Proinflammatory interleukins' production by adipose tissue‐derived mesenchymal stromal cells: the impact of cell culture conditions and cell‐to‐cell interaction

The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL‐6 and IL‐8 production by adipose‐derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O₂ concentrations, some highly up‐regulated at hypoxia. IL‐6 and IL‐8 production was inversely dependent on cell culture density. In early (first–third) passages, IL‐6 and IL‐8 concentration was higher at 20% O₂ and in late (8th‐12th) passages under 5% O₂. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL‐6 and did not result in the elevation of IL‐8 concentration. Thereby, the production of proinflammatory interleukins (IL‐6 and IL‐8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood‐borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. Copyright © 2015 John Wiley & Sons, Ltd. SIGNIFICANCE PARAGRAPH Ex vivo expansion is widely used for increasing the number of adipose‐derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science.     

Induction of insulin‐producing cells from umbilical cord blood‐derived stromal cells by activation of the c‐Met/HGF axis

Efficient and effective therapies are required for diabetes mellitus. The use of adult stem cells for treating diabetes represents a major focus of current research. We have attempted to differentiate adult stem cells produced from umbilical cord blood‐derived stromal cells into insulin‐producing cells (IPCs). By activating the c‐Met/HGF axis through temporal hypoxia treatment and hepatocyte growth factor (HGF) supplementation, our protocol resulted in the differentiation of cells into functional pancreatic endocrine cells with increased viability. Glucose stimulation test results showed that significantly greater amounts of C‐peptide and insulin were released from the differentiated cells than from undifferentiated cells. These IPCs were capable of reversing the hyperglycemia of diabetic mice. In conclusion, targeting the c‐Met/HGF axis can be considered an effective and efficient means of obtaining IPCs from adult stem cells.     

Exosomes from miR‐20b‐3p‐overexpressing stromal cells ameliorate calcium oxalate deposition in rat kidney

Hyperoxaluria‐induced calcium oxalate (CaOx) deposition is the key factor in kidney stone formation, for which adipose‐derived stromal cells (ADSCs) have been used as a therapeutic treatment. Studies revealed that miR‐20b‐3p is down‐regulated in hypercalciuric stone‐forming rat kidney. To investigate whether ADSC‐derived miR‐20b‐3p‐enriched exosomes protect against kidney stones, an ethylene glycol (EG)‐induced hyperoxaluria rat model and an in vitro model of oxalate‐induced NRK‐52E cells were established to explore the protective mechanism of miR‐20b‐3p. The results showed that miR‐20b‐3p levels were decreased following hyperoxaluria in the urine of patients and in kidney tissues from animal models. Furthermore, treatment with miR‐20b‐3p‐enriched exosomes from ADSCs protected EG‐induced hyperoxaluria rats, and cell experiments confirmed that co‐culture with miR‐20b‐3p‐enriched exosomes alleviated oxalate‐induced cell autophagy and the inflammatory response by inhibiting ATG7 and TLR4. In conclusion, ADSC‐derived miR‐20b‐3p‐enriched exosomes protected against kidney stones by suppressing autophagy and inflammatory responses.     

Monitoring the ex‐vivo expansion of human mesenchymal stem/stromal cells in xeno‐free microcarrier‐based reactor systems by MIR spectroscopy

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell‐based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex‐vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi‐analytical methods, some of them time‐consuming. The present work evaluates the use of mid‐infrared (MIR) spectroscopy, through rapid and economic high‐throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno‐free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R² ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R² of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:447–455, 2016     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Primate‐specific regulation of natural killer cells

Natural killer (NK) cells are circulating lymphocytes that function in innate immunity and placental reproduction. Regulating both development and function of NK cells is an array of variable and conserved receptors that interact with major histocompatibility complex (MHC) class I molecules. Families of lectin‐like and immunoglobulin‐like receptors are determined by genes in the natural killer complex (NKC) and leukocyte receptor complex (LRC), respectively. As a consequence of the strong, varying pressures on the immune and reproductive systems, NK cell receptors and their MHC class I ligands evolve rapidly, are highly diverse and exhibit dramatic species‐specific differences. The variable, polymorphic family of killer cell immunoglobulin‐like receptors (KIR) that regulate human NK cell development and function arose recently, from a single‐copy gene during the evolution of simian primates. Our studies of KIR and MHC class I genes in representative species show how these two unlinked but functionally intertwined genetic complexes have co‐evolved. In humans, combinations of KIR and HLA class I factors are associated with infectious diseases, including HIV/AIDS, autoimmunity, reproductive success and the outcome of therapeutic transplantation. The extraordinary, and unanticipated, divergence of human NK cell receptors and MHC class I ligands from their mouse counterparts can in part explain the difficulties experienced in finding informative mouse models for human diseases. Non‐human primate models have far greater potential, but to realize their promise will first require more complete definition of the genetics and function of KIR and MHC variation in non‐human primate species, at a level comparable to that achieved for the human species.     

MicroRNA‐98 regulates osteogenic differentiation of human bone mesenchymal stromal cells by targeting BMP2

To study the effects of microRNA‐98 (miR‐98) on human bone mesenchymal stromal cells (hBMSCs). The patients undergoing hip arthroplasty were selected by inclusion/exclusion criteria for this study. The extracted hBMSCs were detected of osteogenic differentiation by alizarin red S staining, and of cell phenotype by flow cytometry. Bioinformatics, dual luciferase report, western blotting, RT‐PCR and immunoblotting were used in our study. The hBMSCs were divided into miR‐98 mimics, miR‐98 negative control (NC), miR‐98 inhibitors, Mock and miR‐98 inhibitors + siBMP2 groups. Human bone mesenchymal stromal cells were extracted and purified in vitro and had specific cytological morphology, surface markers and abilities of self‐renewal and differentiation. Compared with the NC group and Mock group, the miR‐98 mimics group showed increased miR‐98 level while the miR‐98 inhibitors group decreased miR‐98 level (both P < 0.01). Dual luciferase reporter showed BMP2 was the target gene of miR‐98. The levels of mRNA and protein expression of BMP2, protein expression of RUNX2, alkaline phosphatase activity and osteocalcin content significantly decreased in the miR‐98 mimics group while increased in the miR‐98 inhibitors group and showed no changes in the NC group and Mock group (all P < 0.05). The miR‐98 mimics group showed obviously declined stained red particles and the miR‐98 inhibitors group showed opposite result. After lowering the expression of miR‐98, osteogenic differentiation ability of hBMSCs rose, which was weakened by the transfection with siBMP2. miR‐98 may regulate osteogenic differentiation of hBMSCs by targeting BMP2.     

Astrocyte‐like cells differentiated from a novel population of CD45‐positive cells in adult human peripheral blood

We have previously reported a novel CD45‐positive cell population called peripheral blood insulin‐producing cells (PB‐IPCs) and its unique potential for releasing insulin in vitro. Despite the CD45‐positive phenotype and self‐renewal ability, PB‐IPCs are distinguished from hemopoietic and endothelial progenitor cells (EPCs) by some characteristics, such as a CD34‐negative phenotype and different culture conditions. We have further identified the gene profiles of the embryonic and neural stem cells, and these profiles include Sox2, Nanog, c‐Myc, Klf4, Notch1 and Mash1. After treatment with all‐trans retinoic acid (ATRA) in vitro, most PB‐IPCs exhibited morphological changes that included the development of elongated and branched cell processes. In the process of induction, the mRNA expression of Hes1 was robustly upregulated, and a majority of cells acquired some astrocyte‐associated specific phenotypes including anti‐glial fibrillary acidic protein (GFAP), CD44, Glutamate‐aspartate transporter (GLAST) and S100β. In spite of the deficiency of glutamate uptaking, the differentiated cells significantly relaxed the regulation of the expression of brain‐derived neurotrophic factor (BDNF) mRNA. This finding demonstrates that PB‐IPCs could be induced into a population of astrocyte‐like cells and enhanced the neurotrophic potential when the state of proliferation was limited by ATRA, which implies that this unique CD45+ cell pool may have a protective role in some degenerative diseases of the central nervous system (CNS).