Dataset Information

Neural Differentiation of Spheroids Derived from Human Induced Pluripotent Stem Cells-Mesenchymal Stem Cells Coculture.

ABSTRACT: Organoids, the condensed three-dimensional (3D) tissues emerged at the early stage of organogenesis, are a promising approach to regenerate functional and vascularized organ mimics. While incorporation of heterotypic cell types, such as human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cells (hiPSCs)-derived neural progenitors aid neural organ development, the interactions of secreted factors during neurogenesis have not been well understood. The objective of this study is to investigate the impact of the composition and structure of 3D hybrid spheroids of hiPSCs and hMSCs on dorsal cortical differentiation and the secretion of extracellular matrices and trophic factors in vitro. The hybrid spheroids were formed at different hiPSC:hMSC ratios (100:0, 75:25, 50:50, 25:75, 0:100) using direct mixing or pre-hiPSC aggregation method, which generated dynamic spheroid structure. The cellular organization, proliferation, neural marker expression, and the secretion of extracellular matrix proteins and the cytokines were characterized. The incorporation of MSCs upregulated Nestin and β-tubulin III expression (the dorsal cortical identity was shown by Pax6 and TBR1 expression), matrix remodeling proteins, and the secretion of transforming growth factor-β1 and prostaglandin E2. This study indicates that the appropriate composition and structure of hiPSC-MSC spheroids promote neural differentiation and trophic factor and matrix secretion due to the heterotypic cell-cell interactions.

SUBMITTER: Song L

PROVIDER: S-EPMC5984568 | biostudies-other | 2018 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Neural Differentiation of Spheroids Derived from Human Induced Pluripotent Stem Cells-Mesenchymal Stem Cells Coculture.

Song Liqing L Tsai Ang-Chen AC Yuan Xuegang X Bejoy Julie J Sart Sébastien S Ma Teng T Li Yan Y

Tissue engineering. Part A 20180103 11-12

Organoids, the condensed three-dimensional (3D) tissues emerged at the early stage of organogenesis, are a promising approach to regenerate functional and vascularized organ mimics. While incorporation of heterotypic cell types, such as human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cells (hiPSCs)-derived neural progenitors aid neural organ development, the interactions of secreted factors during neurogenesis have not been well understood. The objective of this study is ...[more]

PMID: 29160172

Similar Datasets

Project description:Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation. Highlights • Induced pluripotent stem cells (iPSCs) were generated from MPS IIIA skin fibroblasts.• MPS IIIA iPSCs were successfully differentiated into neural progenitor cells (NPCs).• MPS IIIA NPC proliferation was reduced due to interference with FGF2 signalling.• Neurogenesis from MPS IIIA iPSC-derived NPCs is reduced via an unknown mechanism.

Project description:Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

Dataset Information

Neural Differentiation of Spheroids Derived from Human Induced Pluripotent Stem Cells-Mesenchymal Stem Cells Coculture.

Publications

Neural Differentiation of Spheroids Derived from Human Induced Pluripotent Stem Cells-Mesenchymal Stem Cells Coculture.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets