapsed into a diabetic state up to the 20th day after transplantation. On the other hand, 6 of 8 rats in the IP group remained normoglycemic throughout the experimental period. Weight gain and diminution of urinary glucose excretion in the IU group were significantly lower than those in the IP group (P < 0.01). The glycosylated hemoglobin level in the IU group did not differ significantly from that in the IP group, but the serum level of fructosamine in the IU group was significantly higher than that in the IP group (P < 0.01). These results indicate that the response to fluctuations of blood glucose of islets in the uterine cavity is less than that of islets in the peritoneal cavity. Histologically, islets were observed to be aggregated in the uterine cavity, however the number of cells decreased markedly with time. Although this study demonstrated that blood glucose was normalized by transplantation of islets into the uterine cavity of diabetic rats, long-term survival of the islets in this location was not obtained.

Monitoring bovine embryo viability with early pregnancy factor.

The viability of the bovine embryo was monitored by measuring the early pregnancy factor (EPF). The EPF activity was measured by the rosette inhibition test before and after artificial insemination (AI) at natural estrus (n = 14), and after superovulatory treatment followed by embryo removal on day 7 after AI (n = 5). In the cows inseminated artificially at natural estrus, there were significant differences (p < 0.01) in the rosette inhibition titer (RIT) between pregnant and non-pregnant cows on day 13-16 and day 20-25 after AI. In the 8 pregnant cows, the RIT remained more than 5 from day 6-9 after AI. In the 6 non-pregnant cows, two patterns were observed. In one pattern, RIT rose transiently to more than 5 and decreased to less than 4 thereafter. In the other pattern, RIT remained less than 4 throughout the experimental period. The former pattern suggested early embryonic death, while the latter suggested that fertilization had not taken place or that early embryonic death had occurred before the first blood collection on day 6-9 after AI. In the cows superovulated followed by embryo removal on day 7 after AI, the RIT values were all less than 4 on the day of AI (day 0), rose to more than 5 on day 3 and thereafter then until the day of embryo removal on day 7. In 4 cows, the RIT decreased to less than 4 by 3 days after embryo removal, and in the remaining one cow, the RIT decreased to less than 4 by 7 days after embryo removal. These findings suggest that the measurement of EPF activity is useful for monitoring the viability of bovine embryos.