Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics.

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.

Handel AE ，Chintawar S ，Lalic T ，Whiteley E ，Vowles J ，Giustacchini A ，Argoud K ，Sopp P ，Nakanishi M ，Bowden R ，Cowley S ，Newey S ，Akerman C ，Ponting CP ，Cader MZ ... -  《-》

Comparative Transcriptomic Analysis of Cerebral Organoids and Cortical Neuron Cultures Derived from Human Induced Pluripotent Stem Cells.

Kathuria A ，Lopez-Lengowski K ，Watmuff B ，Karmacharya R ... -  《-》

Transcription factor programming of human ES cells generates functional neurons expressing both upper and deep layer cortical markers.

Miskinyte G ，Grønning Hansen M ，Monni E ，Lam M ，Bengzon J ，Lindvall O ，Ahlenius H ，Kokaia Z ... -  《-》

Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks.

Miskinyte G ，Devaraju K ，Grønning Hansen M ，Monni E ，Tornero D ，Woods NB ，Bengzon J ，Ahlenius H ，Lindvall O ，Kokaia Z ... -  《-》

NGN2 induces diverse neuron types from human pluripotency.

Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.

Lin HC ，He Z ，Ebert S ，Schörnig M ，Santel M ，Nikolova MT ，Weigert A ，Hevers W ，Kasri NN ，Taverna E ，Camp JG ，Treutlein B ... -  《-》