Advances in human pluripotent stem cell reporter systems.

The capability of human pluripotent stem cells (hPSCs) to self-renew and differentiate into any cell type has greatly contributed to the advancement of biomedicine. Reporter lines derived from hPSCs have played a crucial role in elucidating the mechanisms underlying human development and diseases by acting as an alternative reporter system that cannot be used in living humans. To bring hPSCs closer to clinical application in transplantation, scientists have generated reporter lines for isolating the desired cell populations, as well as improving graft quality and treatment outcomes. This review presents an overview of the applications of hPSC reporter lines and the important variables in designing a reporter system, including options for gene delivery and editing tools, design of reporter constructs, and selection of reporter genes. It also provides insights into the prospects of hPSC reporter lines and the challenges that must be overcome to maximize the potential of hPSC reporter lines.

Autophagy controls neuronal differentiation by regulating the WNT-DVL signaling pathway.

Macroautophagy/autophagy dysregulation is associated with various neurological diseases, including Vici syndrome. We aimed to determine the role of autophagy in early brain development. We generated neurons from human embryonic stem cells and developed a Vici syndrome model by introducing a loss-of-function mutation in the EPG5 gene. Autophagy-related genes were upregulated at the neuronal progenitor cell stage. Inhibition of autolysosome formation with bafilomycin A1 treatment at the neuronal progenitor cell stage delayed neuronal differentiation. Notably, WNT (Wnt family member) signaling may be part of the underlying mechanism, which is negatively regulated by autophagy-mediated DVL2 (disheveled segment polarity protein 2) degradation. Disruption of autolysosome formation may lead to failure in the downregulation of WNT signaling, delaying neuronal differentiation. EPG5 mutations disturbed autolysosome formation, subsequently inducing defects in progenitor cell differentiation and cortical layer generation in organoids. Disrupted autophagy leads to smaller organoids, recapitulating Vici syndrome-associated microcephaly, and validating the disease relevance of our study.Abbreviations: BafA1: bafilomycin A1; co-IP: co-immunoprecipitation; DVL2: dishevelled segment polarity protein 2; EPG5: ectopic P-granules 5 autophagy tethering factor; gRNA, guide RNA; hESC: human embryonic stem cells; KO: knockout; mDA, midbrain dopamine; NIM: neural induction media; NPC: neuronal progenitor cell; qPCR: quantitative polymerase chain reaction; UPS: ubiquitin-proteasome system; WNT: Wnt family member; WT: wild type.

Generation of caudal-type serotonin neurons and hindbrain-fate organoids from hPSCs.

Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on anatomical location and axonal projection, 5-HT neurons are coarsely divided into rostral and caudal groups. Here, we propose a novel strategy to generate hindbrain 5-HT neurons from human pluripotent stem cells (hPSCs), which involves the formation of ventral-type neural progenitor cells and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid, was used to direct the cells into the hindbrain cell fate. Approximately 30%-40% of hPSCs successfully developed into 5-HT-expressing neurons using our protocol, with the majority acquiring a caudal rhombomere identity (r5-8). We further modified our monolayer differentiation system to generate 5-HT neuron-enriched hindbrain-like organoids. We also suggest downstream applications of our 5-HT monolayer and organoid cultures to study neuronal response to gut microbiota. Our methodology could become a powerful tool for future studies related to 5-HT neurotransmission.