Neurogenin 3 (NGN3) is a key transcription factor in the cell fate determination of endocrine progenitors (EPs) in the developing pancreas. Previous studies have shown that the stability and activity of NGN3 are regulated by phosphorylation. However, the role of NGN3 methylation is poorly understood. Here, we report that protein arginine methyltransferase-1 (PRMT1)-mediated arginine 65 methylation of NGN3 is required for the pancreatic endocrine development of human embryonic stem cells (hESCs) in vitro. We found that inducible PRMT1-knockout (P-iKO) hESCs did not differentiate from EPs into endocrine cells (ECs) in the presence of doxycycline. Loss of PRMT1 caused NGN3 accumulation in the cytoplasm of EPs and decreased the transcriptional activity of NGN3. We found that PRMT1 specifically methylates NGN3 arginine 65 and that this modification is a prerequisite for ubiquitin-mediated degradation. Our findings demonstrate that arginine 65 methylation of NGN3 is a key molecular switch in hESCs permitting their differentiation into pancreatic ECs.

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Noonan syndrome (NS) is a genetic disorder mainly caused by gain-of-function mutations in Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2). Although diverse neurological manifestations are commonly diagnosed in NS patients, the mechanisms as to how SHP2 mutations induce the neurodevelopmental defects associated with NS remain elusive. Here, we report that cortical organoids (NS-COs) derived from NS-induced pluripotent stem cells (iPSCs) exhibit developmental abnormalities, especially in excitatory neurons (ENs). Although NS-COs develop normally in their appearance, single-cell transcriptomic analysis revealed an increase in the EN population and overexpression of cortical layer markers in NS-COs. Surprisingly, the EN subpopulation co-expressing the upper layer marker SATB2 and the deep layer maker CTIP2 was enriched in NS-COs during cortical development. In parallel with the developmental disruptions, NS-COs also exhibited reduced synaptic connectivity. Collectively, our findings suggest that perturbed cortical layer identity and impeded neuronal connectivity contribute to the neurological manifestations of NS.

Previously we observed that human adipose-derived stem cells (hADSCs) could form aggregation during culture in the presence of human serum (HS). In the present study, we have examined if the aggregation might result from the cell migration and analyzed the difference of cell adhesivity after culture in various conditions. When cells were cultured in fetal bovine serum (FBS) alone, there was no morphological change. Similarly, cells pretreated with FBS for 1 day or cultured in a mixture of FBS and HS showed little change. In contrast, cells cultured in HS alone exhibited formation of cell-free area (spacing) and/or cell aggregation. When cells cultured in FBS or pretreated with FBS were treated with 0.06% trypsin, almost cells remained attached to the dish surfaces. In contrast, when cells cultured in HS alone were examined, most cells detached from the dish by the same treatment. Treatment of cells with forskolin, isobutylmethyl xanthine (IBMX) or LY294002 inhibited the formation of spacing whereas H89 or Y27632 showed little effect. When these cells were treated with 0.06% trypsin after culture, most cells detached from the dishes as cells cultured in HS alone did. However, cells treated with IBMX exhibited weaker adhesivity than HS alone. Based on these observations, it is suggested that HS treatment might decrease the adhesivity and induce three-dimensional migration of hADSCs, in the latter of which cAMP signaling could be involved.

In the developing pancreas, Neurogenin 3 (NGN3) is a key transcription factor in the cell fate determination of endocrine progenitors (EPs). Although the activation and stability of NGN3 are regulated by phosphorylation, the role of arginine methylation of NGN3 is poorly understood. Here, we report arginine 65 methylation of NGN3 is absolutely required for the pancreatic lineage development of human embryonic stem cells (hESCs) in vitro. First, we found inducible protein arginine methyltransferase 1 (PRMT1)-knockout (P-iKO) hESCs did not differentiate from EPs to endocrine cells (ECs) in the presence of doxycycline. Loss of PRMT1 caused an accumulation of NGN3 in the cytoplasm of EPs and blocked NGN3’s transcriptional activity in PRMT1-KO EPs. We also found that PRMT1 specifically methylates NGN3 arginine 65, and this modification is a prerequisite for ubiquitin-mediated NGN3 degradation. Our findings indicate arginine 65 methylation of NGN3 is a key molecular switch in hESCs in vitro permitting the differentiation into pancreatic endocrine lineages.