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

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.

Production of human entorhinal stellate cell-like cells by forward programming shows an important role of Foxp1 in reprogramming

“…Interestingly, the iNeurons also expressed Reelin, BCL11B and SATB2 at D10 and retained Reelin and BCL11B expression even at D30 ( Figure 5B ). SATB2 /SATB2 expression however, decreased by D20 to low levels ( Figures 5B,C ), which is similar as previously reported in iNeurons ( Miskinyte et al, 2018 ). However, a few iNeurons co-expressed Reelin, BCL11B and SATB2 at D30 ( Figure 5B ).…”

Activity in grafted human iPS cell–derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior

“…For a successful outcome not only in a xenograft situation, as used here, but also in the clinical setting, the transplanted human-derived neurons must be able to establish afferent and efferent synaptic connections with host neurons in the adult human brain. Recent studies with transplantation of human-derived neurons onto long-term organotypic cultures of adult human cortex provide supportive evidence that this could be possible (37). It will now be necessary, e.g., to explore further the involvement of neuronal activity in well-integrated grafts for recovery following stroke using a broader repertoire of behavioral tasks.…”

Induction of dopaminergic neurons for neuronal subtype-specific modeling of psychiatric disease risk

“…iGLUTs, which most closely resemble fetal cortical pyramidal neurons ( Miskinyte Id, Hansen, Monni, Lam, Bengzon, Lindvall, et al, 2018 ), were generated via transduction of hiPSCs (donors C-1 and C-2) with tetO-NGN2-eGFP-PuroR ( Ho, Hartley, TCW, Beaumont, Stafford, Slesinger, et al, 2016 ; Zhang, Pak, Han, Ahlenius, Zhang, Chanda, et al, 2013 ) using the same protocol steps used to produce iGANs, with the exception that cells were dissociated and replated on DIV3 or DIV4 and matured until DIV21, at which time they were harvested for RNA extraction and RNA-sequencing library-generation.…”