Three-dimensional culture models of the brain enable the study of neuroinfection in the context of a complex interconnected cell matrix. Depending on the differentiation status of the neural cells, two models exist: 3D spheroids also called neurospheres and cerebral organoids. Here, we describe the preparation of 3D spheroids and cerebral organoids and give an outlook on their usage to study Rift Valley fever virus and other neurotropic viruses.

暂无摘要。

Neuromuscular disorders are a heterogeneous group of diseases ranging from mild to devastating phenotypes depending on the disorder\'s origin. Pathophysiologies for many of these disorders are not fully understood and efficient therapies are urgently needed. Recent advances in the field of induced pluripotent stem cells and organ-on-a-chip technologies have brought enormous improvement in modeling neuromuscular diseases. Even complex units, like the neuromuscular junction, can now be built, enabling researchers to study each component of the motor unit by itself or interacting with others, allowing the identification of disease mechanisms. This article aims to introduce these new modeling systems to study neuromuscular disorders and the possibilities of organ-on-a-chip platforms to shed light on disease pathologies and their use for therapy development.

Despite two decades of research on silver nanoparticle (AgNP) toxicity, a safe threshold for exposure has not yet been established, albeit being critically needed for risk assessment and regulatory decision-making. Traditionally, a point-of-departure (PoD) value is derived from dose response of apical endpoints in animal studies using either the no-observed-adverse-effect level (NOAEL) approach, or benchmark dose (BMD) modeling. To develop new approach methodologies (NAMs) to inform human risk assessment of AgNPs, we conducted a concentration response modeling of the transcriptomic changes in hepatocytes derived from human induced pluripotent stem cells (iPSCs) after being exposed to a wide range concentration (0.01-25 μg/ml) of AgNPs for 24 h. A plausible transcriptomic PoD of 0.21 μg/ml was derived for a pathway related to the mode-of-action (MOA) of AgNPs, and a more conservative PoD of 0.10 μg/ml for a gene ontology (GO) term not apparently associated with the MOA of AgNPs. A reference dose (RfD) could be calculated from either of the PoDs as a safe threshold for AgNP exposure. The current study illustrates the usefulness of in vitro transcriptomic concentration response study using human cells as a NAM for toxicity study of chemicals that lack adequate toxicity data to inform human risk assessment.

暂无摘要。

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are frequently used for preclinical cardiotoxicity testing and remain an important tool for confirming model-based predictions of drug effects in accordance with the comprehensive in vitro proarrhythmia assay (CiPA). Despite the considerable benefits hiPSC-CMs provide, concerns surrounding experimental reproducibility have emerged. We investigated the effects of temporal changes and experimental parameters on hiPSC-CM electrophysiology. iCell cardiomyocytes2 were cultured and biosignals were acquired using a microelectrode array (MEA) system (2-14 days). Continuous recordings revealed a 22.6% increase in the beating rate and 7.7% decrease in the field potential duration (FPD) during a 20-min equilibration period. Location-specific differences across a multiwell plate were also observed, with iCell cardiomyocytes2 in the outer rows beating 8.8 beats/min faster than the inner rows. Cardiac endpoints were also impacted by cell culture duration; from 2 to 14 days, the beating rate decreased (-12.7 beats/min), FPD lengthened (+257 ms), and spike amplitude increased (+3.3 mV). Cell culture duration (4-10 days) also impacted cardiomyocyte drug responsiveness (E-4031, nifedipine, isoproterenol). qRT-PCR results suggest that daily variations in cardiac metrics may be linked to the continued maturation of hiPSC-CMs in culture (2-30 days). Daily experiments were also repeated using a second cell line (Cor.4U). Collectively, our study highlights multiple sources of variability to consider and address when performing hiPSC-CM MEA studies. To improve reproducibility and data interpretation, MEA-based studies should establish a standardized protocol and report key experimental conditions (e.g., cell line, culture time, equilibration time, electrical stimulation settings, and raw data values).NEW & NOTEWORTHY We demonstrate that iCell cardiomyocytes2 electrophysiology measurements are impacted by deviations in experimental techniques including electrical stimulation protocols, equilibration time, well-to-well variability, and length of hiPSC-CM culture. Furthermore, our results indicate that hiPSC-CM drug responsiveness changes within the first 2 wk following defrost.

BACKGROUND: Succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a model neurometabolic disease at the fulcrum of translational research within the Boston Children\'s Hospital Intellectual and Developmental Disabilities Research Centers (IDDRC), including the NIH-sponsored natural history study of clinical, neurophysiological, neuroimaging, and molecular markers, patient-derived induced pluripotent stem cells (iPSC) characterization, and development of a murine model for tightly regulated, cell-specific gene therapy.
METHODS: SSADHD subjects underwent clinical evaluations, neuropsychological assessments, biochemical quantification of γ-aminobutyrate (GABA) and related metabolites, electroencephalography (standard and high density), magnetoencephalography, transcranial magnetic stimulation, magnetic resonance imaging and spectroscopy, and genetic tests. This was parallel to laboratory molecular investigations of in vitro GABAergic neurons derived from induced human pluripotent stem cells (hiPSCs) of SSADHD subjects and biochemical analyses performed on a versatile murine model that uses an inducible and reversible rescue strategy allowing on-demand and cell-specific gene therapy.
RESULTS: The 62 SSADHD subjects [53% females, median (IQR) age of 9.6 (5.4-14.5) years] included in the study had a reported symptom onset at ∼ 6 months and were diagnosed at a median age of 4 years. Language developmental delays were more prominent than motor. Autism, epilepsy, movement disorders, sleep disturbances, and various psychiatric behaviors constituted the core of the disorder\'s clinical phenotype. Lower clinical severity scores, indicating worst severity, coincided with older age (R= -0.302, p = 0.03), as well as age-adjusted lower values of plasma γ-aminobutyrate (GABA) (R = 0.337, p = 0.02) and γ-hydroxybutyrate (GHB) (R = 0.360, p = 0.05). While epilepsy and psychiatric behaviors increase in severity with age, communication abilities and motor function tend to improve. iPSCs, which were differentiated into GABAergic neurons, represent the first in vitro neuronal model of SSADHD and express the neuronal marker microtubule-associated protein 2 (MAP2), as well as GABA. GABA-metabolism in induced GABAergic neurons could be reversed using CRISPR correction of the pathogenic variants or mRNA transfection and SSADHD iPSCs were associated with excessive glutamatergic activity and related synaptic excitation.
CONCLUSIONS: Findings from the SSADHD Natural History Study converge with iPSC and animal model work focused on a common disorder within our IDDRC, deepening our knowledge of the pathophysiology and longitudinal clinical course of a complex neurodevelopmental disorder. This further enables the identification of biomarkers and changes throughout development that will be essential for upcoming targeted trials of enzyme replacement and gene therapy.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

The extrapolability of the current tumorigenicity test performed by transplanting human cell product into immunodeficient (NOG) mice was investigated. For this purpose, the susceptibility to form teratomas of NOG mice was assessed by transplanting undifferentiated human-induced pluripotent stem cells (hiPSCs) as positive control cells via the liver, striatum, or tail vein and evaluating the TPD50 value (dose required to form teratomas in half of the transplanted mice). This was then compared to the TPD50 of syngeneic or allogeneic mouse models. The TPD50 of C57/BL/6(B6)-iPSC or 129/Ola(129)-embryonic stem cell (ESC) transplanted into the liver of syngeneic mice was 4.08 × 105 and 4.64 × 104 cells, respectively, while the TPD50 of hiPSC administered into the liver of NOG mice was 4.64 × 104 cells. The TPD50 of B6-miPSC-synergic, 129-mESC-synergic, or 129-cell/B6 allogeneic transplantation into the striatum was 5.09 × 102, 1.0 × 104, and 3.73 × 104 cells, respectively, while that of hiPSC/NOG mice was 1.0 × 103 cells. The TPD50 for B6-miPSC or 129-mESC syngeneic tail vein infusion was 3.16 × 106 or 5.62 × 106 cells, respectively, while no incidence was observed from 1 × 107 B6-miPSCs in 129 mice or hiPSCs in NOG mice infusion study. Although the number of data sets was limited, these data indicate that the teratoma formation from transplanted undifferentiated hiPSCs via the liver or striatum in NOG mice is comparable to that in syngeneic or allogeneic mouse transplantation model, suggesting that the result of the current tumorigenicity test in NOG mice would provide useful information to infer the incidence of teratoma from residual undifferentiated hPSCs in hPSC-derived products after transplantation.

Many oncology drugs have been found to induce cardiotoxicity in a subset of patients, which significantly limits their clinical use and impedes the benefit of lifesaving anticancer treatments. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carry donor-specific genetic information and have been proposed for exploring the interindividual difference in oncology drug-induced cardiotoxicity. Herein, we evaluated the inter- and intraindividual variability of iPSC-CM-related assays and presented a proof of concept to prospectively predict doxorubicin (DOX)-induced cardiotoxicity (DIC) using donor-specific iPSC-CMs. Our findings demonstrated that donor-specific iPSC-CMs exhibited greater line-to-line variability than the intraindividual variability in impedance cytotoxicity and transcriptome assays. The variable and dose-dependent cytotoxic responses of iPSC-CMs resembled those observed in clinical practice and largely replicated the reported mechanisms. By categorizing iPSC-CMs into resistant and sensitive cell lines based on their time- and concentration-related phenotypic responses to DOX, we found that the sensitivity of donor-specific iPSC-CMs to DOX may predict in vivo DIC risk. Furthermore, we identified a differentially expressed gene, DND microRNA-mediated repression inhibitor 1 (DND1), between the DOX-resistant and DOX-sensitive iPSC-CMs. Our results support the utilization of donor-specific iPSC-CMs in assessing interindividual differences in DIC. Further studies will encompass a large panel of donor-specific iPSC-CMs to identify potential novel molecular and genetic biomarkers for predicting DOX and other oncology drug-induced cardiotoxicity.