�����ƽ��

Embryonic stem cells (ESC) are pluripotent, with the potential to differentiate into multiple cell types, making them a valuable tool for regenerative medicine and disease therapy. However, common culture methods face challenges, including strict operating procedures and high costs. Currently, Leukemia inhibitory factor (LIF), an indispensable bioactive protein for ESC culture, is typically applied to maintain self-renewal and pluripotency, but its instability and high cost limit its effectiveness in stable culture conditions. Hence, we have developed an innovative strategy using a soluble nanomaterial, metal-organic polyhedra (MOPs), to effectively maintain the self-renewal and pluripotency of ESC. The selected amino-modified vanadium-based MOP not only exhibits excellent biocompatibility and high stability but also possesses similar or even superior biological functions compared to commercial LIF. Due to the precise structure of MOPs, the active site responsible for maintaining ESC pluripotency has been identified and regulated at the molecular level. The new ESC culture method significantly reduces costs, simplifies preparation, and enhances the practicality of biopharmaceutical preparation and storage. This represents the first case of using MOPs to maintain self-renewal of ECS, opening an avenue for introducing advanced materials into the development of innovative ESC culture methods. Subject terms: Biomaterials - cells, Chemical biology

Biological mechanisms are inherently dynamic, requiring precise and rapid manipulations for effective characterization. Traditional genetic manipulations operate on long timescales, making them unsuitable for studying dynamic processes or characterizing essential genes, where chronic depletion can cause cell death. We compare five inducible protein degradation systems—dTAG, HaloPROTAC, IKZF3, and two auxin-inducible degrons (AID) using OsTIR1 and AtFB2—evaluating degradation efficiency, basal degradation, target recovery after ligand washout, and ligand impact. This analysis identifies OsTIR1-based AID 2.0 as the most robust system. However, AID 2.0’s higher degradation efficiency comes with target-specific basal degradation and slower recovery rates. To address these limitations, we employ base-editing-mediated mutagenesis followed by several rounds of functional selection and screening. This directed protein evolution generates several gain-of-function OsTIR1 variants, including S210A, that significantly enhance the overall degron efficiency. The resulting degron system, named AID 2.1, maintains effective target protein depletion with minimal basal degradation and faster recovery after ligand washout, enabling characterization and rescue experiments for essential genes. Our comparative assessment and directed evolution approach provide a reference dataset and improved degron technology for studying gene functions in dynamic biological contexts. Subject terms: Genetic engineering, CRISPR-Cas9 genome editing, Peptides

To produce pluripotent stem cells from peripheral blood mononuclear cells (PBMCs) of a patient with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and culture and differentiate them into vascular organoids, producing a disease model for cerebral small vessel disease (CSVD). (1) PMBCs from patients clinically diagnosed with CADASIL ( NOTCH3 p.R141C) were induced to differentiate into pluripotent stem cells (iPSCs); the quality and differentiation ability of the iPSCs were determined. (2) CADASIL-derived iPSCs and control iPSCs were cultured and differentiated into vascular organoids. The differences in the morphological structure of the two differentiated groups of vascular organoids were observed, and both were identified. (1) No mycoplasma infections were detected in the iPSCs prepared from the PBMCs of patients with CADASIL. The short tandem repeat (STR) identification verified that the iPSCs originated from the patient, and the karyotype was normal. Flow cytometry and immunofluorescence detection revealed that the iPSCs expressed SSEA4, OCT4, and NANOG stem proteins. Tri-germ differentiation testing confirmed that the iPSCs expressed the endoderm markers SOX17 and FOXA2, the mesoderm markers Brachyury and α-SMA, and the ectoderm markers Pax6 and β-III Tubulin. (2) CADASIL-derived iPSCs and control iPSCs were induced to differentiate and produce endothelial networks and vascular networks, ultimately forming vascular organoids. Compared with control vascular organoids, CADASIL vascular organoids exhibited lower growth density, earlier blood vessel sprouting, longer and thinner vascular filaments, and smaller final vascular organoids. The vascular organoids from the two sources expressed the endothelial cell marker CD31, the vascular smooth muscle marker α-SMA, and the pericyte marker PDGFR-β. Reprogramming technology can be used to induce PBMCs to become iPSCs, and a CSVD disease model can be successfully constructed by culturing and differentiating the iPSCs into CADASIL vascular organoids. The NOTCH3 p.R141C mutation suppresses the vascular differentiation process in CADASIL.