Biology subjects

Lee, M.-Y.

Publications and source records attributed to Lee, M.-Y..

10 recordsLinked to original sources

bioRxiv2026

Differential Neurodevelopmental Disruption by Bisphenol A (BPA) and Valproic Acid (VPA) in Human Forebrain Organoids

Neurodevelopmental disorders, including autism spectrum disorder (ASD), are influenced by both genetic abnormalities and environmental toxicants. Among environmental risk factors, endocrine-disrupting chemicals such as bisphenol A (BPA) and pharmaceutical drugs such as valproic acid (VPA) have been associated with an increased risk of autism. In this study, human induced pluripotent stem cell (iPSC)-derived forebrain organoids were used to model early neurodevelopmental disruptions induced by BPA and VPA exposure. On day 62 of differentiation, forebrain organoids were treated with physiologically relevant concentrations of BPA or VPA for 28 days. Following treatment, morphological, molecular, and electrophysiological changes were assessed across experimental conditions. Both compounds produced distinct alterations in organoid morphology, neurodevelopmental gene expression, and network electrical activity, with VPA inducing markedly stronger effects. Overall, these data suggest forebrain organoids as a robust, physiologically relevant in vitro model system for studying neurodevelopment. This platform enables systematic investigation of environmental and pharmacological risk factors implicated in the pathogenesis of neurodevelopmental disorders.

neuroscience↗

bioRxiv2024

Enhanced Maturity and Functionality of Vascularized Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture

Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM+ endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 L BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.

bioengineering↗

bioRxiv2024

Cryopreservation of neuroectoderm on a pillar plate and in situ differentiation into human brain organoids

Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To illustrate cryopreservation application to human brain organoids (HBOs), early-stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NEs) in an ultralow atachement (ULA) 384-well plate. These NEs were transferred and encapsulated in Matrigel on the pillar plate. The early-stage HBOs on the pillar plate were exposed to four commercially available CPAs, including PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80{degrees}C and subsequently stored in a liquid nitrogen dewar. We examined the impact of CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of early-stage HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving these HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability post-cryopreservation than larger ones. An incubation period of 80 minutes with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400 - 600 {micro}m. These cryopreserved early-stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to non-cryopreserved HBOs. The cryopreserved early-stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.

bioengineering↗

bioRxiv2024

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays

Human liver organoids (HLOs) differentiated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can recapitulate the structure and function of human fetal liver tissues, thus being considered as a promising tissue model for liver diseases and predictive compound screening. However, the adoption of HLOs in drug discovery faces several technical challenges, which include the lengthy differentiation process with multiple culture media leading to batch-to-batch variation, short-term maintenance of hepatic functions post-maturation, low assay throughput due to Matrigel dissociation and HLO transfer to a microtiter well plate, and insufficient maturity levels compared to primary hepatocytes. To address these issues, expandable HLOs (Exp-HLOs) derived from human iPSCs were generated by optimizing differentiation protocols, which were rapidly printed on a 144-pillar plate with sidewalls and slits (144PillarPlate) and dynamically cultured for up to 20 days into differentiated HLOs (Diff-HLOs) in a 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for in situ organoid culture and analysis. The dynamically cultured Diff-HLOs exhibited greater maturity and reproducibility than those cultured statically, especially after a 10-day differentiation period. In addition, Diff-HLOs in the pillar/perfusion plate were tested with acetaminophen and troglitazone for 3 days to assess drug-induced liver injury (DILI) and then incubated in an expansion medium for 10 days to evaluate liver recovery from DILI. The assessment of liver regeneration post-injury is critical to understanding the mechanism of recovery and determining the threshold drug concentration beyond which there will be a sharp decrease in the livers regenerative capacity. We envision that bioprinted Diff-HLOs in the pillar/perfusion plate could be used for high-throughput screening (HTS) of hepatotoxic compounds due to the short-term differentiation of passage-able Exp-HLOs, stable hepatic function post-maturation, high reproducibility, and high throughput with capability of in situ organoid culture, testing, staining, imaging, and analysis. Graphical abstractThe overall process of dynamic liver organoid culture and in situ analysis in the 144PillarPlate/144PerfusionPlate for high-throughput hepatotoxicity assays. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/586638v3_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1f2902aorg.highwire.dtl.DTLVardef@1d6e074org.highwire.dtl.DTLVardef@2a0dc0org.highwire.dtl.DTLVardef@eccde0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15 - 18%, without any detrimental effect on cell viability. Despite utilizing 10 - 50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 {+/-} 1.6 {micro}M and 25.4 {+/-} 8.3 {micro}M, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

bioengineering↗

bioRxiv2024

Dynamic culture of cerebral organoids using a pillar/perfusion plate for the assessment of developmental neurotoxicity

Despite the potential toxicity of commercial chemicals to the development of the nervous system (known as developmental neurotoxicity or DNT), conventional in vitro cell models have primarily been employed for the assessment of acute neuronal toxicity. On the other hand, animal models used for the assessment of DNT are not physiologically relevant due to the heterogenic difference between humans and animals. In addition, animal models are low-throughput, time-consuming, expensive, and ethically questionable. Recently, human brain organoids have emerged as a promising alternative to assess the detrimental effects of chemicals on the developing brain. However, conventional organoid culture systems have several technical limitations including low throughput, lack of reproducibility, insufficient maturity of organoids, and the formation of the necrotic core due to limited diffusion of nutrients and oxygen. To address these issues and establish predictive DNT models, cerebral organoids were differentiated in a dynamic condition in a unique pillar/perfusion plate, which were exposed to test compounds to evaluate DNT potential. The pillar/perfusion plate facilitated uniform, dynamic culture of cerebral organoids with improved proliferation and maturity by rapid, bidirectional flow generated on a digital rocker. Day 9 cerebral organoids in the pillar/perfusion plate were exposed to ascorbic acid (DNT negative) and methylmercury (DNT positive) in a dynamic condition for 1 and 3 weeks, and changes in organoid morphology and neural gene expression were measured to determine DNT potential. As expected, ascorbic acid didnt induce any changes in organoid morphology and neural gene expression. However, exposure of day 9 cerebral organoids to methylmercury resulted in significant changes in organoid morphology and neural gene expression. Interestingly, methylmercury did not induce adverse changes in cerebral organoids in a static condition, thus highlighting the importance of dynamic organoid culture in DNT assessment.

bioengineering↗

bioRxiv2023

SOX7: Novel Autistic Gene Identified by Analysis of Multi-Omics Data

Genome-wide association studies and next generation sequencing data analyses based on DNA information have identified thousands of mutations associated with autism spectrum disorder (ASD). However, more than 99% of identified mutations are non-coding. Thus, it is unclear which of these mutations might be functional and thus potentially causal variants. Transcriptomic profiling using total RNA-sequencing has been one of the most utilized approaches to link protein levels to genetic information at the molecular level. The transcriptome captures molecular genomic complexity that the DNA sequence solely does not. Some mutations alter a genes DNA sequence but do not necessarily change expression and/or protein function. To date, few common variants reliably associated with the diagnosis status of ASD despite consistently high estimates of heritability. In addition, reliable biomarkers used to diagnose ASD or molecular mechanisms to define the severity of ASD do not exist. Therefore, it is necessary to integrate DNA and RNA testing together to identify true causal genes and propose useful biomarkers for ASD. We performed gene-based association studies with adaptive test using genome-wide association studies (GWAS) summary statistics with two large GWAS datasets (ASD 2019 data: 18,382 ASD cases and 27,969 controls [discovery data]; ASD 2017 data: 6,197 ASD cases and 7,377 controls [replication data]) which were obtained from the Psychiatric Genomics Consortium (PGC). In addition, we investigated differential expression between ASD cases and controls for genes identified in gene-based GWAS with two RNA-seq datasets (GSE211154: 20 cases and 19 controls; GSE30573: 3 cases and 3 controls). We identified 5 genes significantly associated with ASD in ASD 2019 data (KIZ-AS1, p=8.67x10-10; KIZ, p=1.16x10-9; XRN2, p=7.73x10-9; SOX7, p=2.22x10-7; LOC101929229 also known as PINX1-DT, p=2.14x10-6). Among these 5 genes, gene SOX7 (p=0.00087) and LOC101929229 (p=0.009) were replicated in ASD 2017 data. KIZ-AS1 (p=0.059) and KIZ (p=0.06) were close to the boundary of replication in ASD 2017 data. Genes SOX7 (p=0.036 in all samples; p=0.044 in white samples) indicated significant expression differences between cases and controls in the GSE211154 RNA-seq data. Furthermore, gene SOX7 was upregulated in cases than in controls in the GSE30573 RNA-seq data (p=0.0017; Benjamini-Hochberg adjusted p=0.0085). SOX7 encodes a member of the SOX (SRY-related HMG-box) family of transcription factors pivotally contributing to determining of the cell fate and identity in many lineages. The encoded protein may act as a transcriptional regulator after forming a protein complex with other proteins leading to autism. Gene SOX7 in the transcription factor family could be associated with ASD. This finding may provide new diagnostic and therapeutic strategies for ASD.

bioinformatics↗

bioRxiv2023

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate

Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. By differentiating the EBs on the pillar plate, we achieved robust generation of cerebral organoids with a coefficient of variation (CV) below 19%. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

bioengineering↗

bioRxiv2023

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis

Human organoids have potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, we overcome these challenges by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

bioengineering↗

bioRxiv2023

A pillar/perfusion plate enhances cell growth, reproducibility, throughput, and user friendliness in dynamic 3D cell culture

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from the necrotic core due to limited nutrient and oxygen diffusion and waste removal and have limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids have been loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow rates were maintained within perfusion wells, and the pillar plate could be separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in dynamic 3D cell culture.

bioengineering↗