The mammalian nervous system is comprised of an unknown, but recognizably large, number of diverse neuronal subtypes. Recently, direct reprogramming (also known as transdifferentiation) has become an established method to rapidly produce “induced” neurons of numerous different subtypes directly from fibroblasts by overexpressing specific combinations of transcription factors and/or microRNAs. This technique not only provides the means to study various neuronal subtype populations that are not easily accessible, particularly in humans, but it also serves as a tool to interrogate the transcriptional codes that regulate neuronal subtype identity and maintenance. Both in vivo studies and direct reprogramming protocols have demonstrated that basic helix-loop-helix (bHLH) and Pit-Oct-Unc (POU) transcription factors can aid in the specification of distinct neuronal subtypes. Therefore, we set out to comprehensively and systematically address whether first, additional bHLH and POU factor pairings could reprogram fibroblasts into functional neurons and second, dissect out the discrete and synergistic roles of these factors in neuronal subtype specification. We discovered over 70 novel pairs of bHLH and POU (and non-POU) transcription factors sufficient to generate candidate induced neurons (iNs) from mouse embryonic fibroblasts. Transcriptomic analysis of 35 of these candidate iN populations revealed gene expression profiles similar to those of endogenous neuronal populations. Additionally, differences between iN populations were observed at both a transcriptional and functional level.

Genomic mutations pose a serious risk to the health of individuals both in terms of somatic cells and in stem cells used for clinical and research applications. Here we outline novel approaches for studying genome mutations in the context of human induced pluripotent stem cells and neurons. To definitively establish the extent of reprogramming-associated mutations, we utilize the fact that a single fibroblast can give rise to two iPSC colonies separated by at most two divisions. Comparison of the two colonies allows us to distinguish mutations present in the original cell (those found in both colonies) from mutations arising during the reprogramming process. We find on average 150-450 single nucleotide variants per iPSC line, with iPSCs derived by episomal method being significantly more mutated than iPSCs derived with lentivirus. Further, we find that the mutations from episomal reprogramming show unique signatures compared to lentiviral and somatic reprogramming methods. We also find that reprogramming does not contribute significant number of structural variant or mobile element insertion classes of mutation. In the context of neurons, we utilize somatic cell nuclear transfer to reprogram rod photoreceptors from young and aged mice , allowing us to amplify single neuronal genomes without error-prone PCR methods. We show that these neurons accumulate 20-40 SNVs per year, and that these mutations are enriched in nucleotiode contexts that implicate APOBEC deaminase, as well as potential regions of somatic hypermutation.

The brain is a complex organ that contains hundreds of diverse cellular subtypes which organize into unique regions and build intricate neural circuits. Neurons all transition through developmental stages where they must specify into cellular subtypes, migrate to appropriate brain regions, and extend axons to innervate postsynaptic targets as well as elaborate dendritic trees to receive incoming information. These stages are shaped by a balance of intracellular transcriptional programs and extracellular signals such as guidance molecules, adhesion proteins, and neuronal activity. While an extensive list of factors contributing to these processes has been catalogued, the details remain unclear on how they converge within a cell to direct its development. Therefore, we developed novel genetic systems to decipher the rules that shape neuronal development and circuit formation. In one set of studies, we selectively blocked synaptic activity from a subset of neurons within the rodent olfactory bulb to investigate their role in shaping olfactory circuit development. We observed a dramatic impact on the maturation of newborn inhibitory neurons which could not be completely rescued by inhibiting cell death. By assessing the transcriptome of these developmentally-stalled neurons, we identified gene networks that regulate the maturation and integration of neurons into established circuits. For the second set of experiments, we injected rat stem cells into mouse blastocysts to generate rat-mouse brain chimeras and determine whether rat neurons are flexible to develop into, and contribute to foreign neural circuits. In brain-complemented chimeras, we observed diverse rat neuronal subtypes that adopt their host’s developmental timeline and functionally integrate into the mouse brain. Furthermore, we identified species-specific barriers to rat complementation when these neurons are challenged to reconstitute degenerated mouse circuits. Together, these studies provide insights into the mechanisms governing neuronal integration into foreign and compromised neural circuits, which will inform efforts in regenerative medicine.