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Use of cisplatin,a chemotherapeutic agent,is associated with toxicity as a significant number of patients develop a decline in renal function. The mechanisms by which cisplatin produces renal injury are not well understood. It has been suggested that free radical-catalyzed lipid peroxidation can induce apoptosis or necrosis leading to renal injury. This study examined whether low concentrations of cisplatin induce apoptosis in LLC-PK1 cells and whether caspases 1,2,3,8,and 9 are activated during this event. Our results show a dose- and time-dependent induction of apoptosis by micromolar concentrations of cisplatin. Expression of oncogenes c-myc and p53 was induced,and except for caspase 1,all the other caspases tested were activated. Z-VAD,the broad-spectrum inhibitor of caspases,prevented caspase activation and apoptosis,but not c-myc and p53 induction. On the other hand,N-acetylcysteine prevented cisplatin-induced apoptosis as well as c-myc induction but not p53 induction. The antioxidant trolox also prevented cisplatin-induced apoptosis. The results suggest that antioxidants and caspase inhibitors may alleviate cisplatin-associated nephrotoxicity. View Publication

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study,we observed that TPEN [N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine],a zinc chelator,protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor,diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition,we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX),p38 mitogen-activated protein kinase (MAPK),and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX,generation of reactive oxygen species (ROS),and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX,ROS accumulation,p38 activation,and caspase-3 activation. Therefore,therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation,stroke,and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death. View Publication

Three-dimensional (3D) culture systems have been developed that can re-capitulate organ level responses,simulate compound diffusion through complex structures,and assess cellular heterogeneity of tissues,making them attractive models for advanced in vitro research and discovery. Organoids are a unique subtype of 3D cell culture that are grown from stem cells,are self-organizing,and closely replicate in vivo pathophysiology. Organoids have been used to understand tissue development,model diseases,test drug sensitivity and toxicity,and advance regenerative medicine. However,traditional organoid culture methods are inadequate because they are low throughput and ill-suited for single organoid imaging,phenotypic assessment,and isolation from heterogenous organoid populations. To address these bottlenecks,we have adapted our tissue culture consumable and instrumentation to enable automated imaging,identification,and isolation of individual organoids. Organoids grown on the 3D CytoSort? Array can be reliably tracked,imaged,and phenotypically analyzed using brightfield and fluorescent microscopy as they grow over time,then released and transferred fully intact for use in downstream applications. Using mouse hepatic and pancreatic organoids,we have demonstrated the use of this technology for single-organoid imaging,clonal organoid generation,parent organoid subcloning,and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis. The results validate the ability of the CellRaft AIR? System to facilitate efficient,user-friendly,and automated workflows broadly applicable to organoid research by overcoming several pain points: 1) single organoid time-course imaging and phenotypic assessment,2) establishment of single cell-derived organoids,and 3) isolation and retrieval of single organoids for downstream applications. View Publication

BACKGROUND During the COVID-19 pandemic,thousands of pregnant women have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The implications of maternal SARS-CoV-2 infection on fetal and childhood well-being need to be characterized. We aimed to characterize the fetal immune response to maternal SARS-CoV-2 infection. METHODS We performed single-cell RNA-sequencing and T cell receptor sequencing on cord blood mononuclear cells (CBMCs) from newborns of mothers infected with SARS-CoV-2 in the third trimester (cases) or without SARS-CoV-2 infection (controls). RESULTS We identified widespread gene expression changes in CBMCs from cases,including upregulation of interferon-stimulated genes and major histocompatibility complex genes in CD14+ monocytes,transcriptional changes suggestive of activation of plasmacytoid dendritic cells,and activation and exhaustion of natural killer cells. Lastly,we observed fetal T cell clonal expansion in cases compared to controls. CONCLUSIONS As none of the infants were infected with SARS-CoV-2,our results suggest that maternal SARS-CoV-2 infection might modulate the fetal immune system in the absence of vertical transmission. IMPACT The implications of maternal SARS-CoV-2 infection in the absence of vertical transmission on fetal and childhood well-being are poorly understood. Maternal SARS-CoV-2 infection might modulate the fetal immune system in the absence of vertical transmission. This study raises important questions about the untoward effects of maternal SARS-CoV-2 on the fetus,even in the absence of vertical transmission. View Publication

Oxidative stress,caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses,is one of the characteristic features of cancer. Here,we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo,when compared with blood or normal subcutaneous fluid. Therefore,we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells,and by comparing T,B,and NK cells' sensitivities to redox stress and their antioxidant capacities,we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However,the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore,we genetically modified NK cells to stably overexpress PRDX1,which led to increased survival and NK cell activity in redox stress conditions. Finally,we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide,at concentrations detected in the TME,suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors. View Publication

The intradermal lipopolysaccharide (LPS) challenge in healthy volunteers has proven to be a valuable tool to study local inflammation in vivo. In the current study the inhibitory effects of oral and topical corticosteroid treatment on intradermal LPS responses were evaluated to benchmark the challenge for future investigational drugs. Twenty-four healthy male volunteers received a two-and-a-half-day twice daily (b.i.d.) pretreatment with topical clobetasol propionate 0.05% and six healthy volunteers received a two-and-a-half-day b.i.d. pretreatment with oral prednisolone at 0.25 mg/kg body weight per administration. Participants received one injection regimen of either 0,2,or 4 intradermal LPS injections (5 ng LPS in 50 µL 0.9% sodium chloride solution). The LPS response was evaluated by noninvasive (perfusion,skin temperature,and erythema) and invasive assessments (cellular and cytokine responses) in suction blister exudate. Both corticosteroids significantly suppressed the clinical inflammatory response (erythema P = 0.0001 for clobetasol and P = 0.0016 for prednisolone; heat P = 0.0245 for clobetasol,perfusion P < 0.0001 for clobetasol and P = 0.0036 for prednisolone). Clobetasol also significantly reduced the number of monocytes subsets,dendritic cells,natural killer cells,and T cells in blister exudate. A similar effect was observed for prednisolone. No relevant corticosteroid effects were observed on the cytokine response to LPS. We successfully demonstrated that the anti-inflammatory effects of corticosteroids can be detected using our intradermal LPS challenge model,validating it for evaluation of future investigational drugs,as an initial assessment of the anti-inflammatory effects of such compounds in a minimally invasive manner. View Publication

Genome‐wide association studies (GWAS) have identified APOE2 allele as linked to exceptional longevity,with carriers exhibiting a reduced risk of Alzheimer's disease (AD). Apolipoprotein E (APOE),a glycoprotein involved in lipid transport,has three major alleles. However,alterations in lipid metabolism alone do not fully explain APOE2's protective effects. In contrast,APOE4 is the strongest genetic risk factor for AD. To investigate how APOE2 promotes neuronal longevity and confers neuroprotection,we generated human isogenic APOE iPSC‐derived models of both inhibitory GABAergic and excitatory neurons. In GABAergic neurons,APOE alleles differentially influenced endogenous DNA damage,DNA repair,and neuronal motility. Single‐cell RNA sequencing revealed APOE4‐specific gene expression signatures associated with AD,whereas APOE2 GABAergic neurons were enriched for DNA repair and signaling pathways. Consistent with this,APOE2 neurons exhibited significantly lower levels of DNA damage. APOE4 GABAergic neurons exhibit increased expression of repetitive ribosomal RNA,which is associated with DNA damage and cellular senescence. To determine whether the effects extended to excitatory neurons,we used a separate human model of Ngn2‐induced glutamatergic neurons,and found that APOE2 excitatory neurons were more resistant to cellular senescence and DNA damage than isogenic APOE3 and APOE4 neurons. Similarly,we found human APOE2‐targeted replacement mice exhibited less nucleolar enlargement and increased nuclear Lamin A/C,Hmgb1,and H3K9me3 compared to APOE4 counterparts. Together,our findings identify DNA repair and suppression of senescence‐associated processes as key mechanisms by which APOE2 is associated with neuronal resilience,providing mechanistic insight into its association with exceptional longevity and protection against AD. Neurons expressing APOE2 were more resistant to endogenous DNA damage,activated transcriptional signaling pathways associated with DNA repair,and were resilient to stress‐induced DNA damage and cellular senescence. In contrast,APOE4 neurons exhibited elevated expression of rRNA repetitive elements and were prone to becoming senescent. View Publication

The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system,but it involutes with age,resulting in reduced naive T-cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative medicine strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro,but functional thymic epithelial cells (TECs) develop only after transplantation of TEPs in vivo. Functional human reaggregated thymic organoid cultures (RTOCs) and artificial thymic organoids (ATOs) cultured at the air–liquid interface support T-cell development in vitro and in vivo and permit the interrogation of human thymic function and T-cell development. However,these approaches require access to primary human tissues or murine bone marrow stromal cells,are allogeneic,and do not support negative selection. Recently,we reported the directed differentiation of induced PSCs (iPSCs) to functional thymic epithelial progenitors (TEPs) that support murine T-cell development after transplantation in nude mice. Here,we combined hPSC-derived TEPs,hematopoietic progenitor cells (HPCs),and mesenchymal cells,differentiated from the same hPSC line,and generated functional isogenic stem cell–derived thymic organoids (sTOs). Our revised protocol improves our TEP differentiation process and allows the generation of functional isogenic,patient-specific thymic organoids in vitro. View Publication

Stem cell therapy has been widely studied as a promising treatment for spinal cord injury (SCI). However,a lack of functional scaffolds for stem cell therapy to address SCI leads to low therapeutic efficacy due to poor survival of transplanted cells. To address these challenges,this study aims to enhance regenerative potential of spinal cord organoids (SCOs) by employing extracellular matrix (ECM) recapitulating spinal cord-specific microenvironment. Decellularized spinal cord-derived ECM (ScEM) supports 3D culture for development,maturation,and functionality of human induced pluripotent stem cell-derived SCOs,comparable to standard organoid culture matrix such as Matrigel. Transplantation of SCOs using ScEM hydrogel promotes axonal regeneration with neovascularization in lesions,likely because of enhanced engraftment and integration of transplanted SCOs into defective tissues facilitated by ScEM. Accordingly,this approach induces early locomotor recovery of animals with SCI. These findings suggest that functional ECM scaffold capable of providing microenvironmental complexity of spinal cord can potentiate organoid-based therapeutics for SCI treatment. Graphical abstract Highlights•A decellularized spinal cord-derived ECM (ScEM) exhibits protein profiles resembling native spinal cord tissue.•Functional proteins related to neurodevelopment and regeneration are highly enriched in ScEM.•ScEM hydrogel supports development and maturation of spinal cord organoids (SCOs) comparable to Matrigel.•ScEM hydrogel enhances SCO-mediated axonal regrowth,immune modulation,and early recovery after spinal cord injury. View Publication

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including,synthetic and structural biology,cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity,thus impeding unrestricted multigene expression. We developed MultiPrime,a modular,non-cytotoxic,non-integrating,baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types,including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming,and for genome editing and engineering by CRISPR/Cas9. Moreover,we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool,to deliver multiple genes for a wide range of applications in primary and established mammalian cells. View Publication