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Summary: Here, we show that a NOT gated cell therapy (Tmod) can exploit antigens such as epidermal growth factor receptor (EGFR) and human leukocyte antigen-E (HLA-E) which are widely expressed on cancer cells. Noncancerous cells—despite high expression of these antigens—are protected from cytotoxicity by the action of an inhibitory receptor (“blocker”) via a mechanism that involves blocker modulation of CAR surface expression. The blocker is triggered by the product of a polymorphic HLA allele (e.g., HLA-A∗02) deleted in a significant subset of solid tumors via loss of heterozygosity. Moreover, Tmod constructs that target mouse homologs of EGFR or HLA-E for activation, and a mouse-equivalent of HLA-A∗02 for inhibition, protect mice from toxicity caused by the CAR alone. The blocker also controls graft vs. host response in allogeneic T cells in vitro, consistent with the use of Tmod cells for off-the-shelf therapy without additional gene-editing.

Self-organization of pluripotent stem cells during tissue formation is directed by the adhesion microenvironment, which defines the resulting tissue topography. Although the influence of tissue topography on pluripotency state has been inferred, this aspect of self-organization remains largely unexplored. In this study, to determine the effect of self-organized tissue topography on pluripotency loss, we designed novel island mesh substrates to confine the self-organization process of mouse embryonic stem cells, enabling us to generate isolated cell layers with an island-like topography and overhanging edges. Using immunofluorescence microscopy, we determined that cells at the tissue edge exhibited deformed nuclei associated with low OCT3/4, in contrast with cells nested in the tissue interior which had round-shaped nuclei and exhibited sustained OCT3/4 expression. Interestingly, F-actin and phospho-myosin light chain were visibly enriched at the tissue edge where ERK activation and elevated AP-2γ expression were also found to be localized, as determined using both immunofluorescence microscopy and RT-qPCR analysis. Since actomyosin contractility is known to cause ERK activation, these results suggest that mechanical condition at the tissue edge can contribute to loss of pluripotency leading to differentiation. Thus, our study draws attention to the influence of self-organized tissue topography in stem cell culture and differentiation.

Summary: Metabolism is a key regulator of hematopoietic stem cell (HSC) functions. There is a lack of real-time, non-invasive approaches to evaluate metabolism in single HSCs. Using fluorescence lifetime imaging microscopy, we developed a set of metabolic optical biomarkers (MOBs) from the auto-fluorescent properties of metabolic coenzymes NAD(P)H and FAD. The MOBs revealed the enhanced glycolysis, low oxidative metabolism, and distinct mitochondrial localization of HSCs. Importantly, the fluorescence lifetime of enzyme-bound NAD(P)H (τbound) can non-invasively monitor the glycolytic/lactate dehydrogenase activity in single HSCs. As a proof of concept for metabolism-based cell sorting, we further identified HSCs within the Lineage-cKit+Sca1+ (KLS) hematopoietic stem/progenitor population using MOBs and a machine-learning algorithm. Moreover, we revealed the dynamic changes of MOBs, and the association of longer τbound with enhanced glycolysis under HSC stemness-maintaining conditions during HSC culture. Our work thus provides a new paradigm to identify and track the metabolism of single HSCs non-invasively and in real time. : Optical Imaging; Biological Sciences; Cellular Physiology; Stem Cells Research Subject Areas: Optical Imaging, Biological Sciences, Cellular Physiology, Stem Cells Research

Solid tumors in advanced cancer often feature a structurally and functionally abnormal vasculature through tumor angiogenesis, which contributes to cancer progression, metastasis, and therapeutic resistances. Hypoxia is considered a major driver of angiogenesis in tumor microenvironments. However, there remains a lack of in vitro models that recapitulate both the vasculature and hypoxia in the same model with physiological resemblance to the tumor microenvironment, while allowing for high-content spatiotemporal analyses for mechanistic studies and therapeutic evaluations. We have previously constructed a hypoxia microdevice that utilizes the metabolism of cancer cells to generate an oxygen gradient in the cancer cell layer as seen in solid tumor sections. Here, we have engineered a new composite microdevice-microfluidics platform that recapitulates a vascularized hypoxic tumor. Endothelial cells were seeded in a collagen channel formed by viscous fingering, to generate a rounded vascular lumen surrounding a hypoxic tumor section composed of cancer cells embedded in a 3-D hydrogel extracellular matrix. We demonstrated that the new device can be used with microscopy-based high-content analyses to track the vascular phenotypes, morphology, and sprouting into the hypoxic tumor section over a 7-day culture, as well as the response to different cancer/stromal cells. We further evaluated the integrity/leakiness of the vascular lumen in molecular delivery, and the potential of the platform to study the movement/trafficking of therapeutic immune cells. Therefore, our new platform can be used as a model for understanding tumor angiogenesis and therapeutic delivery/efficacy in vascularized hypoxic tumors.

The cell adhesion microenvironment plays contributory roles in the induction of self-organized tissue formation and differentiation of pluripotent stem cells (PSCs). However, physical factors emanating from the adhesion microenvironment have been less investigated largely in part due to overreliance on biochemical approaches utilizing cytokines to drive in vitro developmental processes. Here, we report that a mesh culture technique can potentially induce mouse embryonic stem cells (mESCs) to self-organize and differentiate into cells expressing key signatures of primordial germ cells (PGCs) even with pluripotency maintained in the culture medium. Intriguingly, mESCs cultured on mesh substrates consisting of thin (5 μm-wide) strands and considerably large (200 μm-wide) openings which were set suspended in order to minimize the cell-substrate adhesion area, self-organized into cell sheets relying solely on cell-cell interactions to fill the large mesh openings by Day 2, and further into dome-shaped features around Day 6. Characterization using microarray analysis and immunofluorescence microscopy revealed that sheet-forming cells exhibited differential gene expressions related to PGCs as early as Day 2, but not other lineages such as epiblast, primitive endoderm, and trophectoderm, implying that the initial interaction with the mesh microenvironment and subsequent self-organization into cells sheets might have triggered PGC-like differentiation to occur differently from the previously reported pathway via epiblast-like differentiation. Overall, considering that the observed differentiation occurred without addition of known biochemical inducers, this study highlights that bioengineering techniques for modulating the adhesion microenvironment alone can be harnessed to coax PSCs to self-organize and differentiate, in this case, to a PGC-like state.

The Polar Night Jet (PNJ) is a strong stratospheric westerly circumpolar wind at around 65° N in winter, and the strength of the climatological PNJ is widely recognized to increase monotonically from October through late December. Remarkably, the climatological PNJ temporarily stops increasing during late November. We examined this short break in terms of the atmospheric dynamical balance and found that it results from an increase in the upward propagation of climatological planetary waves from the troposphere to the stratosphere in late November, which coincides with a maximum of the climatological Eliassen-Palm flux convergence in the lower stratosphere. The upward propagation of planetary waves at 100 hPa, which is strongest over Siberia, is related to the climatological strengthening of the tropospheric trough over Siberia. We suggest that longitudinally asymmetric forcing by land-sea heating contrasts caused by their different heat capacities can account for the strengthening of the trough. [ABSTRACT FROM AUTHOR]

Between 1950 and 2012, boreal summer (rainy season) rainfall in the Sahel changed from a multi-decadal decreasing trend to an increasing trend (positive trend shift) in the mid-1980s. We found that this trend shift was synchronous with similar trend shifts in global oceanic evaporation and in land precipitation on all continents except the Americas. The trend shift in oceanic evaporation occurred mainly in the Southern Hemisphere (SH) and the subtropical oceans of the Northern Hemisphere (NH). Because increased oceanic evaporation strengthens the atmospheric moisture transport toward land areas, the synchrony of oceanic evaporation and land precipitation is reasonable. Surface scalar winds over the SH oceans also displayed a positive trend shift. Sea surface temperature (SST) displayed a trend shift in the mid-1980s that was negative (increasing, then decreasing) in the SH and positive in the NH. Although SST had opposite trend shifts in both hemispheres, the trend shift in evaporation was positive in both hemispheres. We infer that because strong winds promote evaporative cooling, the trend shift in SH winds strengthened the trend shifts in both SST and evaporation in the SH. Because high SST promotes evaporation, the trend shift in NH SST strengthened the NH trend shift in evaporation. Thus differing oceanic roles in the SH and NH generated the positive trend shift in evaporation; however, the details of moisture transport toward the Sahel are still unclear, or perhaps there is no single determining influence. [ABSTRACT FROM AUTHOR]

Tracking stem cell fate transition is crucial for understanding their development and optimizing biomanufacturing. Destructive single-cell methods provide a pseudotemporal landscape of stem cell differentiation but cannot monitor stem cell fate in real time. We established a metabolic optical metric using label-free fluorescence lifetime imaging microscopy (FLIM), feature extraction and machine learning-assisted analysis, for real-time cell fate tracking. From a library of 205 metabolic optical biomarker (MOB) features, we identified 56 associated with hematopoietic stem cell (HSC) differentiation. These features collectively describe HSC fate transition and detect its bifurcate lineage choice. We further derived a MOB score measuring the "metabolic stemness" of single cells and distinguishing their division patterns. This score reveals a distinct role of asymmetric division in rescuing stem cells with compromised metabolic stemness and a unique mechanism of PI3K inhibition in promoting ex vivo HSC maintenance. MOB profiling is a powerful tool for tracking stem cell fate transition and improving their biomanufacturing from a single-cell perspective. [ABSTRACT FROM AUTHOR]

To understand the effect of bandgap grading on carrier recombination for Cu(In,Ga)Se2 (CIGS)-based solar cells in detail, samples with different bandgaps at the CIGS surface were fabricated by changing the Ga/(Ga + In) (GGI) ratio from 0.4 to 0 at the third stage of the conventional three-stage growth process. Optoelectronic characterizations, such as photoluminescence, temperature-dependent open-circuit voltage measurement and light-intensity-dependent current–voltage measurement, indicate that the photo-generated carriers move rapidly towards the location of the bandgap minimum, and the carrier recombination occurs mainly at this location. From simulation using a one-dimensional solar cell capacitance simulator (SCAPS-1D), a single-grade sample with the smallest bandgap on the surface of CIGS showed high recombination current at the surface, while the location of the maximum recombination current moved from the surface to the bulk for double-grade samples. This study suggests that controlling the bandgap grading is one way of suppressing recombination at the interface in CIGS-based solar cells. [ABSTRACT FROM AUTHOR]