Project description:Here we assess the reliability of human cortical spheroid differentiation cultured in xeno-free, feeder-free conditions. We find high reliability in differentiation across multiple hiPSC lines as well as across experiments, and we anticipate that this directed differentiation approach will be useful for large scale generation of brain-region specific spheroids and disease modeling.
Project description:Single-cell RNA-seq: We used single-cell RNAseq to investigate the maturation of astrocytes within human cortical spheroids Bulk RNA-seq: Bulk sequencing from astrocytes and neurons purified (via immunopanning) from iPSC-derived coritical spheroids at varying in vitro differentiation states
Project description:Expression profiles of human cortical spheroids (hCSs) derived from pluripotent stem cells using Illumina cDNA microarrays. The goal of the study is to assess the developmental maturity and the regional identity of those hCSs at two distinct time points with expression profiles and compare the expression pattern with in vivo data. Results show that those hCSs can map transcriptionally to in vivo fetal development. Use transcriptional profiling to assess the developmental maturity and the regional identity of hCSs at two distinct time points.
Project description:Organoid technologies provide an accessible system in which to examine the generation, self-organization,and 3-dimensional cellular interactions during development of the human cerebral cortex. However, oligodendrocytes, the myelinating glia of the central nervous system and third major neural cell type, are conspicuously absent from current protocols. Here we reproducibly generate human oligodendrocytes and myelin in pluripotent stem cell-derived cortical spheroids. Transcriptional and immunohistochemical analysis of the spheroids demonstrates molecular features consistent with maturing human oligodendrocytes within 14 weeks of culture, including expression of MyRF, PLP1, and MBP proteins. Histological analysis by electron microscopy shows initial wrapping of human neuronal axons with myelin by 20 weeks and maturation to compact myelin by 30 weeks in culture. Treatment of spheroids with previously identified promyelinating drugs enhances the rate and extent of human oligodendrocyte generation and myelination. Furthermore, generation of spheroids from patients with a severe genetic myelin disorder, Pelizaeus-Merzbacher disease, demonstrates the ability to recapitulate human disease phenotypes, which were in turn improved with both pharmacologic and CRISPR-based approaches. Collectively, these 3-dimensional, multi-lineage cortical spheroids provide a versatile platform to observe and perturb the complex cellular interactions that occur during developmental myelination of the brain and offer new opportunities for disease modeling and therapeutic development in human tissue.
Project description:Expression profiles of human cortical spheroids (hCSs) derived from pluripotent stem cells using Illumina cDNA microarrays. The goal of the study is to assess the developmental maturity and the regional identity of those hCSs at two distinct time points with expression profiles and compare the expression pattern with in vivo data. Results show that those hCSs can map transcriptionally to in vivo fetal development.
Project description:HT-29 and HCT-116 cells were barcoded using the CloneTracker lentiviral barcode library and then dabrafenib and irinotecan resistant derivatives of these cell lines were established, respectively.10 million barcoded HT-29 and HCT-116 cells were seeded equally onto poly-HEMA coated 4xT75 flask (DMSO Control, Replica A, B, C for each drug). After seeding, cells were allowed to form spheroids and barcoded 3D-HT-29 spheroids were treated with dabrafenib at increasing doses starting from IC50/10 dose until IC50/2 dose with monthly doubling of the dosing (16 weeks), and barcoded 3D-HCT-116 cells were treated with irinotecan at increasing doses starting from IC50/4 dose until IC50 dose with weekly doubling of the dosing (4 weeks). Following the end points of treatment for each cell line, DNA was isolated from harvested cell lines and barcode sequencing and whole exome sequencing were carried out.
Project description:Liver-derived cells from the surface and cave-adapted morphs of Astyanax mexicanus are valuable in vitro resources to explore the metabolism of these unique fish. However, 2D cultures have not yet fully mimicked the metabolic profile of the fish liver. Also, 3D culturing can modulate the transcriptomic profile of cells when compared to its 2D counterpart. Hence, to widen the range of metabolic pathways that can be depicted in vitro, we cultured the liver-derived SFL and CFL into 3D spheroids. We 3D cultured the cells at various cell seeding densities for 4 weeks and characterized the resultant transcriptome. The 3D cultured SFL and CFL cells indeed depicted a wider range of metabolic pathways as compared to the 2D culture. Further, the 3D spheroids also showed surface and cave-specific responses, making the spheroids an exciting system to study cave adaptation. Taken together, SFL and CFL spheroids prove to be a promising model for overall understanding of altered metabolism in Astyanax mexicanus.
Project description:3D spheroids of primary human hepatocytes (3D PHH) keep a differentiated phenotype with retained metabolic function and proteome fingerprint for weeks in culture. As a result, 3D PHH are gaining ground as a model for mechanistic studies on liver homeostasis and in vitro to in vivo (IVIV)predictions in drug discovery. However, the function of drug-transporting proteins has not yet been studied in the 3D PHH. Here we used the organic cation transporter 1 (OCT1/SLC22A1) as a model to explore both transporter kinetics and long-term regulation of transporter activity via different pathways. The 3D PHH were cultured for one week and then used for short- and long-term studies. The OCT1 transporter kinetics was assessed using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, the 3D PHH were exposed to xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomics analysis was used to track prototypical changes of other regulated proteins. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14.14±3.97 using three donors. Established OCT1 inhibitors reduced the uptake of ASP+ in the 3D PHH spheroids, resulting in 40-60% of the maximal uptake rate of ASP+. The long-term exposure studies showed that OCT1 is relatively stable to the activation of a broad range of pathways known to regulate ADME proteins. Importantly, while expression levels of other ADME proteins, such as CYP3A4 and MDR1 were regulated as expected, OCT1 levels remained stable. In conclusion, our results show for the first time that 3D PHH spheroids express fully active OCT1 and that transporter kinetics can be studied in 3D PHH. We also confirm that OCT1 remains stable and functional during activation of various pathways that alter the expression and function of other ADME proteins.