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Dynamic Characterization of Structural, Molecular, and Electrophysiological Phenotypes of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoids, and Comparison with Fetal and Adult Gene Profiles.

ABSTRACT: BACKGROUND:The development of 3D cerebral organoid technology using human-induced pluripotent stem cells (iPSCs) provides a promising platform to study how brain diseases are appropriately modeled and treated. So far, understanding of the characteristics of organoids is still in its infancy. The current study profiled, for the first time, the electrophysiological properties of organoids at molecular and cellular levels and dissected the potential age equivalency of 2-month-old organoids to human ones by a comparison of gene expression profiles among cerebral organoids, human fetal and adult brains. RESULTS:Cerebral organoids exhibit heterogeneous gene and protein markers of various brain cells, such as neurons, astrocytes, and vascular cells (endothelial cells and smooth muscle cells) at 2 months, and increases in neural, glial, vascular, and channel-related gene expression over a 2-month differentiation course. Two-month organoids exhibited action potentials, multiple channel activities, and functional electrophysiological responses to the anesthetic agent propofol. A bioinformatics analysis of 20,723 gene expression profiles showed the similar distance of gene profiles in cerebral organoids to fetal and adult brain tissues. The subsequent Ingenuity Pathway Analysis (IPA) of select canonical pathways related to neural development, network formation, and electrophysiological signaling, revealed that only calcium signaling, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signaling in neurons, glutamate receptor signaling, and synaptogenesis signaling were predicted to be downregulated in cerebral organoids relative to fetal samples. Nearly all cerebral organoid and fetal pathway phenotypes were predicted to be downregulated compared with adult tissue. CONCLUSIONS:This novel study highlights dynamic development, cellular heterogeneity and electrophysiological activity. In particular, for the first time, electrophysiological drug response recapitulates what occurs in vivo, and neural characteristics are predicted to be highly similar to the human brain, further supporting the promising application of the cerebral organoid system for the modeling of the human brain in health and disease. Additionally, the studies from these characterizations of cerebral organoids in multiple levels and the findings from gene comparisons between cerebral organoids and humans (fetuses and adults) help us better understand this cerebral organoid-based cutting-edge platform and its wide uses in modeling human brain in terms of health and disease, development, and testing drug efficacy and toxicity.

SUBMITTER: Logan S

PROVIDER: S-EPMC7291286 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Dynamic Characterization of Structural, Molecular, and Electrophysiological Phenotypes of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoids, and Comparison with Fetal and Adult Gene Profiles.

Logan Sarah S Arzua Thiago T Yan Yasheng Y Jiang Congshan C Liu Xiaojie X Yu Lai-Kang LK Liu Qing-Song QS Bai Xiaowen X

Cells 20200523 5

<h4>Background</h4>The development of 3D cerebral organoid technology using human-induced pluripotent stem cells (iPSCs) provides a promising platform to study how brain diseases are appropriately modeled and treated. So far, understanding of the characteristics of organoids is still in its infancy. The current study profiled, for the first time, the electrophysiological properties of organoids at molecular and cellular levels and dissected the potential age equivalency of 2-month-old organoids ...[more]

PMID: 32456176

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Project description:BackgroundDNA methylation is an epigenetic modification that plays an important role during mammalian development. Around birth in humans, the main site of red blood cell production moves from the fetal liver to the bone marrow. DNA methylation changes at the β-globin locus and a switch from fetal to adult hemoglobin production characterize this transition. Understanding this globin switch may improve the treatment of patients with sickle cell disease and β-thalassemia, two of the most common Mendelian diseases in the world. The goal of our study was to describe and compare the genome-wide patterns of DNA methylation in fetal and adult human erythroblasts.MethodsWe used the Illumina HumanMethylation 450 k BeadChip to measure DNA methylation at 402,819 CpGs in ex vivo-differentiated erythroblasts from 12 fetal liver and 12 bone marrow CD34+ donors.ResultsWe identified 5,937 differentially methylated CpGs that overlap with erythroid enhancers and binding sites for erythropoiesis-related transcription factors. Combining this information with genome-wide association study results, we show that erythroid enhancers define particularly promising genomic regions to identify new genetic variants associated with fetal hemoglobin (HbF) levels in humans. Many differentially methylated CpGs are located near genes with unanticipated roles in red blood cell differentiation and proliferation. For some of these new candidate genes, we confirm the correlation between DNA methylation and gene expression levels in red blood cell progenitors. We also provide evidence that DNA methylation and genetic variation at the β-globin locus independently control globin gene expression in adult erythroblasts.ConclusionsOur DNA methylome maps confirm the widespread dynamic changes in DNA methylation that occur during human erythropoiesis. These changes tend to happen near erythroid enhancers, further highlighting their importance in erythroid regulation and HbF production. Finally, DNA methylation may act independently of the transcription factor BCL11A to repress fetal hemoglobin production. This provides cues on strategies to more efficiently re-activate HbF production in sickle cell disease and β-thalassemia patients.

Project description:Underdeveloped lungs are the primary cause of death in premature infants, however, little is known about stem and progenitor cell maintenance during human lung development. In this study, we have identified that FGF7, Retinoic Acid and CHIR-99021, a small molecule that inhibits GSK3 to activate Wnt signaling, support in vitro maintenance of primary human fetal lung bud tip progenitor cells in a progenitor state. Furthermore, these factors are sufficient to derive a population of human bud tip-like progenitor cells in 3D organoid structures from human pluripotent stem cells (hPSC). Functional studies showed that hPSC-derived bud tip progenitor organoids do not contain any mesenchymal cell types, maintain multilineage potential in vitro and are able to engraft into the airways of injured mice and respond to systemic factors. We performed RNA-sequencing to assess the degree of similarity in global gene expression profiles between the full human fetal lung (59-127 days gestation), isolated human fetal bud tip progenitors, organoids grown from primary fetal bud tip progenitors, and hPSC-derived bud tip organoids. Results showed that hPSC-derived organoids have molecular profiles similar to organoids generated from primary human fetal lung tissue. Gene expression differences between hPSC-derived bud tip organoids and fetal progenitor organoids may be related to the presence of contaminating mesenchymal cells in primary cultures. hPSC-derived bud tip organoids are generated from a well-defined human cell sources, offering a distinct advantage over rare primary tissue as a means to study human specific lung development, homeostasis and disease.<br>Sample Nomenclature - Description<br> -------------------------------------------------------------------------<br> Peripheral fetal lung the distal/peripheral portion of the fetal lung (i.e., distal 0.5 cm) was excised from the rest of the lung using a scalpel. This includes all components of the lung (e.g., epithelial, mesenchymal, vascular). <br>Isolated fetal bud tip the bud peripheral portion of the fetal lung was excised with a scalpel and subjected to enzymatic digestion and microdissection. The epithelium was dissected and separated from the mesenchyme, but a small amount of associated mesenchyme likely remained. <br>Fetal progenitor organoid 3D organoid structures that arose from culturing isolated fetal epithelial bud tips. <br>Foregut spheroid 3D foregut endoderm structure as described in Dye et al. (2015). Gives rise to patterned lung organoid (PLO) when grown in 3F medium. <br> Patterned lung organoid (PLO) lung organoids that were generated by differentiating hPSCs, as described throughout the manuscript. <br> Bud tip organoid organoids derived from PLOs, enriched for SOX2/SOX9 co-expressing cells, and grown/passaged in 3F medium.

Dataset Information

Dynamic Characterization of Structural, Molecular, and Electrophysiological Phenotypes of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoids, and Comparison with Fetal and Adult Gene Profiles.

Publications

Dynamic Characterization of Structural, Molecular, and Electrophysiological Phenotypes of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoids, and Comparison with Fetal and Adult Gene Profiles.

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