Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology

Abstract

Human neural progenitors derived from pluripotent stem cells develop into electrophysiologically active neurons at heterogeneous rates, which can confound disease-relevant discoveries in neurology and psychiatry. By combining patch clamping, morphological and transcriptome analysis on single human neurons in vitro, we defined a continuum of poor to highly functional electrophysiological states of differentiated neurons. The strong correlations between action potentials, synaptic activity, dendritic complexity and gene expression highlight the importance of methods for isolating functionally comparable neurons for in vitro investigations of brain disorders. While whole-cell electrophysiology is the gold standard for functional evaluation, it often lacks the scalability required for disease modeling studies. Here, we demonstrate a multimodal machine-learning strategy to identify new molecular features that predict the physiological states of single neurons, independently of the time spent in vitro. As further proof of concept, we selected one of the potential neurophysiological biomarkers identified in this study – GDAP1L1 – to isolate highly functional live human neurons in vitro.

Disrupted neuronal maturation in Angelman syndrome-derived induced pluripotent stem cells

“…In support of this, we continue to see maturation of AP firing and half-width, transient outward currents, proportion of synaptically active neurons and frequency of spontaneous synaptic currents up to 20 weeks in vitro , which has not been reported in other studies. At our latest time points, most of our control neurons best match what Bardy et al 27. defines as Type 5 cells, in both level of synaptic activity and type of firing.…”

Improved neural differentiation of normal and abnormal induced pluripotent stem cell lines in the presence of valproic acid

“…In the present study, the functional properties of iPSC‐derived neural like cells were tested for both the iPSC lines with the whole cell patch clamp technique by multiple injections of currents. Bardy et al reported an extensive characterization of neurons based on the AP profile (Bardy et al, ). Mostly, the Types 1 and 2 APs from this classification were observed in our cultures.…”

Next‐generation disease modeling with direct conversion: a new path to old neurons

“…This raises the question as to whether directly converted neurons have the neuron-specific ability to create and integrate into a neuronal network. Conversely, gene expression changes related to synapse formation, voltage-gated potassium channels, or mitochondrial oxidative phosphorylation critically define mature neuronal identity [122], and transcriptomes of single human iPSCderived neurons can clearly predict neuronal functionality [123]. Outstandingly, iNs are capable of long-distance axonal outgrowth, following axonal growth cues specific for the neuronal subtype and connecting with target brain regions, providing evidence of a stable conversion of cells into functional, fully differentiated neurons [57].…”

“…Immunocytochemical data are further quite variable between experiments, antibody batches, and laboratories, thus not well-quantifiable, low-dimensional, and thus less ideal to be directly compared by statistically powerful (meta-)analytical means. Conversely, gene expression changes related to synapse formation, voltage-gated potassium channels, or mitochondrial oxidative phosphorylation critically define mature neuronal identity [122], and transcriptomes of single human iPSCderived neurons can clearly predict neuronal functionality [123]. As epigenomic, transcriptomic, proteomic, and metabolic profiles of human brain samples are emerging, we can now use these data and map iN and iPSC-derived data to them.…”