Dataset Information

Human iPSC-Derived 2D and 3D Platforms for Rapidly Assessing Developmental, Functional, and Terminal Toxicities in Neural Cells.

ABSTRACT: With increasing global health threats has come an urgent need to rapidly develop and deploy safe and effective therapies. A common practice to fast track clinical adoption of compounds for new indications is to repurpose already approved therapeutics; however, many compounds considered safe to a specific application or population may elicit undesirable side effects when the dosage, usage directives, and/or clinical context are changed. For example, progenitor and developing cells may have different susceptibilities than mature dormant cells, which may yet be different than mature active cells. Thus, in vitro test systems should reflect the cellular context of the native cell: developing, nascent, or functionally active. To that end, we have developed high-throughput, two- and three-dimensional human induced pluripotent stem cell (hiPSC)-derived neural screening platforms that reflect different neurodevelopmental stages. As a proof of concept, we implemented this in vitro human system to swiftly identify the potential neurotoxicity profiles of 29 therapeutic compounds that could be repurposed as anti-virals. Interestingly, many compounds displayed high toxicity on early-stage neural tissues but not on later stages. Compounds with the safest overall viability profiles were further evaluated for functional assessment in a high-throughput calcium flux assay. Of the 29 drugs tested, only four did not modulate or have other potentially toxic effects on the developing or mature neurospheroids across all the tested dosages. These results highlight the importance of employing human neural cultures at different stages of development to fully understand the neurotoxicity profile of potential therapeutics across normal ontogeny.

SUBMITTER: Slavin I

PROVIDER: S-EPMC7918576 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Human iPSC-Derived 2D and 3D Platforms for Rapidly Assessing Developmental, Functional, and Terminal Toxicities in Neural Cells.

Slavin Ileana I Dea Steven S Arunkumar Priyanka P Sodhi Neha N Montefusco Sandro S Siqueira-Neto Jair J Seelke Janet J Lofstrom Mary Anne MA Anson Blake B Zanella Fabian F Carromeu Cassiano C

International journal of molecular sciences 20210214 4

With increasing global health threats has come an urgent need to rapidly develop and deploy safe and effective therapies. A common practice to fast track clinical adoption of compounds for new indications is to repurpose already approved therapeutics; however, many compounds considered safe to a specific application or population may elicit undesirable side effects when the dosage, usage directives, and/or clinical context are changed. For example, progenitor and developing cells may have differ ...[more]

PMID: 33672998

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Project description:BACKGROUND:Pericytes of the blood-brain barrier (BBB) are embedded within basement membrane between brain microvascular endothelial cells (BMECs) and astrocyte end-feet. Despite the direct cell-cell contact observed in vivo, most in vitro BBB models introduce an artificial membrane that separates pericytes from BMECs. In this study, we investigated the effects of pericytes on BMEC barrier function across a range of in vitro platforms with varied spatial orientations and levels of cell-cell contact. METHODS:We differentiated RFP-pericytes and GFP-BMECs from hiPSCs and monitored transendothelial electrical resistance (TEER) across BMECs on transwell inserts while pericytes were either directly co-cultured on the membrane, indirectly co-cultured in the basolateral chamber, or embedded in a collagen I gel formed on the transwell membrane. We then incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. RESULTS:We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm2). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes restored optimal TEER. Conversely, directly co-cultured pericytes resulted in a decrease in TEER by interfering with BMEC monolayer continuity. In the microvessel model, we observed direct pericyte-BMEC contact, abluminal pericyte localization, and physiologically-low Lucifer yellow permeability comparable to that of BMEC microvessels. In addition, pericyte motility decreased during the first 48 h of co-culture, suggesting progression towards pericyte stabilization. CONCLUSIONS:We demonstrated that monocultured BMECs do not require co-culture to achieve physiological TEER, but that suboptimal TEER in stressed monolayers can be increased through co-culture with hiPSC-derived pericytes or conditioned media. We also developed the first BBB microvessel model using exclusively hiPSC-derived BMECs and pericytes, which could be used to examine vascular dysfunction in the human CNS.

Project description:While there is a growing appreciation of three-dimensional (3D) neural tissues (i.e., hydrogel-based, organoids, and spheroids), shown to improve cellular health and network activity to mirror brain-like activity in vivo, functional assessment using current electrophysiology techniques (e.g., planar multi-electrode arrays or patch clamp) has been technically challenging and limited to surface measurements at the bottom or top of the 3D tissue. As next-generation MEAs, specifically 3D MEAs, are being developed to increase the spatial precision across all three dimensions (X, Y, Z), development of improved computational analytical tools to discern region-specific changes within the Z dimension of the 3D tissue is needed. In the present study, we introduce a novel computational analytical pipeline to analyze 3D neural network activity recorded from a "bottom-up" 3D MEA integrated with a 3D hydrogel-based tissue containing human iPSC-derived neurons and primary astrocytes. Over a period of ~6.5 weeks, we describe the development and maturation of 3D neural activity (i.e., features of spiking and bursting activity) within cross sections of the 3D tissue, based on the vertical position of the electrode on the 3D MEA probe, in addition to network activity (identified using synchrony analysis) within and between cross sections. Then, using the sequential addition of postsynaptic receptor antagonists, bicuculline (BIC), 2-amino-5-phosphonovaleric acid (AP-5), and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), we demonstrate that networks within and between cross sections of the 3D hydrogel-based tissue show a preference for GABA and/or glutamate synaptic transmission, suggesting differences in the network composition throughout the neural tissue. The ability to monitor the functional dynamics of the entire 3D reconstructed neural tissue is a critical bottleneck; here we demonstrate a computational pipeline that can be implemented in studies to better interpret network activity within an engineered 3D neural tissue and have a better understanding of the modeled organ tissue.

Project description:Background: Bladder cancer is the fourth most commonly diagnosed cancer among males worldwide. Current treatment strategies established for bladder cancer mainly consist of cystectomy yet advances in radiation therapy have pointed to the value of organ-preserving strategies in preserving patients' quality of life. Aim: To study and compare the radiosensitivity in two-dimension (2D) and physiologically-relevant three-dimension (3D) in vitro culture of three human bladder cancer cell lines, RT4, T24, and UM-UC-3. Materials and Methods: Clonogenic assay was performed to assess cells' radiosensitivity in 2D. Employing the 3D Matrigel™-based cultures to enrich for cancer stem cells (CSCs) allowed us to assess the survival of this subpopulation of cells via evaluating the number, i.e., sphere forming unit (SFU), and the sizes of cultured spheres, formed from cells exposed to different radiation doses compared to non-irradiated cells. Results: Irradiating cells with increasing radiation doses revealed highest survival rates with RT4 cells in 2D, followed by T24 and UM-UC-3. In 3D, however, UM-UC-3 cells were shown to be the most radio-resistant as evidenced by the number of spheres formed, yet they displayed the least efficient volume reduction/regression (VR), whilst the volume decreased significantly for both RT4 and T24 cells. Sphere VR and sphere ratio (SR) values were then plotted against each other demonstrating a linear correlation between volume and number with RT4 and UM-UC-3 cell lines, but not T24. Lastly, multiple regression model was employed to evaluate the possibility of obtaining a function combining both 3D parameters, SR and VR, with the surviving fraction (SF) in 2D, and showed a linear regression for T24 cells only, with a correlation coefficient of 0.97 for the combined parameters. Conclusion: We were able to radiobiologically characterize 3 human bladder cancer cell lines showing differential effects of radiation between 2D and 3D culture systems, paving the way for achieving better assessment of radiosensitivity of bladder cancer in vitro.

Dataset Information

Human iPSC-Derived 2D and 3D Platforms for Rapidly Assessing Developmental, Functional, and Terminal Toxicities in Neural Cells.

Publications

Human iPSC-Derived 2D and 3D Platforms for Rapidly Assessing Developmental, Functional, and Terminal Toxicities in Neural Cells.

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