Assembly of gut-derived bacterial communities follows "early-bird" resource utilization dynamics.

Diet can impact host health through changes to the gut microbiota, yet we lack mechanistic understanding linking nutrient availability and microbiota composition. Here, we use thousands of microbial communities cultured in vitro from human feces to uncover simple assembly rules and develop a predictive model of community composition upon addition of single nutrients from central carbon metabolism to a complex medium. Community membership was largely determined by the donor feces, whereas relative abundances were determined by the supplemental carbon source. The absolute abundance of most taxa was independent of the supplementing nutrient, due to the ability of fast-growing organisms to quickly exhaust their niche in the complex medium and then exploit and monopolize the supplemental carbon source. Relative abundances of dominant taxa could be predicted from the nutritional preferences and growth dynamics of species in isolation, and exceptions were consistent with strain-level variation in growth capabilities. Our study reveals that community assembly follows simple rules of nutrient utilization dynamics and provides a predictive framework for manipulating gut commensal communities through nutritional perturbations.

Engineering of the microenvironment to accelerate vascular regeneration.

Blood vessels are crucial for tissue development, functionality, and homeostasis and are typically a determinant in the progression of healing and regeneration. The tissue microenvironment provides physicochemical cues that affect cellular function, and the study of the microenvironment can be accelerated by the engineering of approaches capable of mimicking various aspects of the microenvironment. In this review, we introduce the major components of the vascular niche and focus on the roles of oxygen and the extracellular matrix (ECM). We demonstrate how vascular engineering approaches enhance our understanding of the microenvironment's impact on the vasculature towards vascular regeneration and describe the current limitations and future directions towards clinical utilization.

Myoblast deactivation within engineered human skeletal muscle creates a transcriptionally heterogeneous population of quiescent satellite-like cells.

Satellite cells (SCs), the adult Pax7-expressing stem cells of skeletal muscle, are essential for muscle repair. However, in vitro investigations of SC function are challenging due to isolation-induced SC activation, loss of native quiescent state, and differentiation to myoblasts. In the present study, we optimized methods to deactivate in vitro expanded human myoblasts within a 3D culture environment of engineered human skeletal muscle tissues ("myobundles"). Immunostaining and gene expression analyses revealed that a fraction of myoblasts within myobundles adopted a quiescent phenotype (3D-SCs) characterized by increased Pax7 expression, cell cycle exit, and activation of Notch signaling. Similar to native SCs, 3D-SC quiescence is regulated by Notch and Wnt signaling while loss of quiescence and reactivation of 3D-SCs can be induced by growth factors including bFGF. Myobundle injury with a bee toxin, melittin, induces robust myofiber fragmentation, functional decline, and 3D-SC proliferation. By applying single cell RNA-sequencing (scRNA-seq), we discover the existence of two 3D-SC subpopulations (quiescent and activated), identify deactivation-associated gene signature using trajectory inference between 2D myoblasts and 3D-SCs, and characterize the transcriptomic changes within reactivated 3D-SCs in response to melittin-induced injury. These results demonstrate the ability of an in vitro engineered 3D human skeletal muscle environment to support the formation of a quiescent and heterogeneous SC population recapitulating several aspects of the native SC phenotype, and provide a platform for future studies of human muscle regeneration and disease-associated SC dysfunction.

Development and quantitative analysis of a biosensor based on the Arabidopsis SWEET1 sugar transporter.

SWEETs are transporters with homologs in Archeae, plants, some fungi, and animals. As the only transporters known to facilitate the cellular release of sugars in plants, SWEETs play critical roles in the allocation of sugars from photosynthetic leaves to storage tissues in seeds, fruits, and tubers. Here, we report the design and use of genetically encoded biosensors to measure the activity of SWEETs. We created a SweetTrac1 sensor by inserting a circularly permutated green fluorescent protein into the Arabidopsis SWEET1, resulting in a chimera that translates substrate binding during the transport cycle into detectable changes in fluorescence intensity. We demonstrate that a combination of cell sorting and bioinformatics can accelerate the design of biosensors and formulate a mass action kinetics model to correlate the fluorescence response of SweetTrac1 with the transport of glucose. Our analysis suggests that SWEETs are low-affinity, symmetric transporters that can rapidly equilibrate intra- and extracellular concentrations of sugars. This approach can be extended to SWEET homologs and other transporters.

Establishment and characterization of stable, diverse, fecal-derived in vitro microbial communities that model the intestinal microbiota.

Efforts to probe the role of the gut microbiota in disease would benefit from a system in which patient-derived bacterial communities can be studied at scale. We addressed this by validating a strategy to propagate phylogenetically complex, diverse, stable, and highly reproducible stool-derived communities in vitro. We generated hundreds of in vitro communities cultured from diverse stool samples in various media; certain media generally preserved inoculum composition, and inocula from different subjects yielded source-specific community compositions. Upon colonization of germ-free mice, community composition was maintained, and the host proteome resembled the host from which the community was derived. Treatment with ciprofloxacin in vivo increased susceptibility to Salmonella invasion in vitro, and the in vitro response to ciprofloxacin was predictive of compositional changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. These findings demonstrate that stool-derived in vitro communities can serve as a powerful system for microbiota research.

Neuromuscular Development and Disease: Learning From in vitro and in vivo Models.

The neuromuscular junction (NMJ) is a specialized cholinergic synaptic interface between a motor neuron and a skeletal muscle fiber that translates presynaptic electrical impulses into motor function. NMJ formation and maintenance require tightly regulated signaling and cellular communication among motor neurons, myogenic cells, and Schwann cells. Neuromuscular diseases (NMDs) can result in loss of NMJ function and motor input leading to paralysis or even death. Although small animal models have been instrumental in advancing our understanding of the NMJ structure and function, the complexities of studying this multi-tissue system in vivo and poor clinical outcomes of candidate therapies developed in small animal models has driven the need for in vitro models of functional human NMJ to complement animal studies. In this review, we discuss prevailing models of NMDs and highlight the current progress and ongoing challenges in developing human iPSC-derived (hiPSC) 3D cell culture models of functional NMJs. We first review in vivo development of motor neurons, skeletal muscle, Schwann cells, and the NMJ alongside current methods for directing the differentiation of relevant cell types from hiPSCs. We further compare the efficacy of modeling NMDs in animals and human cell culture systems in the context of five NMDs: amyotrophic lateral sclerosis, myasthenia gravis, Duchenne muscular dystrophy, myotonic dystrophy, and Pompe disease. Finally, we discuss further work necessary for hiPSC-derived NMJ models to function as effective personalized NMD platforms.