New methods for investigating human astrocytes are urgently needed, given their critical role in the central nervous system. Here we show that CD49f is a novel marker for human astrocytes, expressed ...in fetal and adult brains from healthy and diseased individuals. CD49f can be used to purify fetal astrocytes and human induced pluripotent stem cell (hiPSC)-derived astrocytes. We provide single-cell and bulk transcriptome analyses of CD49f+ hiPSC-astrocytes and demonstrate that they perform key astrocytic functions in vitro, including trophic support of neurons, glutamate uptake, and phagocytosis. Notably, CD49f+ hiPSC-astrocytes respond to inflammatory stimuli, acquiring an A1-like reactive state, in which they display impaired phagocytosis and glutamate uptake and fail to support neuronal maturation. Most importantly, we show that conditioned medium from human reactive A1-like astrocytes is toxic to human and rodent neurons. CD49f+ hiPSC-astrocytes are thus a valuable resource for investigating human astrocyte function and dysfunction in health and disease.
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•CD49f is a novel, reactivity-independent marker for human astrocytes•CD49f can be used to purify human fetal astrocytes and iPSC-derived astrocytes•CD49f+ hiPSC-astrocytes acquire an A1-like reactive state upon cytokine stimulation•CD49f+ A1-like reactive astrocytes are dysfunctional and toxic to neurons in vitro
Barbar et al. identify CD49f as a novel surface marker expressed by human astrocytes that can purify hiPSC-astrocytes and primary fetal astrocytes. CD49f+ hiPSC-astrocytes respond to pro-inflammatory stimuli and become A1 reactive astrocytes, which are dysfunctional and secrete neurotoxic factors that induce apoptosis in human and rodent neurons.
Cells integrate mechanical cues to direct fate specification to maintain tissue function and homeostasis. While disruption of these cues is known to lead to aberrant cell behavior and chronic ...diseases, such as tendinopathies, the underlying mechanisms by which mechanical signals maintain cell function are not well understood. Here, we show using a model of tendon de-tensioning that loss of tensile cues in vivo acutely changes nuclear morphology, positioning, and expression of catabolic gene programs, resulting in subsequent weakening of the tendon. In vitro studies using paired ATAC/RNAseq demonstrate that the loss of cellular tension rapidly reduces chromatin accessibility in the vicinity of Yap/Taz genomic targets while also increasing expression of genes involved in matrix catabolism. Concordantly, the depletion of Yap/Taz elevates matrix catabolic expression. Conversely, overexpression of Yap results in a reduction of chromatin accessibility at matrix catabolic gene loci, while also reducing transcriptional levels. The overexpression of Yap not only prevents the induction of this broad catabolic program following a loss of cellular tension, but also preserves the underlying chromatin state from force-induced alterations. Taken together, these results provide novel mechanistic details by which mechanoepigenetic signals regulate tendon cell function through a Yap/Taz axis.
Fetal bone development occurs through the conversion of avascular cartilage to vascularized bone at the growth plate. This requires coordinated mobilization of osteoblast precursors with blood ...vessels. In adult bone, vessel-adjacent osteoblast precursors are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Here, we show that the mechanoresponsive transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) spatially couple osteoblast precursor mobilization to angiogenesis, regulate vascular morphogenesis to control cartilage remodeling, and mediate mechanoregulation of embryonic murine osteogenesis. Mechanistically, YAP and TAZ regulate a subset of osteoblast-lineage cells, identified by single-cell RNA sequencing as vessel-associated osteoblast precursors, which regulate transcriptional programs that direct blood vessel invasion through collagen-integrin interactions and Cxcl12. Functionally, in 3D human cell co-culture, CXCL12 treatment rescues angiogenesis impaired by stromal cell YAP/TAZ depletion. Together, these data establish functions of the vessel-associated osteoblast precursors in bone development.
Purpose of Review
The development of the skeleton is controlled by cellular decisions determined by the coordinated activation of multiple transcription factors. Recent evidence suggests that the ...transcriptional regulator proteins, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), could have important roles in directing the activity of these transcriptional programs. However, in vitro evidence for the roles of YAP and TAZ in skeletal cells has been hopelessly contradictory. The goals of this review are to provide a cross-sectional view on the state of the field and to synthesize the available data toward a unified perspective.
Recent Findings
YAP and TAZ are regulated by diverse upstream signals and interact downstream with multiple transcription factors involved in skeletal development, positioning YAP and TAZ as important signal integration nodes in an hourglass-shaped signaling pathway. Here, we provide a survey of putative transcriptional co-effectors for YAP and TAZ in skeletal cells. Synthesizing the in vitro data, we conclude that TAZ is consistently pro-osteogenic in function, while YAP can exhibit either pro- or anti-osteogenic activity depending on cell type and context. Synthesizing the in vivo data, we conclude that YAP and TAZ combinatorially promote developmental bone formation, bone matrix homeostasis, and endochondral fracture repair by regulating a variety of transcriptional programs depending on developmental stage.
Summary
Here, we discuss the current understanding of the roles of the transcriptional regulators YAP and TAZ in skeletal development, and provide recommendations for continued study of molecular mechanisms, mechanotransduction, and therapeutic implications for skeletal disease.
Mechanobiology is an interdisciplinary field that aims to understand how physical forces impact biological systems. Enhancing our knowledge of mechanobiology has become increasingly important for ...understanding human disease and developing novel therapeutics. There is a societal need to teach diverse students principles of mechanobiology so that we may collectively expand our knowledge of this subject and apply new principles to improving human health. Toward this goal, we designed, implemented, and evaluated a hands-on, inquiry-based learning (IBL) module to teach students principles of cell–biomaterial interactions. This module was designed to be hosted in two 3-h sessions, over two consecutive days. During this time, students learned how to synthesize and mechanically test biomaterials, culture bacteria cells, and assess effects of matrix stiffness on bacteria cell proliferation. Among the 73 students who registered to participate in our IBL mechanobiology module, 40 students completed both days and participated in this study. A vast majority of the participants were considered underrepresented minority (URM) students based on race/ethnicity. Using pre/post-tests, we found that students experienced significant learning gains of 33 percentage points from completing our IBL mechanobiology module. In addition to gaining knowledge of mechanobiology, validated pre/post-surveys showed that students also experienced significant improvements in scientific literacy. Instructors may use this module as described, increase the complexity for an undergraduate classroom assignment, or make the module less complex for K-12 outreach. As presented, this IBL mechanobiology module effectively teaches diverse students principles of mechanobiology and scientific inquiry. Deploying this module, and similar IBL modules, may help advance the next generation of mechanobiologists.
Mechanosensitivity of Human Oligodendrocytes Espinosa-Hoyos, Daniela; Burstein, Suzanne R.; Cha, Jaaram ...
Frontiers in cellular neuroscience,
07/2020, Letnik:
14
Journal Article
Recenzirano
Odprti dostop
Oligodendrocytes produce and repair myelin, which is critical for the integrity and function of the central nervous system. Oligodendrocyte and oligodendrocyte progenitor cell biology is modulated in ...vitro by mechanical cues within the magnitudes observed in vivo. In some cases, these cues are sufficient to accelerate or inhibit terminal differentiation of murine oligodendrocyte progenitors. However, our understanding of oligodendrocyte lineage mechanobiology has been restricted primarily to animal models to date, due to the inaccessibility and challenges of human oligodendrocyte cell culture. Here we probe the mechanosensitivity of human oligodendrocyte lineage cells derived from human induced pluripotent stem cells. We target phenotypically distinct stages of the human oligodendrocyte lineage and quantify the effect of substratum stiffness on cell migration and differentiation, within the range documented in vivo. We find that human oligodendrocyte lineage cells exhibit mechanosensitive migration and differentiation. Further, we identify two patterns of human donor line-dependent mechanosensitive differentiation. Our findings illustrate the variation among human oligodendrocyte responses, otherwise not captured by animal models, that are important for translational research. Moreover, these findings highlight the importance of studying glia under conditions that better approximate in vivo mechanical cues. Despite significant progress in human oligodendrocyte derivation methodology, the extended duration, low yield, and low selectivity of human induced pluripotent stem cell-derived oligodendrocyte protocols significantly limit the scale-up and implementation of these cells and protocols for in vivo and in vitro applications. We propose that mechanical modulation, in combination with traditional soluble and insoluble factors, provides a key avenue to address these challenges in cell production and in vitro analysis.
Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small ...molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.
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•Compounds impacting particular genes’ function are highly sought•Most chemicals and overexpressed genes impact cell morphology in the Cell Painting assay•Matching these image profiles can find chemicals that impact a particular gene’s function•This virtual screen using public data found new chemical regulators of several pathways
If a chemical compound and a gene overexpression yield the same cell morphology in the microscopy-based assay Cell Painting, then they are likely to impact the same functions. This principle is exploited to retrieve useful compounds for particular query genes in public Cell Painting datasets.
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