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In mammals, early embryonic gastrulation process is high energy demanding. Previous studies showed that, unlike endoderm and mesoderm cells, neuroectoderm differentiated from human embryonic stem cells relied on aerobic glycolysis as the major energy metabolic process, which generates lactate as the final product. Here we explored the function of intracellular lactate during neuroectoderm differentiation. Our results revealed that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Changing intracellular lactate levels by sodium lactate or LDHA inhibitors had no obvious effect on BMP or WNT/?-catenin signaling during neuroectoderm differentiation. Notably, histone lactylation, especially H3K18 lactylation was significant upregulated during this process. We further performed CUT&Tag experiments and the results showed that H3K18la is highly enriched at gene promoter regions. By analyzing data from CUT&Tag and RNA-seq experiments, we further identified that four genes, including PAX6, were transcriptionally upregulated by lactate during neuroectoderm differentiation. A H3K18la modification site at PAX6 promoter was verified and exogenous lactate could also rescue the level of PAX6 after shPAX6 inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05510-x.

Pancreatic islets are densely packed cellular aggregates containing various hormonal cell types essential for blood glucose regulation. Interactions among these cells markedly affect the glucoregulatory functions of islets along with the surrounding niche and pancreatic tissue-specific geometrical organization. However, stem cell (SC)-derived islets generated in vitro often lack the three-dimensional extracellular microenvironment and peri-vasculature, which leads to the immaturity of SC-derived islets, reducing their ability to detect glucose fluctuations and insulin release. Here, we bioengineer the in vivo-like pancreatic niches by optimizing the combination of pancreatic tissue-specific extracellular matrix and basement membrane proteins and utilizing bioprinting-based geometrical guidance to recreate the spatial pattern of islet peripheries. The bioprinted islet-specific niche promotes coordinated interactions between islets and vasculature, supporting structural and functional features resembling native islets. Our strategy not only improves SC-derived islet functionality but also offers significant potential for advancing research on islet development, maturation, and diabetic disease modeling, with future implications for translational applications. The glucoregulatory functions of pancreatic islets are affected by their surrounding niche and spatial organization. Here, bioengineered stem-cell derived islet niches use bioprinting-based geometrical guidance to promote islet maturation for improved functionality and diabetes research.

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by repeat expansion of the CAG trinucleotide within exon 10 of the ATXN3 gene. This mutation results in the production of an abnormal ataxin-3 protein containing an extended polyglutamine tract, referred to as mutant ataxin-3. In this study, we investigated the therapeutic potential of CRISPR/Cas9-mediated genome editing for SCA3. First, we designed a specific single-guide RNA targeting the ATXN3 gene and constructed the corresponding targeting vector. Induced pluripotent stem cells (iPSCs) derived from a SCA3 patient were then electroporated with the CRISPR/Cas9 components. Positive clones were screened and validated by PCR and Sanger sequencing to obtain genome-editing iPSCs (GE-iPSCs). Subsequently, the pluripotency of GE-iPSCs was confirmed, and the effects of genome editing on mutant ataxin-3 protein expression and Golgi apparatus morphology were assessed using Western blotting and immunofluorescence analyses. Our results demonstrated that targeted insertion of polyadenylation signals (PAS) upstream of the abnormal CAG repeats effectively suppressed the production of mutant ataxin-3. This intervention also reduced the formation of neuronal nuclear inclusions in differentiated neurons, restored the structural integrity of the Golgi apparatus (which exhibited a loose and enlarged morphology in SCA3 cells), and increased the expression levels of Golgi structural proteins (GM130 and GORASP2). In conclusion, our findings indicate that the targeted insertion of PAS upstream of the abnormal CAG repeats in the ATXN3 gene represents a promising therapeutic strategy for SCA3 through genome editing.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-93369-8.