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.

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:Purpose: The population of muscle-derived stem cells called MuStem cells is presented as promising candidate for cell-based therapy of muscle diseases. To validate if this agent can be really presented as therapeutic product and so to be eligible to a future clinical use, it is now required to demonstrate beforehand an efficacy with cells prepared in compliance with good manufacturing practices (GMPs). The aim of the current study was to evaluate the use of two xeno-free blood derivatives corresponding to human serum (HS) and human platelet lysate (hPL) as alternatives to controverted but until now used fetal bovine serum (FBS) for isolation and expansion of human MuStem (hMuStem) cells. Methods: A comparative study was performed with hMuStem cells isolated and in vitro expanded by using commercially available HS and hPL to determine its impact on their proliferation rates, clonogenicity, myogenic commitment level and oligopotency with regard to results obtained under FBS-based medium. Also, their respective phenotype and global gene expression patterns were investigated by flow cytometry and high throughput 3’ digital gene expression RNA-sequencing in order to define a possible differential impact of the human nutrients tested. Results: Comparatively to FBS-based medium, use of HS- and hPL-supplemented ones efficiently supported long-term proliferation of hMuStem cells and enhanced clonogenicity, without main modification of their expression profile and allowing besides limiting the supplementation in growth factors. In vitro differentiation assay combined to transforming growth factor β1 (TGF-β1)-depletion experiments showed a lower myogenic commitment level as well as fusion ability of hMuStem cells when cultured with hPL-based medium according to a TGF-β1-independent process. Use of hPL-derived 3D hydrogel or fibrinogen-depleted hPL demonstrated that heparin-free hPL derivatives maintain consequent myogenic differentiation potential. In addition, the reduced myogenicity was shown to be rapidly reversible following replacement of hPL by HS or fibrinogen-depleted hPL. Conclusions: All together, our original findings position HS and hPL as efficient and suitable alternatives to FBS for preparation of hMuStem cell batch in compliance with GMPs.

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Expression data from human adipose stem cells expanded in allogeneic human serum and fetal bovine serum

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