Project description:Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by PCR and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change .2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change .0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analysing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-sepcific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo culture in human supplements and thus may contribute to the therapeutic potential. Microarray hybridizations (Agilent: Whole Human Genome Oligo Microarray; 41k unique probe) were carried out with 5 µg Cy3 labeled cRNA and 5 µg Cy5 labeled cRNA, both prepared from each sample.

Project description:Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by PCR and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change .2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change .0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analysing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-sepcific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo culture in human supplements and thus may contribute to the therapeutic potential.

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Expression profiling of human peripheral blood before and after omega 3 dietary supplement.

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