Cortical spheroid on perfusable microvascular network in a microfluidic device.

Human induced pluripotent stem cell (hiPSC)-derived brain spheroids can recapitulate the complex cytoarchitecture of the brain, as well as the genetic/epigenetic footprint of human brain development. However, hiPSC-derived 3D models such as spheroid and organoids does not have a perfusable microvascular network, which plays a vital role in maintaining homeostasis in vivo. With the critical balance of positive and negative angiogenic modulators, 3D microvascular network can be achieved by angiogenesis. This paper reports on a microfluidic-based three-dimensional, cortical spheroid grafted on the vascular-network. Vascular network was formed by inducing angiogenic sprouting using concentration gradient-driven angiogenic factors in the microfluidic device. We investigate critical factors for angiogenic vascular network formation with spheroid placement, including 1) a PKCα activator, phorbol-12-myristate-13-acetate (PMA); 2) orientation of endothelial cells under perfusion and permeability of vascular network; 3) effect of extracellular matrix (ECM) types and their densities on angiogenesis; and 4) integration with cortical spheroid on vascular network. This paper demonstrates proof of concept for the potential utility of a membrane-free in vitro cortical spheroid tissue construct with perfusable microvascular network that can be scaled up to a high throughput platform. It can provide a cost-effective alternative platform to animal testing by modeling brain diseases and disorders, and screening drugs.

Studying the Inflammatory Responses to Amyloid Beta Oligomers in Brain-Specific Pericyte and Endothelial Co-culture from Human Stem Cells.

Background: Recently, the in vitro blood brain barrier (BBB) models derived from human pluripotent stem cells have been given extensive attention in therapeutics due to the implications it has with the health of the central nervous system. It is essential to create an accurate BBB model in vitro in order to better understand the properties of the BBB and how it can respond to inflammatory stimulation and be passed by targeted or non-targeted cell therapeutics, more specifically extracellular vesicles. Methods: Brain-specific pericytes (iPCs) were differentiated from iPSK3 cells using dual SMAD signaling inhibitors and Wnt activation plus fibroblast growth factor 2 (FGF-2). The derived cells were characterized by immunostaining, flow cytometry and RT-PCR. In parallel, blood vessels organoids were derived using Wnt activation, BMP4, FGF2, VEGF and SB431542. The organoids were replated and treated with retinoic acid to enhance the blood brain barrier (BBB) features in the differentiated brain endothelial cells (iECs). Co-culture was performed for the iPCs and iECs in transwell system and 3-D microfluidics channels. Results: The derived iPCs expressed common markers PDGFRb and NG2, as well as brain-specific genes FOXF2, ABCC9, KCNJ8, and ZIC1. The derived iECs expressed common endothelial cell markers CD31, VE-cadherin, as well as BBB-associated genes BRCP, GLUT-1, PGP, ABCC1, OCLN, SLC2A1. The co-culture of the two cell types responded to the stimulation of amyloid ß42 oligomers by the upregulation of expression of TNFa, IL6, NFKB, Casp3, SOD2 and TP53. The co-culture also showed the property of trans-endothelial electrical resistance. The proof-of-concept vascularization strategy was demonstrated in a 3-D microfluidics-based device. Conclusion: The derived iPCs and iECs have brain-specific properties and the co-culture of iPCs and iECs provides an in vitro BBB model that show inflammatory response. This study has significance in establishing micro-physiological systems for neurological disease modeling and drug screening.

A Three-Dimensional Brain-on-a-Chip Using Human iPSC-Derived GABAergic Neurons and Astrocytes.

Brain-on-a-chip is a miniaturized engineering platform to mimic the structural and functional aspects of brain tissue. We describe a method to construct a three-dimensional (3D) brain-on-a-chip in this chapter. We firstly portray the method of a brain-on-a-chip model with cocultured mice neurons, microglia, and astrocytes to mimic brain tissue and membrane-free perfusion with endothelial cells, in which we successfully build the blood-brain barrier to screen neurotoxicity. Then we describe a method to construct a brain-on-a-chip with human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes to simulate human brain behavior. This platform consists of neuronal tissue with extracellular matrix (ECM)-embedded GABAergic neurons and astrocytes and a perfusion channel with dynamic flow. We also include the broader applicability test of this model using an organophosphate (OP), malathion, to induce acute and chronic neurotoxicity, and then using butyrylcholinesterase (BuChE) as an exogenous bioscavenger of OP. Following the methods listed in this chapter, we are able to measure the neurotoxic effects on construct integrity, viability, and total AChE and BuChE activity.

Mechanically Tunable Extracellular Matrix of Genipin Crosslinked Collagen and Its Effect on Endothelial Function.

Mechanical rigidity of a matrix, to which cells adhere, plays a significant role in regulating phenotypic cellular behaviors such as spreading and junction formation because vascular cells sense and respond to changes in their mechanical environment. Controlling mechanical properties of extracellular matrix by using a crosslinker is important for cell and tissue mechanobiology. In this paper, we explored genipin, a natural plant extract, to crosslink collagen-I in order to enhance mechanical properties with low cytotoxicity. We characterized the effects of genipin concentration on the mechanical properties, color change, degradation, structure, cell viability, and endothelial function such as transendothelial electrical resistance (TEER). Through the analysis of both material properties and endothelial response, it was found that genipin-based glycation caused an increase in viscoelastic moduli in collagen hydrogels, as well as increased fiber density in their structural morphology. Endothelial cells were found to form better barriers, express higher levels of tight junction proteins, and exhibit better adhesion on stiffer matrices.

Activation and degranulation of CAR-T cells using engineered antigen-presenting cell surfaces.

Adoptive cell transfer of Chimeric Antigen Receptor (CAR)-T cells showed promising results in patients with B cell malignancies. However, the detailed mechanism of CAR-T cell interaction with the target tumor cells is still not well understood. This work provides a systematic method for analyzing the activation and degranulation of second-generation CAR-T cells utilizing antigen-presenting cell surfaces. Antigen-presenting cell surfaces composed of circular micropatterns of CAR-specific anti-idiotype antibodies have been developed to mimic the interaction of CAR-T cells with target tumor cells using micro-contact printing. The levels of activation and degranulation of fixed non-transduced T cells (NT), CD19.CAR-T cells, and GD2.CAR-T cells on the antigen-presenting cell surfaces were quantified and compared by measuring the intensity of the CD3ζ chain phosphorylation and the Lysosome-Associated Membrane Protein 1 (LAMP-1), respectively. The size and morphology of the cells were also measured. The intracellular Ca2+ flux of NT and CAR-T cells upon engagement with the antigen-presenting cell surface was reported. Results suggest that NT and CD19.CAR-T cells have comparable activation levels, while NT have higher degranulation levels than CD19.CAR-T cells and GD2.CAR-T cells. The findings show that antigen-presenting cell surfaces allow a quantitative analysis of the molecules involved in synapse formation in different CAR-T cells in a systematic, reproducible manner.

Three-dimensional brain-on-chip model using human iPSC-derived GABAergic neurons and astrocytes: Butyrylcholinesterase post-treatment for acute malathion exposure.

Organophosphates (OPs) induce acute and chronic neurotoxicity, primarily by inhibiting acetylcholinesterase (AChE) activity as well as by necrosis, and apoptosis. Butyrylcholinesterase (BuChE), an exogenous bioscavenger of OPs, can be used as a treatment for OP exposure. It is prerequisite to develop in vitro brain models that can study BuChE post-treatment for acute OP exposure. In this study, we developed a three-dimensional (3D) brain-on-chip platform with human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes to simulate human brain behavior. The platform consists of two compartments: 1) a hydrogel embedded with human iPSC-derived GABAergic neurons and astrocytes and 2) a perfusion channel with dynamic medium flow. The brain tissue constructs were exposed to Malathion (MT) at various concentrations and then treated with BuChE after 20 minutes of MT exposure. Results show that the iPSC-derived neurons and astrocytes directly interacted and formed synapses in the 3D matrix, and that treatment with BuChE improved viability after MT exposure up to a concentration of 10-3 M. We conclude that the 3D brain-on-chip platform with human iPSC-derived brain cells is a suitable model to study the neurotoxicity of OP exposure and evaluate therapeutic compounds for treatment.

Three-dimensional (3D) brain microphysiological system for organophosphates and neurochemical agent toxicity screening.

We investigated a potential use of a 3D tetraculture brain microphysiological system (BMPS) for neurotoxic chemical agent screening. This platform consists of neuronal tissue with extracellular matrix (ECM)-embedded neuroblastoma cells, microglia, and astrocytes, and vascular tissue with dynamic flow and membrane-free culture of the endothelial layer. We tested the broader applicability of this model, focusing on organophosphates (OPs) Malathion (MT), Parathion (PT), and Chlorpyrifos (CPF), and chemicals that interact with GABA and/or opioid receptor systems, including Muscimol (MUS), Dextromethorphan (DXM), and Ethanol (EtOH). We validated the BMPS platform by measuring the neurotoxic effects on barrier integrity, acetylcholinesterase (AChE) inhibition, viability, and residual OP concentration. The results show that OPs penetrated the model blood brain barrier (BBB) and inhibited AChE activity. DXM, MUS, and EtOH also penetrated the BBB and induced moderate toxicity. The results correlate well with available in vivo data. In addition, simulation results from an in silico physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) model that we generated show good agreement with in vivo and in vitro data. In conclusion, this paper demonstrates the potential utility of a membrane-free tetraculture BMPS that can recapitulate brain complexity as a cost-effective alternative to animal models.

Comparison of endothelial cell attachment on surfaces of biodegradable polymer-coated magnesium alloys in a microfluidic environment.

Polymeric coatings can provide temporary stability to bioresorbable metallic stents at the initial stage of deployment by alleviating rapid degradation and providing better interaction with surrounding vasculature. To understand this interfacing biocompatibility, this study explored the endothelial-cytocompatibility of polymer-coated magnesium (Mg) alloys under static and dynamic conditions compared to that of non-coated Mg alloy surfaces. Poly (carbonate urethane) urea (PCUU) and poly (lactic-co-glycolic acid) (PLGA) were coated on Mg alloys (WE43, AZ31, ZWEKL, ZWEKC) and 316L stainless steel (316L SS, control sample), which were embedded into a microfluidic device to simulate a vascular environment with dynamic flow. The results from attachment and viability tests showed that more cells were attached on the polymer-coated Mg alloys than on non-coated Mg alloys in both static and dynamic conditions. In particular, the attachment and viability on PCUU-coated surfaces were significantly higher than that of PLGA-coated surfaces of WE43 and ZWEKC in both static and dynamic conditions, and of AZ31 in dynamic conditions (P<0.05). The elementary distribution map showed that there were relatively higher Carbon weight percentages and lower Mg weight percentages on PCUU-coated alloys than PLGA-coated alloys. Various levels of pittings were observed underneath the polymer coatings, and the pittings were more severe on the surface of Mg alloys that corroded rapidly. Polymer coatings are recommended to be applied on Mg alloys with relatively low corrosion rates, or after pre-stabilizing the substrate. PCUU-coating has more selective potential to enhance the biocompatibility and mitigate the endothelium damage of Mg alloy stenting.

A forward genetic screen reveals novel independent regulators of ULBP1, an activating ligand for natural killer cells.

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.

Activin/nodal signaling switches the terminal fate of human embryonic stem cell-derived trophoblasts.

Human embryonic stem cells (hESCs) have been routinely treated with bone morphogenetic protein and/or inhibitors of activin/nodal signaling to obtain cells that express trophoblast markers. Trophoblasts can terminally differentiate to either extravillous trophoblasts or syncytiotrophoblasts. The signaling pathways that govern the terminal fate of these trophoblasts are not understood. We show that activin/nodal signaling switches the terminal fate of these hESC-derived trophoblasts. Inhibition of activin/nodal signaling leads to formation of extravillous trophoblast, whereas loss of activin/nodal inhibition leads to the formation of syncytiotrophoblasts. Also, the ability of hESCs to form bona fide trophoblasts has been intensely debated. We have examined hESC-derived trophoblasts in the light of stringent criteria that were proposed recently, such as hypomethylation of the ELF5-2b promoter region and down-regulation of HLA class I antigens. We report that trophoblasts that possess these properties can indeed be obtained from hESCs.