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Chronic compressive cervical spinal cord injury (cCSCI),a debilitating condition,lacks effective treatment options. Addressing this gap,our study introduces a novel rat model of cCSCI developed through spinal cord compression via synthetic polyether sheet implantation,closely mimicking human pathology. We evaluated the model’s fidelity utilizing a comprehensive series of behavioral,electrophysiological,and histological assessments. Our research also explored the therapeutic potential of oligodendrogenic neural progenitor cells (oNPCs) derived from induced pluripotent stem cells. Transplanted oNPCs successfully integrated into the host spinal cord,differentiated into neurons,astrocytes,and oligodendrocytes,and demonstrated a remarkable capacity for enhancing neuroplasticity. Electrophysiological analyses revealed significant improvements in motor evoked potentials and a rectification of the excitability imbalance posttransplantation,indicating substantial recovery of motor circuits. Histological findings complemented these results,showing enhanced remyelination and a reduction in excitatory transmitter expression in the residual gray matter. Functionally,the transplantation of oNPCs led to marked improvements in grip strength,locomotor abilities,and sensory functions,surpassing those seen with standard treatments. This study not only provides a novel and reliable rat model of cCSCI for further research but also highlights the potential of oNPCs as a transformative approach for spinal cord injury therapy,suggesting their significant role in neural regeneration and repair. View Publication

Management of chronic kidney disease (CKD) remains a major challenge due limited therapeutic options to reverse fibrosis,which is a critical feature in CKD. Partial epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs) is a key driver of fibrosis,and has become an important focus for kidney protection strategies. Blood platelets,a major source of circulating transforming growth factor beta (TGF-β),are implicated in pathogenesis of CKD,but their involvement in EMT and kidney fibrosis remains uncertain. Methods: We used two mouse models of renal fibrosis—diabetic kidney disease (DKD) and unilateral ureter obstruction (UUO)—to examine the connection between platelets,partial EMT,and fibrosis. Platelet inhibition or depletion was performed to assess EMT,cell cycle arrest,and fibrosis. In vitro,platelets were applied to TECs and kidney organoids. To determine the role of TGF-β signaling,we used TGF-βRI inhibitor. Expression of EMT,and fibrosis markers,as well as TGF-β1 signaling,were analyzed using western blot,reverse transcription quantitative PCR (RT-qPCR),enzyme-linked immunosorbent assay (ELISA),and immunostaining. Results: In both animal models,platelet inhibition or depletion resulted in reduced expression of cell cycle arrest marker p21,partial EMT and fibrosis. In vitro,activated platelets stimulated cell cycle arrest,EMT,and fibrosis in TECs and kidney organoids. Chronically injured TECs experience cell-cycle arrest which promote a paracrine EMT program in TECs,jointly leading to fibrosis. This platelet-mediated effect on cell cycle arrest and EMT was driven by TGF-β1 signaling,as selective inhibition of the TGF-β receptor rescued these dysfunctional phenotypes. Conclusions: Our study demonstrates that platelets activate the TGF-β1 pathway,leading to cell cycle arrest,EMT and renal fibrosis. These findings suggest that antiplatelet therapies may have potential renoprotective effects by protecting tubular homeostasis,attenuating partial EMT and fibrosis. View Publication

Bipolar disorder (BD) is a complex psychiatric condition usually requiring long-term treatment. Lithium (Li) remains the most effective mood stabilizer for BD,yet it benefits only a subset of patients,and its precise mechanism of action remains elusive. Exome sequencing has identified AKAP11 (A-kinase anchoring protein 11) as a shared risk gene for BD and schizophrenia (SCZ). Given that both the AKAP11-Protein Kinase A (PKA) complex and Li target and inhibit Glycogen Synthase Kinase-3 beta (GSK3β),we hypothesize that Li may partially normalize the transcriptomic and/or epigenomic alterations observed in heterozygous AKAP11-knockout (Het-AKAP11-KO) iPSC-derived neurons. In this study,we employed genome-wide approaches to assess the effects of Li on the transcriptome and epigenome of human iPSC-derived Het-AKAP11-KO neuronal culture. We show that chronic Li treatment in this cellular model upregulates key pathways that were initially downregulated by Het-AKAP11-KO,several of which have also been reported as downregulated in synapses of BD and SCZ post-mortem brain tissues. Moreover,we demonstrated that Li treatment partially rescues certain transcriptomic alterations resulting from Het-AKAP11-KO,bringing them closer to the WT state. We suggest two possible mechanisms underlying these transcriptomic effects: (1) Li modulates histone H3K27ac levels at intergenic and intronic enhancers,influencing enhancer activity and transcription factor binding,and (2) Li enhances GSK3β serine 9 phosphorylation,impacting WNT/β-catenin signaling and downstream transcription. These findings underscore Li’s potential as a therapeutic agent for BD and SCZ patients carrying AKAP11 loss-of-function variants or exhibiting similar pathway alterations to those observed in Het-AKAP11-KO models. View Publication

Alzheimer’s disease (AD) involves cognitive decline,amyloid-beta (Aβ) accumulation,tau hyperphosphorylation,and neuroinflammation. CREB1,a key transcription factor for memory,is downregulated in AD,contributing to disease progression. Phosphodiesterases 4 and 10 (PDE4 and PDE10) are key enzymes that degrade cAMP,a second messenger involved in CREB signaling,synaptic plasticity,and neuroprotection. Dysregulation of PDE activity has been implicated in AD and other neurodegenerative disorders. Methods: We used human iPSC-derived cortical neurons and microglia,along with the APP/PS1 mouse model,to investigate the role of CREB1 and assess the therapeutic potential of dual PDE4/10A inhibition in AD. Results: CREB1 deficiency in neurons increased Aβ and p-tau231 accumulation. Dual inhibition of PDE4 and PDE10A activated the cAMP-PKA-CREB pathway,restoring CREB1 activity,reducing Aβ and p-tau231,and mitigating neuroinflammation. This intervention improved synaptic plasticity and cognitive performance in vivo. Conclusions: Our findings demonstrate that dual PDE4/10A inhibition synergistically enhances the cAMP-PKA-CREB signaling,promoting neuroprotection and synaptic remodeling. This approach offers a promising therapeutic strategy for modifying AD pathology and restoring cognitive function. View Publication

Pluripotent cancer stem cells play a pivotal role in inducing phenotypic plasticity across various cancer types,including bladder cancer. This plasticity,crucial for cancer progression,is largely regulated by epigenetic modifications including N6-methyladenosine (m6A) in RNAs. However,the role of the m6A reader protein YTHDC2 in this process remains poorly understood. In this study,we uncovered that the depletion of YTHDC2 significantly increased the pool of bladder cancer stem cells (BCSCs),resulting in a phenotypic shift towards a more invasive subtype of bladder cancer. This shift was characterized by enhanced proliferation,migration,invasion,and self-renewal capabilities of cancer cells,highlighting YTHDC2’s function as a tumor suppressor. Mechanistically,YTHDC2 recognized and bound to m6A-modified SOX2 mRNA,resulting in translational inhibition of SOX2. In conclusion,our study identifies YTHDC2 as a tumor suppressor in bladder cancer through inhibiting SOX2-mediated cell pluripotency and underscores the therapeutic potential of targeting the YTHDC2-SOX2 axis in bladder cancer. View Publication

Objective: To develop a rapid functional assay to validate variants of uncertain significance (VUS) in the MEFV gene. Methods: Overactivity of the pyrin inflammasome pathway and ASC speck oligomerisation in response to stimulation with low concentrations of Clostridium difficile toxin A was directly visualised by immunofluorescence microscopy. A semi‐automated algorithm was developed to count cells and ASC specks. Results: The semi‐automated ASC speck assay is able to discriminate between healthy controls and patients with familial Mediterranean fever (FMF) and pyrin inflammasome overactivity with high sensitivity. It is also able to discriminate pyrin inflammasome overactivity from other autoinflammatory disease controls with high specificity. Conclusion: The semi‐automated ASC speck assay may be a useful test to functionally validate VUS in the MEFV gene and screen for pyrin inflammasome overactivity. A semi‐automated ASC speck assay using machine learning is able to discriminate between healthy controls and patients with familial Mediterranean fever (FMF) with high sensitivity. It is also able to discriminate FMF from other autoinflammatory diseases with high specificity. View Publication

Tolerogenic dendritic cells (tolDCs) that dampen T cell responses can be induced from blood monocytes in vitro using factors such as Vitamin D3 (VitD),dexamethasone,IL‐10,or rapamycin. However,challenges remain in obtaining robust and efficient generation of cell therapy‐based tolDCs without compromising their viability. We recently reported that CCR2‐dependent recruitment of monocytic cells,with the capacity to dampen T‐helper responses,occurs in mice treated with a single‐stranded oligonucleotide (ssON). Here,we investigated the effects of this immunomodulatory noncoding ssON on differentiating human monocytes towards DC in the presence of IL‐4 and GM‐CSF (moDC). The moDC differentiated in the presence of ssON upregulated CD1a but also increased their expression of PD‐L1. The differentiation of monocytes to moDC in the presence of ssON introduced transcriptomic changes,many of which overlapped with VitD‐moDC and resulted in moDCs with altered lipopolysaccharide (LPS)‐responsiveness. Moreover,ssON‐moDC exhibited a low capacity to stimulate alloreactive T cells in vitro and instead promoted the induction of CD4+FoxP3+CD25+ T cells. Experiments using chemical reagents support a role for PPAR‐γ in the generation of ssON‐moDC. Collectively,our data show that monocytes differentiated with IL‐4,GM‐CSF,and ssON generate cells with phenotypic and functional characteristics of tolDCs. In this article,the authors elucidated the immunoregulatory role of an oligonucleotide (ssON) that favors the induction of human tolerogenic dendritic cells (DC). The tolerogenic profile was evidenced by reduced responsiveness to lipopolysaccharides (LPS) (A). Importantly,the tolerogenic DCs had upregulated PD‐L1 molecules and functionally inhibited the proliferation of alloreactive T cells and induced FoxP3+ Tregs (B). This study envisions the development of ssON as therapeutic for rebalancing overactive T‐helper cell responses. View Publication

In stem cell biology,a long-held structure–function relationship is the domed colony morphology and naïve pluripotency for mouse or human pluripotent stem cells. This link has provided a convenient way to recognize bona fide naïve pluripotent cells during derivation,passaging and characterization. However,the molecular basis of this link remains poorly understood. Results: We show that a loss of domed morphology may not impact the overall genetic architecture of naïve pluripotency in mouse embryonic stem cells (mESCs). We first generated stable mESC lines by knocking out Myh9 that encodes non-muscle myosin heavy chain IIA,resulting in colonies deprived of the typical domed morphology,but competent to differentiate into the three germ layers and chimeric mice. Modulating cell morphologies with inhibitors against kinases known to regulate myosin pathway also phenocopy the knockout in wild type mESCs. Conclusions: These results provide evidence that the domed morphology and potency can be uncoupled and suggest that domed structure is not a pre-requisite for acquiring and maintaining naïve pluripotency. View Publication

EVI1-rearranged acute myeloid leukemia (AML) with inv(3)(q21q26) or t(3;3)(q21q26) represents a distinct and aggressive subtype characterized by poor prognosis and limited treatment options. However,the optimal strategy to overcome resistance to conventional therapy remains elusive. Building upon observations correlating EVI1 overexpression with reduced sensitivity to venetoclax,a BH3-mimetic BCL-2 inhibitor,we investigated the mechanisms of resistance to venetoclax in combination with hypomethylating agents in inv(3)/t(3;3) AML cells. Utilizing novel murine models recapitulating inv(3) AML with concomitant SF3B1 mutations,we conducted comprehensive phenotypic and transcriptomic analyses in the presence or absence of venetoclax-containing therapy. Despite initial therapeutic responses,manifested as partially prolonged survival and myeloid differentiation,resistant leukemic cells demonstrated enhanced dependency on BRD4 and MYB pathways with a dormant phenotype. Notably,inhibition of either BRD4 or MYB significantly augmented the efficacy of venetoclax and hypomethylating agents in both murine and patient-derived AML models harboring inv(3) and SF3B1 mutations. These findings elucidate the transcriptional dynamics underlying venetoclax resistance and propose alternative therapeutic strategies targeting BRD4 and MYB as promising avenues for improving outcomes in patients with EVI1-rearranged AML. Our work highlights the necessity for innovative combination therapies to address the multifaceted mechanisms of resistance in this high-risk leukemia subtype. View Publication

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment landscape of hematologic cancers by engineering T cells to specifically target and destroy cancer cells. Monitoring CAR T cell activity and function is essential for optimizing therapeutic outcomes,but existing tools for CAR detection are often limited in specificity and functional assessment capability. Methods: We developed dextran multimers by conjugating multiple CAR-specific antigens to a dextran backbone. The multimers were compared to previously reported antigen tetramers for their ability to stain and detect CAR T cells. Because these multimers incorporate the CAR target antigen,they uniquely enable assessment of CAR T cell functionality. We tested the staining and functional properties of the multimers across a range of CAR constructs with different affinities,using flow cytometry and microscopy. Results: The dextran multimers demonstrated high specificity and sensitivity in staining CAR T cells,with adjustable antigen density to optimize binding. Dextran multimers also enabled effective clustering and subsequent activation of CARs,showing their utility as both a staining and functional assessment tool. The multimers revealed that CARs with different affinities and clustering tendencies displayed varied binding and activation in response to different antigen densities. Conclusions: Dextran multimers offer a dual advantage as versatile reagents for both staining and functional analysis of CAR T cells. Their capacity to engage CARs with the specific antigen provides a valuable platform for evaluating CAR functionality,informing CAR design improvements,and enhancing therapeutic precision. View Publication

Assessment of Fc receptors and immune cells in breast cancer enables development of tailored engineering strategies for tumor-targeting monoclonal antibodies with enhanced immune-stimulating and anticancer attributes by combining glycoengineering and Fc mutations. AbstractFc engineering to enhance antibody effector functions harbors the potential to improve therapeutic effects. Understanding FcγR expression and distribution in the tumor microenvironment prior to and following treatment may help guide immune-engaging antibody design and patient stratification. In this study,we investigated FcR-expressing immune effector cells in HER2+ and triple-negative breast cancers (TNBC),including neoadjuvant chemotherapy–resistant disease. FcγRIIIa expression,FcγRIIIa+ NK cells,and classically activated (M1-like) macrophages correlated with improved anti-HER2 antibody efficacy. FcγRIIIa protein and FcγRIIIa+ NK cells and macrophages were present in primary TNBC and retained in treatment-resistant tumors. FcγRIIIa was spatially associated with folate receptor alpha–positive (FRα+) tumor areas at baseline and in residual tumors following neoadjuvant chemotherapy. Wild-type and Fc-engineered antibodies recognizing two breast cancer–associated antigens,HER2 and the emerging TNBC target FRα,were designed and generated to have increased FcγRIIIa-expressing effector cell engagement. The combination of glycoengineering,including fucose removal from the N-linked Fc glycan,and Fc point mutations greatly increased antibody affinity for and retention on FcγRIIIa. The Fc-engineered antibodies enhanced immune effector activity against HER2+ breast cancer and TNBC,altering proinflammatory cytokine production by NK cells and tumor-conditioned macrophages and skewing macrophages toward proinflammatory states. Furthermore,the Fc-engineered antibodies restricted orthotopic HER2+ and FRα+ breast cancer xenograft growth at doses suboptimal for equivalent wild-type antibodies and recruited FcγRIIIa-expressing cells into tumors. Antibody design through combined glycoengineering and Fc point mutations to enhance FcγRIIIa engagement of tumor-infiltrating effector cells may be a promising strategy for developing therapies for patients with aggressive and treatment-resistant breast cancers.Significance:Assessment of Fc receptors and immune cells in breast cancer enables development of tailored engineering strategies for tumor-targeting monoclonal antibodies with enhanced immune-stimulating and anticancer attributes by combining glycoengineering and Fc mutations. View Publication