Directed neuronal differentiation of human pluripotent stem cells (hPSCs), neural progenitors, or fibroblasts using transcription factors has allowed for the rapid and highly reproducible differentiation of mature and functional neurons. Exogenous expression of the transcription factor Neurogenin-2 (NGN2) has been widely used to generate different populations of neurons, which have been used in neurodevelopment studies, disease modeling, drug screening, and neuronal replacement therapies. Could NGN2 be a ''one-glove-fits-all'' approach for neuronal differentiations? This review summarizes the cellular roles of NGN2 and describes the applications and limitations of using NGN2 for the rapid and directed differentiation of neurons.

Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies in vitro involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs via a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons (NGN2iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.

Tissue fibrosis, an accumulation of extracellular matrix proteins such as collagen, accompanies cardiac ageing in humans and this is linked to an increased risk of cardiac failure. The mechanisms driving age-related tissue fibrosis and cardiac dysfunction are unclear, yet clinically important. Drosophila is amenable to the study of cardiac ageing as well as collagen deposition; however it is unclear whether collagen accumulates in the ageing Drosophila heart.This work examined collagen deposition and cardiac function in ageing Drosophila, in the context of reduced expression of collagen-interacting protein SPARC (Secreted Protein Acidic and Rich in Cysteine) an evolutionarily conserved protein linked with fibrosis. Heart function was measured using high frame rate videomicroscopy. Collagen deposition was monitored using a fluorescently-tagged collagen IV reporter (encoded by the Viking gene) and staining of the cardiac collagen, Pericardin.The Drosophila heart accumulated collagen IV and Pericardin as flies aged. Associated with this was a decline in cardiac function. SPARC heterozygous flies lived longer than controls and showed little to no age-related cardiac dysfunction. As flies of both genotypes aged, cardiac levels of collagen IV (Viking) and Pericardin increased similarly. Over-expression of SPARC caused cardiomyopathy and increased Pericardin deposition.The findings demonstrate that, like humans, the Drosophila heart develops a fibrosis-like phenotype as it ages. Although having no gross impact on collagen accumulation, reduced SPARC expression extended Drosophila lifespan and cardiac health span. It is proposed that cardiac fibrosis in humans may develop due to the activation of conserved mechanisms and that SPARC may mediate cardiac ageing by mechanisms more subtle than gross accumulation of collagen.