Donor Age of Human Platelet Lysate Affects Proliferation and Differentiation of Mesenchymal Stem Cells

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation

Generation and characterization of a mitotane-resistant adrenocortical cell line

Mitotane is the only drug approved for the therapy of adrenocortical carcinoma (ACC). Its clinical use is limited by the occurrence of relapse during therapy. To investigate the underlying mechanisms in vitro, we here generated mitotane-resistant cell lines. After long-term pulsed treatment of HAC-15 human adrenocortical carcinoma cells with 70 µM mitotane, we isolated monoclonal cell populations of treated cells and controls and assessed their respective mitotane sensitivities by MTT assay. We performed exome sequencing and electron microscopy, conducted gene expression microarray analysis and determined intracellular lipid concentrations in the presence and absence of mitotane. Clonal cell lines established after pulsed treatment were resistant to mitotane (IC50 of 102.2 ± 7.3 µM (n = 12) vs 39.4 ± 6.2 µM (n = 6) in controls (biological replicates, mean ± s.d., P = 0.0001)). Unlike nonresistant clones, resistant clones maintained normal mitochondrial and nucleolar morphology during mitotane treatment. Resistant clones largely shared structural and single nucleotide variants, suggesting a common cell of origin. Resistance depended, in part, on extracellular lipoproteins and was associated with alterations in intracellular lipid homeostasis, including levels of free cholesterol, as well as decreased steroid production. By gene expression analysis, resistant cells showed profound alterations in pathways including steroid metabolism and transport, apoptosis, cell growth and Wnt signaling. These studies establish an in vitro model of mitotane resistance in ACC and point to underlying molecular mechanisms. They may enable future studies to overcome resistance in vitro and improve ACC treatment in vivo

Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks

Cells in culture undergo replicative senescence. In this study, we analyzed functional, genetic and epigenetic sequels of long-term culture in human mesenchymal stem cells (MSC). Already within early passages the fibroblastoid colonyforming unit (CFU-f) frequency and the differentiation potential of MSC declined significantly. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow. Notably, all MSC revealed highly consistent senescence-associated modifications at specific CpG sites. These DNA-methylation changes correlated with histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Taken together, culture expansion of MSC has profound functional implications - these are hardly reflected by genomic instability but they are associated with highly reproducible DNA-methylation changes which correlate with repressive histone marks. Therefore replicative senescence seems to be epigenetically controlled

Повышение эффективности противопожарной защиты магазина «Fix Price»

Целью работы является усовершенствование системы противопожарной защиты магазина "Fix Price". В выпускной квалификационной работе проведен обзор литературных источников по вопросам состояния проблем обеспечения пожарной безопасности на предприятиях торговли, дан анализ автоматических систем пожаротушения, обоснован выбор автоматической установки пожаротушения тонкораспылённой водой для объекта исследования, произведены расчёты индивидуального пожарного риска.The aim of the work is to improve the fire protection system of the "Fix Price" store. In the final qualifying work, a review of literature sources on the state of problems of fire safety in commercial enterprises is conducted, an analysis of automatic fire extinguishing systems is given, the choice of an automatic fire extinguishing system with thin-sprayed water for the object of research is justified, and calculations of individual fire risk are made

Serum after Autologous Transplantation Stimulates Proliferation and Expansion of Human Hematopoietic Progenitor Cells

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34+ cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34+, CD133+, CD45−) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool

Optimierung der Kulturbedingungen zur Expansion humaner mesenchymaler Stromazellen

Mesenchymal stromal cells (MSC) show a mesodermal differentiation and immunosuppressive potential and are suitable for various clinical applications like for treatment of bone and connective tissue disorders or autoimmune diseases. Different substrates and media are used for in vitro expansion and manipulation of MSC, but they may affect MSC-heterogenity and characteristics. Currently, there are no optimal culture conditions available for MSC. In this work, the properties of the culture-media supplement human platelet lysate (HPL) and the effect of transforming growth factor (TGF)-b1 on MSC-culture-expansion was systematically tested. Therefore, their effects on MSC-characteristics like vitality, expansion, differentiation potential and replicative senescence were analyzed. An examination of individual platelet lysates for MSC-culture revealed high variations in their potential to support MSC-proliferation, which showed a positive correlation to the PDGF-AB and IGFBP1/2 cytokine concentrations of the platelet lysates. Nevertheless, each individual HPL supported MSC-characteristics and was suitable for efficient MSC-expansion. An improvement of MSC-proliferation could be achieved by using preferably low concentrations of preservative-free heparin (0.61 IU/mL). This anti-coagulant is necessary to prevent co-agulation of HPL-media, but showed an anti-proliferative and in high concentrations (625 IU/mL) a cytotoxic effect that was enhanced by benzyl alcohol as a preservative. On the basis of its content of fibrinogen and factors of the coagulation cascade coagulated HPL-media, could be further established as a more efficient three-dimensional substrate for the isolation and multi-layered and rapid culture-expansion of MSC. The viscosity allowed for a non-enzymatic passaging method by fragmentation of the gel, thereby facilitating this critical cell-culture step. MSC-proliferation could be further promoted over a short period by addition of recombinant TGF-b1. Yet, TGF-b1 induced reversible changes in MSC growth-pattern and impaired adipogenic and osteogenic differentiation. A comparative transcriptome-wide analysis of the effect of TGF-b1 on MSC of early and late passage additionally indicated that culture-expansion does not change the response towards this cytokine. Taken together, the results of this thesis offer valuable information that can be used for further optimization of MSC-culture-expansion

Human platelet lysate gel provides a novel three dimensional-matrix for enhanced culture expansion of mesenchymal stromal cells.

Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that-apart from heparin-consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and reseeding procedure by pipetting cells together with their HPL-matrix-this method does not require washing steps and can easily be automated. The immunophenotype and in vitro differentiation potential toward adipogenic, osteogenic, and chondrogenic lineage were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities, and nonenzymatic passaging procedures for culture expansion of MSCs