Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

<p>Abstract</p> <p>Background</p> <p>It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.</p> <p>Results</p> <p>A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice.</p> <p>Conclusion</p> <p>Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.</p

Engraftment of human adipose derived stem cells delivered in a hyaluronic acid preparation in mice Implante de células tronco do tecido adiposo humano numa preparação de ácido hialurônico em camundongos

PURPOSE: To evaluate the implant of human adipose derived stem cells (ADSC) delivered in hyaluronic acid gel (HA), injected in the subcutaneous of athymic mice. METHODS: Control implants -HA plus culture media was injected in the subcutaneous of the left sub scapular area of 12 athymic mice. ADSC implants: HA plus ADSC suspended in culture media was injected in the subcutaneous, at the contra lateral area, of the same animals. With eight weeks, animals were sacrificed and the recovered implants were processed for extraction of genomic DNA, and histological study by hematoxilin-eosin staining and immunufluorescence using anti human vimentin and anti von Willebrand factor antibodies. RESULTS: Controls: Not visualized at the injection site. An amorphous substance was observed in hematoxilin-eosin stained sections. Human vimentin and anti von Willebrand factor were not detected. No human DNA was detected. ADSC implants - A plug was visible at the site of injection. Fusiform cells were observed in sections stained by hematoxilin- eosin and both human vimentin and anti von Willebrand factor were detected by immunofluorescence. The presence of human DNA was confirmed. CONCLUSION: The delivery of human adipose derived stem cells in preparations of hyaluronic acid assured cells engraftment at the site of injection.<br>OBJETIVO: Avaliar o implante de células tronco do tecido adiposo humano (CTTAH) em gel de ácido hialurônico (AH), injetados no tecido subcutâneo de camundongos atímicos. MÉTODOS: Implantes controle - HA com meio de cultura foram injetados no tecido subcutâneo da região infraescapular esquerda de 12 camundongos atímicos. Implantes de CTTAH: HA com CTTAH suspensas em meio de cultura foi injetado no subcutâneo da região contra lateral, dos mesmos animais. Com oito semanas, os animais foram sacrificados e os implantes recuperados foram processados para extração de DNA genômico, estudo histológico por coloração por hematoxilina eosina e imnuoflurescência utilizando anticorpos anti vimentina humana e anti fator de von Willebrand. RESULTADOS: Controles - implantes não visualizados no local da injeção. Uma substância amorfa foi observada nos cortes corados por hematoxilina eosina. Vimentina humana e fator anti von Willebrand não foram identificados. DNA humano não foi detectado. Implantes de CTTAH - Uma massa era visível no local da injeção. Células fusiformes foram observadas nos corte corados com hematoxilina eosina. Tanto vimentina humana quanto fator de von Willebrand foram identificados pela imunofluorescência. A presença de DNA humano foi confirmada. CONCLUSÃO: O implante de células tronco do tecido adiposo humano em veículo de ácido hialurônico gel assegurou a manutenção das células no local do implante

Impact of neonatal digestion on the physiology of breast milk bacteria and their immunomodulation capacities

International audienceExclusive breastfeeding in the first months of life has protective effects on infant health compared to formula-fed infants including a lower risk of gastrointestinal and respiratory infections and of metabolic and immune disorders. These observable differences in 'health effect' are likely due to differences in composition between breast milk and infant formula. In particular, breast milk contains a wide variety of bacterial species that are present at low dose. This microbial counterpart contributes to the development of the newborn's gut microbiota after digestion of milk matrix. It was also suggested to influence more largely intestinal homeostasis, acting on the gut immunity and intestinal barrier, and thus to contribute to breastmilk health promoting effects [1]. Several studies have investigated the impact of commensal bacteria on gut homeostasis. However, they generally do not include the different phases of digestion, like gastric (G) or intestinal (I) phases, which could have an impact on the physiological state and properties of the bacteria. The aim of this study was to evaluate the impact of newborn digestion on the physiology of breastmilk bacteria and on their mmunomodulatory potential. For this study, six strains representative of the prevalent bacteria in breast milk were selected. The strains were digested in a static in vitro model of digestion, at full-term infant stage, in a milk matrix. Following the G and I phases, bacterial cultivability was measured and the immunomodulation properties were determined through the quantification of IL-10 and TNF-α released by macrophages (THP-1 line: human monocytic cell line differentiated into macrophages). The impact of the G and I digestion phases on both viability and immunomodulation properties was strain-dependent, pointing out the interest to consider these steps for the determination of ingested bacteria properties.This study is part of the PROLIFIC project and was financially supported by the Régions Bretagne and Pays de Loire and the Milkvalley-BBA consortium

Comparaison of dynamic in vitro digestion of human milk vs standard infant formula to better understand their digestive kinetics

International audienceHuman milk (HM) and Infant formula (IF) are assumed to present different digestion kinetics, while rarely directly compared. The study aimed to address this question using a dynamic digestion model (DIDGI®) at the infant stage.Fresh HM (pool from 50 mothers) and standard IF with similar total nitrogen content were digested in triplicate. Digesta were sampled regularly in gastric and intestinal phase from 0 to 180 min to evaluate structural changes (confocal microscopy and laser light scattering), proteolysis (SDS-PAGE and densitometry, OPA), and nitrogen digestibility (µ-Kjeldahl). Differences between groups were assessed using a two-way repeated measures ANOVA.The microstructure of the digesta differed between HM and IF along the gastric digestion. Proteolysis of the common HM and IF proteins, caseins and α-lactalbumin, was significantly slower for HM than for IF. The intestinal degree of proteolysis was lower for HM than IF, at least during the first two hours. Total N digestibility was lower with HM, such as observed in vivo. Peptidomic and lipolysis data will complete the dataset.Despite nutritional similarity, this study highlights the influence of the matrix on the digestion kinetics and gives some further understanding to the global value of digestibility, such as determined in vivo

Comparaison of dynamic in vitro digestion of human milk vs standard infant formula to better understand their digestive kinetics

International audienceHuman milk (HM) and Infant formula (IF) are assumed to present different digestion kinetics, while rarely directly compared. The study aimed to address this question using a dynamic digestion model (DIDGI®) at the infant stage.Fresh HM (pool from 50 mothers) and standard IF with similar total nitrogen content were digested in triplicate. Digesta were sampled regularly in gastric and intestinal phase from 0 to 180 min to evaluate structural changes (confocal microscopy and laser light scattering), proteolysis (SDS-PAGE and densitometry, OPA), and nitrogen digestibility (µ-Kjeldahl). Differences between groups were assessed using a two-way repeated measures ANOVA.The microstructure of the digesta differed between HM and IF along the gastric digestion. Proteolysis of the common HM and IF proteins, caseins and α-lactalbumin, was significantly slower for HM than for IF. The intestinal degree of proteolysis was lower for HM than IF, at least during the first two hours. Total N digestibility was lower with HM, such as observed in vivo. Peptidomic and lipolysis data will complete the dataset.Despite nutritional similarity, this study highlights the influence of the matrix on the digestion kinetics and gives some further understanding to the global value of digestibility, such as determined in vivo

The in vitro dynamic digestion : a suitable model to mimic the in vivo digestion of infant foods in terms of food deconstruction and protein hydrolysis

International audienceHuman infants are preferably fed human milk (HM), but a majority still receive infant formula (IF) as a HM substitute. Optimization of IF is still required to improve HM biomimetics, including digestion behavior biomimetics. While this can be studied in vivo, such experiments have to be reduced and appropriate in vitro models are needed. The present study aimed to compare food deconstruction and protein digestion of HM vs. IF using two infant digestion models, the mini-piglet and the in vitro dynamic gastrointestinal system (DIDGI®). Mini-piglets (Yucatan) were fed either a mature HM (n=9) or a standard IF (n=9) during 6 days. Piglet digesta were collected along the digestive tract 30 min after the last meal. The same foods were digested in triplicate using a term infant in vitro dynamic model with regular digesta sampling along time. Microstructure (confocal microscopy and laser diffraction) and protein digestion (SDS-PAGE, hydrolysis degree, peptidomics) were investigated in both digestion models. Data were statistically analyzed thanks to ANOVA and multidimensional analyses (hierarchical classification and multiple factor analyses). The microstructure of the digesta differed between HM and IF in a similar manner in vitro and in vivo along digestion. The meal dilution and emptying were similar between both digestion models, with a faster emptying for HM. Proteolysis, as investigated by SDS-PAGE, were similar between digestion models, with a lower hydrolysis level for HM caseins. Peptide mapping along the sequence of the major proteins was well correlated between models, particularly in the stomach and the proximal jejunum (r > 0.6). Similar result was found for bioactive peptide release. The ratio between bioaccessibility (in vitro) and bioavailability (in vivo) of amino acids was high (50-80%) at the cleavage sites of the pancreatic enzymes, more precisely for Arg, Tyr, Lys, Phe and Leu, but was much lower for the other amino acids (<30%). In overall, the in vitro dynamic gastrointestinal digestion model well predicted the in vivo digestion of HM and IF, particularly for protein hydrolysis, peptidomics and food deconstruction, while further improvement is needed to better correlate bioaccessibility and bioavailability

Current agri-environmental policies dismiss varied perceptions and discourses on management of traditional rural biotopes

Traditional rural biotopes (TRBs) are threatened habitats that host significant biodiversity and several ecosystem services, and depend on active management such as low-intensity grazing. The current study explores private landowners’ decision-making on TRB management and abandonment within a social-ecological system framework. We provide insight into supporting resilience of TRB systems in the face of agricultural modernization. Using a mixed methods approach with content analysis and Q analysis, we demonstrate that TRB management fosters cultural, biological, aesthetic, and utilitarian values. These are reflected in different ways through conservationist’s, profit-oriented farmer’s, landscape manager’s, and landscape admirer’s discourses on TRB management. Overall, management reinforces landowners’ place attachment, and reflects an approach to landscapes as spatial representations of cultural heritage and identity over multiple generations. Landowners consider TRB pasturage and its social-ecological outcomes motivating and rewarding. Giving up grazing cattle and perceived bureaucracy of national agri-environment scheme contribute to TRB abandonment. Landowners point out that current policies detach TRB management from what is seen as “regular agriculture”, and the focus on monetary compensation bypasses the multiple values tied to TRB management. Based on our results, we suggest that promoting TRBs requires reconfiguring the current arrangement of remedial management payments and adopting a more participatory governance approach. Locally, resilience of TRB systems relies on the connections between landowners and landscapes that foster sense of place and landscape identity, which can be supported by knowledge sharing and collaborative grazing efforts among landowners.peerReviewe

Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes

International audienceIn contrast to the earlier contention, adult humans have been recently shown to possess active brown adipose tissue (BAT) with a potential of being of metabolic significance. Up to now, brown fat precursor cells have not been available for human studies. We have shown previously that human multipotent adipose-derived stem (hMADS) cells exhibit a normal karyotype and high self-renewal ability; they are known to differentiate into cells which exhibit the key properties of human white adipocytes, i.e. UCP2 expression, insulin-stimulated glucose uptake, lipolysis in response to beta-agonists and atrial natriuretic peptide, and release of adiponectin and leptin. Herein we show that, upon chronic exposure to a specific PPARgamma but not to a PPARbeta/delta or a PPARalpha agonist, hMADS cell-derived white adipocytes are able to switch to a brown phenotype by expressing both UCP1 and CIDEA mRNA. This switch is accompanied by an increase in oxygen consumption and uncoupling. The expression of UCP1 protein is associated to stimulation of respiration by beta-AR agonists, including beta3-AR agonist. Thus hMADS cells represent an invaluable cell model to screen for drugs stimulating the formation and/or the uncoupling capacity of human brown adipocytes that could help to dissipate excess caloric intake of individuals