Histomorphometric and scanning electron microscopic analyses were performed on 74 embryos and fetuses and 20 sheep (early postnatal to adult age). Histologic differentiation of the omasum took place at 33 days of fetal life, with the appearance of first-order laminae. Second-, third-, and fourth-order laminae appeared at 39, 50, and 59 days, respectively. Neutral mucopolysaccharides first appeared in epithelial cells at 46 days of fetal life, decreasing quantitatively until birth, before subsequently stabilizing in postnatal life. Acid mucopolysaccharides, mucins, and mucoid compounds were not detected. Growth curves and formulas were constructed for each tissue layer. Initial tests involved multiplicative (y = axb), exponential (y = EXP [a + bx]), linear (y = a + bx), and polynomial models [y = a + bx + cx(2) + dx(3)].

OBJECTIVE To evaluate gene expression and DNA copy number in adipose tissue-derived stromal cells (ADSCs) and in ADSC-derived neurosphere-like cell clusters (ADSC-NSCs) generated from tissues of chronically paraplegic dogs. ANIMALS 14 client-owned paraplegic dogs. PROCEDURES Dorsal subcutaneous adipose tissue (< 1 cm3) was collected under general anesthesia; ADSCs were isolated and cultured. Third-passage ADSCs were cultured in neural cell induction medium to generate ADSC-NSCs. Relative gene expression of mesenchymal cell surface marker CD90 and neural progenitor marker nestin was assessed in ADSCs and ADSC-NSCs from 3 dogs by quantitative real-time PCR assay; expression of these and various neural lineage genes was evaluated for the same dogs by reverse transcription PCR assay. Percentages of cells expressing CD90, nestin, glial fibrillary acidic protein (GFAP), and tubulin β 3 class III (TUJ1) proteins were determined by flow cytometry for all dogs. The DNA copy number stability (in samples from 6 dogs) and neural cell differentiation (14 dogs) were assessed with array-comparative genomic hybridization analysis and immunocytochemical evaluation, respectively. RESULTS ADSCs and ADSC-NSCs expressed neural cell progenitor and differentiation markers; GFAP and microtubule-associated protein 2 were expressed by ADSC-NSCs but not ADSCs. Relative gene expression of CD90 and nestin was subjectively higher in ADSC-NSCs than in ADSCs. Percentages of ADSC-NSCs expressing nestin, GFAP, and TUJ1 proteins were substantially higher than those of ADSCs. Cells expressing neuronal and glial markers were generated from ADSC-NSCs and had no DNA copy number instability detectable by the methods used. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested ADSCs can potentially be a safe and clinically relevant autologous source for canine neural progenitor cells. Further research is needed to verify these findings.

Breast tumors are the most common tumors in dogs and the study of disease prognostic factors is important for establishing the appropriate treatment protocols. The purpose of this study was to clinically stage mammary tumors of bitches and correlate the stages with histological type and grade. The tumors of 63 dogs were clinically staged based on the findings of tumor sizing, lymph node evaluation, and radiographic examination. After surgical excision, the tumors were classified histologically and graded. The relationship between the tumor grade, stage, and histological type was evaluated using a binomial test. Stage I tumors were the most numerous (31.75%), followed by tumors at stages II, III, IV, and V. Animals with histological grade I carcinomas presented stage I, II, or III tumors more frequently and stage IV and V tumors less frequently. The number of animals with simple carcinomas that were at stage I of the disease was greater than that at stage V. Carcinomas in the mixed tumors were less aggressive; however, the small number of animals in stage V of the disease made any statistical association impossible. The complex carcinomas presented with the invasion of the lymph nodes and less cellular differentiation in a larger number of animals than did simple carcinomas. Histological grading proved to be the best parameter for the prognostic evaluation of the breast carcinomas.

OBJECTIVE To produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions. SAMPLE Cultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred. PROCEDURES Cell cultures were transfected by electroporation with a plasmid (pNIT) that expresses the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg), which can be controlled by a doxycycline-responsive promoter. Cells that stably integrated the TAg were selected and expanded to passage 25. For each passage, differentiation and fusion properties of the cells were determined and immunocytochemical analyses were performed to evaluate expression of TAg and other muscle-specific proteins. Optimum conditions that led to cell differentiation into myotubes were also determined. RESULTS Compared with nontransfected control cells, myogenic, desmin-positive cells expressed the TAg when incubated at 33°C and could be maintained in culture for numerous passages. Reduced expression of TAg was identified in cells incubated at 37°C or when incubated with doxycycline at 33°C. Expression of TAg was not detected when cells were incubated with doxycycline at 37°C, and when serum was withdrawn from the culture medium, those clones differentiated into a pure population of multinucleated myotubes. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that production of an immortalized clonal equine skeletal muscle cell line was possible. A clonal equine skeletal muscle cell line will be a valuable in vitro tool for use in equine physiology and disease research.

Objective-To evaluate molecular abnormalities in the c-kit gene of canine mast cell tumors (MCT) with different grades of cellular differentiation. Sample Population-31 normal tissue specimens from dogs and 45 canine MCT classified according to grade of cell differentiation. Procedure-Genomic DNA extractions were made from canine MCT and normal tissues. Parts of exon 11, intron 11, and exon 12 of the c-kit gene were amplified by use of polymerase chain reaction. These regions were cloned, sequenced, and compared with GenBank sequences of the National Center for Biotechnology International. A statistical analysis was used to compare sequences from canine MCT and normal tissues. Results-A significantly higher percentage of homozygous intron 11 deletion was found in canine MCT (49%) than in normal tissues (13%). This percentage was also higher in moderately and poorly differentiated MCT, compared with well-differentiated MCT. Although no mutations were detected in any of the specimens, a polymorphism at amino acid position 606 of the canine c-kit sequence was found in all the studied sequences. Conclusion and Clinical Relevance-Results indicated a relationship between intron 11 deletion and MCT, and the grade of MCT differentiation. We suggest that intron 11 deletion may be implicated in the pathogenesis of MCT and could be used as a marker for diagnosis and prognosis of canine MCT.

Explants were prepared from skeletal muscle tissue from 5 nondystrophic pups and from 5 pups with X-linked muscular dystrophy; pups were 2 to 17 weeks old. A serial reexplant method resulted in optimal cell density with minimal fibroblast growth. Cultures were examined daily by use of phase-contrast microscopy; differentiated (postfusion) cultures were examined by electron microscopy. Moderate nuclear pleomorphism and cell clustering were observed in cultures of normal and dystrophic muscle cells. Cultures were maintained to 27 days after plating. Minimal myofilament synthesis was observed in multinucleate cells from nondystrophic and dystrophic pups, but spontaneous contraction of myotubes was not observed during this period. Differences in growth, fusion, or differentiation of myogenic cells into multinucleate cells and myotubes were not found between dystrophic and normal muscle.

Bone repair in horses implies invasive surgeries and increased cost. Research on musculoskeletal disorders therapy in horses includes cell-based therapy with mesenchymal stromal cells (MSCs). Mesenchymal stromal cells can be obtained from bone marrow (BMMSCs). Unfortunately, BMMSCs have limited cell replication in vitro. The objective of this study was to develop a biologically immortalized equine stem cell line derived from bone marrow, with unlimited in-vitro proliferation and the ability to differentiate into bone cells. Equine BMMSCs were transfected and immortalized with human telomerase reverse transcriptase (hTERT) gene. Cell passages from equine immortal BMMSCs were characterized by the presence of stemness CD markers and expression of multi-potent differentiation genes (OCT-4, SOX2, and NANOG). Equine immortal BMMSCs were incubated in osteogenic medium and bone cell differentiation was determined by alkaline phosphatase and von Kossa staining, and osteogenic gene expression (osteocalcin, Runx2, and osterix). Telomerase activity was determined by telomeric repeat amplification technique. Results showed that equine immortal BMMSCs were able to replicate in-vitro up to passage 50 and maintain stem cell characteristics by the presence of CD90 and expression of multi-potent genes. Equine immortal BMMSCs were able to differentiate into bone cells, which was confirmed by the positive osteogenic staining and gene expression. Equine BMMSCs were successfully immortalized and maintained characteristics of stem cells and readily differentiated into osteogenic cells. Extending the life span of equine BMMSCs by transfection of the hTERT gene will revolutionize the clinical use of MSCs by making them available to orthopedic surgeons "off the shelf."

Using lung tissues separated from 12-day-old chicken embryos, we attempted to obtain a novel population of stem cells, namely, chicken lung-derived mesenchymal stem cells (LMSCs), which exhibit spindle-like morphology. The results of colony-forming assay and population doubling assay demonstrated that LMSCs had enormous colony-forming, self-renewal, and proliferative potential. When appropriately induced, LMSCs could differentiate into osteoblasts, adypocytes, chondrocytes, and neurons; in other words, LMSCs had cross-embryonic layer differentiation potential under corresponding induction conditions. Aside from colony-forming, self-renewal, and multilineage differentiation capabilities, LMSCs were characterized by specific cell phenotypes. The results of immunohistochemistry and flow cytometry demonstrated that LMSCs consistently expressed OCT-4 - a specific gene marker expressed in pluripotent stem cells - and markers associated with MSCs such as CD29, CD73, CD90, and CD105. However, LMSCs lacked hematopoietic cell surface molecules such as CD34 and CD45. Primary LMSCs could be subcultured to passage 24 at most in vitro and karyotype analysis demonstrated that LMSCs possessed genomic stability. These unique characteristics were consistent with the characteristics of MSCs, which had been isolated from other tissues. This provides a foundation for LMSCs as a promising avenue for cellular transplantation therapy, regenerative medicine, and tissue engineering.

The in vitro differentiative potential of mouse parthenogenetic (PG) embryonic stem (PGES) cells were investigated in the formation of embryoid bodies (EBs). EBs derived from PGES cells retarded in growth and showed restricted differentiation compared to their fertilized counterpart. In chimeric EBs from the aggregation of PGES and fertilized ES cells, morphological examination revealed that PGES cells were reduced in their population and distributed in endodermal layer as culture periods proceeded. These findings were comparable to those in aggregation chimeras of fertilized and PG embryos, and suggest that the differentiation of PGES cells in vitro is restricted in the formation of EBs

Murine parthenogenetic embryonic stem (ES) cell lines expressing lac zeta reporter gene were isolated after co-transfection with lac zeta reporter gene (pENL) and neoR gene (pSTneo) to TMA-48P cell line of 129/Sv origin. Karyotype analyses showed that all of four transfected cell lines examined contained 41 chromosomes with trisomy 8. Bacterial neoR transgene required for G418 selection were integrated into several chromosomes including chromosome 8. Histological studies of teratomas formed in syngenic mice and embryoid bodies grown in vitro showed that the differentiative potential remained almost identical in chromosomally normal parental cell line and its derivative cell lines trisomic for chromosome 8