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Accelerated differentiation of human pluripotent stem cells into neural lineages via an early intermediate ectoderm population.

ABSTRACT: Differentiation of human pluripotent stem cells (hPSCs) into ectoderm provides neurons and glia useful for research, disease modeling, drug discovery, and potential cell therapies. In current protocols, hPSCs are traditionally differentiated into an obligate rostro-dorsal ectodermal fate expressing PAX6 after 6 to 12?days in?vitro when protected from mesendoderm inducers. This rate-limiting step has performed a long-standing role in hindering the development of rapid differentiation protocols for ectoderm-derived cell types, as any protocol requires 6 to 10?days in?vitro to simply initiate. Here, we report efficient differentiation of hPSCs into a naive early ectodermal intermediate within 24?hours using combined inhibition of bone morphogenic protein and fibroblast growth factor signaling. The induced population responds immediately to morphogen gradients to upregulate rostro-caudal neurodevelopmental landmark gene expression in a generally accelerated fashion. This method can serve as a new platform for the development of novel, rapid, and efficient protocols for the manufacture of hPSC-derived neural lineages.

SUBMITTER: Walsh P

PROVIDER: S-EPMC7693041 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Accelerated differentiation of human pluripotent stem cells into neural lineages via an early intermediate ectoderm population.

Walsh Patrick P Truong Vincent V Nayak Sushmita S Saldías Montivero Marietta M Low Walter C WC Parr Ann M AM Dutton James R JR

Stem cells (Dayton, Ohio) 20200819 11

Differentiation of human pluripotent stem cells (hPSCs) into ectoderm provides neurons and glia useful for research, disease modeling, drug discovery, and potential cell therapies. In current protocols, hPSCs are traditionally differentiated into an obligate rostro-dorsal ectodermal fate expressing PAX6 after 6 to 12 days in vitro when protected from mesendoderm inducers. This rate-limiting step has performed a long-standing role in hindering the development of rapid differentiation protocols fo ...[more]

PMID: 32745311

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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.

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.

Dataset Information

Accelerated differentiation of human pluripotent stem cells into neural lineages via an early intermediate ectoderm population.

Publications

Accelerated differentiation of human pluripotent stem cells into neural lineages via an early intermediate ectoderm population.

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