Deciphering potential vascularization factors of on-chip co-cultured hiPSC-derived cerebral organoids.

The lack of functional vascular system in stem cell-derived cerebral organoids (COs) limits their utility in modeling developmental processes and disease pathologies. Unlike other organs, brain vascularization is poorly understood, which makes it particularly difficult to mimic in vitro. Although several attempts have been made to vascularize COs, complete vascularization leading to functional capillary network development has only been achieved via transplantation into a mouse brain. Understanding the cues governing neurovascular communication is therefore imperative for establishing an efficient in vitro system for vascularized cerebral organoids that can emulate human brain development. Here, we used a multidisciplinary approach combining microfluidics, organoids, and transcriptomics to identify molecular changes in angiogenic programs that impede the successful in vitro vascularization of human induced pluripotent stem cell (iPSC)-derived COs. First, we established a microfluidic cerebral organoid (CO)-vascular bed (VB) co-culture system and conducted transcriptome analysis on the outermost cell layer of COs cultured on the preformed VB. Results revealed coordinated regulation of multiple pro-angiogenic factors and their downstream targets. The VEGF-HIF1A-AKT network was identified as a central pathway involved in the angiogenic response of cerebral organoids to the preformed VB. Among the 324 regulated genes associated with angiogenesis, six transcripts represented significantly regulated growth factors with the capacity to influence angiogenic activity during co-culture. Subsequent on-chip experiments demonstrated the angiogenic and vasculogenic potential of cysteine-rich angiogenic inducer 61 (CYR61) and hepatoma-derived growth factor (HDGF) as potential enhancers of organoid vascularization. Our study provides the first global analysis of cerebral organoid response to three-dimensional microvasculature for in vitro vascularization.

Long-term effect of sodium selenite on the integrity and permeability of on-chip microvasculature.

Development of the robust and functionally stable three-dimensional (3D) microvasculature remains challenging. One often-overlooked factor is the presence of potential anti-angiogenic agents in culture media. Sodium selenite, an antioxidant commonly used in serum-free media, demonstrates strong anti-angiogenic properties and has been proposed as an anticancer drug. However, its long-term effects on in vitro microvascular systems at the concentrations used in culture media have not been studied. In this study, we used a five-channel microfluidic device to investigate the concentration and temporal effects of sodium selenite on the morphology and functionality of on-chip preformed microvasculature. We found that high concentrations (∼3.0 µM) had adverse effects on microvasculature perfusion, permeability, and overall integrity within the first few days. Moreover, even at low concentrations (∼3.0 nM), a long-term culture effect was observed, resulting in an increase in vascular permeability without any noticeable changes in morphology. A further analysis suggested that vessel leakage may be due to vascular endothelial growth factor dysregulation, disruption of intracellular junctions, or both. This study provides important insight into the adverse effects caused by the routinely present sodium selenite on 3D microvasculature in long-term studies for its application in disease modeling and drug screening.

Automated Capillary-Based Vacuum Pulse-Assisted Instrument for Single-Cell Acquisition and Concurrent Detachment/Adhesion Assay, A-picK.

A large body of evidence points to the importance of cell adhesion molecules in cancer metastasis. Alterations in adhesion and attachment properties of neoplastic cells are important biomarkers of the metastatic potential of cancer. Loss of intracellular adhesion is correlated with more invasive phenotype by increasing the chances of malignant cells escaping from their site of origin, promoting metastasis. Therefore, there is great demand for rapid and accurate measurements of individual cell adhesion and attachment. Current technologies that measure adhesion properties in either suspension or bulk (microfluidics) remain very complex (e.g., atomic force microscopy [AFM], optical tweezers). Moreover, existing tools cannot provide measurements for fully attached individual adherent cells as they operate outside of such a force range. Even more importantly, none of the existing approaches permit concurrent and automated single-cell adhesion measurement and collection, which prohibits direct correlation between single-cell adhesion properties and molecular profile. Here, we report a fully automated and versatile platform, A-picK, that offers single-cell adhesion assay and isolation in parallel. We demonstrate the use of this approach for a time course analysis of human lung carcinoma A549 cells and substrate-specific adhesion potential using seven different substrates, including fibronectin, laminin, poly-l-lysine, carboxyl, amine, collagen, and gelatin.

Developing a MEMS Device with Built-in Microfluidics for Biophysical Single Cell Characterization.

This study combines the high-throughput capabilities of microfluidics with the sensitive measurements of microelectromechanical systems (MEMS) technology to perform biophysical characterization of circulating cells for diagnostic purposes. The proposed device includes a built-in microchannel that is probed by two opposing tips performing compression and sensing separately. Mechanical displacement of the compressing tip (up to a maximum of 14 µm) and the sensing tip (with a quality factor of 8.9) are provided by two separate comb-drive actuators, and sensing is performed with a capacitive displacement sensor. The device is designed and developed for simultaneous electrical and mechanical measurements. As the device is capable of exchanging the liquid inside the channel, different solutions were tested consecutively. The performance of the device was evaluated by introducing varying concentrations of glucose (from 0.55 mM (0.1%) to 55.5 mM (10%)) and NaCl (from 0.1 mM to 10 mM) solutions in the microchannel and by monitoring changes in the mechanical and electrical properties. Moreover, we demonstrated biological sample handling by capturing single cancer cells. These results show three important capabilities of the proposed device: mechanical measurements, electrical measurements, and biological sample handling. Combined in one device, these features allow for high-throughput multi-parameter characterization of single cells.

Microfluidic Tissue Mesodissection in Molecular Cancer Diagnostics.

We present a mesodissection platform that retains the advantages of laser-based dissection instrumentation with the speed and ease of manual dissection. Tissue dissection in clinical laboratories is often performed by manually scraping a physician-selected region from standard glass slide mounts. In this manner, costs associated with dissection remain low, but spatial resolution is compromised. In contrast, laser microdissection methods maintain spatial resolution that matches the requirements for analysis of important tissue heterogeneity but remains costly and labor intensive. We demonstrate a microfluidic tool for rapid extraction of histological regions of interest from formalin-fixed paraffin-embedded tissue, which uses a simple and automated method that is compatible with most downstream enzymatic reactions, including protocols used for next-generation DNA sequencing.

Direct electrical and mechanical characterization of in situ generated DNA between the tips of silicon nanotweezers (SNT).

Previously, we reported the application of micromachined silicon nanotweezers (SNT) integrated with a comb-drive actuator and capacitive sensors for capturing and mechanical characterization of DNA bundles. Here, we demonstrate direct DNA amplification on such a MEMS structure with subsequent electrical and mechanical characterization of a single stranded DNA (ssDNA) bundle generated between the tips of SNT via isothermal rolling circle amplification (RCA) and dielectrophoresis (DEP). An in situ generated ssDNA bundle was visualized and evaluated via electrical conductivity (I-V) and mechanical frequency response measurements. Colloidal gold nanoparticles significantly enhanced (P < 0.01) the electrical properties of thin ssDNA bundles. The proposed technology allows direct in situ synthesis of DNA with a predefined sequence on the tips of a MEMS sensor device, such as SNT, followed by direct DNA electrical and mechanical characterization. In addition, our data provides a "proof-of-principle" for the feasibility of the on-chip label free DNA detection device that can be used for a variety of biomedical applications focused on sequence specific DNA detection.

On-chip microtubule gliding assay for parallel measurement of tau protein species.

Tau protein is a well-established biomarker for a group of neurodegenerative diseases collectively called tauopathies. So far, clinically relevant detection of tau species in cerebrospinal fluid (CSF) cannot be achieved without immunological methods. Recently, it was shown that different tau isoforms including the ones carrying various types of mutations affect microtubule (MT)-kinesin binding and velocity in an isoform specific manner. Here, based on these observations, we developed a microfluidic device to analyze tau mutations, isoforms and their ratios. The assay device consists of three regions: a MT reservoir which captures MTs from a solution to a kinesin-coated surface, a microchannel which guides gliding MTs, and an arrowhead-shaped collector which concentrates MTs. Tau-bound fluorescently labeled MTs (tau-MTs) were assayed, and the increase in fluorescence intensity (FI) corresponding to the total number of MTs accumulated was measured at the collector. We show that our device is capable of differentiating 3R and 4R tau isoform ratios and effects of point mutations within 5 minutes. Furthermore, radially oriented collector regions enable simultaneous FI measurements for six independent assays. Performing parallel assays in the proposed device with minimal image processing provides a cost-efficient, easy-to-use and fast tau detection platform.

Point-of-care (POC) devices by means of advanced MEMS.

Microelectromechanical systems (MEMS) have become an invaluable technology to advance the development of point-of-care (POC) devices for diagnostics and sample analyses. MEMS can transform sophisticated methods into compact and cost-effective microdevices that offer numerous advantages at many levels. Such devices include microchannels, microsensors, etc., that have been applied to various miniaturized POC products. Here we discuss some of the recent advances made in the use of MEMS devices for POC applications.

Intestinal tumor suppression in ApcMin/+ mice by prostaglandin D2 receptor PTGDR.

Our earlier work showed that knockout of hematopoietic prostaglandin D synthase (HPGDS, an enzyme that produces prostaglandin D2) caused more adenomas in Apc(Min/+) mice. Conversely, highly expressed transgenic HPGDS allowed fewer tumors. Prostaglandin D2 (PGD2) binds to the prostaglandin D2 receptor known as PTGDR (or DP1). PGD2 metabolites bind to peroxisome proliferator-activated receptor γ (PPARG). We hypothesized that Ptgdr or Pparg knockouts may raise numbers of tumors, if these receptors take part in tumor suppression by PGD2. To assess, we produced Apc(Min/+) mice with and without Ptgdr knockouts (147 mice). In separate experiments, we produced Apc(Min/+) mice expressing transgenic lipocalin-type prostaglandin D synthase (PTGDS), with and without heterozygous Pparg knockouts (104 mice). Homozygous Ptgdr knockouts raised total numbers of tumors by 30-40% at 6 and 14 weeks. Colon tumors were not affected. Heterozygous Pparg knockouts alone did not affect tumor numbers in Apc(Min/+) mice. As mentioned above, our Pparg knockout assessment also included mice with highly expressed PTGDS transgenes. Apc(Min/+) mice with transgenic PTGDS had fewer large adenomas (63% of control) and lower levels of v-myc avian myelocytomatosis viral oncogene homolog (MYC) mRNA in the colon. Heterozygous Pparg knockouts appeared to blunt the tumor-suppressing effect of transgenic PTGDS. However, tumor suppression by PGD2 was more clearly mediated by receptor PTGDR in our experiments. The suppression mechanism did not appear to involve changes in microvessel density or slower proliferation of tumor cells. The data support a role for PGD2 signals acting through PTGDR in suppression of intestinal tumors.

Biosensing MAPs as "roadblocks": kinesin-based functional analysis of tau protein isoforms and mutants using suspended microtubules (sMTs).

The concept of a reconstructed microtubule kinesin-based transport system was originally introduced for studies of underlying biophysical mechanisms of intracellular transport and its potential applications in bioengineering at micro- and nanoscale levels. However, several technically challenging shortcomings prohibit its use in practical applications. One of them is the propensity of microtubules to bind various protein molecules creating "roadblocks" for kinesin molecule movement and subsequently preventing efficient delivery of the molecular cargo. The interruption in kinesin movement strictly depends on the specific type of "roadblock", i.e. the microtubule associated protein (MAP). Therefore, we propose to use the "roadblock" effect as a molecular sensor that may be used for functional characterization of particular MAPs with respect to their role in MT-based transport and associated pathologies, such as neurodegeneration. Here, we applied a kinesin-based assay using a suspended MT design (sMT assay) to functionally characterize known MAP tau protein isoforms and common mutations found in familial frontotemporal dementia (FTD). The proposed sMT assay is compatible with an on-chip format and may be used for the routine characterization of MT associated molecules applicable to diagnostics and translational research.

Nano bioresearch approach by microtechnology.

To progress in basic science and drug development, convenient methodology for detecting specific biological molecules and their interaction in living organism is in high demand. After more than 20 years of increasing research efforts, micro and nanotechnologies are now mature to propose a new class of miniature devices and principles enabling compartmentalized bioassays. Among them, this review proposes various examples that include array of electro-active microwells for highly parallel single cell analysis, cost-effective nanofluidic for DNA separation, parallel enzymatic reaction in 100pL droplet and high-throughput platform for membrane proteins assays. The micro devices are presented with relevant experiments to foresee their future contribution to translational research and drug discovery.

Suspended microtubules demonstrate high sensitivity and low experimental variability in kinesin bead assay.

Microtubule (MT) based intraneuronal transport deficiency is directly linked to neurodegeneration. Hence, the development of a reliable and sensitive in vitro approach permitting efficient analysis of MT-based transport is essential for our understanding of the underlying molecular mechanisms that may lead to novel therapeutic approaches for treating neurodegenerative diseases. Here, based on previously developed reconstructed MT-kinesin assay, we propose its "suspended" modification that shows higher sensitivity and lower experimental variability.

Application of a new microcantilever biosensor resonating at the air-liquid interface for direct insulin detection and continuous monitoring of enzymatic reactions.

Here we describe the application of a recently developed high-resolution microcantilever biosensor resonating at the air-liquid interface for the continuous detection of antigen-antibody and enzyme-substrate interactions. The cantilever at the air-liquid interface demonstrated 50% higher quality factor and a 5.7-fold increase in signal-to-noise-ratio (SNR) compared with one immersed in the purified water. First, a label-free detection of a low molecular weight protein (insulin, 5.8 kDa) in physiological concentration was demonstrated. The liquid facing side of the cantilever was functionalized by coating its surface with insulin antibodies, while the opposite side was exposed to air. The meniscus membrane at the micro-slit around the cantilever sustained the liquid in the microchannel. After optimizing the process of surface functionalization, the resonance frequency shift was successfully measured for insulin solutions of 0.4, 2.0, and 6.3 ng ml(-1). To demonstrate additional application of the device for monitoring enzymatic protein degradation, the liquid facing microcantilever surface was coated with human recombinant SOD1 (superoxide dismutase 1) and exposed to various concentrations of proteinase K solution, and the kinetics of the SOD1 digestion was continuously monitored. The results showed that it is a suitable tool for sensitive protein detection and analysis.

Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues.

We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

A systems level, functional genomics analysis of chronic epilepsy.

Neither the molecular basis of the pathologic tendency of neuronal circuits to generate spontaneous seizures (epileptogenicity) nor anti-epileptogenic mechanisms that maintain a seizure-free state are well understood. Here, we performed transcriptomic analysis in the intrahippocampal kainate model of temporal lobe epilepsy in rats using both Agilent and Codelink microarray platforms to characterize the epileptic processes. The experimental design allowed subtraction of the confounding effects of the lesion, identification of expression changes associated with epileptogenicity, and genes upregulated by seizures with potential homeostatic anti-epileptogenic effects. Using differential expression analysis, we identified several hundred expression changes in chronic epilepsy, including candidate genes associated with epileptogenicity such as Bdnf and Kcnj13. To analyze these data from a systems perspective, we applied weighted gene co-expression network analysis (WGCNA) to identify groups of co-expressed genes (modules) and their central (hub) genes. One such module contained genes upregulated in the epileptogenic region, including multiple epileptogenicity candidate genes, and was found to be involved the protection of glial cells against oxidative stress, implicating glial oxidative stress in epileptogenicity. Another distinct module corresponded to the effects of chronic seizures and represented changes in neuronal synaptic vesicle trafficking. We found that the network structure and connectivity of one hub gene, Sv2a, showed significant changes between normal and epileptogenic tissue, becoming more highly connected in epileptic brain. Since Sv2a is a target of the antiepileptic levetiracetam, this module may be important in controlling seizure activity. Bioinformatic analysis of this module also revealed a potential mechanism for the observed transcriptional changes via generation of longer alternatively polyadenlyated transcripts through the upregulation of the RNA binding protein HuD. In summary, combining conventional statistical methods and network analysis allowed us to interpret the differentially regulated genes from a systems perspective, yielding new insight into several biological pathways underlying homeostatic anti-epileptogenic effects and epileptogenicity.

Cu, Zn-superoxide dismutase 1 (SOD1) is a novel target of Puromycin-sensitive aminopeptidase (PSA/NPEPPS): PSA/NPEPPS is a possible modifier of amyotrophic lateral sclerosis.

Accumulation of misfolded neurotoxic Cu, Zn-superoxide dismutase-1 (SOD1) protein found in both familial and sporadic amyotrophic lateral sclerosis (ALS) is recognized as an important contributing factor of neuronal cell death. However, little is known about the mechanisms controlling the accumulation and turnover of SOD1 protein. Puromycin-sensitive aminopeptidase (PSA/NPEPPS) was recently identified as a major peptidase acting on neurotoxic TAU protein and protecting against TAU-induced neurodegeneration. In addition, recent report implicated PSA/NPEPPS in the direct removal of neurotoxic polyglutamine repeats. These combined data suggest that PSA/NPEPPS might represent a novel degradation pathway targeting pathologically aggregating neurotoxic protein substrates including SOD1. Here, we report that PSA/NPEPPS directly regulates SOD1 protein abundance and clearance via proteolysis. In addition, PSA/NPEPPS expression is significantly decreased in motor neurons of both SODG93A transgenic mice and sporadic ALS patients, suggesting its possible contribution to the disease pathogenesis. These results implicate SOD1 as a new target protein of PSA/NPEPPS and point to the possible neuroprotective role of PSA/NPEPPS in ALS.

Use of peripheral blood transcriptome biomarkers for epilepsy prediction.

There are currently no predictive methods to identify patients who suffered an initial brain injury and are at high risk of developing chronic epilepsy. Consequently, treatments aimed at epilepsy prevention that would target the underlying epileptogenic process are neither available nor being developed. After a brain injury or any other initial precipitating event (IPE) to the development of epilepsy, pathological changes may occur in forms of inflammation, damage in the blood brain barrier, neuron loss, gliosis, axon sprouting, etc., in multiple brain areas. Recent studies provide connections between various kinds of brain pathology and alterations in the peripheral blood transcriptome. In this review we discuss the possibility of using peripheral blood transcriptome biomarkers for the detection of epileptogenesis and consequently, subjects at high risk of developing epilepsy.

Puromycin-sensitive aminopeptidase (PSA/NPEPPS) impedes development of neuropathology in hPSA/TAU(P301L) double-transgenic mice.

Accumulation of neurotoxic hyperphosphorylated TAU protein is a major pathological hallmark of Alzheimer disease and other neurodegenerative dementias collectively called tauopathies. Puromycin-sensitive aminopeptidase (PSA/NPEPPS) is a novel modifier of TAU-induced neurodegeneration with neuroprotective effects via direct proteolysis of TAU protein. Here, to examine the effects of PSA/NPEPPS overexpression in vivo in the mammalian system, we generated and crossed BAC-PSA/NPEPPS transgenic mice with the TAU(P301L) mouse model of neurodegeneration. PSA/NPEPPS activity in the brain and peripheral tissues of human PSA/NPEPPS (hPSA) mice was elevated by ∼2-3-fold with no noticeable deleterious physiological effects. Double-transgenic animals for hPSA and TAU(P301L) transgenes demonstrated a distinct trend for delayed paralysis and showed significantly improved motor neuron counts, no gliosis and markedly reduced levels of total and hyperphosphorylated TAU in the spinal cord, brain stem, cortex, hippocampus and cerebellum of adult and aged animals when compared with TAU(P301L) mice. Furthermore, endogenous TAU protein abundance in human neuroblastoma SH-SY5Y cells was significantly reduced or augmented by overexpression or knockdown of PSA/NPEPPS, respectively. This study demonstrated that without showing neurotoxic effects, elevation of PSA/NPEPPS activity in vivo effectively blocks accumulation of soluble hyperphosphorylated TAU protein and slows down the disease progression in the mammalian system. Our data suggest that increasing PSA/NPEPPS activity may be a feasible therapeutic approach to eliminate accumulation of unwanted toxic substrates such as TAU.

Integrative gene-tissue microarray-based approach for identification of human disease biomarkers: application to amyotrophic lateral sclerosis.

Advances in genomics and proteomics permit rapid identification of disease-relevant genes and proteins. Challenges include biological differences between animal models and human diseases, high discordance between DNA and protein expression data and a lack of experimental models to study human complex diseases. To overcome some of these limitations, we developed an integrative approach using animal models, postmortem human material and a combination of high-throughput microarray methods to identify novel molecular markers of amyotrophic lateral sclerosis (ALS). We used laser capture microdissection coupled with microarrays to identify early transcriptome changes occurring in spinal cord motor neurons or surrounding glial cells. Two models of familial motor neuron disease, SOD1(G93A) and TAU(P301L), transgenic mice were used at the presymptomatic stage. Identified gene expression changes were predominantly model-specific. However, several genes were regulated in both models. The relevance of identified genes as clinical biomarkers was tested in the peripheral blood transcriptome of presymptomatic SOD1(G93A) animals using custom-designed ALS microarray. To confirm the relevance of identified genes in human sporadic ALS (SALS), selected corresponding protein products were examined by high-throughput immunoassays using tissue microarrays constructed from human postmortem spinal cord tissues. Genes that were identified by these experiments and located within a linkage region associated with familial ALS/frontotemporal dementia were sequenced in several families. This large-scale gene and protein expression study pointing to distinct molecular mechanisms of TAU- and SOD1-induced motor neuron degeneration identified several new SALS-relevant proteins (CNGA3, CRB1, OTUB2, MMP14, SLK, DDX58, RSPO2) and putative blood biomarkers, including Nefh, Prph and Mgll.

Sanfilippo syndrome type B, a lysosomal storage disease, is also a tauopathy.

Sanfilippo syndrome type B (mucopolysaccharidosis III B, MPS III B) is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of alpha-N-acetylglucosaminidase and lysosomal accumulation of heparan sulfate. In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate, but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances. We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu(-/-) and Naglu(+/-) mice. Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls. The highest increase in gene expression (6- to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs, which encodes lysozyme, but accumulation of lysozyme protein was seen in MEC neurons only. Because of a report that lysozyme induced the formation of hyperphosphorylated tau (P-tau) in cultured neurons, we searched for P-tau by immunohistochemistry. P-tau was found in MEC of Naglu(-/-) mice, in the same neurons as lysozyme. In older mutant mice, it was also seen in the dentate gyrus, an area important for memory. Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments, P-tau aggregates characteristic of tauopathies-a group of age-related dementias that include Alzheimer disease. Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy.