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Using single-cell multiomics to study stem cell differentiation

Human cortical spheroids for largescale differentiation experiments and disorder modelling1

Generating three-dimensional (3D) cultures, so-called organoids, from human pluripotent stem cells (hPSCs) can be used to investigate brain development and disorders.


For the central nervous system, organoids demonstrate high-cell diversity, while reproducing more cell-cell interactions and developing to later stages than 2D cultures. However, one of the challenges of using brain organoids has been the lack of reproducability of differentiation.


The authors previously developed brain-region-specific organoid cultures that resemble the cerebral cortex by differentiating pyramidal cortical neurons from human-induced pluripotent stem cells (hiPSCs) in a 3D culture, called human cortical spheroids (hCSs).


In this study, they used single-cell analyses, transcriptional profiling and immunocytochemistry during long-term in vitro differentiation to assess the reliability of hCS derivation across multiple hiPSC lines and experiments.




To assess the overall success of differentiation across hiPSC lines and experiments, 4–11 differentiations were carried out with each of the 12 hiPSC lines, for a total of 85 experiments. Spheroids expressed cortical neuron markers and demonstrated an internal site architecture that include proliferative zones.


hiPSC-derived hCSs were characterised using single-cell transcriptional profiling on the BD Rhapsody™ Single-Cell Analysis System after 105 days. Sequencing data was processed using BD Rhapsody™ Analysis Pipelines.


Cell distribution in the eight main clusters, that were previously identified in forebrain spheroids (the glutamatergic neuron cluster, the intermediate progenitor cluster, the radial glia cluster, the astroglia cluster, the ventral progenitor cluster, the GABAergic neuron cluster, the oligodendrocyte progenitor cell (OPC) cluster and the chloride plexus cluster), was similar across hCS conditions.




Using transcriptional time course, immunocytochemistry and single-cell analyses, it was shown that the derivation of hCS is reliable across multiple hiPSC lines and differentiation experiments.


This methodology could be adapted to other regions of the brain. The use of this approach for largescale differentiation experiments and disease modelling is anticipated in the future.


Read the article entitled “Reliability of human cortical organoid generation” and learn in more detail how the BD Rhapsody™ Single-Cell Analysis System enabled the study of stem-cell differentiation.


1. Yoon SJ, Elahi LS, Pașca AM, et al. Reliability of human cortical organoid generation. Nat Methods. 2019;16(1):75-78. doi:10.1038/s41592-018-0255-0

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