Characterization of teratogenic potential and gene expression in canine and feline amniotic membrane-derived stem cells.

The biosafety of innovative procedures that utilize stem cells in regenerative medicine has been addressed in several studies. Previous work has showed no tumour formation following the use of feline and human amniotic membrane-derived stem cells (AMSCs). In contrast, tumour formation was observed when canine AMSCs were utilized. These findings suggested that feline and human, but not canine, AMSCs are suitable for cell transplantation trials. This study aimed to further evaluate the feasibility of utilizing canine AMSCs for transplantation purposes as well as for felines. We tested teratoma formation following cell injection into BALB/c nude mice and then assessed expression of haematopoietic, mesenchymal, tumorigenic, pluripotency and cellular regulation markers using flow cytometry and qPCR. The use of canine AMSCs did not result in macroscopic tumour formation as determined 60 days after transplantation. The immunophenotypic characterization by flow cytometry revealed expression of mesenchymal markers (CD73 and CD90) and expression of the pluripotent marker OCT4 and SOX2. Quantitative PCR analysis revealed that there were no differences in the patterns of gene expression (CD34, CD73, OCT4, CD30 and P53) between canine and feline AMSCs, with the exception of the expression of SOX2 and CD90.

Occurrence of Smut Caused by a Ustilago sp. on Dry-Beans.

In September 1997, after the tropical winter season and following a long warm (28 to 33°C) and rainy (27 mm) period, smut symptoms were observed on two dry-bean (Phaseolus vulgaris) fields in Brazil. Brownish to blackish powdery masses of spores were observed on stems, pods, and roof green and senescent plants. No galls were observed. Hyphae in infected tissue were intercellular; no clamp connections were observed. Typical globose teliospores were observed that measured 7 to 11 µm. Spores were uninucleate or binucleate; predominantly brownish with flattened poles and equatorial band; the exospore wall was thick and surface ornamented (echinulate-type). Teliospores incubated in water drops generated transversely septate promycelia, unbranched, producing terminal and lateral basidiospores. The fungus did not grow well on potato dextrose agar (PDA) medium, generating yeastlike colonies. To confirm pathogenicity of a Ustilago sp., spores collected from field-infected plants were inoculated on 8- and 20-day old seedlings of dry-bean cultivars Rosinha and Perola. Spore suspensions of 105 teliospores were sprayed all over the plants. After inoculation, plants were kept in a moist chamber (relative humidity [RH] 100%) for 2 days. At the third day, inoculated plants were covered with clear polyethylene bags and transferred to the greenhouse (RH 50%, temperature 25 ± 2°C). Noninoculated controls were included for comparisons. Within 2 weeks, all inoculated plants reproduced field symptoms of darkened tissues covered by profuse sori containing teliospores and basidiospores. Eight-day-old plants died 2 weeks after inoculation. Noninoculated plants did not develop any symptoms. Teliospores obtained from dead plants were identical to those used for inoculation, thus completing Koch's postulates. Fischer (1), and later Fischer and Shaw (2), proposed a species concept for Ustilago based on morphology (primarily of the teliospore), symptomatology, and host specialization at the host-family level. Although the teliospores found on dry-beans, in Brazil, are much like those of Ustilago maydis, the fungus did not cause galls on plants, was able to infect roots and kill seedlings. and is very well adapted to a legume plant. Cross inoculation tests indicated that spores from dry-beans cannot cause disease symptoms on maize. We therefore regard the bean smut as a new species. We are not aware of any other Ustilago sp. being reported on a legume host. References: (1) G. W. Fischer. 1953. Manual of the North America Smut Fungi. (2) G. W. Fischer and C. G. Shaw. Phytopathology 43:181, 1953.