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Human neural progenitor cell differentiation in vitro

ABSTRACT: This experiment was designed to characterize the temporal gene expression dynamics of differentiating human neural progenitor cells through time in culture. In vitro models of neuronal differentiation are emerging as an important tool for high-throughput and high-content screening in neurodevelopmental toxicology. However, little has been done to characterize normal temporal pathway dynamics of differentiation in vitro or to anchor processes captured in vitro to developmental processes in vivo that are vulnerable to toxicant perturbation. We cultured human neural progenitor cell (hNPCs) up to 21 days in differentiation conditions, examining changes in morphology, protein expression and global gene expression. Over time, hNPCs acquired morphological characteristics of mature neuronal networks and increased protein expression of neuronal markers, including beta tubulin III, MAP2, and alpha-synuclein. Significantly changed genes were organized according to temporal expression patterns using K-means clustering, revealing 3 phases of gene expression. Quantitative pathway analysis identified gene ontology (GO) terms enriched among genes expressed in each of these phases and created a quantitative summary of temporal pathway trends in vitro. These observations of morphology, protein and gene expression provide a timeline of progression through differentiation, facilitating identification of key phases of sensitivity. We compared gene expression in vitro with publicly available gene expression data from developing human brain tissue in vivo and found substantial concordance in relative gene expression intensity. Genes highly expressed in both samples were enriched for key processes of brain development, including proliferation, migration, differentiation, synapse formation, and neurotransmission. GO terms enriched among genes highly expressed only in vivo or only in vitro reveal important differences between systems. For example, genes highly expressed in vitro are enriched for more stress and apoptosis pathways. This analysis provides a temporal roadmap of in vitro neuronal differentiation and anchors gene expression patterns in vitro to gene expression during sensitive windows of in vivo development. By anchoring in vitro dynamics to in vivo reference points, this work clarifies the extent to which fundamental processes of brain development are captured in our model.

ORGANISM(S): Homo sapiens

PROVIDER: GSE78919 | GEO | 2019/02/01

REPOSITORIES: GEO

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Project description:All established protocols for differentiation of mouse and human pluripotent stem cells into specific neural subpopulations generate a considerable cellular heterogeneity that hampers experimental and clinical progress. In order to obtain a homogenous population of neuronal precursor cells and to streamline the differentiation of embryonic stem cells (ESCs), we assessed PSA-NCAM, a surface glycoprotein that is specifically expressed on immature neurons. We developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from differentiated ESC cultures and characterized their neuronal differentiation potential in vitro. PSA-NCAM enrichment at an early step of neural differentiation increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. The in vivo potential of in vitro derived PSA-NCAM+ enriched precursors was functionally characterized by grafting into the forebrain of adult mice. Analysis for several neuronal and glia markers at 10 or 40 days post graft showed a distinct differentiation pattern. While unsorted control cells gave rise to a mixed population composed of immature precursors, early postmitotic neurons or glial cells, the majority of PSA-NCAM+ enriched cells differentiated into NeuN positive neurons. Furthermore, when in contact with the rostral migratory stream, higher numbers of cells integrated into the stream and migrated towards the olfactory bulb when the PSA-NCAM enriched population was grafted. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells. Two conditions (step 4, step 5), each represented by three biological replicates of control and enriched cells (Cy5); mESC was used as common reference (Cy3)

Project description:The self-renewal and differentiation potential of human embryonic stem cells (hESCs) suggests that hESCs could be used for regenerative medicine, especially for restoring neuronal functions in brain diseases. However, the functional properties of neurons derived from hESC are largely unknown. Moreover, since hESCs were derived under diverse conditions, the possibility arises that neurons derived from different hESC lines exhibit distinct properties, but this possibility remains unexplored. To address these issues, we developed a protocol that allows step-wise generation from hESCs of ~70-80% pure human neurons that form spontaneously active synaptic networks in culture. Comparison of neurons derived from the well-characterized HSF1 and HSF6 hESC lines revealed that HSF1- but not HSF6-derived neurons exhibit forebrain properties. Accordingly, HSF1-derived neurons initially form primarily GABAergic synaptic networks, whereas HSF6-derived neurons initially form glutamatergic networks. microRNA profiling revealed significant expression differences between the two hESC lines, suggesting that microRNAs may influence their distinct differentiation properties. These observations indicate that although both HSF1 and HSF6 hESCs differentiate into functional neurons, the two hESC lines exhibit distinct differentiation potentials, suggesting that they are pre-programmed. Information on hESC line-specific differentiation biases is crucial for neural stem cell therapy and establishment of novel disease models using hESCs. Experiment Overall Design: We directly compared the transcriptome of hNPCs derived from HSF-1 and HSF-6 hESC lines under two different neural induction conditions (embryonic body formation in presence of caudalizing factors (retinoic acid and bFGF) and monolayer conversion in presence of BMP signaling inhibitor (Noggin)). Since HSF6 hESC cannot undergo efficient neural differentiation without caudalizing factors, we cannot obtain RNA samples for HSF6 (Noggin). Experiment Overall Design: In summary, consistent with our immunostaining results, HSF1 hNPCs display more forebrain properties as compared to HSF6 hNPCs, indicated by expression of multiple forebrain specific markers. Conversely, HSF6 hNPCs show regional identity towards posterior central nervous system.

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Human neural progenitor cell differentiation in vitro

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