Cell-cell and cell-substratum contacts in the regulation of MAPK and Akt signalling: Importance in therapy, biopharmacy and bioproduction.

The use of cultured cells as a tool for research, precision medicine, biopharmacy, and biomanufacturing is constantly increasing. In parallel, the role of cell-cell and cell-substratum contacts in cell functioning is increasingly validated. Adhesion signalling plays a key role here. The activity of cell fate-regulating signalling molecules is an important factor in determining cell behaviour, as well as their response to treatment, depending on cell phenotypic status and location in the body. Three cellular state models (adherent, single cells in suspension, and aggregated cells) were compared for cell signalling, including focal adhesion (FAK), mitogen-activated (MAPK), as well as Akt protein kinases, and transcription factor cJun, by using lung adenocarcinoma A549, muscle-derived stem Myo, as well as primary lung cancer cell lines. Survival of both A549 and Myo cells was dependent on kinases Akt and ERK in detached conditions. Intercellular contacts in aggregates promoted activation of Akt and ERK, and cell survival. Loss of contacts with the substrate increased phosphorylation of MAP kinases JNK and p38, while decreased Akt phosphorylation by processes involving FAK. Unexpectedly, detachment increased phosphorylation of antiapoptotic kinase ERK in A549, while in Myo stem cells ERK phosphorylation was downregulated. JNK target transcription factor cJun protein level was markedly diminished by contacts between cells possibly involving mechanism of proteasomal degradation. Furthermore, studies revealed the opposite dependence of molecules of the same signalling pathway - phospho-cJun and phospho-JNK - on cell culture density. Differences in ERK activation under detachment conditions indicate that targeting of prosurvival kinases during anoikis should be different in different cells. Moreover, the outcome of JNK activation in cells may depend on the amount of cJun, which is determined by cell-cell contacts.

Paracrine Potential of the Human Adipose Tissue-Derived Stem Cells to Modulate Balance between Matrix Metalloproteinases and Their Inhibitors in the Osteoarthritic Cartilage In Vitro.

Adipose tissue represents an abundant source of stem cells. Along with anti-inflammatory effects, ASC secrete various factors that may modulate metabolism of extracellular matrix in osteoarthritic (OA) cartilage, suggesting that the presence of ASC could be advantageous for OA cartilage due to the recovery of homeostasis between matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs). To evaluate these effects, cartilage explants (CE) were cocultured with ASC for 3 and 7 days under stimulation with or without IL-1ß. The pattern of gene expression in CE was modified by ASC, including the upregulation of COL1A1 and COL3A1 and the downregulation of MMP13 and COL10A1. The production of MMP-1, MMP-3, and MMP-13 by ASC was not significant; moreover, cocultures with ASC reduced MMP-13 production in CE. In conclusion, active production of TIMP-1, TIMP-2, TIMP-3, IL-6, IL-8, and gelatinases MMP-2 and MMP-9 by ASC may be involved in the extracellular matrix remodelling, as indicated by the altered expression of collagens, the downregulated production of MMP-13, and the reduced chondrocyte apoptosis in the cocultured CE. These data suggest that ASC modulated homeostasis of MMPs/TIMPs in degenerated OA cartilage in vitro and might be favourable in case of the intra-articular application of ASC therapy for the treatment of OA.

Epigenetic regulation of human adipose-derived stem cells differentiation.

Adult stem cells have more restricted differentiation potential than embryonic stem cells (ESCs), but upon appropriate stimulation can differentiate into cells of different germ layers. Epigenetic factors, including DNA modifications, take a significant part in regulation of pluripotency and differentiation of ESCs. Less is known about the epigenetic regulation of these processes in adult stem cells. Gene expression profile and location of DNA modifications in adipose-derived stem cells (ADSCs) and their osteogenically differentiated lineages were analyzed using Agilent microarrays. Methylation-specific PCR and restriction-based quantitative PCR were applied for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) detection in selected loci. The level of DNA modifications in the POU5F1 locus was quantified with deep sequencing. Expression levels of selected genes were assayed by real-time PCR. ADSCs differentiation into osteogenic lineages involved marked changes in both 5mC and 5hmC profiles, but 5hmC changes were more abundant. 5mC losses and 5hmC gains were the main events observed during ADSCs differentiation, and were accompanied by increased expression of TET1 (P = 0.009). In ADSCs, POU5F1 was better expressed than NANOG or SOX2 (P ≤ 0.001). Both 5mC and 5hmC marks were present in the POU5F1 locus, but only hydroxymethylation of specific cytosine showed significant effect on the gene expression. In summary, the data of our study suggest significant involvement of changes in 5hmC profile during the differentiation of human adult stem cells.

Effects of different sera on adipose tissue-derived mesenchymal stromal cells.

Current cell therapy protocols require considerable numbers of mesenchymal stromal cells (MSCs), which can be obtained only by in vitro expansion. The most important issue is a choice of optimal growth supplements for cell culture. Ideally, these should be of known composition, free of animal components and allow production of large homogenic populations of MSCs in a considerably short period of time. Since this standard has not been achieved to date, we aimed to assess the molecular responses of MSCs to different growth supplements commonly in use. MSCs were isolated from breast or abdominal adipose tissue and plated into DMEM supplemented with one of four different sera: fetal calf serum (FCS), pretested fetal calf serum (FCS-Sp), human allogeneic serum (HS) or artificial serum substitute (AS). MSCs cultivated with different serum supplements demonstrated distinct morphologies, high adipogenic and osteogenic differentiation potential and expressed characteristic antigens. Using real-time PCR, we found a large increase in PPARγ and Msx2 gene expression in both lines of proliferating MSCs cultivated with AS. We found that MSCs cultivated in the presence of different sera had similar global proteomic expression patterns, but comparisons of identified proteins revealed most differences in the MSCs cultivated with AS. Our results indicate that MSCs cultivated in the presence of FCS and HS display similar growth, differentiation, immunophenotypic and proteomic properties, while AS induces more profound changes in the physiology of MSCs, suggesting that further fundamental studies should be done before its introduction into clinical practice.