Date Published: October 11, 2018
Publisher: Public Library of Science
Author(s): Giedre Miskinyte, Marita Grønning Hansen, Emanuela Monni, Matti Lam, Johan Bengzon, Olle Lindvall, Henrik Ahlenius, Zaal Kokaia, Jialin Charles Zheng.
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.
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) [3–5]. Efficient production of corticofugal projection neurons (CfuPNs) from ES cells has also been reported . 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 . In addition, most available protocols are time-consuming, inefficient and the generated neurons are often immature and with incomplete functional properties. It also remains to be assessed how closely the ES cell-derived cortical neurons resemble their in vivo counterparts.
Human fetal tissue was obtained with informed consent from patients from Lund and Malmö University Hospitals according to guidelines approved by the Lund-Malmö Ethical Committee, Sweden (Dnr. 6,1,8-2887/2017). Adult human cortical tissue was obtained with informed consent from patients or LAR/guardians of patients undergoing elective surgery for temporal lobe epilepsy according to guidelines approved by the Regional Ethical Committee, Lund (Dnr. H15 642/2008). All animal related procedures in the present study were conducted in accordance with the European Union Directive (2010/63/EU) on the subject of animal rights, and were approved by the committee for the use of laboratory animals at Lund University and the Swedish Board of Agriculture (Dnr. M68-16).
Here we demonstrate that forced expression of three different combinations of the transcription factors NGN2, FEZF2 and SATB2 directs human ES cells to become pyramidal-shaped cells, morphologically resembling adult human cortical neurons, expressing cortical PN markers, and with mature electrophysiological properties. Our findings after grafting onto organotypic cultures suggest that these hES-iNs are also capable of integrating into adult human cortical neural circuitry. However, direct comparison between the different hES-iNs, derived with the three combinations of TFs, did not reveal any striking differences in either morphology, marker expression, molecular signature or electrical activity. Thus, our findings provided no evidence that the hES-iNs had acquired a distinct cortical layer phenotype depending on the TF combination used. Instead, our single-cell data showed that the hES-iNs expressed both upper and deep layer cortical neuronal markers.