Hybrid spheroids containing mesenchymal stem cells promote therapeutic angiogenesis by increasing engraftment of co-transplanted endothelial colony-forming cells in vivo.

BACKGROUND: Peripheral artery disease is an ischemic vascular disease caused by the blockage of blood vessels supplying blood to the lower extremities. Mesenchymal stem cells (MSCs) and endothelial colony-forming cells (ECFCs) have been reported to alleviate peripheral artery disease by forming new blood vessels. However, the clinical application of MSCs and ECFCs has been impeded by their poor in vivo engraftment after cell transplantation. To augment in vivo engraftment of transplanted MSCs and ECFCs, we investigated the effects of hybrid cell spheroids, which mimic a tissue-like environment, on the therapeutic efficacy and survival of transplanted cells. METHODS: The in vivo survival and angiogenic activities of the spheroids or cell suspension composed of MSCs and ECFCs were measured in a murine hindlimb ischemia model and Matrigel plug assay. In the hindlimb ischemia model, the hybrid spheroids showed enhanced therapeutic effects compared with the control groups, such as adherent cultured cells or spheroids containing either MSCs or ECFCs. RESULTS: Spheroids from MSCs, but not from ECFCs, exhibited prolonged in vivo survival compared with adherent cultured cells, whereas hybrid spheroids composed of MSCs and ECFCs substantially increased the survival of ECFCs. Moreover, single spheroids of either MSCs or ECFCs secreted greater levels of pro-angiogenic factors than adherent cultured cells, and the hybrid spheroids of MSCs and ECFCs promoted the secretion of several pro-angiogenic factors, such as angiopoietin-2 and platelet-derived growth factor. CONCLUSION: These results suggest that hybrid spheroids containing MSCs can serve as carriers for cell transplantation of ECFCs which have poor in vivo engraftment efficiency.

Esophageal wound healing by aligned smooth muscle cell-laden nanofibrous patch.

The esophagus exhibits peristalsis via contraction of circularly and longitudinally aligned smooth muscles, and esophageal replacement is required if there is a critical-sized wound. In this study, we proposed to reconstruct esophageal tissues using cell electrospinning (CE), an advanced technique for encapsulating living cells into fibers that allows control of the direction of fiber deposition. After treatment with transforming growth factor-ß, mesenchymal stem cell-derived smooth muscle cells (SMCs) were utilized for cell electrospinning or three-dimensional bioprinting to compare the effects of aligned micropatterns on cell morphology. CE resulted in SMCs with uniaxially arranged and elongated cell morphology with upregulated expression levels of SMC-specific markers, including connexin 43, smooth muscle protein 22 alpha (SM22α), desmin, and smoothelin. When SMC-laden nanofibrous patches were transplanted into a rat esophageal defect model, the SMC patch promoted regeneration of esophageal wounds with an increased number of newly formed blood vessels and enhanced the SMC-specific markers of SM22α and vimentin. Taken together, CE with its advantages, such as guidance of highly elongated, aligned cell morphology and accelerated SMC differentiation, can be an efficient strategy to reconstruct smooth muscle tissues and treat esophageal perforation.

Transfer-Tattoo-Like Cell-Sheet Delivery Induced by Interfacial Cell Migration.

A direct transfer of a cell sheet from a culture surface to a target tissue is introduced. Commercially available, flexible parylene is used as the culture surface, and it is proposed that the UV-treated parylene offers adequate and intermediate levels of cell adhesiveness for both the stable cell attachment during culture and for the efficient cell transfer to a target surface. The versatility of this cell-transfer process is demonstrated with various cell types, including MRC-5, HDFn, HULEC-5a, MC3T3-E1, A549, C2C12 cells, and MDCK-II cells. The novel cell-sheet engineering is based on a mechanism of interfacial cell migration between two surfaces with different adhesion preferences. Monitoring of cytoskeletal dynamics and drug treatments during the cell-transfer process reveals that the interfacial cell migration occurs by utilizing the existing transmembrane proteins on the cell surface to bind to the targeted surface. The re-establishment and reversal of cell polarity after the transfer process are also identified. Its unique capabilities of 3D multilayer stacking, freeform design, and curved surface application are demonstrated. Finally, the therapeutic potential of the cell-sheet delivery system is demonstrated by applying it to cutaneous wound healing and skin-tissue regeneration in mice models.

Mesenchymal stem cell spheroids alleviate neuropathic pain by modulating chronic inflammatory response genes.

Chronic neuropathic pain is caused by dysfunction of the peripheral nerves associated with the somatosensory system. Mesenchymal stem cells (MSCs) have attracted attention as promising cell therapeutics for chronic pain; however, their clinical application has been hampered by the poor in vivo survival and low therapeutic efficacy of transplanted cells. Increasing evidence suggests enhanced therapeutic efficacy of spheroids formed by three-dimensional culture of MSCs. In the present study, we established a neuropathic pain murine model by inducing a chronic constriction injury through ligation of the right sciatic nerve and measured the therapeutic effects and survival efficacy of spheroids. Monolayer-cultured and spheroids were transplanted into the gastrocnemius muscle close to the damaged sciatic nerve. Transplantation of spheroids alleviated chronic pain more potently and exhibited prolonged in vivo survival compared to monolayer-cultured cells. Moreover, spheroids significantly reduced macrophage infiltration into the injured tissues. Interestingly, the expression of mouse-origin genes associated with inflammatory responses, Ccl11/Eotaxin, interleukin 1A, tumor necrosis factor B, and tumor necrosis factor, was significantly attenuated by the administration of spheroids compared to that of monolayer. These results suggest that MSC spheroids exhibit enhanced in vivo survival after cell transplantation and reduced the host inflammatory response through the regulation of main chronic inflammatory response-related genes.

Shift of nitrate sources in groundwater due to intensive livestock farming on Jeju Island, South Korea: With emphasis on legacy effects on water management.

Time lags between anthropogenic nitrogen inputs and their impacts to nitrate levels cause a misunderstanding for sources and subsequently misguide the groundwater management.We investigated the hydrochemical data of groundwater samples (n = 172 from 49 wells) with chlorofluorocarbons (CFCs)-based groundwater age dating and stable N (δ15N) and O isotopes (δ18O) of nitrate to assess the legacy effect of livestock farming to groundwater in an agricultural area where intensive livestock farming started in the 1970s and illegal dumping of manure wastewater in a lava cave was revealed in 2015. Approximately 90% of the groundwater samples had nitrate concentrations exceeding the natural threshold (5.5 mg/L NO3-) for nitrate contamination and 34% exceeded the World Health Organization's guideline for drinking water quality (44.3 mg/L), indicating severe nitrate contamination. The δ15NNO3 values (5.5 to 24.3‰) in groundwater exceeding the threshold of nitrate showed that livestock manure was a major nitrate source, while ammonium fertilizer also seemed influential given the δ15NNO3 values in the overlapping fields of N sources. Factor analysis of hydrochemical data also supported nitrate contamination by manure as well as by plant farming in the study area. Based on the spatial distribution of nitrate levels and δ15NNO3, livestock farming affected nitrate contamination by illegal manure dumping in the leakage cave. According to a Bayesian mixing model, the contribution of manure wastewater was 33.5 to 81.8% as of 2015-2018, with the rest from fertilizers. Meanwhile, the groundwater ages showed negative correlations with both nitrate levels (r = -0.90) and δ15NNO3 values (r = -0.74) on a log scale, consistent with the increasing N release from livestock farming since the 1960s. In particular, the median value of δ15NNO3 rapidly increased to 9.2‰ in groundwater recharged between the late 1970s and early 1990s when N production exponentially increased, implying a significant effect of livestock farming after the 1980s. Groundwater quality is expected to deteriorate over the next several decades based on the groundwater ages (> 23.5 years), the increased N production from livestock farming, and the legacy effect of N. Long-term groundwater management plans (> 25 years) are required to decrease N loads in the study area, because it takes time for management practices to take effect. The study results are a good reference for groundwater management in regions with a source shift to livestock farming under intensive livestock production systems. Moreover, the chronological study using historical N production, groundwater age data, and dual nitrate isotopes can be applied to other regions with multiple N sources and their shifting for identifying sources and estimating time lags.

Stable carbon and nitrogen isotopic characterization and tracing nutrient sources of Ulva blooms around Jeju coastal areas.

The present investigation was aimed to characterize the Ulva blooms and to identify the probable sources for Ulva blooms along the Jeju Island coast for pertinent control measures. Algal isotope signatures (δ13C, δ15N and δ18O) and tissue nitrogen and carbon were analyzed to map nutrient sources around the Jeju coastal areas. The algal δ13C values were ranged from -20.52 to -4.39‰, while δ15N and δ18O values ranged from 4.26 to 8.29‰ and 12.80-17.34‰, respectively. Moreover, site-specific significant differences were observed in algal stable isotope (δ13C, δ15N and δ18O) values. The bi-plot (δ15N vs δ18O) diagram indicated four dominant nitrogen sources along the Jeju coast, with 1) soil organic nitrogen mixed with livestock wastes (spring water samples and E), the 2) synthetic fertilizer input (A3 and B2), 3) sewage discharge (D1, D2 and I3) and 4) aquaculture waste (fish farm samples, A4, A5, B1, G and I2). Present findings revealed the different potential nitrogen sources for localized increase of algal growth along the Jeju coast. Finally, the present findings could be used as baseline data for efficient nutrient management to remediate Ulva blooms along Jeju coastal environment.

Preparation of gold- and chlorine-impregnated bead-type activated carbon for a mercury sorbent trap.

This study aimed to develop a mercury (Hg) adsorption trap, which can be used to measure the concentration of elemental Hg in emissions from a Hg discharge facility, and evaluate its adsorption efficiency. The Hg spiking efficiency was compared by impregnating metallic and halogen materials that have high affinity for Hg into activated carbon (AC) to determine an accurate spiking method for Hg on AC. The Hg spiking efficiency was compared according to the type and content of the impregnated substances. AC impregnated with Cl and Au had a 15-20% higher Hg spiking efficiency compared to virgin AC. For Au impregnation at weight ratios of 0-20 wt% of adsorbent, spiking efficiencies of over 97% were observed under certain conditions. The Hg adsorption properties of the above adsorbent were determined experimentally, and the results were used to test the adsorption performance of Hg adsorption traps.

Free radical scavengers from the heartwood of Juniperus chinensis.

The antioxidant activity of Juniperus chinensis (Cupressaceae) was determined by measuring the radical scavenging effect on DPPH (1,1-diphenyl-2-picrylhydrazyl). The methanolic extract of J. chinensis heartwood showed the strong antioxidant activity. The antioxidant activity of n-BuOH soluble fraction was stronger than that of the others, and the fraction was subjected to purification by repeated silica gel and Sephadex LH-20 column chromatography. Quercetin, naringenin, taxifolin, aromadendrin and isoquercitrin were isolated from the n-BuOH fraction. Their structures were elucidated by physico-chemical and spectroscopic studies.