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Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However,our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore,we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types,n = 792) by immunohistochemical staining. Using the ALDEFUOR assay,ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition,an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct,and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers,we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439,p = 0.0036). Finally,ALDH(br) tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker,ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast,lung,ovarian or colon cancer. View Publication

The embryonic mouse lung is a widely used substitute for human lung development. For example,attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology,gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover,we identify mouse-human differences,including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated. View Publication

BackgroundMicroglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells,such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2),as the strongest risk factors for Alzheimer’s disease (AD) highlights the importance of microglial biology in the brain. The sequence,structure and function of several microglial proteins are poorly conserved across species,which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs).MethodsIn this study,we identified,produced,and tested novel,selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models,as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo.ResultsWe proved their efficacy in human iPSC microglia in vitro,as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-? plaques in vivo in a model of AD.ConclusionsThis study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-024-00725-9. View Publication

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS,but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore,we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2,KIF5A,NEK1,TBK1,and TUBA4A) which highlighted many novel binding partners,and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system,and of ALS-associated C21ORF2 variants is largely unknown. Therefore,we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First,our data show C21ORF2 expression in ALS-relevant mouse and human neurons,such as spinal and cortical motor neurons. Further,the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients,but not isogenic controls,show increased apoptosis,and changes in DNA damage response,mitochondria and neuronal excitability. In addition,C21ORF2-V58L induced post-transcriptional downregulation of NEK1,an ALS-associated protein implicated in apoptosis and DDR. In all,our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS. The online version contains supplementary material available at 10.1186/s40478-024-01852-6. View Publication

Cerebral cavernous malformations (CCMs) are clusters of thin-walled enlarged blood vessels in the central nervous system that are prone to recurrent hemorrhage and can occur in both sporadic and familial forms. The familial form results from loss-of-function variants in the CCM1,CCM2,or CCM3 gene. Despite a better understanding of CCM pathogenesis in recent years,it is still unclear why CCM3 mutations often lead to a more aggressive phenotype than CCM1 or CCM2 variants. By combining high-throughput differentiation of blood vessel organoids from human induced pluripotent stem cells (hiPSCs) with a CCM1,CCM2,or CCM3 knockout,single-cell RNA sequencing,and high-content imaging,we uncovered both shared and distinct functions of the CCM proteins. While there was a significant overlap of differentially expressed genes in fibroblasts across all three knockout conditions,inactivation of CCM1,CCM2,or CCM3 also led to specific gene expression patterns in neuronal,mesenchymal,and endothelial cell populations,respectively. Taking advantage of the different fluorescent labels of the hiPSCs,we could also visualize the abnormal expansion of CCM1 and CCM3 knockout cells when differentiated together with wild-type cells into mosaic blood vessel organoids. In contrast,CCM2 knockout cells showed even reduced proliferation. These observations may help to explain the less severe clinical course in individuals with a pathogenic variant in CCM2 and to decode the molecular and cellular heterogeneity in CCM disease. Finally,the excellent scalability of blood vessel organoid differentiation in a 96-well format further supports their use in high-throughput drug discovery and other biomedical research studies. The online version contains supplementary material available at 10.1007/s10456-025-09985-5. View Publication

Spinal cord injury (SCI) remains a significant clinical challenge and poses a dramatic threat to the life quality of patients due to limited neural regeneration and detrimental post-injury alternations in tissue microenvironment. We developed a therapeutic approach by transplanting spinal neural progenitor cells (spNPGs),derived from human induced pluripotent stem cell (iPSC)-generated neuromesodermal progenitors,into a contusive SCI model in NOD-SCID mice. Single-cell RNA sequencing mapped the in vitro differentiation of iPSC-spNPGs,confirming their specification into spinal neuronal lineages. Single-nucleus transcriptomics at 1 week post-transplantation showed that the grafted cells differentiated in vivo into motor neurons and two interneuron subtypes (V2 and dI4). Additionally,spNPGs integrated into host neural circuits,enhancing synaptic connectivity,while simultaneously modulating the injury microenvironment by shifting microglia and astrocyte polarization toward anti-inflammatory and neuroprotective phenotypes. This dual mechanism promoted axonal regrowth,remyelination,and significant sensorimotor recovery,as evidenced by improved locomotor scores. Our findings highlight the therapeutic potential of human iPSC-spNPGs in reconstructing neural networks and mitigating secondary damage,providing compelling preclinical evidence for advancing stem cell-based SCI therapies. Subject terms: Stem-cell differentiation,Spinal cord injury View Publication

We evaluated the feasibility and efficacy of a reduced-intensity conditioning (RIC) regimen of fludarabine and melphalan to achieve rapid complete donor chimerism after allogeneic stem cell transplantation (SCT) in patients with metastatic solid tumors. Between January 1999 and January 2003,8 patients with metastatic breast cancer (BC) and 15 with metastatic renal cell carcinoma (RCC) underwent allogeneic SCT after an RIC regimen of 5 days of fludarabine and 2 days of melphalan. Filgrastim-mobilized stem cells from HLA-identical related or unrelated donors were infused. Prophylaxis for graft-versus-host disease (GVHD) consisted of tacrolimus and methotrexate. All 22 evaluable patients had 100% donor chimerism at day 30 and at all measurement times thereafter. One patient died 19 days after SCT. Nine patients (39%) had grades II to IV acute GVHD and 10 patients (43%) had chronic GVHD. Five patients (22%) died of nonrelapse treatment-related complications. Treatment-related disease response was seen in 10 patients (45%),with 3 complete responses,2 partial responses,and 5 minor responses. Fludarabine-melphalan is a feasible and effective RIC regimen for allogeneic SCT in metastatic BC and RCC. It induces rapid complete donor chimerism without the need for donor lymphocyte infusion. Tumor regression associated with GVHD is consistent with graft-versus-tumor effect. View Publication

Outcomes of 159 young patients with inherited metabolic disorders (IMDs) undergoing transplantation with partially HLA-mismatched unrelated donor umbilical cord blood were studied to investigate the impact of graft and patient characteristics on engraftment,overall survival (OS),and graft-versus-host disease (GVHD). Patients received myeloablative chemotherapy (busulfan,cyclophosphamide,ATG) and cyclosporine-based GVHD prophylaxis. Infused cell doses were high (7.57 x 10(7)/kg) because of the patients' young age (median,1.5 years) and small size (median,12 kg). Median follow-up was 4.2 years (range,1-11 years). The cumulative incidences of neutrophil and platelet engraftment were 87.1% (95% confidence interval [CI],81.8%-92.4%) and 71.0% (95% CI,63.7%-78.3%). A total of 97% achieved high (textgreater 90%) donor chimerism. Serum enzyme normalized in 97% of patients with diseases for which testings exist. Grade III/IV acute GVHD occurred in 10.3% (95% CI,5.4%-15.2%) of patients. Extensive chronic GVHD occurred in 10.8% (95% CI,5.7%-15.9%) of patients by 1 year. OS at 1 and 5 years was 71.8% (95% CI,64.7%-78.9%) and 58.2% (95% CI,49.7%-66.6%) in all patients and 84.5% (95% CI,77.0%-92.0%) and 75.7% (95% CI,66.1%-85.3%) in patients with high (80-100) performance score. In multivariate analysis,favorable factors for OS were high pretransplantation performance status,matched donor/recipient ethnicity,and higher infused colony forming units. View Publication

HOX genes,notably members of the HOXA cluster,and HOX cofactors have increasingly been linked to human leukemia. Intriguingly,HOXD13,a member of the HOXD cluster not normally expressed in hematopoietic cells,was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t(2; 11) fusion gene in the murine hematopoietic model. NUP98-HOXD13 strongly promoted growth and impaired differentiation of early hematopoietic progenitor cells in vitro; this effect was dependent on the NUP98 portion and an intact HOXD13 homeodomain. Expression of the NUP98-HOXD13 fusion gene in vivo resulted in a partial impairment of lymphopoiesis but did not induce evident hematologic disease until late after transplantation (more than 5 months),when some mice developed a myeloproliferative-like disease. In contrast,mice transplanted with bone marrow (BM) cells cotransduced with NUP98-HOXD13 and the HOX cofactor Meis1 rapidly developed lethal and transplantable acute myeloid leukemia (AML),with a median disease onset of 75 days. In summary,this study demonstrates that NUP98-HOXD13 can be directly implicated in the molecular process leading to leukemic transformation,and it supports a model in which the transforming properties of NUP98-HOXD13 are mediated through HOX-dependent pathways. View Publication

T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however,the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3zeta chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3zeta that lacks a 562-bp region in its 3'-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3zeta transcript are critical for the mRNA stability and protein expression of CD3zeta. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3zeta 3'-UTR. Knockdown of HuR resulted in decreased expression of the CD3zeta chain,whereas overexpression led to the increase of CD3zeta chain levels. Additionally,overexpression of HuR in human T cells resulted in increased mRNA stability of CD3zeta. Our results identify the 3'-UTR of CD3zeta as a novel target for the mRNA-stabilizing protein HuR. Thus,the absence of two critical AREs in the alternatively spliced CD3zeta 3'-UTR found in SLE T cells may result in decreased HuR binding,representing a possible molecular mechanism contributing to the reduced stability and expression of CD3zeta in SLE. View Publication

Efficient differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to a variety of lineages requires step-wise approaches replicating the key commitment stages found during embryonic development. Here we show that expression of PdgfR-α segregates mouse ESC-derived Flk-1 mesoderm into Flk-1(+)PdgfR-α(+) cardiac and Flk-1(+)PdgfR-α(-) hematopoietic subpopulations. By monitoring Flk-1 and PdgfR-α expression,we found that specification of cardiac mesoderm and cardiomyocytes is determined by remarkably small changes in levels of Activin/Nodal and BMP signaling. Translation to human ESCs and iPSCs revealed that the emergence of cardiac mesoderm could also be monitored by coexpression of KDR and PDGFR-α and that this process was similarly dependent on optimal levels of Activin/Nodal and BMP signaling. Importantly,we found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation,illustrating a principle that may well apply in other contexts. View Publication

Interleukin (IL)-23 is thought to be critical in the pathogenesis of psoriasis,and anti-IL-23 monoclonal antibodies (mAbs) have been approved for the treatment of psoriasis. We speculated that an anti-IL-23 receptor mAb might have greater efficacy than an anti-IL-23 mAb in the treatment of local inflamed lesions with high IL-23 levels. We previously generated an anti-human IL-23 receptor mAb,AS2762900-00,which potently blocked IL-23-induced cell proliferation,regardless of the concentration of IL-23. Here,we evaluated the therapeutic potential of AS2762900-00 in the treatment of psoriasis. Compared with untreated control,AS2762900-00 significantly reduced the epidermal thickness of lesions in a clinically relevant psoriatic human skin xenograft model. The expression of inflammatory genes including genes downstream of IL-23 signaling in the lesion tended to be lower in the AS2762900-00 group than the untreated group,suggesting that the inhibitory effects of AS2762900-00 in the psoriatic human skin xenograft model might occur via blockade of IL-23 signaling pathways. Further,AS2762900-00 showed an inhibitory effect on signal transducer and activator of transcription 3 (STAT3) phosphorylation as a downstream signal of IL-23 receptor activation in whole blood from patients with psoriasis. We also confirmed that AS2762900-00 inhibited IL-23-induced STAT3 phosphorylation in a concentration-dependent manner using whole blood from healthy donors. These data suggest that AS2762900-00 is a promising drug candidate for the treatment of psoriasis. In addition,STAT3 phosphorylation in whole blood may be a useful biomarker for the evaluation of the pharmacodynamic effects of AS2762900-00 in healthy volunteers in clinical development. View Publication