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Unveiling dynamic metabolic signatures in human induced pluripotent and neural stem cells.

ABSTRACT: Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to human induced pluripotent stem cells (hiPSC) and human neural stem cells (hNSC) subjected to a sudden extracellular glutamine step. The fold-changes of steady-states and the transient profiles of metabolic pools revealed that dynamic responses were reproducible and cell type-specific. Importantly, many amino acids had conserved dynamics and readjusted their steady state concentration in response to the increased glutamine influx. Overall, we propose a novel methodology for systematic metabolic characterization and identification of potential metabolic regulators.

SUBMITTER: Vasconcelos E Sa J

PROVIDER: S-EPMC7188302 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Unveiling dynamic metabolic signatures in human induced pluripotent and neural stem cells.

Vasconcelos E Sá João J Simão Daniel D Terrasso Ana P AP Silva Marta M MM Brito Catarina C Isidro Inês A IA Alves Paula M PM Carrondo Manuel J T MJT

PLoS computational biology 20200416 4

Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to ...[more]

PMID: 32298259

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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:IntroductionNeural induction of human induced pluripotent stem cells represents a critical switch in cell state during which pluripotency is lost and commitment to a neural lineage is initiated. Although many of the key transcription factors involved in neural induction are known, we know little of the temporal and causal relationships that are required for this state transition.MethodsHere, we have carried out a longitudinal analysis of the transcriptome of human iPSCs undergoing neural induction. Using the temporal relationships between the changing profile of key transcription factors and subsequent changes in their target gene expression profiles, we have identified distinct functional modules operative throughout neural induction.ResultsIn addition to modules that govern loss of pluripotency and gain of neural ectoderm identity, we discover other modules governing cell cycle and metabolism. Strikingly, some of these functional modules are retained throughout neural induction, even though the gene membership of the module changes. Systems analysis identifies other modules associated with cell fate commitment, genome integrity, stress response and lineage specification. We then focussed on OTX2, one of the most precociously activated transcription factors during neural induction. Our temporal analysis of OTX2 target gene expression identified several OTX2 regulated gene modules representing protein remodelling, RNA splicing and RNA processing. Further CRISPRi inhibition of OTX2 prior to neural induction promotes an accelerated loss of pluripotency and a precocious and aberrant neural induction disrupting some of the previously identified modules.DiscussionWe infer that OTX2 has a diverse role during neural induction and regulates many of the biological processes that are required for loss of pluripotency and gain of neural identity. This dynamical analysis of transcriptional changes provides a unique perspective of the widespread remodelling of the cell machinery that occurs during neural induction of human iPSCs.

Project description:BackgroundNeural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).Methodology/principal findingsWe performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.Conclusions/significanceThese findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.

Dataset Information

Unveiling dynamic metabolic signatures in human induced pluripotent and neural stem cells.

Publications

Unveiling dynamic metabolic signatures in human induced pluripotent and neural stem cells.

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