Research into adipose tissue-derived mesenchymal stem cells (AD-MSCs) has demonstrated the feasibility of their use in clinical applications due to their ease of isolation and abundance in adipose tissue. We isolated AD-MSCs from young and old dogs, and the cells were subjected to sequential sub-passaging from passage 1 (P1) to P7. Canine AD-MSCs (cAD-MSCs) were examined for proliferation kinetics, expression of molecules associated with self-renewal, expression of cell surface markers, and differentiation potentials at P3. Cumulative population doubling level was significantly higher in cAD-MSCs of young donors than in those of old donors. In addition, expressions of CD73, CD80, Oct3/4, Nanog, cell survival genes and differentiation potentials were significantly higher in young donors than in old donors. The present study suggests that donor age should be considered when developing cell-based therapies for clinical application of cAD-MSCs.

Until now, the isolation and characterisation of bovine bone marrow-derived mesenchymal stem cells (bBM-MSCs) have not been established, which prompted us to optimise the differentiation protocol for bBM-MSCs. In this study, bBM-MSCs were freshly isolated from three six-month-old cattle and used for pluripotent or phenotypic characterisation by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometer. Initially, the cells were adherent to plastic surfaces and exhibited spindle-like morphology. The cells expressed pluripotent as well as typical MSC markers. In addition, bBM-MSCs were differentiated into chondrocytes and osteocytes, but less efficiently into adipocytes. Accordingly, we conducted this study to establish the optimal adipogenic differentiation protocol under specific culture conditions. For this purpose, we formulated the basal differentiation medium using low-glucose Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin (P/S), 1 mu M dexamethasone, 0.5 mM indomethacin and 0.5 mM 3-isobutyl-l-methylxanthine, and added with three levels of insulin at 10, 15 and 20 mu g/mL as insulin is the key adipogenesis inducer. The level of differentiation was evaluated by Oil Red O staining and by analysing the expression of adipocyte lineage-specific genes by a quantitative real time RT-PCR. In this study, we found that the bBM-MSCs were effectively differentiated into adipocytes as manifested by the presence of Oil Red O-stained lipid droplets. The mRNA levels of adipocyte-specific genes in the differentiated cells were highly expressed as compared with the non-differentiated bBM-MSCs. In conclusion, we successfully isolated and characterised bBM-MSCs with multipotent and differentiation potential. Additionally, enhanced in vitro adipogenic differentiation protocol for bBM-MSCs was established.

Mesenchymal stem cells (MSCs) have the ability to differentiate into multi-lineage cells, which confers great promise for use in regenerative medicine. In this study, MSCs were isolated from adipose tissue, bone marrow, ear skin, lung, and abdominal skin of miniature pigs (mpMSCs), and the optimal medium (DMEM/F12-Glutamax) was selected for the culturing of mpMSCs. As a result, proliferation of the mpMSCs derived from all tissues was steadily increased when cultured with DMEM/F12-Glutamax during 14 consecutive passages. The cells harbored MSC surface markers (CD34-, CD45-, CD29+, CD44+, CD90+, and CD105+), whose levels of expression differed among the tissue sources and declined over sub-passaging. In addition, the expression of stemness markers (Oct4, Sox2, and Nanog) and differentiation into mesoderm (adipocytes, chondrocytes, and osteoblasts) were clearly represented at early passage; however, expression of stemness markers decreased, and differentiation potential was lost over sequential sub-passaging, which should be considered in the selection of mpMSC for MSC-based application.

Adipose-derived mesenchymal stem cells (AD-MSCs) are abundant in adipose tissue from animals of all ages, are easily isolated, can differentiate into multi-lineage cells, and have a clinical application. This promising potential may only be achieved if the cells are expanding in a large number while maintaining their stemness in sequential passages. In this study, canine AD-MSCs (cAD-MSCs) were individually isolated from five dogs and subjected to proliferative culture with seven sub-passages. The cells at each sub-passage were characterized for properties associated with multipotent MSCs such as proliferation kinetics, expression of MSCs-specific surface markers, expression of molecules associated with self-renewal and differentiation capabilities into mesodermal lineage cells. Proliferation of the cells plateaued at passage 5 by cumulative population doubling level, while cell doubling time gradually increased with passage. MSCs surface markers (CD44, CD90, and CD105) and molecules (Oct 3/4, Sox-2, Nanog and HMGA2) associated with self-renewal were all expressed in the cells between passages 1 to 6 by RT-PCR. In addition, the cells at passage 1, 3 or 6 underwent adipogenic and chondrogenic differentiation under specific induction conditions. However, the level of adipogenic and chondrogenic differentiation was negatively correlated with the number of sub-passage. The present study suggests that sequential sub-passages affect multipotent properties of cAD-MSCs, which should be considered in their therapeutic application in regenerative medicine.

The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers. OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.