Vesicles were made from amphiphilic diblock copolymers and characterized by micromanipulation. The average molecular weight of the specific polymer studied, polyethyleneoxide-polyethylethylene (EO40-EE37), is several times greater than that of typical phospholipids in natural membranes. Both the membrane bending and area expansion moduli of electroformed polymersomes (polymer-based liposomes) fell within the range of lipid membrane measurements, but the giant polymersomes proved to be almost an order of magnitude tougher and sustained far greater areal strain before rupture. The polymersome membrane was also at least 10 times less permeable to water than common phospholipid bilayers. The results suggest a new class of synthetic thin-shelled capsules based on block copolymer chemistry.

The terms MSC and MSCs have become the preferred acronym to describe a cell and a cell population of multipotential stem/progenitor cells commonly referred to as mesenchymal stem cells, multipotential stromal cells, mesenchymal stromal cells, and mesenchymal progenitor cells. The MSCs can differentiate to important lineages under defined conditions in vitro and in limited situations after implantation in vivo. MSCs were isolated and described about 30 years ago and now there are over 55,000 publications on MSCs readily available. Here, we have focused on human MSCs whenever possible. The MSCs have broad anti-inflammatory and immune-modulatory properties. At present, these provide the greatest focus of human MSCs in clinical testing; however, the properties of cultured MSCs in vitro suggest they can have broader applications. The medical utility of MSCs continues to be investigated in over 950 clinical trials. There has been much progress in understanding MSCs over the years, and there is a strong foundation for future scientific research and clinical applications, but also some important questions remain to be answered. Developing further methods to understand and unlock MSC potential through intracellular and intercellular signaling, biomedical engineering, delivery methods and patient selection should all provide substantial advancements in the coming years and greater clinical opportunities. The expansive and growing field of MSC research is teaching us basic human cell biology as well as how to use this type of cell for cellular therapy in a variety of clinical settings, and while much promise is evident, careful new work is still needed.

Inflammatory gene expression following genotoxic cancer therapy is well documented, yet the events underlying its induction remain poorly understood. Inflammatory cytokines modify the tumor microenvironment by recruiting immune cells and are critical for both local and systemic (abscopal) tumor responses to radiotherapy1. An enigmatic feature of this phenomenon is its delayed onset (days), in contrast to the acute DNA damage responses that occur in minutes to hours. Such dichotomous kinetics implicate additional rate limiting steps that are essential for DNA-damage induced inflammation. Here, we show that cell cycle progression through mitosis following DNA double-strand breaks (DSBs) leads to the formation of micronuclei, which precede activation of inflammatory signaling and are a repository for the pattern recognition receptor cGAS. Inhibiting progression through mitosis or loss of pattern recognition by cGAS-STING impaired interferon signaling. Moreover, STING loss prevented the regression of abscopal tumors in the context of ionizing radiation and immune checkpoint blockade in vivo. These findings implicate temporal modulation of the cell cycle as an important consideration in the context of therapeutic strategies that combine genotoxic agents with immune checkpoint blockade.

Vesicles prepared in water from a series of diblock copolymers -"polymersomes" -are physically characterized and compared to lipid vesicles. With increasing molecular weightM n , the hydrophobic core thickness d for the self-assembled bilayers of poly(ethylene oxide)-polybutadiene (PEO-PBD) increases up to ≃20 nm -considerably greater than any previously studied lipid system. The mechanical responses of these membranes, specifically, the area elastic modulus K a and maximal areal strain α c are measured by micromanipulation. As expected for interface-dominated elasticity, K a (≃100 pN/nm) is found to be independent ofM n , but lower than the usual values for zwitterionic lipid membranes. Experiments on polymersomes show α c increases in a nearly linear fashion withM n , approaching a limiting value predicted by mean-field ideas which is universal and about 10-fold above that typical of lipids. Nonlinear responses and memory effects generally emerge with increasingM n , indicating the onset of chain entanglements at higherM n . The effects ofM n thus suggest a compromise between stability and fluidity for biomembranes. More generally, the results highlight the interfacial limits of self-assemblies at the nanoscale.

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Radiotherapy and many chemotherapeutics rely on DNA double strand break (DSB) formation to drive the killing of tumor cells over several cell division cycles 2,3 . Concomitant with this protracted cell death schedule, inflammatory cytokine production increases over days following the insult. As a host of cytokines and inflammatory signals are produced following ionizing radiation (IR) 4,5 , we used STAT1 phosphorylation at Y701 as a surrogate for inflammatory pathway activation ( Fig. 1a and 1b). MCF10A mammary epithelial cells showed STAT1activation between 3 and 6 days post-IR with a dose-dependent threshold of at least 5Gy. TotalAll rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/156414 doi: bioRxiv preprint first posted online Jun. 27, 2017; STAT1 protein and the mRNA levels of multiple inflammatory genes were also detected in a time dependent manner (Fig. 1c. and Extended data 1a). Using inducible nucleases ( Fig. 1d and Extended data 1b and 1c), we observed a delayed accumulation of active STAT1 and inflammatory gene expression, confirming these signals are driven by DSBs.We reasoned that if residual DSBs were driving inflammatory signals then failure of nonhomologous end-joining (NHEJ) DSB repair should amplify the response. Paradoxically, we observed that inhibition of DNA-PKcs (DNA-PKi) or CRISPR-Cas9 knockout of multiple NHEJ components diminished STAT1 activation in MCF10A and prostate epithelial cells (Fig 1e and Extended data 1c and 1d). STAT1 activation through exogenous IFNβ1 was unaffected by DNAPKi (Extended data 1e), ruling out a direct role in STAT1 phosphorylation. Inhibition of ATM kinase on the other hand had little influence over the level of STAT1 activation (Extended data 1f).DSB-induced STAT1 activation correlated with the appearance of aberrantly shaped nuclei and micronuclei (Fig 1f and 2a). The DSB marker γH2AX was increased in micronuclei however 53BP1 was absent, consistent with their reported defects in DSB signaling (Fig 1f) 6,7 . Nuclei of NHEJ knockout and DNA-PKi treated cells were morphologically normal despite being replete with DSBs (Fig 1f and 2a). As micronuclei are byproducts of mitotic progression, these data suggest that STAT1 activation occurred following mitosis 8 . Flow cytometry showed progression of parental cells from G2 into G1 between 24 and 48h post-IR, whereas NHEJ knockouts were static over this time (Extended data 2a). This corresponds to a wave of parental cells moving into mitosis as evidenced by phospho-histone H3 staining that is absent in the NHEJ knockout cellsAll rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/156414 doi: bioRxiv preprint first posted online Jun....

Vesicles made completely from diblock copolymers-polymersomes-can be stably prepared by a wide range of techniques common to liposomes. Processes such as film rehydration, sonication, and extrusion can generate many-micron giants as well as monodisperse, approximately 100 nm vesicles of PEO-PEE (polyethyleneoxide-polyethylethylene) or PEO-PBD (polyethyleneoxide-polybutadiene). These thick-walled vesicles of polymer can encapsulate macromolecules just as liposomes can but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal transitions and a subpopulation even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an extended time with an excess of polymersomes. The effects are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG-driven micellization. The results potentiate a broad new class of technologically useful, polymer-based vesicles.

Narrow‐dispersity thermoresponsive block copolymers of poly(ethylene oxide)‐block‐poly(N‐isopropylacrylamide) self‐assemble into vesicles at temperatures above 32 °C. The vesicles integrate a hydrophobic fluorescent dye into their membranes and encapsulate the hydrophilic anticancer drug doxorubicin. Temperature‐controlled release of the dye through disintegration of the vesicles takes place at temperatures below 32 °C, as shown in the figure.