Iacono Eleonora, Pascucci Luisa, Bazzucchi Cinzia, Cunto Marco, Ricci Francesca, Rossi Barbara, Merlo Barbara
Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064, Ozzano Emilia (Bologna), Italy.
Department of Veterinary Medicine, University of Perugia, via San Costanzo 4, 06126, Perugia, Italy.
Vet Res Commun. 2018 Dec;42(4):297-308. doi: 10.1007/s11259-018-9738-9. Epub 2018 Sep 20.
The aim of the present study was to compare canine adipose tissue mesenchymal stem cells cultured under normoxic (20% O) and not severe hypoxic (7% O) conditions in terms of marker expression, proliferation rate, differentiation potential and cell morphology. Intra-abdominal fat tissue samples were recovered from 4 dogs and cells isolated from each sample were cultured under hypoxic and normoxic conditions. Proliferation rate and adhesion ability were determined, differentiation towards chondrogenic, osteogenic and adipogenic lineages was induced; the expression of CD44, CD34, DLA-DQA1, DLA-DRA1 was determined by PCR, while flow cytometry analysis for CD90, CD105, CD45 and CD14 was carried out. The morphological study was performed by transmission electron microscopy. Canine AT-MSCs, cultured under different oxygen tensions, maintained their basic biological features. However, under hypoxia, cells were not able to form spheroid aggregates revealing a reduction of their adhesivness. In both conditions, MSCs mainly displayed the same ultrastructural morphology and retained the ability to produce membrane vesicles. Noteworthy, MSCs cultivated under hypoxya revealed a huge shedding of large complex vesicles, containing smaller round-shaped vesicles. In our study, hypoxia partially influences the basic biological properties and the ultrastructural features of canine mesenchymal stem /stromal cells. Further studies are needed to clarify how hypoxia affects EVs production in term of amount and content in order to understand its contribution in tissue regenerative mechanisms and the possible employment in clinical applications. The findings of the present work could be noteworthy for canine as well as for other mammalian species.
本研究的目的是比较在常氧（20% O₂）和非严重低氧（7% O₂）条件下培养的犬脂肪组织间充质干细胞在标志物表达、增殖率、分化潜能和细胞形态方面的差异。从4只犬获取腹腔内脂肪组织样本，将每个样本分离出的细胞分别在低氧和常氧条件下培养。测定增殖率和黏附能力，诱导其向软骨生成、成骨和成脂谱系分化；通过PCR测定CD44、CD34、DLA - DQA1、DLA - DRA1的表达，同时对CD90、CD105、CD45和CD14进行流式细胞术分析。通过透射电子显微镜进行形态学研究。在不同氧张力下培养的犬脂肪组织间充质干细胞保持其基本生物学特性。然而，在低氧条件下，细胞无法形成球状体聚集体，显示其黏附性降低。在两种条件下，间充质干细胞主要呈现相同的超微结构形态，并保留产生膜泡的能力。值得注意的是，在低氧条件下培养的间充质干细胞显示出大量含有较小圆形囊泡的大型复合囊泡脱落。在我们的研究中，低氧部分影响犬间充质干/基质细胞的基本生物学特性和超微结构特征。需要进一步研究以阐明低氧如何在数量和内容方面影响细胞外囊泡的产生，以便了解其在组织再生机制中的作用以及在临床应用中的可能用途。本研究结果对犬以及其他哺乳动物物种可能具有重要意义。
