�����ƽ��

Neuronal differentiation requires extensive metabolic remodeling to support increased energetic and biosynthetic demands. Here, we present an integrated multi-omics and functional characterization of metabolic transitions during early differentiation of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons using doxycycline-inducible overexpression of neurogenin-2 (NGN2). We analyzed parental iPSCs and induced neurons (iNs) at days 7 and 14 of differentiation, integrating gene expression profiling, label-free quantitative proteomics, high-resolution respirometry, fluorescence lifetime imaging microscopy (FLIM), and 13C₆-glucose metabolic flux analysis. Our data reveal progressive metabolic remodeling associated with neuronal maturation, including enhanced oxidative phosphorylation, increased mitochondrial content, and respiratory capacity. Proteomic analyses showed upregulation of mitochondrial and antioxidant pathways, while FLIM indicated a progressive increase in enzyme-bound NAD(P)H, consistent with a shift toward oxidative metabolism. Notably, 13C₆-glucose tracing revealed delayed labeling of the intracellular pool of fully labeled glucose and tricarboxylic acid cycle metabolites, together with enhanced labeling of pentose phosphate pathway intermediates and glutathione in iNs, indicating a shift toward biosynthetic and antioxidant glucose utilization during differentiation. Despite this enhancement in mitochondrial function, differentiated neurons maintained glycolytic activity, suggesting metabolic flexibility. Our results define the first week of differentiation as a critical window of metabolic specialization and establish NGN2-iPSC-derived cortical neurons as a versatile and well-characterized model system for investigating bioenergetic remodeling during early human neurodevelopment. It provides a robust foundation for mechanistic insights and high-throughput evaluation of metabolic pathways relevant to human disease.

Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity, their large size often leads to necrotic core formation, limiting reproducibility and challenging the integration of microglia. Here, we present a detailed, reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons, astrocytes, and optionally microglia, all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells, generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling), microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications, including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories, demonstrating its robustness and translational potential for studying neuron–glia interactions and modeling neurodegenerative processes.

The differentiation of pluripotent stem cells into neurons is an essential area of biomedical research, with significant implications for understanding neural development and treating neurological diseases. This study compares neural cultures derived from a common induced pluripotent stem cell line (KYOU-DXR0109B) generated by two widely adopted methods: DUAL SMAD inhibition and exogenous NGN2 overexpression. The DUAL SMAD inhibition method, which differentiates through the neural stem cell stage, produces heterogeneous cultures containing a mix of neurons, neural precursors, and glial cells. Conversely, NGN2 overexpression generates more homogeneous cultures composed predominantly of mature neurons. Transcriptomic analysis revealed significant differences in neural gene markers expression profiles, with cultures from the DUAL SMAD inhibition method enriched in neural stem cell and glial markers, while NGN2 overexpression cultures showed elevated markers for cholinergic and peripheral sensory neurons. This study underscores the importance of choosing appropriate differentiation protocols based on the desired cell types, as each method yields neural cultures with distinct cellular compositions. Understanding these differences can help optimize protocols for specific research and therapeutic applications.

Proteomics of dystrophic muscle samples is limited by the amount of protein that can be extracted from patient biopsies. Cells and tissues derived from patient-derived induced pluripotent stem cells (iPSCs) can be an expandable alternative source. We have patterned iPSCs from three Duchenne muscular dystrophy (DMD) patient lines into skeletal muscle cells using a two-dimensional as well as our three-dimensional organoid differentiation system. Probes with sufficient protein amounts could be extracted and prepared for mass spectrometry. In total, 3007 proteins in 2D and 2709 proteins in 3D were detected in DMD patient probes. A total of 83 proteins in 2D and 338 proteins in 3D can be described as differentially expressed between DMD and control patient probes in a post hoc test. We have identified and we propose Myosin-9, Collagen 18A, Tropomyosin 1, BASP1, RUVBL1, and NCAM1 as proteins specifically altered in their expression in DMD for further investigation. Proteomics of skeletal muscle organoids resulted in greater consistency of results between cell lines in comparison to the two-dimensional myogenic differentiation protocol.

Human microglia are central regulators and actors in brain infections and neuro-inflammatory pathologies. However, access to such cells is limited, and studies systematically mapping the spectrum of their inflammatory states are scarce. Here, we generated microglia-like cells (MGLCs) from human induced pluripotent stem cells and characterized them as a robust, accessible model system for studying inflammatory activation. We validated lineage identity through transcriptome profiling, revealing selective upregulation of microglial signature genes and enrichment of microglia/macrophage-related gene sets. MGLCs displayed distinct morphologies and produced stimulus- and time-dependent cytokine secretion profiles upon exposure to diverse inflammatory stimuli, including pro-inflammatory cytokines (TNFα, interferon-γ) and agonists of the Toll-like receptors TLR2 (FSL-1), TLR3 (Poly(I:C)), TLR4 (lipopolysaccharide, LPS), and TLR7 (imiquimod). Transcriptome profiling and bioinformatics analysis revealed distinct activation signatures. Functional assays demonstrated stimulus-specific engagement of NFκB and JAK-STAT signaling pathways. The shared NFκB nuclear translocation response of TLR ligands and TNFα was reflected in overlapping transcriptome profiles: they shared modules (e.g., oxidative stress response and TNFα-related signaling) identified by weighted gene co-expression network analysis. Finally, the potential consequences of microglia activation for neighboring cells were studied on the example of microglia-astrocyte crosstalk. The capacity of MGLC supernatants to stimulate astrocytes was measured by quantifying astrocytic NFκB translocation. MGLCs stimulated with FSL-1, LPS, or Poly(I:C) indirectly activated astrocytes via a strictly TNFα-dependent mechanism, highlighting the role of soluble mediators in the signal propagation. Altogether, this platform enables a dissection of microglia activation states and multi-parametric characterization of subsequent neuroinflammation.

Hepatocellular carcinoma (HCC) is the most prevalent primary malignancy of the liver. Characterized by rapid progression and poor overall survival rates, HCC requires effective and streamlined treatment regimens. It predominantly occurs in East Asia and sub-Saharan Africa, where it has historically been managed with herbal formulas. We previously observed that the herbal formula HO-1089 exerts potent anti-HCC effects both in vitro and in vivo. In this study, we investigated the anticancer efficacy and mechanisms of HO-1197, a reconstituted herbal formulation derived from HO-1089. HO-1197 selectively inhibited the viability of HCC cell lines without hepatotoxicity and demonstrated superior anticancer activity compared with both HO-1089 and sorafenib. Mechanistically, HO-1197 induced apoptosis and G2/M arrest through reactive oxygen species-mediated DNA damage, independent of p53 status. Transcriptomic analysis revealed downregulation of mitosis-related genes, particularly those regulated by FOXM1, a key driver of HCC proliferation and metastasis. HO-1197 suppressed FOXM1 expression and nuclear translocation, reducing its downstream targets and diminishing angiogenic and metastatic potential. Furthermore, HO-1197 modulated the tumor immune microenvironment by promoting pro-inflammatory macrophage polarization and enhancing natural killer cell-mediated cytotoxicity. HO-1197 exhibited potent antitumor efficacy, and combination therapy with HO-1197 and sorafenib exhibited synergistic effects in both two-dimensional and immune-activated multicellular spheroid models. These findings suggest that HO-1197 is a promising multifunctional therapeutic candidate with antitumor, antiangiogenic, antimetastatic, and immunomodulatory properties. Its combination with sorafenib may offer effective treatment for HCC. HO-1197, which demonstrated strong efficacy, is a novel herbal medicine developed by H&O Biosis and is referred to as an Integrated Natural Medicine.

Osteoporosis (OP) could lead to the alteration of bone marrow microenvironment and non-homeostasis of hematopoiesis, which could increase the incidence of hematologic malignancies. However, whether myeloid-biased hematopoiesis occurred and contributed to the leukemogenesis under the condition of OP remains unclear. Results: This study successfully induced a mouse model for OP by hindlimb unloading, which shows increased myeloid cells and decreased B cells in the peripheral blood (PB). Furthermore, our study demonstrates that the myeloid-biased subset of HSPCs (hematopoietic stem and progenitor cells) with reduced differentiation and apoptosis, including multipotent progenitor 3 (MPP3) and granulocyte-monocyte progenitors (GMPs), were expanded in the OP mice. The expansion of myeloid-biased HSPCs contributes to the accumulation of HSPCs in the bone marrow and increased myeloid cells in the PB of OP mice. In the expanded pool of HSPCs, OP mice specifically enriched subsets were identified and profiled by single cell RNA-seq, including subHSCs from primitive HSCs, MPP3-1 from MPP3, GMP5 from GMPs, MkP2 from megakaryocyte progenitors and EryP1 from erythrocyte progenitors. Meanwhile, those OP-HU mice enriched subsets shared significantly up- and down-regulated genes enriched in chromatin modification and cell differentiation and apoptosis such as Bromodomain-containing protein 4 (Brd4), encoding an important chromatin remodeling protein, and Proteinase 3 (Prtn3). Moreover, the specific transcription factors corresponding to the expansion of subHSCs, MPP3-1, GMP5 and EryP1 in OP-HU mice were identified as Zfp951, Nfic, Maz and Ezh2. Finally, inhibition of BRD4 in vivo could partially restore the phenotype of OP-HU mice and the expression of genes regulating HSPC expansion, differentiation and apoptosis. Conclusions: First of all, our study shows that OP could induce the unbalanced hematopoiesis and enhances the myeloid-biased hematopoiesis. Secondly, OP mice enriched subsets of HSPCs were identified and characterized with enhanced chromatin remodeling, reduced differentiation and resistance to apoptosis. Finally, this study demonstrate that Brd4 regulated gene programs endow the myeloid-biased subsets of HSPCs with tumor cell-like characters in OP mice, which may increase the incidence of the leukemic evolution. This study sheds light on the importance for the prevention of myeloid leukemogenesis in human with OP.

Effective therapies for Alzheimer’s disease (AD) remain to be developed. APOE4 is the strongest genetic risk factor for late-onset AD. Pericyte degeneration and blood–brain barrier (BBB) disruption are thought to be early biomarkers of AD and contribute to cognitive decline in APOE4 carriers, representing potential therapeutic targets. Our previous studies have shown that pericyte transplantation is one of the most effective strategies for BBB restoration, exhibiting great therapeutic potential for APOE4-related BBB damage and AD phenotypes. Methods: APOE4/4 mice were treated with pericytes derived from APOE3/3 human induced pluripotent stem cells (hiPSCs). Behavioral tests, AD pathologies, and BBB integrity were assessed. Subsequently, temporal and spatial distribution of the transplanted pericytes was analyzed using tdTomato+ lentivirus labeling. Next, therapeutic effects of apoptotic vesicles (ApoVs) generated from APOE3/3 pericytes were evaluated in APOE4/4 pericytes in vitro. Additionally, transcriptomic and proteomic profiling were performed to identify key effector molecules in pericyte-derived ApoVs. Finally, the therapeutic effects of ApoVs derived from pericytes were evaluated in APOE4/4 mice. Results: Early, multiple transplantations of pericytes derived from APOE3/3 hiPSCs robustly rescued cognitive decline and AD pathologies, restored BBB integrity, and prevented in situ pericyte degeneration in aged APOE4/4 mice. Intriguingly, ApoVs released from the infused cells, rather than the transplanted pericytes, were predominantly distributed in the brain, which were ingested by in situ APOE4/4 pericytes and then promoted functional recovery. We further characterized insulin growth factor-2 (IGF-2) as a key factor in APOE3/3 pericyte-derived ApoVs. Infusion of the in vitro generated ApoVs from APOE3/3 pericytes demonstrated distinct therapeutic effects in APOE4/4 mice, which were reversed by IGF2 knockout. Conclusions: APOE3/3 pericytes or APOE3/3 pericyte-derived IGF2-rich ApoVs may offer promising therapeutic strategies for APOE4-associated AD.

Interleukin-1 receptor accessory protein (IL1RAP) is selectively expressed on both bulk blasts and leukemic stem cells (LSCs) in acute myeloid leukemia (AML), while its expression is virtually absent on normal hematopoietic stem cells (HSCs), making it an appealing target for chimeric antigen receptor (CAR) T cell therapy. Methods: We developed a novel IL1RAP-targeting CAR-T cells using a single-chain Fab (24scFab) fused to CD28 and CD3ζ costimulatory domains. CAR-T cells with a mutated IL1RAP-binding paratope were also generated as a control by introducing two point-mutations in the complementarity determining region (CDR) loops of the 24scFab domain. We tested the CAR-T cells in cell line-derived (CD) and patient-derived (PD) xenografts (X). To address persistence and activity of IL1RAP CAR-T cells, we then tested two approaches. First, we mutated two of the three immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3ζ domain (i.e., IL1RAP-1XX CAR-T). Second, we co-administered a synthetic miR-142 mimic (M-miR-142), previously shown to enhance T cell antileukemic activity, with IL1RAP CAR-T cells to AML xenografted mice. Results: IL1RAP CAR-T cells demonstrated a potent antileukemic activity in both AML CDX and PDX models. Target specificity was confirmed by the complete loss of function of IL1RAP-mutated CAR-T cells. IL1RAP-1XX CAR-T cells improved T cell persistence in vitro but failed to demonstrate therapeutic benefit compared with IL1RAP CAR-T cells in vivo. We previously reported that leukemic cell growth suppresses miR-142 biogenesis, thereby hindering the metabolic switch and impairing host T cell antileukemic activity; this was rescued by administration of M-miR-142. Thus, we hypothesized a similar impact of leukemic cells on CAR-T and that M-miR-142 treatment could rescue it and enhance the IL1RAP CAR-T cell antileukemic activity. We showed that both CDXs and PDXs receiving M-miR-142 and IL1RAP CAR-T lived significantly longer than those receiving scrambled oligonucleotide and IL1RAP CAR-T or mutated CAR-T controls (median survival of PDX: 78 vs 51 vs 24 days). Conclusions: We have identified a potentially novel strategy to enhance CAR-T cell persistence and efficacy in AML by counteracting a leukemia-induced, microRNA-deficiency mediated mechanism of immune suppression.

Tumor patients often exhibit limited responses to immunotherapy owing to the low immunogenicity and immunosuppressive environment of tumors. Our previous studies revealed that cryo-thermal therapy caused tumor cell rupture due to mechanical compression, notably causing the release of a substantial amount of DAMPs (danger-associated molecular patterns), such as heat shock protein 70, calreticulin and high-mobility group box protein 1; the release of these DAMPs increased myeloid cell maturation, thereby reshaping the systemic immune environment and ultimately inducing durable CD4+ T helper type 1 (Th1) cell-dominated antitumor immunity. In fact, under conditions of mechanical stress and rapid temperature changes, the disruption of tumor cells caused by cryo-thermal therapy results in extensive deoxyribonucleic acid (DNA) damage and the rapid release of substantial amounts of DNA. Consequently, tumor-derived DNA, which potently activates innate immunity by engaging multiple DNA sensors, plays a pivotal role in orchestrating antitumor immunity. We hypothesized that cryo-thermal therapy induces the transient release of high levels of DNA, which modulates CD11b+ myeloid cell function, subsequently influencing CD4+ Th1-cell dominated antitumor immune responses. In this study, a B16F10 melanoma model was established, and DNA concentrations were measured at different time points after cryo-thermal therapy. Deoxyribonuclease I (DNase I) was subsequently administered immediately following cryo-thermal therapy to deplete extracellular DNA, allowing an investigation of the role of DNA in regulating CD11b+ myeloid cell function and CD4+ T cell differentiation. The phenotype and function of CD11b+ myeloid cells and CD4+ T cells were assessed by flow cytometry, RNA sequencing, and cell culture in vitro. Our studies confirmed that cryo-thermal therapy triggered a transient release of high levels of DNA, which was internalized by CD11b+ myeloid cells via C-type lectin receptors and subsequently sensed by inflammasomes. Then, the intracellular sensing of DNA induced the production of the mature form of interleukin (IL)-18, ultimately promoting the Th1 differentiation of CD4+ T cells. This study highlights the pivotal role of DNA release after cryo-thermal therapy in driving CD4+ Th1 cell-dominant antitumor immunity.

The efficacy of immunotherapy is often hindered by the suppression of immune responses via the tumor microenvironment (TME). The presence of cancer cells forces other proximal non-cancerous cells to support tumor growth and persistence. A clear example of this cancerous-to-non-cancerous communication is represented by the accumulation of myeloid-derived suppressor cells (MDSCs) within the TME. Several studies have convergently shown that the overexpression of DNA-methyl-transferase-1 (DNMT1) in these cells results in protection from necroptosis and enhanced accumulation in vivo. Conversely, targeting DNMT1 through hypo-methylating agents has shown promising therapeutic potential by not only reducing the levels of MDSCs but also enhancing cancer immunogenicity and the efficacy of immune checkpoint inhibitors (ICI). Methods: Murine 4T1 (triple-negative breast cancer (TNBC)) and CT26 (colon carcinoma) cell lines were cultured under standard conditions and used to generate tumor models in BALB/c mice. An oncolytic adenovirus expressing a DNMT1-targeting short hairpin RNA (OAd.shDNMT1) was engineered and validated for DNMT1 knockdown and genome-wide methylation reduction. Small extracellular vesicles (sEVs) were isolated from virus-infected cancer cells and characterized for RNA content and uptake by MDSCs. MDSC differentiation and suppressive function were assessed in vitro using flow cytometry and co-culture assays with murine splenocytes. In vivo, tumor-bearing mice received intratumoral OAd.shDNMT1, systemic decitabine, or immune checkpoint inhibitors (anti-Programmed cell Death protein-1), and tumor growth, immune infiltration, and systemic MDSC levels were evaluated. Results: In this study, we report that, by using virally infected TNBC murine cells as a source for shDNMT1-loaded sEVs, OAd.shDNMT1 successfully reduced MDSC levels in vitro and in vivo. Furthermore, the co-administration with ICI resulted in a significant tumor growth reduction in mice bearing poorly immunogenic TNBC 4T1 cells. Also, our treatment promoted antitumor immunity, prolonged survival, and complete tumor eradication in modestly immunogenic colon CT26 cancer cells. Conclusions: This multifaceted strategy, based on OV-mediated immune stimulation and reduction of MDSC levels via sEVs, may improve clinical outcomes and the success of immuno-based regimens for patients facing MDSC-rich and highly aggressive cancer subtypes.