Abstract
Direct conversion of human somatic fibroblasts into induced neurons (iNs) allows for the generation of functional neurons while bypassing any stem cell intermediary stages. Although iN technology has an enormous potential for modeling age-related diseases, as well as therapeutic approaches, the technology faces limitations due to variable conversion efficiencies and a lack of thorough understanding of the signaling pathways directing iN conversion. Here, we introduce a new all-in-one inducible lentiviral system that simplifies fibroblast transgenesis for the two pioneer transcription factors, Ngn2 and Ascl1, and markedly improves iN yields. Further, our timeline RNA-Seq data across the course of conversion has identified signaling pathways that become transcriptionally enriched during iN conversion. Small molecular modulators were identified for four signaling pathways that reliably increase the yield of iNs. Taken together, these advances provide an improved toolkit for iN technology and new insight into the mechanisms influencing direct iN conversion.
Highlights
Human somatic cells such as skin fibroblasts can be directly converted into cultures of functional induced neurons by the overexpression of pro-neuronal transcription factors (Pang et al, 2011; Chambers and Studer, 2011)
To test the efficiency of UNA compared to our conventional two-vector system (EtO +N2A), we selected fibroblasts from three individual donors that had not yielded optimal induced neurons (iNs) conversion efficiencies in the past (Figure 1B and Figure 1—source data 1)
Flow cytometry revealed that UNA-derived iNs exhibited significantly more PSANCAM-positive cells than E + N2A, boosting efficiencies by up to 90–100% for the two suboptimal fibroblast lines, and increasing efficiencies of the lines that already converted well by 30 ± 8% (Figure 1D–E)
Summary
Human somatic cells such as skin fibroblasts can be directly converted into cultures of functional induced neurons (iNs) by the overexpression of pro-neuronal transcription factors (Pang et al, 2011; Chambers and Studer, 2011). IN technology has shown promise in vivo as a strategy to replace damaged cells following brain injury by direct conversion of non-neuronal cell types into neurons directly within the nervous system (Karow et al, 2012; Heinrich et al, 2010). Using combinations of pro-neuronal and region-/subtype-specific transcription factors, a variety of neuronal subtypes has been produced via direct conversion (Caiazzo et al, 2011; Son et al, 2011; Victor et al, 2014; Vadodaria et al, 2016; Tsunemoto et al, 2018). The identification and the combination of Ngn with Ascl as two proneuronal pioneer transcription factors that can induce neuronal identity in non-neuronal cells have been key features in the advancement of this technology
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