The multiple faces of self-assembled lipidic systems

Lipids, the building blocks of cells, common to every living organisms, have the propensity to self-assemble into well-defined structures over short and long-range spatial scales. The driving forces have their roots mainly in the hydrophobic effect and electrostatic interactions. Membranes in lamellar phase are ubiquitous in cellular compartments and can phase-separate upon mixing lipids in different liquid-crystalline states. Hexagonal phases and especially cubic phases can be synthesized and observed in vivo as well. Membrane often closes up into a vesicle whose shape is determined by the interplay of curvature, area difference elasticity and line tension energies, and can adopt the form of a sphere, a tube, a prolate, a starfish and many more. Complexes made of lipids and polyelectrolytes or inorganic materials exhibit a rich diversity of structural morphologies due to additional interactions which become increasingly hard to track without the aid of suitable computer models. From the plasma membrane of archaebacteria to gene delivery, self-assembled lipidic systems have left their mark in cell biology and nanobiotechnology; however, the underlying physics is yet to be fully unraveled

Highly Efficient Cardiac Differentiation of Human Embryonic Stem Cells for Cardiac Repair

Heart failure is a leading cause of death in United States. One of the causes of heart failure is associated with the death or loss of cardiomyocytes (CMs). Since adult CMs do not regenerate, their death permanently compromises myocardial contractile function. Stem cell transplantation is one therapeutic strategy to replace damaged or lost myocardial tissue to restore cardiac function. Embryonic stem cells (ESCs) are an attractive population for cardiac repair because they can self-renew unlimitedly and differentiate into all cell types including CMs. Furthermore, ESC derived CMs can functionally integrate with the recipient organ and improve heart function after transplantation. However, a major challenge in ESC-based cardiac therapies is that the differentiation efficiency of ESCs into CMs has been very low (~1%). And large numbers of cells are required for administration for each patient. In this study, a protocol for efficient generation of CMs from hESCs was explored by optimizing various staged components in the microenvironment. Specifically, I 1) developed Honeycomb Microwell chips to generate homogeneous EB for CM differentiation; 2) optimized Actvin A/BMP4 concentration for CM differentiation; 3) optimized effects of extracellular matrix (ECM) signaling and investigated some mechanisms of ECM signaling on CM differentiation. The optimized protocols reproducibly generate approximately 70% CMs from H7 and H9 hESCs. These hESC derived CMs can now be enriched and tested for their ability to enhance cardiac function in preclinical animal models and for utility in drug discovery for future study

Review of the placer mining methods with regard to geotechnical conditions and climate in Yakutia

Abstract The author reviews the current situation in opencast mining placers in Yakutia and characterizes geotechnical conditions and climate in the major mining regions in the Republic. In the most placers under mining, sands and peat have thickness of 1.5–1.6 and 5.2 m, respectively. Dozer cutting is applicable to peat with maximum thickness of 15.2 m; the combinations of dozer–shovel–dump truck and dozer–loader–dump truck are bet suitable t peat thicknesses f 17.5 and 11.5 m, respectively. Permafrost rocks are widely prepared to mining through natural thawing (71–76% in the Aldan, North and East regions). The common method of stripping is dozers. The most popular method of actual extraction is the combination of dozer–loader–dump truck. The most efficient approach to stripping is the combination of dozer–shovel–dump truck as its cost is lower than in striping with dozers or with the dozer– loader combination.</jats:p

High Capacity Immunomagnetic Isolation of Apheresis Samples in an Automated Closed System

Abstract Cell therapy development, including Chimeric antigen receptor (CAR)-T cells, involves many steps and requires processing on average 3 billion white blood cells or more. Automated large scale immunomagnetic cell isolation with high efficiency and throughput in a closed system is desired to enrich T cells from apheresis products. Current automated, high capacity methods are time consuming, high cost, low yield and can potentially influence the physiology of the target cells. We have developed an automated closed magnetic separation platform, MARS® Bar to address these problems. This platform uses three uniquely designed magnetic separation modules and each magnetic module can perform positive or negative selection of target cells in a fluidic chamber at high flow rate (1mL/min per module). On this platform we have developed cell isolation reagents and program with minimal cell preparation. In the pilot study, we isolated 116 million CD3+ T cells from 267million CD45+ cells in a single LSR chamber on a single MARS® module. The entire automated process took less than 15 minutes to complete and resulted in &amp;gt; 95% purity and &amp;gt; 96% recovery of CD3+ T cells. The isolated cells were then activated and expanded, with results showing high proliferation and the dominant population being central memory T cells. The unique MARS® Bar matrix-free design has unlimited capacity because of the automated release of positively captured cells and very low carry-over. This was validated by processing greater than 3 billion leukocytes at 20 million cells per mL within the three modules in parallel and intermittent release of captured cells for collection. The MARS® Bar can be easily integrated into standard T cell production workflow.</jats:p

Combinatorial fibronectin and laminin signaling promote highly efficient cardiac differentiation of human embryonic stem cells.

Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies-primarily based on the optimization of growth factors-have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4(+) and ITGB5(+) cells

Abstract 3779: Incorporating in-line sample pre-enrichment with FACS and single cell mRNA-seq to facilitate isolation and characterization of prostate cancer circulating tumor cells

Abstract For decades, circulating tumor cells (CTCs) have been used as biomarkers to indicate disease progression and survival in patients with metastatic tumors. Detection of CTCs is an attractive, non-invasive blood test for early diagnosis of many types of cancer. However, CTCs are present at extremely low frequency in blood, and it is challenging to recover them in typical multi-step sample processing workflows. EpCAM, the widely accepted marker for CTC, has been found being down regulated in protein expression and RNA expression analysis in many cases thus EpCAM-dependent CTC detection might not be sensitive enough. We have developed a new sample pre-enrichment technology incorporated in line with fluorescence activated cell sorting (BD FACS™) platforms to demonstrate a one-step sample processing workflow to minimize cell loss and maximize CTC recovery. The pre-enrichment technology combines magnetic depletion of white blood cells and acoustic removal of lysed red blood cells, sending enriched tumor cells directly to a cell sorter for single CTC isolation. The in-line enrichment and sorting technology has shown 90% removal of CD45+ cells and over 90% recovery of target cells going through the system. Furthermore, we have incorporated molecular indexing technology for next generation sequencing (NGS) into the single CTC isolation to streamline gene expression analysis. We conducted a pilot study in which prostate cancer cell lines PC3, DU145, and LNCaP were spiked in healthy donors’ peripheral blood, quantitatively recovered, and individually sorted into 96-well BD™ Precise barcoded plates using a BD Influx™ sorter equipped with the in-line pre-enrichment module. The sorted cells were then sequenced for a prostate cancer targeted gene panels with approximate 100 genes, including EpCAM. Single cell sorting and sequencing were also performed on cell line cells directly and confirmed the workflow did not affect the gene expression. In LNCaP and PC3 cells, EpCAM was not the high expression marker in the genes analyzed, and its protein expression was also heterogeneous. The highly expressed genes discovered by single cell mRNA sequencing such as GSTP1 and CAV1 were investigated to provide additional cell surface markers for more sensitive CTC detection. Finally, CTCs from blood samples collected from prostate cancer patients were isolated based on marker panels beyond EpCAM and characterized at the single cell level. The differences between gene expression profiles of patient sample CTCs and cancer cell line cells were revealed. Citation Format: Liping Yu, Silin Sa, Michael Tycon, Xiaoyang Alice Wang. Incorporating in-line sample pre-enrichment with FACS and single cell mRNA-seq to facilitate isolation and characterization of prostate cancer circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3779. doi:10.1158/1538-7445.AM2017-3779</jats:p