In vitro screening system for hepatotoxicity: Comparison of bone‐marrow‐derived mesenchymal stem cells and Placenta‐derived stem cells

Stem cells have unique properties such as self‐renewal, plasticity to generate various cell types, and availability of cells of human origin. The characteristics are attentive in the toxicity screening against chemical toxicants. Placenta‐derived stem cells (PDSCs) have been spotlighted as a new cell source in stem cell research recently because they are characterized by their capacity to differentiate into multilineages. However, the use of PDSCs as an in vitro screening model for potential drug candidates has not yet been studied. Here, we analyzed the potentials for bone‐marrow‐derived mesenchymal stem (BM‐MSCs), which is a representative adult stem cells and PDSCs as an in vitro hepatotoxicity screening system, using well‐known hepatotoxicants. BM‐MSCs and PDSCs were analyzed to the potential for hepatogenic differentiation and were cultured with different concentrations of hepatotoxicants for time courses. The viability and ATP‐binding cassette (ABC) transporters were measured by the MTT assay and RT‐PCR, respectively. The sensitivities of PDSCs to hepatotoxicants are more sensitive than those of BM‐MSCs. The viability (IC₅₀) to in PDSCs was less than that of BM‐MSCs after 48 and 72 h (P < 0.05) of CCl₄ exposure. The toxicities of CCl₄ were decreased by fourfold in hepatogenic differentiation inducing PDSCs compared to the undifferentiated cells. The alteration of ABCGs was observed in PDSCs during differentiation. These findings suggest that the naïve PDSCs expressing ABCGs can be used as a source for in vitro screening system as well as the expression patterns of ABCG1 and ABCG2 might be involved in the sensitivity of PDSCs to hepatotoxicants. J. Cell. Biochem. 112: 49–58, 2011. © 2010 Wiley‐Liss, Inc.     

Gill‐derived glands in species of Astyanax (Teleostei: Characidae)

The presence of a gill‐derived gland is herein reported for the first time in males of species of Astyanax and related genera; they are described through histological cuts and SEM. The gill‐derived glands described for the Characidae, when fully developed, present a similar structure in different species. The main external feature of gill‐derived glands is the fusion of anteriormost gill filaments on the ventral branch of first gill arch. This fusion is caused by squamous stratified epithelial tissue that covers adjacent filaments, forming a series of chambers. In the region where the gill‐derived gland develops, the secondary lamellae of the gill filaments are much reduced or completely atrophied being characterized by the presence of glandular cells forming nests.     

Salmon‐derived nutrients drive diatom beta‐diversity patterns

1. Pacific salmon are a textbook example of migratory animals that transfer nutrients between ecosystems, but little is known about how salmon‐derived nutrients (SDN) affect the biodiversity of recipient freshwater ecosystems. We examined paleolimnological records from six Alaskan lakes to define how changes in SDN from sockeye salmon (Oncorhynchus nerka) influenced sedimentary diatom community structure and beta‐diversity among lakes and through time. 2. Using an isotopic mixing model, we showed that SDN loading could account for >80% of the lake total nitrogen budgets and strongly regulated diatom community composition. Spatial dissimilarity in diatom communities was positively related to differences in SDN among lakes (r² = 0.69, P < 0.01, n = 10). Likewise, temporal dissimilarity in diatom communities was positively related to differences in SDN in a sediment core with substantial variation in salmon spawner dynamics between 1700 and 1950 AD (r² = 0.34, P < 0.01, n = 19). Finally, beta‐diversity metrics quantifying temporal turnover within each lake’s sediment record were also positively related to the variance in SDN loading among lakes (r² = 0.88, P < 0.05, n = 5). Mean SDN was only negatively correlated to temporal diatom beta‐diversity. 3. Spatially subsidised systems often receive temporally variable resource inputs, and thus, it is not surprising that, unlike previous studies, we found that resource variability was the key driver of community composition and beta‐diversity. In habitats that receive strongly fluctuating external nutrient loads, environment heterogeneity may overweigh stochastic community processes. In addition, freshwater diatoms are characterised by great dispersal capabilities and short life cycles and therefore may be a more sensitive indicator for evaluating the role of resource variability than previously used model organisms. These results suggest that productivity–diversity relationship vary with the nature of nutrient loading and the life history of the community studied. 4. Overall, our study highlights that the transport of nutrients by sockeye salmon across ecosystem boundaries is a significant driver of algal community and biodiversity in nursery lakes, mainly through changing the magnitude of nutrient variation. As such, freshwater species diversity in regions like the U.S. Pacific Northwest may become impoverished where there have been long‐term declines in salmon populations and decreases in nutrient variability among lakes.     

Platelet‐derived growth factor promotes the proliferation of human umbilical cord‐derived mesenchymal stem cells

This study was designed to investigate the effect of platelet‐derived growth factor (PDGF) on the proliferation of human umbilical cord mesenchymal stem cells (UC‐MSCs) and further explore the mechanism of PDGF in promoting the proliferation of UC‐MSCs. The human UC‐MSCs were treated with different concentrations of PDGF, and the effects were evaluated by counting the cell number, the cell viability, the expression of PDGF receptors analyzed by RT‐PCR, and the detection of the gene expression of cell proliferation, cell cycle and pluripotency, and Brdu assay by immunofluorescent staining and Quantitative real‐time (QRT‐PCR). The results showed that PDGF could promote the proliferation of UC‐MSCs in vitro in a dose‐dependent way, and 10 to 50 ng/ml PDGF had a significant proliferation effect on UC‐MSCs; the most obvious concentration was 50 ng/ml. Significant inhibition on the proliferation of UC‐MSCs was observed when the concentration of PDGF was higher than 100 ng/ml, and all cells died when the concentration reached 200 ng/ml PDGF. The PDGF‐treated cells had stronger proliferation and antiapoptotic capacity than the control group by Brdu staining. The expression of the proliferation‐related genes C‐MYC, PCNA and TERT and cell cycle–related genes cyclin A, cyclin 1 and CDK2 were up‐regulated in PDGF medium compared with control. However, pluripotent gene OCT4 was not significantly different between cells cultured in PDGF and cells analyzed by immunofluorescence and QRT‐PCR. The PDGF could promote the proliferation of human UC‐MSCs in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.     

EPC‐derived exosomes promote osteoclastogenesis through LncRNA‐MALAT1

Bone repair involves bone resorption through osteoclastogenesis and the stimulation of neovascularization and osteogenesis by endothelial progenitor cells (EPCs). However, the role of EPCs in osteoclastogenesis is unclear. In this study, we assess the effects of EPC‐derived exosomes on the migration and osteoclastic differentiation of primary mouse bone marrow‐derived macrophages (BMMs) in vitro using immunofluorescence, western blotting, RT‐PCR and Transwell assays. We also evaluated the effects of EPC‐derived exosomes on the homing and osteoclastic differentiation of transplanted BMMs in a mouse bone fracture model in vivo. We found that EPCs cultured with BMMs secreted exosomes into the medium and, compared with EPCs, exosomes had a higher expression level of LncRNA‐MALAT1. We confirmed that LncRNA‐MALAT1 directly binds to miR‐124 to negatively control miR‐124 activity. Moreover, overexpression of miR‐124 could reverse the migration and osteoclastic differentiation of BMMs induced by EPC‐derived exosomes. A dual‐luciferase reporter assay indicated that the integrin ITGB1 is the target of miR‐124. Mice treated with EPC‐derived exosome‐BMM co‐transplantations exhibited increased neovascularization at the fracture site and enhanced fracture healing compared with those treated with BMMs alone. Overall, our results suggest that EPC‐derived exosomes can promote bone repair by enhancing recruitment and differentiation of osteoclast precursors through LncRNA‐MALAT1.     

Extracellular vesicle‐enclosed miR‐486‐5p mediates wound healing with adipose‐derived stem cells by promoting angiogenesis

Adipose‐derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC‐secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC‐based therapies. Considering its effects, we aimed to investigate the role of ASC‐EVs in the cutaneous wound healing accompanied with the study on the specific cargo‐medicated effects on wound healing. Two full‐thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α‐SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co‐cultured with EVs isolated from ASC (ASC‐EVs), respectively, followed by the evaluation of their viability and mobility using CCK‐8, scratch test and transwell migration assays. Matrigel‐based angiogenesis assays were performed to evaluate vessel‐like tube formation by HMECs in vitro. ASC‐EVs accelerated the healing of full‐thickness skin wounds, increased re‐epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR‐486‐5p antagomir abrogated the ASC‐EVs‐induced effects. Intriguingly, miR‐486‐5p was found to be highly enriched in ASC‐EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC‐EVs‐secreted miR‐486‐5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR‐486‐5p released by ASC‐EVs could be a critical mediator to develop an ASC‐based therapeutic strategy for wound healing.     

Fibroblast growth factor‐2 and interleukin‐4 synergistically induce eotaxin‐1 expression in adipose tissue‐derived stromal cells

The relationships between eosinophils and adipose tissues are involved in metabolic homeostasis. Eotaxin is a chemokine with potent effects on eosinophil migration. To clarify the mechanisms of eotaxin expression in adipose tissues, we examined the effects of fibroblast growth factor‐2 (FGF‐2) and interleukin‐4 (IL‐4) stimulation on eotaxin expression in adipose tissue‐derived stromal cells (ASCs), a type of adipocyte progenitor, in vitro. ASCs expressed eotaxin‐1 and did not express eotaxin‐2 or ‐3. Eotaxin‐1 expression was increased in a concentration‐dependent manner following FGF‐2 treatment. Additionally, ASCs expressed FGF receptor‐1 (FGFR‐1) and did not express FGFR‐2, ‐3, or ‐4. Eotaxin‐1 expression was inhibited in cells treated with the FGFR tyrosine kinase inhibitor and extracellular signal‐regulated kinase (ERK) inhibitor U0126, even in the presence of FGF‐2. Moreover, eotaxin‐1 expression was synergistically enhanced by combined treatment with FGF‐2 and IL‐4 and inhibited in the presence of U0126. Eotaxin‐1 expression induced by FGF‐2 and IL‐4 was involved in ERK activation via FGFR‐1 in ASCs. Upregulation of eotaxin expression in adipose tissues could increase eosinophil migration, thereby inducing IL‐4 secretion and activation of alternative macrophages and improving glucose homeostasis. These findings provide insights into the mechanisms through which eotaxin mediates metabolic homeostasis in adipose tissues and eosinophils.     

Improving computational protein design by using structure‐derived sequence profile

Designing a protein sequence that will fold into a predefined structure is of both practical and fundamental interest. Many successful, computational designs in the last decade resulted from improved understanding of hydrophobic and polar interactions between side chains of amino acid residues in stabilizing protein tertiary structures. However, the coupling between main‐chain backbone structure and local sequence has yet to be fully addressed. Here, we attempt to account for such coupling by using a sequence profile derived from the sequences of five residue fragments in a fragment library that are structurally matched to the five‐residue segments contained in a target structure. We further introduced a term to reduce low complexity regions of designed sequences. These two terms together with optimized reference states for amino‐acid residues were implemented in the RosettaDesign program. The new method, called RosettaDesign‐SR, makes a 12% increase (from 34 to 46%) in fraction of proteins whose designed sequences are more than 35% identical to wild‐type sequences. Meanwhile, it reduces 8% (from 22% to 14%) to the number of designed sequences that are not homologous to any known protein sequences according to psi‐blast. More importantly, the sequences designed by RosettaDesign‐SR have 2–3% more polar residues at the surface and core regions of proteins and these surface and core polar residues have about 4% higher sequence identity to wild‐type sequences than by RosettaDesign. Thus, the proteins designed by RosettaDesign‐SR should be less likely to aggregate and more likely to have unique structures due to more specific polar interactions. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Fibroblast growth factor‐2/platelet‐derived growth factor enhances atherosclerotic plaque stability

Increased immature neovessels contribute to plaque growth and instability. Here, we investigated a method to establish functional and stable neovessel networks to increase plaque stability. Rabbits underwent aortic balloon injury and were divided into six groups: sham, vector and lentiviral transfection with vascular endothelial growth factor‐A (VEGF)‐A, fibroblast growth factor (FGF)‐2, platelet‐derived growth factor (PDGF)‐BB and FGF‐2 + PDGF‐BB. Lentivirus was percutaneously injected into the media‐adventitia of the abdominal aorta by intravascular ultrasound guidance, and plaque‐rupture rate, plaque‐vulnerability index and plaque neovessel density at the injection site were evaluated. Confocal microscopy, Prussian Blue assay, Evans Blue, immunofluorescence and transmission electron microscopy were used to assess neovessel function and pericyte coverage. To evaluate the effect of FGF‐2/PDGF‐BB on pericyte migration, we used the mesenchymal progenitor cell line 10T1/2 as an in vitro model. VEGF‐A‐ and FGF‐2‐overexpression increased the number of immature neovessels, which caused intraplaque haemorrhage and inflammatory cell infiltration, eventually resulting in the plaque vulnerability; however, FGF‐2/PDGF‐BB induced mature and functional neovessels, through increased neovessel pericyte coverage. Additionally, in vitro analysis of 10T1/2 cells revealed that FGF‐2/PDGF‐BB induced epsin‐2 expression and enhanced the VEGF receptor‐2 degradation, which negatively regulated pericyte function consistent with the in vivo data. These results showed that the combination of FGF‐2 and PDGF‐BB promoted the function and maturation of plaque neovessels, thereby representing a novel potential treatment strategy for vulnerable plaques.     

Differentiation of human adipose‐derived stem cells into beating cardiomyocytes

Human adipose‐derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5‐azacytidine (5‐aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co‐culture with neonatal rat cardiomyocytes. 5‐aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5‐ to 1.9‐fold) and the number of cells co‐expressing nkx2.5/sarcomeric α‐actin (27.2%versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11‐fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA‐treated cells also stained positively for cardiac myosin heavy chain, α‐actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non‐contact co‐culture showed no cardiac differentiation; however, ASCs co‐cultured in direct contact co‐culture exhibited a time‐dependent increase in cardiac actin mRNA expression (up to 33‐fold) between days 3 and 14. Immunocytochemistry revealed co‐expression of GATA4 and Nkx2.5, α‐actin, TropI and cardiac myosin heavy chain in CM‐DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca²⁺ transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell‐to‐cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co‐culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.