Rapid Host Defense against Aspergillus fumigatus Involves Alveolar Macrophages with a Predominance of Alternatively Activated Phenotype

The ubiquitous fungus Aspergillus fumigatus is associated with chronic diseases such as invasive pulmonary aspergillosis in immunosuppressed patients and allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis or severe asthma. Because of constant exposure to this fungus, it is critical for the host to exercise an immediate and decisive immune response to clear fungal spores to ward off disease. In this study, we observed that rapidly after infection by A. fumigatus, alveolar macrophages predominantly express Arginase 1 (Arg1), a key marker of alternatively activated macrophages (AAMs). The macrophages were also found to express Ym1 and CD206 that are also expressed by AAMs but not NOS2, which is expressed by classically activated macrophages. The expression of Arg1 was reduced in the absence of the known signaling axis, IL-4Rα/STAT6, for AAM development. While both Dectin-1 and TLR expressed on the cell surface have been shown to sense A. fumigatus, fungus-induced Arg1 expression in CD11c+ alveolar macrophages was not dependent on either Dectin-1 or the adaptor MyD88 that mediates intracellular signaling by most TLRs. Alveolar macrophages from WT mice efficiently phagocytosed fungal conidia, but those from mice deficient in Dectin-1 showed impaired fungal uptake. Depletion of macrophages with clodronate-filled liposomes increased fungal burden in infected mice. Collectively, our studies suggest that alveolar macrophages, which predominantly acquire an AAM phenotype following A. fumigatus infection, have a protective role in defense against this fungus

An Optimized Protocol for the Generation of Midbrain Dopamine Neurons under Defined Conditions.

Here, we describe a xeno-free, feeder-free, and chemically defined protocol for the generation of ventral midbrain dopaminergic (vmDA) progenitors from human pluripotent stem cells (hPSCs). This simple-to-follow protocol results in high yields of cryopreservable dopamine neurons across multiple hPSC lines. Wnt signaling is the critical component of the differentiation and can be finely adjusted in a line-dependent manner to enhance production of dopamine neurons for the purposes of transplantation, studying development and homeostasis, disease modeling, drug discovery, and drug development. For complete details on the use and execution of this protocol, please refer to Gantner et al. (2020) and Niclis et al. (2017a)

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells

Development of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinson's disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types

Efficiently Specified Ventral Midbrain Dopamine Neurons from Human Pluripotent Stem Cells Under Xeno-Free Conditions Restore Motor Deficits in Parkinsonian Rodents

: Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)-derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson's disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder- and xenogeneic-free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock-in lines (LMX1A-eGFP and PITX3-eGFP) for in-depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this "next generation" protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno-free vmDA progenitors from LMX1A- and PITX3-eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC-derived neurons into the clinic. SIGNIFICANCE: The authors report the generation of highly pure midbrain dopamine cultures under feeder-free, fully defined, and xeno-free conditions from human pluripotent stem cells. Xeno-free differentiated cells display gene, protein, and electrophysiological properties of midbrain neurons, as well as improved grafting outcomes in Parkinsonian rodents, observations enhanced by the use of two novel reporter lines of interest to this research field. Furthermore, for the first time, ventral midbrain dopamine neurons were amenable to scalability and cryopreservation, crucial steps for the advancement of cell replacement therapy in Parkinson's disease

Projection Based Model Reduction for Optimal Design of the Time-Dependent Stokes System

The optimal design of structures and systems described by partial differential equations (PDEs) often gives rise to large-scale optimization problems, in particular if the underlying system of PDEs represents a multi-scale, multi-physics problem. Therefore, reduced order modeling techniques such as balanced truncation model reduction, proper orthogonal decomposition, or reduced basis methods are used to significantly decrease the computational complexity while maintaining the desired accuracy of the approximation. In particular, we are interested in such shape optimization problems where the design issue is restricted to a relatively small portion of the computational domain. In this case, it appears to be natural to rely on a full order model only in that specific part of the domain and to use a reduced order model elsewhere. A convenient methodology to realize this idea consists in a suitable combination of domain decomposition techniques and balanced truncation model reduction. We will consider such an approach for shape optimization problems associated with the time-dependent Stokes system and derive explicit error bounds for the modeling error. As an application in life sciences, we will be concerned with the optimal design of capillary barriers as part of a network of microchannels and reservoirs on microfluidic biochips that are used in clinical diagnostics, pharmacology, and forensics for high-throughput screening and hybridization in genomics and protein profiling in proteomics

Projection Based Model Reduction for Optimal Design of the Time-dependent Stokes System

The optimal design of structures and systems described by partial differential equations (PDEs) often gives rise to large-scale optimization problems, in particular if the underlying system of PDEs represents a multi-scale, multi-physics problem. Therefore, reduced order modeling techniques such as balanced truncation model reduction, proper orthogonal decomposition, or reduced basis methods are used to significantly decrease the computational complexity while maintaining the desired accuracy of the approximation. In particular, we are interested in such shape optimization problems where the design issue is restricted to a relatively small portion of the computational domain. In this case, it appears to be natural to rely on a full order model only in that specific part of the domain and to use a reduced order model elsewhere. A convenient methodology to realize this idea consists in a suitable combination of domain decomposition techniques and balanced truncation model reduction. We will consider such an approach for shape optimization problems associated with the time-dependent Stokes system and derive explicit error bounds for the modeling error. As an application in life sciences, we will be concerned with the optimal design of capillary barriers as part of a network of microchannels and reservoirs on microfluidic biochips that are used in clinical diagnostics, pharmacology, and forensics for high-throughput screening and hybridization in genomics and protein profiling in proteomics