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Modelling human CNS injury with human neural stem cells in 2- and 3-Dimensional cultures.

ABSTRACT: The adult human central nervous system (CNS) has very limited regenerative capability, and injury at the cellular and molecular level cannot be studied in vivo. Modelling neural damage in human systems is crucial to identifying species-specific responses to injury and potentially neurotoxic compounds leading to development of more effective neuroprotective agents. Hence we developed human neural stem cell (hNSC) 3-dimensional (3D) cultures and tested their potential for modelling neural insults, including hypoxic-ischaemic and Ca2+-dependent injury. Standard 3D conditions for rodent cells support neuroblastoma lines used as human CNS models, but not hNSCs, but in all cases changes in culture architecture alter gene expression. Importantly, response to damage differs in 2D and 3D cultures and this is not due to reduced drug accessibility. Together, this study highlights the impact of culture cytoarchitecture on hNSC phenotype and damage response, indicating that 3D models may be better predictors of in vivo response to damage and compound toxicity.

SUBMITTER: Vagaska B

PROVIDER: S-EPMC7176653 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Modelling human CNS injury with human neural stem cells in 2- and 3-Dimensional cultures.

Vagaska Barbora B Gillham Olivia O Ferretti Patrizia P

Scientific reports 20200422 1

The adult human central nervous system (CNS) has very limited regenerative capability, and injury at the cellular and molecular level cannot be studied in vivo. Modelling neural damage in human systems is crucial to identifying species-specific responses to injury and potentially neurotoxic compounds leading to development of more effective neuroprotective agents. Hence we developed human neural stem cell (hNSC) 3-dimensional (3D) cultures and tested their potential for modelling neural insults, ...[more]

PMID: 32321995

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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:Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.

Dataset Information

Modelling human CNS injury with human neural stem cells in 2- and 3-Dimensional cultures.

Publications

Modelling human CNS injury with human neural stem cells in 2- and 3-Dimensional cultures.

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