Influence of the Pd Oxidation State in PdNi Thin Films on Surface Acoustic Wave Hydrogen Sensing Performance.

PdNi alloy thin films demonstrate exceptional hydrogen sensing performance and exhibit significant potential for application in surface acoustic wave (SAW) hydrogen sensors. However, the long-term stability of SAW H2 sensors utilizing PdNi films as catalysts experiences a substantial decrease during operation. In this paper, X-ray photoelectron spectroscopy (XPS) is employed to investigate the failure mechanisms of PdNi thin films under operational conditions. The XPS analysis reveals that the formation of PdO species on PdNi thin films plays a crucial role in the failure of hydrogen sensing. Additionally, density functional theory (DFT) calculations indicate that hydrogen atoms encounter a diffusion energy barrier during the penetration process from the PdNiOx surface to the subsurface region. The identification of PdNi film failure mechanisms through XPS and DFT offers valuable insights into the development of gas sensors with enhanced long-term stability. Guided by these mechanisms, we propose a method to restore the hydrogen sensing response time and magnitude to a certain extent by reducing the partially oxidized surface of the PdNi alloy under a hydrogen atmosphere at 70 °C, thereby restoring Pd to its metallic state with zero valence.

Metabolic-Glycoengineering-Enabled Molecularly Specific Acoustic Tweezing Cytometry for Targeted Mechanical Stimulation of Cell Surface Sialoglycans.

In this study, we developed a novel type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated by metabolic glycoengineering (MGE). This enabled the application of mechanical forces to sialoglycans on hPSCs through molecularly specific acoustic tweezing cytometry (mATC), that is, displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to the acoustic radiation forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force. Furthermore, stimulating sialoglycans on hPSCs using mATC reduced stage-specific embryonic antigen-3 (SSEA-3) and GD3 expression but not OCT4 and SOX2 nuclear localization. Conversely, stimulating integrins decreased OCT4 nuclear localization but not SSEA-3 and GD3 expression, suggesting that mechanically stimulating sialoglycans and integrins initiated distinctive mechanoresponses during the early stages of hPSC differentiation. Taken together, these results demonstrated that MGE-enabled mATC uncovered not only different mechanical properties of sialoglycans on hPSCs and integrins but also their different mechanoregulatory impacts on hPSC differentiation, validating MGE-based mATC as a new, powerful tool for investigating the roles of glycans and other cell surface biomolecules in mechanotransduction.

A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

Derivation of human primordial germ cell-like cells in an embryonic-like culture.

Primordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. They transmit genetic and epigenetic information across generations. Given the prominent role of germline defects in diseases such as infertility, detailed understanding of human PGC (hPGC) development has important implications in reproductive medicine and studying human evolution. Yet, hPGC specification remains an elusive process. Here, we report the induction of hPGC-like cells (hPGCLCs) in a bioengineered human pluripotent stem cell (hPSC) culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs), derived from hPSCs, induce hPGCLC specification from hPSCs through paracrine signaling downstream of ISL1. Our data further show functional roles of NODAL, WNT, and BMP signaling in hPGCLC induction. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived hPSC lines using this biomimetic platform, demonstrating its promise for screening applications.

Thermal Stress Analysis and Control Method for Surface Acoustic Wave Atomizer.

To prevent the potential failure of the surface acoustic wave (SAW) atomizer caused by the concentration of thermal stresses, this study investigates the thermal elevation process inherent to the operation of the surface wave atomizer. Subsequently, a method for temperature regulation is proposed. By collecting the temperature rise data of SAW atomizers with water, olive oil, and glycerol at 5/6/7 Watts (W) of power, the temperature curves of the atomizer surface under different conditions are obtained, and the stress changes in the working process are simulated additionally. The results indicate that although the stress generated by surface acoustic wave atomizers varies for different media, there is always a problem of rapid heating during the initial working stage in all cases. To address the above issues, this study analyzed the time when the maximum stress occurred and proposed control methods based on experimental data. The simulation results show that by controlling the driving power within 4 s after the start of atomization, the problem of excessive stress during the heating stage can be avoided. Finally, the feasibility of the control method was verified through a simple power control method (limiting the driving power to 3 W in the first 2 s), proving that this method can effectively reduce the thermal stress during the working process of the atomizer and prevent the atomizer from cracking.

A human pluripotent stem cell-based somitogenesis model using microfluidics.

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated bio-chemical and -mechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Here we report a new human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on PSM cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the bio-chemical and -mechanical events that guide somite formation.

Rapid responses of human pluripotent stem cells to cyclic mechanical strains applied to integrin by acoustic tweezing cytometry.

Acoustic tweezing cytometry (ATC) is an ultrasound-based biophysical technique that has shown the capability to promote differentiation of human pluripotent stem cells (hPSCs). This study systematically examined how hPSCs respond to cyclic mechanical strains applied by ATC via displacement of integrin-bound microbubbles (averaged diameter of 4.3 µm) using ultrasound pulses (acoustic pressure 0.034 MPa, center frequency 1.24 MHz and pulse repetition frequency 1 Hz). Our data show downregulation of pluripotency marker Octamer-binding transcription factor 4 (OCT4) by at least 10% and increased nuclear localization of Yes-associated protein (YAP) by almost 100% in hPSCs immediately after ATC application for as short as 1 min and 5 min respectively. Analysis of the movements of integrin-anchored microbubbles under ATC stimulations reveals different stages of viscoelastic characteristic behavior and increasing deformation of the integrin-cytoskeleton (CSK) linkage. The peak displacement of integrin-bound microbubbles increased from 1.45 ± 0.16 to 4.74 ± 0.67 µm as the duty cycle of ultrasound pulses increased from 5% to 50% or the duration of each ultrasound pulse increased from 0.05 to 0.5 s. Real-time tracking of integrin-bound microbubbles during ATC application detects high correlation of microbubble displacements with OCT4 downregulation in hPSCs. Together, our data showing fast downregulation of OCT4 in hPSCs in respond to ATC stimulations highlight the unique mechanosensitivity of hPSCs to integrin-targeted cyclic force/strain dependent on the pulse duration or duty cycle of ultrasound pulses, providing insights into the mechanism of ATC-induced accelerated differentiation of hPSCs.

Deep learning-based lung image registration: A review.

Lung image registration can effectively describe the relative motion of lung tissues, thereby helping to solve series problems in clinical applications. Since the lungs are soft and fairly passive organs, they are influenced by respiration and heartbeat, resulting in discontinuity of lung motion and large deformation of anatomic features. This poses great challenges for accurate registration of lung image and its applications. The recent application of deep learning (DL) methods in the field of medical image registration has brought promising results. However, a versatile registration framework has not yet emerged due to diverse challenges of registration for different regions of interest (ROI). DL-based image registration methods used for other ROI cannot achieve satisfactory results in lungs. In addition, there are few review articles available on DL-based lung image registration. In this review, the development of conventional methods for lung image registration is briefly described and a more comprehensive survey of DL-based methods for lung image registration is illustrated. The DL-based methods are classified according to different supervision types, including fully-supervised, weakly-supervised and unsupervised. The contributions of researchers in addressing various challenges are described, as well as the limitations of these approaches. This review also presents a comprehensive statistical analysis of the cited papers in terms of evaluation metrics and loss functions. In addition, publicly available datasets for lung image registration are also summarized. Finally, the remaining challenges and potential trends in DL-based lung image registration are discussed.

Modeling development using microfluidics: bridging gaps to foster fundamental and translational research.

In vitro stem cell-derived embryo and organ models, termed embryoids and organoids, respectively, provide promising experimental tools to study physiological and pathological processes in mammalian development and organ formation. Most of current embryoid and organoid systems are developed using conventional three-dimensional cultures that lack controls of spatiotemporal extracellular signals. Microfluidics, an established technology for quantitative controls and quantifications of dynamic chemical and physical environments, has recently been utilized for developing next-generation embryoids and organoids in a controllable and reproducible manner. In this review, we summarize recent progress in constructing microfluidics-based embryoids and organoids. Development of these models demonstrates the successful applications of microfluidics in establishing morphogen gradients, accelerating medium transport, exerting mechanical forces, facilitating tissue coculture studies, and improving assay throughput, thus supporting using microfluidics for building next-generation embryoids and organoids for fundamental and translational research.

Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development.

Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast toward the amniotic ectoderm, primitive streak/mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.

Single-Cell Sequencing to Unveil the Mystery of Embryonic Development.

Embryonic development is a fundamental physiological process that can provide tremendous insights into stem cell biology and regenerative medicine. In this process, cell fate decision is highly heterogeneous and dynamic, and investigations at the single-cell level can greatly facilitate the understanding of the molecular roadmap of embryonic development. Rapid advances in the technology of single-cell sequencing offer a perfectly useful tool to fulfill this purpose. Despite its great promise, single-cell sequencing is highly interdisciplinary, and successful applications in specific biological contexts require a general understanding of its diversity as well as the advantage versus limitations for each of its variants. Here, the technological principles of single-cell sequencing are consolidated and its applications in the study of embryonic development are summarized. First, the technology basics are presented and the available tools for each step including cell isolation, library construction, sequencing, and data analysis are discussed. Then, the works that employed single-cell sequencing are reviewed to investigate the specific processes of embryonic development, including preimplantation, peri-implantation, gastrulation, and organogenesis. Further, insights are provided on existing challenges and future research directions.

Micro/nanoengineered technologies for human pluripotent stem cells maintenance and differentiation.

Human pluripotent stem cells (hPSCs) are a promising source of cells for cell replacement-based therapies as well as modeling human development and diseases in vitro. However, achieving fate control of hPSC with a high yield and specificity remains challenging. The fate specification of hPSCs is regulated by biochemical and biomechanical cues in their environment. Driven by this knowledge, recent exciting advances in micro/nanoengineering have been leveraged to develop a broad range of tools for the generation of extracellular biomechanical and biochemical signals that determine the behavior of hPSCs. In this review, we summarize such micro/nanoengineered technologies for controlling hPSC fate and highlight the role of biochemical and biomechanical cues such as substrate rigidity, surface topography, and cellular confinement in the hPSC-based technologies that are on the horizon.

Branching development of early post-implantation human embryonic-like tissues in 3D stem cell culture.

Human embryonic stem cells (hESCs) have the intrinsic capacity to self-organize and generate patterned tissues. In vitro models that coax hESCs to form embryonic-like structures by modulating physical environments and priming with chemical signals have become a powerful tool for dissecting the regulatory mechanisms underlying early human development. Here we present a 3D suspension culture system of hESCs that can generate post-implantation, pre-gastrulation embryonic-like tissues in an efficient and controllable manner. The efficiency of the development of asymmetric tissues, which mimic the post-implantation, pre-gastrulation amniotic sac, was about 50% in the 3D suspension culture. Quantitative imaging profiling and unsupervised trajectory analysis revealed that hESC aggregates first entered into a transitional stage expressing Brachyury (or T), before their development branched into different paths to develop into asymmetric embryonic-like tissues, amniotic-like tissues, and mesodermal-like tissues, respectively. Moreover, the branching developmental trajectory of embryonic-like structures was affected by the initial cell seeding density or cluster size of hESCs. A higher percentage of amniotic-like tissues was observed under a small initial cell seeding density of hESCs. Conversely, a large initial cell seeding density of hESCs promoted the development of mesodermal-like tissues. Intermediate cell seeding densities of hESCs in the 3D suspension culture promoted the development of asymmetric embryonic-like tissues. Our results suggest that hESCs have the intrinsic capability to sense the initial cell population size, which in turn regulates their differentiation and self-organization into different embryonic-like tissues. Our 3D suspension culture thus provides a promising experimental tool to study the interplay between tissue topology and self-organization and progressive embryonic development using in vitro hESC-based models.

Mechanical Tension Promotes Formation of Gastrulation-like Nodes and Patterns Mesoderm Specification in Human Embryonic Stem Cells.

Embryogenesis is directed by morphogens that induce differentiation within a defined tissue geometry. Tissue organization is mediated by cell-cell and cell-extracellular matrix (ECM) adhesions and is modulated by cell tension and tissue-level forces. Whether cell tension regulates development by modifying morphogen signaling is less clear. Human embryonic stem cells (hESCs) exhibit an intrinsic capacity for self-organization, which motivates their use as a tractable model of early human embryogenesis. We engineered patterned substrates that recapitulate the biophysical properties of the early embryo and mediate the self-organization of "gastrulation-like" nodes in cultured hESCs. Tissue geometries that generated local nodes of high cell-adhesion tension directed the spatial patterning of the BMP4-dependent "gastrulation-like" phenotype by enhancing phosphorylation and junctional release of ß-catenin to promote Wnt signaling and mesoderm specification. Furthermore, direct force application via mechanical stretching promoted BMP-dependent mesoderm specification, confirming that tissue-level forces can directly regulate cell fate specification in early human development.

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation.

Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca2+ into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca2+ diffusion in hESC colonies. We quantify cell-cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca2+ waves in hESC colonies induced by sonoporation of single cells.

Modeling of human neurulation using bioengineered pluripotent stem cell culture.

Leveraging the developmental potential and self-organizing property of human pluripotent stem (hPS) cells, researchers have developed tractable models of human embryonic development. Owing to their compatibility to live imaging, genome editing, mechanical perturbation and measurement, these models offer promising quantitative experimental platforms to advance human embryology and regenerative medicine. Herein, we provide a review of recent progress in using hPS cells to generate models of early human neural development or neurulation, including neural induction and regional patterning of the neural tube. These models, even in their nascent developmental stages, have already revealed intricate cell-cell signaling and mechanoregulation mechanisms likely involved in tissue patterning during early neural development. We also discuss future opportunities in modeling early neural development by incorporating bioengineering tools to control precisely neural tissue morphology and architecture, morphogen dynamics, intracellular signaling events, and cell-cell interactions to further the development of this emerging field and expand its applications.

Effects of temperature and humidity on the performance of a PECH polymer coated SAW sensor.

The influences of environment, such as temperature, humidity and interfering gases, on the performance of a surface acoustic wave (SAW) sensor in the detection of 2-chloroethyl ethyl sulfide (CEES) were invested. The 150 MHz SAW dual delay lines were used, coated with a poly(epichlorohydrin) (PECH) thin layer, and CEES was detected under different concentrations. Linear correlation between the frequency-shift and the exposure time of the sensor to CEES could be observed, and the limit of CEES could be detected as low as 1.5 mg m-3. Under different temperature (0-50 C°) and humidity (30-80% RH) conditions, CEES was detected by the fabricated SAW sensor coated with PECH, the frequency shifts were measured and the performance of the sensor was evaluated. The results proved that temperature and humidity were the most important factors to influence the performance of SAW sensors; with the decreasing of temperature and the increasing of humidity, there would be larger frequency shifts. In the interference experiments, it was found that most gases existing in the environment in high concentrations would not influence the detection of CEES. Then, the SAW sensor having been fabricated was kept under the conditions of 25 °C and 35% RH for 18 months to further verify the quality, and CEES was detected every so many months. It proved that the performance of the sensor would decrease about 16.39% after 18 months. Although it reflected the attenuation of the sensor to some extent, the sensor was still in good condition. Additionally, the related mechanisms were also discussed.

Controlled modelling of human epiblast and amnion development using stem cells.

Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.

Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping.

During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.

Biophysical phenotypes and determinants of anterior vs. posterior primitive streak cells derived from human pluripotent stem cells.

Formation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, our understanding of cellular mechanism(s) underlying cell fate diversification along the anterior-posterior axis of the PS remains incomplete. Furthermore, differences in biophysical phenotypes between anterior and posterior PS cells, which could affect their functions and regulate their fate decisions, remain uncharacterized. Herein, anterior and posterior PS cells were derived using human pluripotent stem cell (hPSC)-based in vitro culture systems. We observed that anterior and posterior PS cells displayed significantly different biophysical phenotypes, including cell morphology, migration, and traction force generation, which was further regulated by different levels of Activin A- and BMP4-mediated developmental signaling. Our data further suggested that intracellular cytoskeletal contraction could mediate anterior and posterior PS differentiation and phenotypic bifurcation through its effect on Activin A- and BMP4-mediated intracellular signaling events. Together, our data provide new information about biophysical phenotypes of anterior and posterior PS cells and reveal an important role of intracellular cytoskeletal contractility in regulating anterior and posterior PS differentiation of hPSCs. STATEMENT OF SIGNIFICANCE: Formation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, molecular and cellular mechanism(s) underlying functional diversification of embryonic cells along the anterior-posterior axis of the PS remains incompletely understood. This work describes the first study to characterize the biophysical properties of anterior and posterior PS cells derived from human pluripotent stem cells (hPSCs). Importantly, our data showing the important role of cytoskeleton contraction in controlling anterior vs. posterior PS cell phenotypic switch (through its effect on intracellular Smad signaling activities downstream of Activin A and BMP4) should shed new light on biomechanical regulations of the development and anterior-posterior patterning of the PS. Our work will contribute significantly to uncovering new biophysical principles and cellular mechanisms driving cell lineage diversification and patterning during the PS formation.