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

Giedre Miskinyte,Henrik Ahlenius,Johan Bengzon,Marita Grønning Hansen,Emanuela Monni,Matti Lam,Olle Lindvall,Zaal Kokaia,Jialin Charles Zheng

doi:10.1371/journal.pone.0204688

Abstract

Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols for the generation of such neurons for cell replacement, disease modeling and drug screening are highly warranted. Current methods for the production of cortical neurons from human embryonic stem (ES) cells are often time-consuming and inefficient, and the functional properties of the generated cells have been incompletely characterized. Here we have used transcription factor (TF) programming with the aim to induce rapid differentiation of human ES cells to layer-specific cortical neurons (hES-iNs). Three different combinations of TFs, NEUROGENIN 2 (NGN2) only, NGN2 plus Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2), and NGN2 plus Special AT-Rich Sequence-Binding Protein 2 (SATB2), were delivered to human ES cells by lentiviral vectors. We observed only subtle differences between the TF combinations, which all gave rise to the formation of pyramidal-shaped cells, morphologically resembling adult human cortical neurons expressing cortical projection neuron (PN) markers and with mature electrophysiological properties. Using ex vivo transplantation to human organotypic cultures, we found that the hES-iNs could integrate into adult human cortical networks. We obtained no evidence that the hES-iNs had acquired a distinct cortical layer phenotype. Instead, our single-cell data showed that the hES-iNs, similar to fetal human cortical neurons, expressed both upper and deep layer cortical neuronal markers. Taken together, our findings provide evidence that TF programming can direct human ES cells towards cortical neurons but that the generated cells are transcriptionally profiled to generate both upper and deep layer cortical neurons. Therefore, most likely additional cues will be needed if these cells should adopt a specific cortical layer and area identity.

Highlights

The human cortex is affected by several debilitating acute and chronic neurodegenerative disorders such as stroke, traumatic brain injury, amyotrophic lateral sclerosis and Alzheimer’s disease, which target specific types of cortical neurons
In an attempt to specify the fate of these NEUROGENIN 2 (NGN2)-induced cells towards specific cortical neuronal subtypes, we combined NGN2 with either SATB2 or Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2)
Similar to previous studies [24, 25], we delivered the transcription factor (TF) to the human embryonic stem (ES) cells by doxycycline (Dox)-inducible lentiviral vectors carrying TFs coupled to resistance genes, along with a vector for constitutive expression of rtTA

Summary

Introduction

The human cortex is affected by several debilitating acute and chronic neurodegenerative disorders such as stroke, traumatic brain injury, amyotrophic lateral sclerosis and Alzheimer’s disease, which target specific types of cortical neurons. Emerging evidence indicates that stem cells and reprogrammed cells can be used to generate human cortical neurons both for cell replacement by transplantation, and for disease modeling and drug screening [1, 2]. Several laboratories have established in vitro protocols for the derivation of excitatory pyramidal neurons, the principal type of neuron in the adult cortex, from human pluripotent stem cells (hPSCs) [3,4,5]. While the temporal generation of neurons belonging to the different cortical layers is largely maintained in vitro, and the presence of neurons belonging to specific layers has been found, the proportion of cells characteristic of each layer varies considerably depending on the method used [6]. It remains to be assessed how closely the ES cell-derived cortical neurons resemble their in vivo counterparts

Methods

Results

Conclusion