scRNA-Seq of Cultured Human Amniotic Fluid from Fetuses with Spina Bifida Reveals the Origin and Heterogeneity of the Cellular Content

Amniotic fluid has been proposed as an easily available source of cells for numerous applications in regenerative medicine and tissue engineering. The use of amniotic fluid cells in biomedical applications necessitates their unequivocal characterization; however, the exact cellular composition of amniotic fluid and the precise tissue origins of these cells remain largely unclear. Using cells cultured from the human amniotic fluid of fetuses with spina bifida aperta and of a healthy fetus, we performed single-cell RNA sequencing to characterize the tissue origin and marker expression of cultured amniotic fluid cells at the single-cell level. Our analysis revealed nine different cell types of stromal, epithelial and immune cell phenotypes, and from various fetal tissue origins, demonstrating the heterogeneity of the cultured amniotic fluid cell population at a single-cell resolution. It also identified cell types of neural origin in amniotic fluid from fetuses with spina bifida aperta. Our data provide a comprehensive list of markers for the characterization of the various progenitor and terminally differentiated cell types in cultured amniotic fluid. This study highlights the relevance of single-cell analysis approaches for the characterization of amniotic fluid cells in order to harness their full potential in biomedical research and clinical applications

Intrinsic TGF-β signaling attenuates proximal tubule mitochondrial injury and inflammation in chronic kidney disease

Excessive TGF-β signaling and mitochondrial dysfunction fuel chronic kidney disease (CKD) progression. However, inhibiting TGF-β failed to impede CKD in humans. The proximal tubule (PT), the most vulnerable renal segment, is packed with giant mitochondria and injured PT is pivotal in CKD progression. How TGF-β signaling affects PT mitochondria in CKD remained unknown. Here, we combine spatial transcriptomics and bulk RNAseq with biochemical analyses to depict the role of TGF-β signaling on PT mitochondrial homeostasis and tubulo-interstitial interactions in CKD. Male mice carrying specific deletion of Tgfbr2 in the PT have increased mitochondrial injury and exacerbated Th1 immune response in the aristolochic acid model of CKD, partly, through impaired complex I expression and mitochondrial quality control associated with a metabolic rewiring toward aerobic glycolysis in the PT cells. Injured S3T2 PT cells are identified as the main mediators of the maladaptive macrophage/dendritic cell activation in the absence of Tgfbr2. snRNAseq database analyses confirm decreased TGF-β receptors and a metabolic deregulation in the PT of CKD patients. This study describes the role of TGF-β signaling in PT mitochondrial homeostasis and inflammation in CKD, suggesting potential therapeutic targets that might be used to mitigate CKD progression

Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)], and compared them to neurons from an epileptic encephalopathy patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. By contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency, and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations

Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)], and compared them to neurons from an epileptic encephalopathy patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. By contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency, and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations

scRNA-Seq of Cultured Human Amniotic Fluid from Fetuses with Spina Bifida Reveals the Origin and Heterogeneity of the Cellular Content

Amniotic fluid has been proposed as an easily available source of cells for numerous applications in regenerative medicine and tissue engineering. The use of amniotic fluid cells in biomedical applications necessitates their unequivocal characterization; however, the exact cellular composition of amniotic fluid and the precise tissue origins of these cells remain largely unclear. Using cells cultured from the human amniotic fluid of fetuses with spina bifida aperta and of a healthy fetus, we performed single-cell RNA sequencing to characterize the tissue origin and marker expression of cultured amniotic fluid cells at the single-cell level. Our analysis revealed nine different cell types of stromal, epithelial and immune cell phenotypes, and from various fetal tissue origins, demonstrating the heterogeneity of the cultured amniotic fluid cell population at a single-cell resolution. It also identified cell types of neural origin in amniotic fluid from fetuses with spina bifida aperta. Our data provide a comprehensive list of markers for the characterization of the various progenitor and terminally differentiated cell types in cultured amniotic fluid. This study highlights the relevance of single-cell analysis approaches for the characterization of amniotic fluid cells in order to harness their full potential in biomedical research and clinical applications

Intrinsic TGF-β signaling attenuates proximal tubule mitochondrial injury and inflammation in chronic kidney disease

Excessive TGF-β signaling and mitochondrial dysfunction fuel chronic kidney disease (CKD) progression. However, inhibiting TGF-β failed to impede CKD in humans. The proximal tubule (PT), the most vulnerable renal segment, is packed with giant mitochondria and injured PT is pivotal in CKD progression. How TGF-β signaling affects PT mitochondria in CKD remained unknown. Here, we combine spatial transcriptomics and bulk RNAseq with biochemical analyses to depict the role of TGF-β signaling on PT mitochondrial homeostasis and tubulo-interstitial interactions in CKD. Male mice carrying specific deletion of Tgfbr2 in the PT have increased mitochondrial injury and exacerbated Th1 immune response in the aristolochic acid model of CKD, partly, through impaired complex I expression and mitochondrial quality control associated with a metabolic rewiring toward aerobic glycolysis in the PT cells. Injured S3T2 PT cells are identified as the main mediators of the maladaptive macrophage/dendritic cell activation in the absence of Tgfbr2. snRNAseq database analyses confirm decreased TGF-β receptors and a metabolic deregulation in the PT of CKD patients. This study describes the role of TGF-β signaling in PT mitochondrial homeostasis and inflammation in CKD, suggesting potential therapeutic targets that might be used to mitigate CKD progression.ISSN:2041-172

Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel Na(V)1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that Na(V)1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower Na(V)1.2 protein levels. In neurons with the frameshift variant, Na(V)1.2 mRNA and protein levels were reduced by similar to 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying Na(V)1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced Na(V)1.2 levels. In contrast, epilepsy neurons displayed no change in Na(V)1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced Na(V)1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to Na(V)1.2 dysfunction and can guide further investigations.ISSN:0964-6906ISSN:1460-208