The influence of hypoxia, strain and growth differentiation factors on equine adipose tissue derived mesenchymal stem cells : a study to improve stem cell differentiation in vitro for their future application in vivo

摘要

Oxygen tension is an important factor for stem cell culture and differentiation. Since in vivo conditions are mostly hypoxic, but in vitro conditions are generally normoxic, the goal of this study was to examine and compare equine ASCs behaviour and differentiation potential under hypoxic (3% O2) and normoxic (21% O2) conditions. Examined was the differentiation potential towards the adipogenic, osteogenic, chondrogenic and tenogenic lineage, with a special interest and focus on the tenogenic differentiation, since tendon injuries are one of the most common lameness reason in the horse and lack of satisfying treatment options at the same time.While the expression of characteristic stem cell markers did not vary under normoxic or hypoxic culture conditions, the cell proliferation as determined by the MTT assay was higher under normoxic conditions. We were also able to show that adipogenesis and chondrogenesis revealed better histological differentiation results under hypoxic conditions, while osteogenesis was more effective under normoxic culture conditions.For the tenogenic differentiation potential not only the influence of oxygen tension, but also the influence of a 3d collagen scaffold, applied uniaxial tensile strain and supplementation of growth differentiation factors, namely GDF 5, GDF 6, GDF 7, respectively a combination of those three factors was examined in a combined in vitro experiment. Immunohistochemistry revealed that all ASCs of the differently treated collagen constructs formed cell to cell contacts and developed a 3d network in the scaffold. Furthermore it could be demonstrated that tensile strain is necessary to reach matrix stiffness, tendon-typical cell morphology and a coordinated cell alignment in the scaffold. By electron microscopy it could be shown that the examined cell morphology was more tendon-typical under an oxygen tension of 21%, while the gene expression of the tendon relevant markers Col I, Col III, COMP and Scx revealed no big differences under the compared oxygen tensions, even though the results under normoxic conditions were more stable. Compared with equine tendon, the gene expression of Col I and Col III was higher in the samples of the in vitro engineered tendon-like cells, but the gene expression of COMP and Scx was distinct lower. A supplementation of the medium with GDFs, especially GDF 5 and GDF 7 improved the cell morphology and the gene expression. Surprisingly we could show that alone the usage of the 3d collagen scaffold helped to drive the stem cell differentiation towards the tenogenic lineage. An expression of all four examined genes could be detected, even though the gene expression of COMP and Scx was very low. At the end of the pre-differentiation experiment the cells emigrated the collagen construct after taking the scaffold out of the bioreactor and placing it in DMEM. This way, the pre-differentiated cells can be harvested and used for in vivo application. Hopes are, that the in vivo application of in vitro pre-differentiated tenocyte-like cells lead to faster and better tendon repair, if not regeneration compared with the usage of undifferentiated ASCs.