Angiogenesis in Differentiated Placental Multipotent Mesenchymal Stromal Cells Is Dependent on Integrin α5β1

Human placental multipotent mesenchymal stromal cells (hPMSCs) can be isolated from term placenta, but their angiogenic ability and the regulatory pathways involved are not known. hPMSCs were shown to express integrins αv, α4, α5, β1, β3, and β5 and could be induced to differentiate into cells expressing endothelial markers. Increases in cell surface integrins α5 and β1, but not α4, αvβ3, or αvβ5, accompanied endothelial differentiation. Vascular endothelial growth factor-A augmented the effect of fibronectin in enhancing adhesion and migration of differentiated hPMSC through integrin α5β1, but not αvβ3 or αvβ5. Formation of capillary-like structures in vitro from differentiated cells was inhibited by pre-treatment with function-blocking antibodies to integrins α5 and β1. When hPMSCs were seeded onto chick chorioallantoic membranes (CAM), human von Willebrand factor-positive cells were observed to engraft in the chick endothelium. CAMs transplanted with differentiated hPMSCs had a greater number of vessels containing human cells and more incorporated cells per vessel compared to CAMs transplanted with undifferentiated hPMSCs, and overall angiogenesis was enhanced more by the differentiated cells. Function-blocking antibodies to integrins α5 and β1 inhibited angiogenesis in the CAM assay. These results suggest that differentiated hPMSCs may contribute to blood vessel formation, and this activity depends on integrin α5β1

Environmental sustainability framework for plastic waste management—a case study of bubble tea industry in Malaysia

Economic growth and rapid industrialisation have led to enormous increase in municipal solid waste (MSW). Lack of waste management alternatives and ineffective waste policy implementation are the major challenges for government to materialise a sustainable solid waste management framework, especially for plastic waste. Booming of the food and beverage (F&B) industry has aggravated the situation by generating more plastic waste with no economic values. Hence, this study aims to evaluate the overall environmental performance of existing and alternative waste management technologies that are available in Malaysia based on net greenhouse gas (GHG) emission in terms of carbon dioxide equivalent (kg CO(2)-eq) per tonne of plastic waste that are analysed through life cycle assessment (LCA) methodology. LCA result has proven that Scenario B (waste to energy (WTE) incineration) is more environmentally preferable as it had a negative net GHG emission of − 573.80 kg CO(2)-eq as compared to GHG emission of existing Scenario A (sanitary landfill) of 566.15 kg CO(2)-eq. Negative net GHG emission in WTE incineration was mainly due to higher GHG saving achieved through cleaner electricity generation as compared to conventional power production. This alternative technology was proven to have the potential to reduce the dependence on landfills and is served as the basis of environmental sustainability framework development for plastic waste management based on case study in Malaysia. This framework can be served as the baseline for the local authorities or policy makers for other plastic waste generation hotspots other than bubble tea industry to improve plastic waste management via WTE incineration

Adhesion and migration assays were performed with human placental multipotent mesenchymal stromal cells (hPMSCs) and various integrin-blocking antibodies.

<p>(A). hPMSCs were induced to differentiate into endothelial cells, then plated onto surfaces coated with BSA (NC), fibronectin, or vitronectin in the presence or absence of VEGF-A. The differentiated hPMSCs adhered to fibronectin more efficiently than to vitronectin (P<0.001). VEGF-A significantly increased the adhesion of differentiated hPMSCs to fibronectin but not to vitronectin or plates coated with 2.5% BSA solution (NC). (B) Inhibition of differentiated hPMSC adhesion to fibronectin and vitronectin in the presence of blocking antibodies to integrin subunits. A significant inhibition of differentiated hPMSC adhesion to fibronectin was observed in the presence of blocking antibodies to integrin α₅, β₁ or α_vβ₃, but not integrin α_vβ₅. The number of differentiated hPMSCs attached to vitronectin was low. (C) Transwell migration of differentiated hPMSCs induced by VEGF-A (50 ng/ml) with or without fibronectin (0.5 to 50 µg/ml) or vitronectin (0.5 to 50 µg/ml). Fibronectin in the presence of VEGF-A stimulated migration. (D) Inhibition of differentiated hPMSC transwell migration induced by VEGF-A (50 ng/ml) with or without fibronectin (50 µg/ml). Various blocking antibodies to integrin subunits and non-specific IgG were used. Antibodies to integrin subunits α₅ and β₁ were inhibitory. Error bar: SD; Ab: antibody; ECM: extracellular matrix.</p

In vitro angiogenesis: formation of capillary-like structures by differentiated human placental multipotent mesenchymal stromal cells (hPMSCs).

<p>hPMSCs were trypsinized, seeded on wells coated with ECMatrix^TM. (A) Human umbilical vein endothelial cells and (B) undifferentiated hPMSCs were used as positive and negative control (shown after 6 hours of culture). (C) hPMSCs induced to differentiate into endothelial cells form characteristic capillary-like structures. Cell elongation and cell interconnecting cell networks were observed. Differentiated hPMSCs were pretreated with antibodies against (D) integrin β₁, (E) α₄, (F) α₅, (G) α_vβ₃, or (H) α_vβ₅. Scale bar: 200 µm. Representative photomicrographs of 3 different experiments are shown. This ability of differentiated hPMSCs to form capillary-like structures was strongly diminished when integrin β₁ or α₅ was inhibited. Blocking antibodies to integrins α₄, α_vβ₃, or α_vβ₅ did not inhibit the formation of capillary-like structures. The capillary-like structures on ECmatrix^TM gel were immunostained using antibody against (I) non-specific IgG or specific endothelial markers (J) von Willebrand factor, (K) CD31, and (L) CD105. Scale bar: 50 µm. Arrows indicate von Willebrand factor, CD31 and CD105 positive cells present in ECmatrix^TM gel. (M) Quantification of the capillary-like structures by measuring the polygonal network (upper panel) and the cumulative tube length (lower panel) formed by differentiated hPMSCs. A significant inhibition of capillary-like structure formation was observed when integrin β₁ or α₅ antibody was applied to the cells. Error bar: SD.</p

Integrin expression at the surface of human placental multipotent mesenchymal stromal cells (hPMSCs) before and after inducing differentiation into endothelial cells.

<p>Undifferentiated hPMSCs: A, C, E, G, I; differentiated hPMSCs: B, D, F, H, J. The cells were immunostained using antibody against integrin (A, B) β₁, (C, D) α₄, (E, F) α₅, (G, H) α_vβ₃, or (I, J) α_vβ₅. Scale bar: 10 µm. Immunofluorescence staining revealed significant increases in integrin α₅ and β₁. (K) The change of hPMSC surface integrin expression after differentiation induced by VEGF-A was measured by flow cytometry and expressed in fluorescence intensity units. Change in fluorescence intensity is an index of integrin surface concentration per cell. (L) Quantification of specific mean fluorescence intensity (which corresponds to the increase in fluorescence intensity relative to second antibody alone) is shown (±SD, n = 3). The specific mean fluorescence intensity was higher for both integrins α₅ and β₁ in differentiated hPMSCs. C: controls; E: endothelial cell differentiation induced by VEGF-A.</p

A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancers. Many hot-spot mutations of TP53 confer novel functions not found in wild-type p53 and contribute to tumor development and progression. We report on the generation of a H1 human embryonic stem cell line carrying a homozygous TP53 R282W mutation using TALEN-mediated genome editing. The generated cell line demonstrates normal karyotype, maintains a pluripotent state, and is capable of generating a teratoma in vivo containing tissues from all three germ layers

Characterization of human placental multipotent mesenchymal stromal cells (hPMSCs) after inducing differentiation into endothelial cells.

<p>Immunofluorescence staining of CD31 in undifferentiated hPMSCs is shown in (A) as a negative control. Immunofluorescence of endothelial markers in differentiated hPMSCs under induced culture conditions: (B) CD31, (C) CD34, (D) VE-cadherin, (E) VEGFR-1, (F) VEGFR-2, (G) von Willebrand factor, (H) von Willebrand factor under high magnification (Scale bar: 1 µm); Weibel-Palade bodies were visible within the differentiated hPMSC cytoplasm (arrow). CD105 was positive in (I) undifferentiated hPMSCs and (J) hPMSCs differentiated into endothelial cells. Cell nuclei were counterstained with DAPI. Scale bar: 10 µm. (K) Flow cytometry analysis of endothelial cell markers on the hPMSC surface before and after differentiation induced by VEGF-A. (L) mRNA for endothelial cell markers was amplified from 2 different strains of undifferentiated hPMSCs (lane 1 and 2) and hPMSCs differentiated into endothelial cells (lane 3 and 4). mRNA from human umbilical vein endothelial cells was used as a positive control (lane 5). The data shown are representative of 3 different experiments.</p