Cryopreservation and multipotential characteristics evaluation of a novel type of mesenchymal stem cells derived from Small Tailed Han Sheep fetal lung tissue

- L-MSCs were successfully isolated and cryopreserved from the lung tissues of 45-day-old embryos of sheep.
- L-MSCs could be passaged at least 18 passages and Chromosome G-banding analysis showed >91.8% were diploid.
- L-MSCs could express Oct4, Nanog and MSCs-related surface antigens, while CD34 and CD45 were negative.
- L-MSCs can be efficiently induced to transdifferentiate into mesoblastic cells, neural ectoderm and endoderm in vitro.
- Cell cycle of L-MSCs showed that the rate of G0/G1 was about 70.5-91.2%.

Lung mesenchymal stem cells (L-MSCs) characterized by plasticity, reduced relative immune privilege and high anti-fibrosis characteristics play the crucial role in lung tissue regenerative processes. However, up to date, the multi-differentiation potentials and application values of L-MSCs are still uncertain. In the current study, the Small Tailed Han Sheep embryo L-MSCs line from 12 samples, stocking 124 cryogenically-preserved vials, was successfully established by using primary culture and cell cryopreservation techniques. Isolated L-MSCs were morphologically consistent with fibroblasts, could be passaged for at least 18 passages and more than 91.8% of cells were diploid (2n = 54) analyze by G-banding. The majority of cells were in the G0/G1 phase (70.5-91.2%), and the growth curves were all typically sigmoidal. Moreover, L-MSCs were found to express pluripotent genes Oct4, Nanog and MSCs-associated genes β-integrin, CD29, CD44, CD71, CD73 and CD90, while the expressions of hematopoietic cell markers CD34 and CD45 were negative. In addtion, the L-MSCs could be differentiated into cells of three layers with induction medium in vitro, which confirmed their multilineage differentiation potential. The secretion of urea and ALB showed the differentiated hepatocytes still possessed the detoxification function. These results indicated that the isolated L-MSCs displayed typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their maintenance of stemness and their proliferation in vitro.

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