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Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes. Here, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes.

PU.1 ( SPI1 ) is pivotal in hematopoiesis, yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Comparing human in vivo and in vitro EHT transcriptomes revealed SPI1 ’s regulatory role. Knocking down SPI1 during in vitro EHT led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis, we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells, respectively - as downstream targets of SPI1 . Overexpressing KLF1 or LYL1 partially rescues the SPI1 knockdown-induced reduction in HPC formation. Specifically, KLF1 overexpression restores myeloid lineage potential, while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1 - LYL1 axis in the regulatory network in in vivo EHT. Taken together, our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT, potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs). Subject areas: Biological sciences, Molecular biology, Molecular interaction, Cell biology;

Most studies used animal serum-containing medium for bioengineered-root regeneration, but ethical and safety issues raised by animal serum are a potentially significant risk for clinical use. Thus, this study aimed to find a safer method for bioengineered-root regeneration. The biological properties of human dental pulp stem cells (hDPSCs) cultured in animal component-free (ACF) medium or serum-containing medium (5%, 10% serum-containing medium, SCM) were compared in vitro . hDPSCs were cultured in a three-dimensional (3D) environment with human-treated dentin matrix (hTDM). The capacity for odontogenesis was compared using quantitative real-time PCR (qPCR) and Western blot. Subsequently, the hDPSCs/hTDM complexes were transplanted into nude mice subcutaneously. Histological staining was then used to verify the regeneration effect in vivo . ACF medium promoted the migration of hDPSCs, but slightly inhibited the proliferation of hDPSCs in the first three days of culture compared to SCM. However, it had no significant effect on cell aging and apoptosis. After 7 days of 3D culture in ACF medium with hTDM, qPCR showed that DMP1, DSPP, OCN, RUNX2 , and β-tubulin III were highly expressed in hDPSCs. In addition, 3D cultured hDPSCs/hTDM complexes in ACF medium regenerated dentin, pulp, and periodontal ligament-like tissues similar to SCM groups in vivo . ACF medium was proved to be an alternative medium for bioengineered-root regeneration. The strategy of using ACF medium to regenerate bioengineered-root can improve clinical safety for tooth tissue engineering.

Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling. In this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay. We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1 fl/fl Flt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1 -KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo. Together, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance. The online version contains supplementary material available at 10.1186/s13287-024-03836-8.

Delivering macromolecules across biological barriers such as the blood–brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii , a parasite that naturally travels from the human gut to the central nervous system (CNS), can deliver proteins to host cells. Here we engineered T. gondii ’s endogenous secretion systems, the rhoptries and dense granules, to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells, brain organoids and in vivo, and probe protein activity using imaging, pull-down assays, scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept, we demonstrate GRA16-mediated brain delivery of the MeCP2 protein, a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system, we aim to guide future improvements that will be required for broader application. Subject terms: Parasitology, Biologics, Synthetic biology

Intestinal epithelial cells line the luminal surface to establish the intestinal barrier, where the cells play essential roles in the digestion of food, absorption of nutrients and water, protection from microbial infections, and maintaining symbiotic interactions with the commensal microbial populations. Maintaining and coordinating all these functions requires tight regulatory signaling, which is essential for intestinal homeostasis and organismal health. Dysfunction of intestinal epithelial cells, indeed, is linked to gastrointestinal disorders such as irritable bowel syndrome, inflammatory bowel disease, and gluten-related enteropathies. Emerging evidence suggests that peroxisome metabolic functions are crucial in maintaining intestinal epithelial cell functions and intestinal epithelium regeneration and, therefore, homeostasis. Here, we investigated the molecular mechanisms by which peroxisome metabolism impacts enteric health using the fruit fly Drosophila melanogaster and murine model organisms and clinical samples. We show that peroxisomes control cellular cholesterol, which in turn regulates the conserved yes-associated protein-signaling and contributes to intestinal epithelial structure and epithelial barrier function. Moreover, analysis of intestinal organoid cultures derived from biopsies of patients affected by Crohn’s Disease revealed that the dysregulation of peroxisome number, excessive cellular cholesterol, and inhibition of Yap-signaling are markers of disease and could be novel diagnostic and/or therapeutic targets for treating Crohn’s Disease. Our studies provided mechanistic insights on peroxisomal signaling in intestinal epithelial cell functions and identified cholesterol as a novel metabolic regulator of yes-associated protein-signaling in tissue homeostasis. Subject terms: Cell biology, Medical research

Previous studies have shown that aggregated alpha-synuclein (α-s) protein, a key pathological marker of Parkinson’s disease (PD), can propagate between cells, thus participating in disease progression. This prion-like propagation has been widely studied using in vivo and in vitro models, including rodent and human cell cultures. In this study, our focus was on temporal assessment of functional changes during α-s aggregation and propagation in human induced pluripotent stem cell (hiPSC)-derived neuronal cultures and in engineered networks. Here, we report an engineered circular tripartite human neuronal network model in a microfluidic chip integrated with microelectrode arrays (MEAs) as a platform to study functional markers during α-s aggregation and propagation. We observed progressive aggregation of α-s in conventional neuronal cultures and in the exposed (proximal) compartments of circular tripartite networks following exposure to preformed α-s fibrils (PFF). Furthermore, aggregated forms propagated to distal compartments of the circular tripartite networks through axonal transport. We observed impacts of α-s aggregation on both the structure and function of neuronal cells, such as in presynaptic proteins, mitochondrial motility, calcium oscillations and neuronal activity. The model enabled an assessment of the early, middle, and late phases of α-s aggregation and its propagation during a 13-day follow-up period. While our temporal analysis suggested a complex interplay of structural and functional changes during the in vitro propagation of α-s aggregates, further investigation is required to elucidate the underlying mechanisms. Taken together, this study demonstrates the technical potential of our introduced model for conducting in-depth analyses for revealing such mechanisms. Subject terms: Parkinson's disease, Neurological models

Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of cancer chemotherapy that can often limit treatment options for cancer patients or have life-long neurodegenerative consequences that reduce the patient’s quality of life. CIPN is caused by the detrimental actions of various chemotherapeutic agents on peripheral axons. Currently, there are no approved preventative measures or treatment options for CIPN, highlighting the need for the discovery of novel therapeutics and improving our understanding of disease mechanisms. In this study, we utilized human-induced pluripotent stem cell (hiPSC)-derived motor neurons as a platform to mimic axonal damage after treatment with vincristine, a chemotherapeutic used for the treatment of breast cancers, osteosarcomas, and leukemia. We screened a total of 1902 small molecules for neuroprotective properties in rescuing vincristine-induced axon growth deficits. From our primary screen, we identified 38 hit compounds that were subjected to secondary dose response screens. Six compounds showed favorable pharmacological profiles – AZD7762, A-674563, Blebbistatin, Glesatinib, KW-2449, and Pelitinib, all novel neuroprotectants against vincristine toxicity to neurons. In addition, four of these six compounds also showed efficacy against vincristine-induced growth arrest in human iPSC-derived sensory neurons. In this study, we utilized high-throughput screening of a large library of compounds in a therapeutically relevant assay. We identified several novel compounds that are efficacious in protecting different neuronal subtypes from the toxicity induced by a common chemotherapeutic agent, vincristine which could have therapeutic potential in the clinic. The online version contains supplementary material available at 10.1007/s00018-024-05340-x.