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Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures.

ABSTRACT: Investigating human oligodendrogenesis and the interaction of oligodendrocytes with neurons and astrocytes would accelerate our understanding of the mechanisms underlying white matter disorders. However, this is challenging because of the limited accessibility of functional human brain tissue. Here, we developed a new differentiation method of human induced pluripotent stem cells to generate three-dimensional brain organoids that contain oligodendrocytes as well as neurons and astrocytes, called human oligodendrocyte spheroids. We found that oligodendrocyte lineage cells derived in human oligodendrocyte spheroids transitioned through developmental stages similar to primary human oligodendrocytes and that the migration of oligodendrocyte lineage cells and their susceptibility to lysolecithin exposure could be captured by live imaging. Moreover, their morphology changed as they matured over time in vitro and started myelinating neurons. We anticipate that this method can be used to study oligodendrocyte development, myelination, and interactions with other major cell types in the CNS.

SUBMITTER: Marton RM

PROVIDER: S-EPMC6788758 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures.

Marton Rebecca M RM Miura Yuki Y Sloan Steven A SA Li Qingyun Q Revah Omer O Levy Rebecca J RJ Huguenard John R JR Pașca Sergiu P SP

Nature neuroscience 20190128 3

Investigating human oligodendrogenesis and the interaction of oligodendrocytes with neurons and astrocytes would accelerate our understanding of the mechanisms underlying white matter disorders. However, this is challenging because of the limited accessibility of functional human brain tissue. Here, we developed a new differentiation method of human induced pluripotent stem cells to generate three-dimensional brain organoids that contain oligodendrocytes as well as neurons and astrocytes, called ...[more]

PMID: 30692691

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Project description:IntroductionStem cells have the ability to self-renew or to differentiate into numerous cell types; however, our understanding of how to control and exploit this potential is currently limited. An emerging hypothesis is that microRNAs (miRNAs) play a central role in controlling stem cell-fate determination. Herein, we have characterized the effects of miRNAs in differentiated human neural stem cells (hNSCs) by using a cell line currently being tested in clinical trials for stroke disability (NCT01151124, Clinicaltrials.gov).MethodsHNSCs were differentiated on 2- (2D) and 3-dimensional (3D) cultures for 1 and 3 weeks. Quantification of hNSC differentiation was measured with real-time PCR and axon outgrowth. The miRNA PCR arrays were implemented to investigate differential expression profiles in differentiated hNSCs. Evaluation of miRNA effects on hNSCs was performed by using transfection of miRNA mimics, real-time PCR, Western blot, and immunocytochemistry.ResultsThe 3D substrate promoted enhanced hNSC differentiation coupled with a loss of cell proliferation. Differentiated hNSCs exhibited a similar miRNA profiling. However, in 3D samples, the degree and timing of regulation were significantly different in miRNA members of cluster mi-R17 and miR-96-182, and hsa-miR-302a. Overall, hNSC 3D cultures demonstrated differential regulation of miRNAs involved in hNSC stemness, cell proliferation, and differentiation. The miRNA mimic analysis of hsa-miR-146b-5p and hsa-miR-99a confirmed induction of lineage-committed progenitors. Downregulated miRNAs were more abundant; those most significantly downregulated were selected, and their putative target mRNAs analyzed with the aim of unraveling their functionality. In differentiated hNSCs, downregulated hsa-miR-96 correlated with SOX5 upregulation of gene and protein expression; similar results were obtained for hsa-miR-302a, hsa-miR-182, hsa-miR-7, hsa-miR-20a/b, and hsa-miR-17 and their target NR4A3. Moreover, SOX5 was identified as a direct target gene of hsa-miR-96, and NR43A, a direct target of hsa-miR-7 and hsa-mir-17 by luciferase reporter assays. Therefore, the regulatory role of these miRNAs may occur through targeting NR4A3 and SOX5, both reported as modulators of cell-cycle progression and axon length.ConclusionsThe results provide new insight into the identification of specific miRNAs implicated in hNSC differentiation. These strategies may be exploited to optimize in vitro hNSC differentiation potential for use in preclinical studies and future clinical applications.

Project description:Human neural stem cells (NSCs) are self-renewing, multipotent cells of the central nervous system (CNS). They are characterized by their ability to differentiate into a range of cells, including oligodendrocytes (OLs), neurons, and astrocytes, depending on exogenous stimuli. An efficient and easy directional differentiation method was developed for obtaining large quantities of high-quality of human OL progenitor cells (OPCs) and OLs from NSCs. RNA sequencing, immunofluorescence staining, flow cytometry, western blot, label-free proteomic sequencing, and qPCR were performed in OL lines differentiated from NSC lines. The changes in the positive rate of typical proteins were analyzed expressed by NSCs, neurons, astrocytes, OPCs, and OLs. We assessed Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of differentially expressed (DE) messenger RNAs (mRNAs) related to the differentiation of NSCs and the maturation of OLs. The percentage of NSCs differentiated into neurons, astrocytes, and OLs was 82.13%, 80.19%, and 90.15%, respectively. We found that nestin, PAX6, Musashi, and vimentin were highly expressed in NSCs; PDGFR-α, A2B5, NG2, OLIG2, SOX10, and NKX2-2 were highly expressed in OPCs; and CNP, GALC, PLP1, and MBP were highly expressed in OLs. RNA sequencing, western blot and qPCR revealed that ERBB4 and SORL1 gradually increased during NSC-OL differentiation. In conclusion, NSCs can differentiate into neurons, astrocytes, and OLs efficiently. PDGFR-α, APC, ID4, PLLP, and other markers were related to NSC differentiation and OL maturation. Moreover, we refined a screening method for ERBB4 and SORL1, which may underlie NSC differentiation and OL maturation. Potential unreported genes and proteins may regulate differentiation of human neural stem cells into oligodendrocyte lineage. Neural stem cells (NSCs) can differentiate into neurons, astrocytes, and oligodendrocyte (OLs) efficiently. By analyzing the DE mRNAs and proteins of NSCs and OLs lineage, we could identify reported markers and unreported markers of ERBB4 and SORL1 that may underlie regulate NSC differentiation and OL maturation.

Project description:Alzheimer's disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-? plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer's disease posits that the excessive accumulation of amyloid-? peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer's disease (FAD) mutations exhibit amyloid-?-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer's disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer's disease patients have shown elevated levels of toxic amyloid-? species and phosphorylated tau but did not demonstrate amyloid-? plaques or neurofibrillary tangles. Here we report that FAD mutations in ?-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-?, including amyloid-? plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-? generation with ?- or ?-secretase inhibitors not only decreased amyloid-? pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-?-mediated tau phosphorylation. We have successfully recapitulated amyloid-? and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer's disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

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Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures.

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Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures.

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