Project description:Limbal stromal cells were reported to resemble mesenchymal stem cells (MSCs) with multipotential differentiation cability. However, little is known about their gene expression profiles compared to MSC derived from various sources. In this study, the gene expression profile of limbal stromal cells was compared to bone marrow, adipose stromal cells and foreskin fibroblasts. In addition, we also explored the gene expression changes of ex vivo expanded limbal stromal cells when cultured in two different systems. Expanded limbal stromal cells were divided into two groups; each cultured separately on a matrigel-coated plate in DMEM/F12 medium supplemented with bFGF and LIF and the other on a normal plate in DMEM medium supplemented with 10% fetal bovine serum (FBS). Cryopreserved bone marrow mesenchymal cells, adipose stromal cells and foreskin fibroblasts were cultured-expanded until confluent. Total RNA was extracted from all the samples and subjected to microarray experiments with an Agilent platform by using Human GE 8x60k microarrays. Data analysis was carried out with GeneSpring software. A total of 871 genes were upregulated when the limbal stromal cells were cultured in the matrigel system, whereas 58 genes were consistently differentially expressed in limbal stromal cells compared to other lineages. Besides the long intergenic non-coding RNA and unknown genes, these genes represent gene ontology for cellular components, molecular function and biological process. Samples derived from the same source were closely clustered by Hierachical clustering analysis. The limbal stromal cells have a distinct molecular signature compared to MSCs from other lineages. The culture system affected the gene expression profile of limbal stromal cells tremendously. Derived limbal stromal cells were cultured using two different methods, one with matrigel and the other with FBS. Their gene expression profiles were compared. The gene expression profile of limbal stromal cells that were cultured with FBS also was compared to the gene expression profiles of bone marrow mesenchymal stem cells, adipose stromal cells and foreskin fibroblasts.

Project description:The treatment of bone defects caused by infection, trauma or neoplasms remains a clinical challenge. Autologous bone transplantation is limited by availability, donor site morbidity and surgical risk factors. This has given rise to stromal/stem-cell based therapy. Bone marrow derived stromal cells (BMSCs) have been studied to a large extent and show high regenerative potential but their use is limited by availability, donor site morbidity and the relatively low cell yield as they represent only <0.1% of cell harvested from bone marrow aspirate. At the same time, they are the closest mesenchymal stromal cells for bone tissue engineering given their tissue origin and, unlike other mesenchymal stromal cells, can support the formation of hematopoietic marrow. Adipose tissue derived stromal cells (ASCs) as part of the stromal vascular fraction of adipose tissue can as well undergo osteogenic differentiation but can be additionally isolated in a sufficient quantity from lipoaspirate after liposuction of abundant subcutaneous fat tissue. Here, it has been shown that there are no major differences in regard to proliferation or differentiation capacity of ASCs derived from subcutaneous fat of different anatomical regions. It has been shown that BMSCs are more prone to senescence during expansion and passage than ASCs and that ageing impacts proliferative capabilities of BMSCs more than that of ASCs while it has also been reported that osteogenic differentiation capacity is least impacted by age. Multiple studies have compared the characteristics of these two mesenchymal stromal cells in regard to bone tissue engineering in vitro. Most studies point to inferior extracellular matrix mineralization and lower expression of key osteogenic transcription markers like Runx2 in osteogenic differentiated ASCs compared to BMSCs. On the other hand, a study by Rath et al. found contrary results using particular culturing conditions like 3D bioglass scaffolds. An intraindividual comparison of human MSCs of three donors cultured on decellularized porcine bone confirmed superior osteogenic capacity of BMSCs compared to ASCs. In contrast to BMSCs, ASCs were not able to induce heterogenic ossification in a mouse model. In a sheep tibia defect model application of BMSCs resulted in a significantly higher amount of newly formed bone tissue. Importantly, Osteogenic differentiated ASCs do not support the formation of a hematopoietic marrow. Proteomics enables large-scale analysis of proteins present in a cell type and can be used to identify differentially regulated key proteins in a comparative approach. A comparative proteomic analysis of BMSCs and ASCs by Roche et al. in 2009 identified 556 proteins with 78% of these not being differentially regulated between these two cell populations, regarded as high similarity. Another comparative proteomic study of 2016 by Jeon et al. found 90 differentially regulated proteins out of 3000 total identified proteins. Both studies do not specify a number of different tissue donors and in part using cell lines. Looking for differences upon osteogenic differentiation, transcriptomic comparison of osteogenic differentiated porcine ASCs and BMSCs has been performed, resulting in 21 differentially expressed genes after 21 days of osteogenic culture conditions. Still, it remains unanswered, which are the key distinctive features of osteogenic differentiated ASCs and BMSCs at protein level that might help address the abovementioned weaknesses of ASCs in bone tissue engineering/regeneration for translational research. To overcome this need, an intraindividual comparative DIA based proteomic analysis of osteogenic differentiated human BMSC and ASCs was performed in this study.

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