Inertial flow focusing: a case study in optimizing cellular trajectory through a microfluidic MEMS device for timing-critical applications.

Although microfluidic micro-electromechanical systems (MEMS) are well suited to investigate the effects of mechanical force on large populations of cells, their high-throughput capabilities cannot be fully leveraged without optimizing the experimental conditions of the fluid and particles flowing through them. Parameters such as flow velocity and particle size are known to affect the trajectories of particles in microfluidic systems and have been studied extensively, but the effects of temperature and buffer viscosity are not as well understood. In this paper, we explored the effects of these parameters on the timing of our own cell-impact device, the µHammer, by first tracking the velocity of polystyrene beads through the device and then visualizing the impact of these beads. Through these assays, we find that the timing of our device is sensitive to changes in the ratio of inertial forces to viscous forces that particles experience while traveling through the device. This sensitivity provides a set of parameters that can serve as a robust framework for optimizing device performance under various experimental conditions, without requiring extensive geometric redesigns. Using these tools, we were able to achieve an effective throughput over 360 beads/s with our device, demonstrating the potential of this framework to improve the consistency of microfluidic systems that rely on precise particle trajectories and timing.

Spatiotemporal variation of endogenous cell-generated stresses within 3D multicellular spheroids.

Multicellular spheroids serve as an excellent platform to study tissue behavior and tumor growth in a controlled, three-dimensional (3D) environment. While molecular and cellular studies have long used this platform to study cell behavior in 3D, only recently have studies using multicellular spheroids shown an important role for the mechanics of the microenvironment in a wide range of cellular processes, including during tumor progression. Despite the well-established relevance of mechanical cues to cell behavior and the numerous studies on mechanics using 2D cell culture systems, the spatial and temporal variations in endogenous cellular forces within growing multicellular aggregates remain unknown. Using cell-sized oil droplets with controlled physicochemical properties as force transducers in mesenchymal cell aggregates, we show that the magnitude of cell-generated stresses varies only weakly with spatial location within the spherical aggregate, but it increases considerably over time during aggregate compaction and growth. Moreover, our results indicate that the temporal increase in cellular stresses is due to increasing cell pulling forces transmitted via integrin-mediated cell adhesion, consistent with the need for larger intercellular pulling forces to compact cell aggregates.

Fixed single-cell transcriptomic characterization of human radial glial diversity.

The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors, particularly radial glia (RG), are rare and are defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed Fixed and Recovered Intact Single-cell RNA (FRISCR), a method for profiling the transcriptomes of individual fixed, stained and sorted cells. Using FRISCR, we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles, an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers.

Human mesenchymal stem cells form multicellular structures in response to applied cyclic strain.

Mesenchymal stem cells (MSCs) are a component of many cardiovascular cell-based regenerative medicine therapies. There is little understanding, however, of the response of MSCs to mechanical cues present in cardiovascular tissues. The objectives of these studies were to identify a model system to study the effect of well-defined applied cyclic strain on MSCs and to use this system to determine the effect of cyclic equibiaxial strain on the cellular and cytoskeletal organization of MSCs. When exposed to 10%, 1 Hz cyclic equibiaxial strain for 48 h, MSCs remained viable, retained characteristic gene and protein markers, and rearranged to form multicellular structures defined as clusters and knobs. This novel observation of cluster (overlapping cells surrounded by radial cellular projections) and knob (more dome-like structure containing significantly more cells than a cluster) formation did not involve changes in cytoskeletal proteins and resulted from cellular rearrangements initiated within 8 h of applied strain. Observed cellular responses were found to be dependent on substrate coating, but not on cell density for the 8-fold ranges tested. This system can thus be used to study the mechanoresponse over hours to days of MSCs exposed to applied cyclic strain in the context of cell-cell and cell-matrix interactions.

Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development.

The bone morphogenetic protein (BMP) signaling pathway plays an essential role during gastrointestinal (GI) tract development in vertebrates. In the present study, we use an antibody that recognizes the phosphorylated and activated form of Smad1, 5, and 8 to examine (by immunohistochemistry) the endogenous patterns of BMP signaling pathway activation in the developing GI tract. We show that the endogenous BMP signaling pathway is activated in the mesoderm, the endoderm, and the enteric nervous system (ENS) of the developing chick GI tract and is more widespread than BMP ligand expression patterns. Using an avian-specific retroviral misexpression technique to activate or inhibit BMP signaling pathway activity in the mesoderm of the gut, we show that BMP activity is required for the pattern, the development, and the differentiation of all three tissue types of the gut: mesoderm (that forms the visceral smooth muscle), endoderm (that forms the epithelium), and ectoderm (that forms the ENS). These results demonstrate that BMP signaling is activated in all the tissue layers of the GI tract during the development and plays a role during interactions and reciprocal communications of these tissue layers.

BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system.

The enteric nervous system (ENS) is derived from neural crest cells that migrate along the gastrointestinal tract to form a network of neurons and glia that are essential for regulating intestinal motility. Despite the number of genes known to play essential roles in ENS development, the molecular etiology of congenital disorders affecting this process remains largely unknown. To determine the role of bone morphogenetic protein (BMP) signaling in ENS development, we first examined the expression of bmp2, bmp4, and bmprII during hindgut development and find these strongly expressed in the ENS. Moreover, functional BMP signaling, demonstrated by the expression of phosphorylated Smad1/5/8, is present in the enteric ganglia. Inhibition of BMP activity by noggin misexpression within the developing gut, both in ovo and in vitro, inhibits normal migration of enteric neural crest cells. BMP inhibition also leads to hypoganglionosis and failure of enteric ganglion formation, with crest cells unable to cluster into aggregates. Abnormalities of migration and ganglion formation are the hallmarks of two human intestinal disorders, Hirschsprung's disease and intestinal neuronal dysplasia. Our results support an essential role for BMP signaling in these aspects of ENS development and provide a basis for further investigation of these proteins in the etiology of neuro-intestinal disorders.

Enteric nervous system patterning in the avian hindgut.

The enteric nervous system (ENS) is principally derived from vagal and sacral neural crest cells that migrate throughout the gastrointestinal tract before differentiating into neurons and glia. These cells form two concentric rings of ganglia and regulate intestinal motility, absorption, and secretion. Abnormalities of ENS development can lead to disorders of intestinal function, including Hirschsprung's disease. These disorders are generally limited to the distal hindgut, suggesting unique features to development of this region. This study characterized the normal spatiotemporal development of the ENS within the avian hindgut. Neural crest cells begin to populate the hindgut at E8, with patterning of both plexuses complete by embryonic day 9. Crest-derived cells arrive in the submucosal layer before the myenteric layer, as well as differentiate to a neuronal phenotype first. The cloaca demonstrates a unique pattern, characterized by a disorganized myenteric plexus and a flattened nerve of Remak. Detailed understanding of normal avian hindgut ENS development will allow better utilization of this model system to study abnormalities of the intestinal nervous system.