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We investigated the expression status of periostin in breast cancer stem cells and its clinical implications in order to lay a foundation for managing breast cancer. CD44+/CD24-/line- tumor cells (CSC) from clinical specimens were sorted using flow cytometry. Periostin expression status was detected in CSC cells and 1,086 breast cancer specimens by Western blot and immunohistochemistry staining,with the CSC ratio determined by immunofluorescence double staining. The relationship between the periostin protein and clinico-pathological parameters and prognosis was subsequently determined. As a result,CSC cells are more likely to generate new tumors in mice and cell microspheres that are deficient in NOD/SCID compared to the control group. Periostin protein was expressed higher in CSC cells compared to the control cells and was found to be related to CSC chemotherapy resistance. Moreover,periostin expression was found to be related to the CSC ratio in 1,086 breast cancer specimens (P = 0.001). In total,334 (30.76%) of the 1,086 breast cases showed high periostin expression. After universal and Spearman regression correlation analysis,periostin was observed to be related to histological grade,CSC ratio,lymph node metastasis,tumor size,and triple-negative breast cancer (all Ptextless0.05). Furthermore,periostin was shown to attain a significantly more distant bone metastasis and worse disease-specific survival than those with none or low-expressed periostin protein (P = 0.001). In the Cox regression test,periostin protein was detected as an independent prognostic factor (P = 0.001). In conclusion,periostin was found to be related to the CSC and an independent prognostic factor for breast cancer. It is also perhaps a potential target to breast cancer. View Publication

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death,but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis,or of its underlying transcriptome network. Therefore,the ‘human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes,whereas MeHg altered fewer transcripts. To attenuate batch effects,analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (backslashtextless20 % overlap). Moreover,within one test system,little overlap between the PS changed by the two compounds has been observed. However,using TFBS enrichment,a relatively large ‘common response' to VPA and MeHg could be distinguished from ‘compound-specific' responses. In conclusion,the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles. View Publication

Asthma is a complex and heterogeneous respiratory disorder characterized by chronic airway inflammation. It has generally been associated with allergic mechanisms related to type 2 airway inflammation. Nevertheless,between 10 and 33{\%} of asthmatic individuals have nonallergic asthma (NA). Several targeted treatments are in clinical development for patients with Th2 immune response,but few biomarkers are been defined for low or non-Th2-mediated inflammation asthma. We have recently defined by gene expression a set of genes as potential biomarkers of NA,mainly associated with disease severity: IL10,MSR1,PHLDA1,SERPINB2,CHI3L1,IL8,and PI3. Here,we analyzed their protein expression and specificity using sera and isolated peripheral blood mononuclear cells (PBMCs). First,protein quantification was carried out using ELISA (in sera) or Western blot (proteins extracted from PBMCs by Trizol procedure),depending on the biomarker in 30 healthy controls (C) subjects and 30 NA patients. A receiver operating characteristic curve analysis was performed by using the R program to study the specificity and sensitivity of the candidate biomarkers at a gene- and protein expression level. Four kinds of comparisons were performed: total NA group vs C group,severe NA patients vs C,moderate-mild NA patients vs C,and severe NA patients vs moderate-mild NA patients. We found that all the single genes showed good sensitivity vs specificity for some phenotypic discrimination,with CHI3L1 and PI3 exhibiting the best results for C vs NA: CHI3L1 area under the curve (AUC) (CI 95{\%}): 0.95 (0.84-1.00) and PI3 AUC: 0.99 (0.98-1.00); C vs severe NA: PI3 AUC: 1 (0.99-1.00); and C vs moderate-mild NA: CHI3L1 AUC: 1 (0.99-1.00) and PI3 AUC: 0.99 (0.96-1.00). However,the results for discriminating asthma disease and severity with protein expression were better when two or three biomarkers were combined. In conclusion,individual genes and combinations of proteins have been evaluated as reliable biomarkers for classifying NA subjects and their severity. These new panels could be good diagnostic tests. View Publication

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study,we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively,while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs,multiple uniquely shared amino acid configurations were identified,warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes,which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation. View Publication

Huntington’s disease and other disorders of the basal ganglia create challenges for biomolecule-based medicines given the poor accessibility of these deep brain structures following intracerebral or intravascular delivery. Here,we found that low dose,low volume delivery of unbiased AAV libraries into the globus pallidus allowed recovery of novel capsids capable of broad access to key deep brain and cortical structures relevant for human therapies. One such capsid,AAV-DB-3,provided transduction of up to 45% of medium spiny neurons in the adult NHP striatum,along with substantial transduction of relevant deep layer neurons in the cortex. Notably,AAV-DB-3 behaved similarly in mice as in NHPs and potently transduced human neurons derived from induced pluripotent stem cells. Thus,AAV-DB-3 provides a unique AAV for network level brain gene therapies that translates up and down the evolutionary scale for preclinical studies and eventual clinical use. To date,brain gene therapies require high vector doses. Here,authors devised an AAV capsid screen and found variants with unprecedented potency for transduction of deep brain and cortical neurons and human iPSC-neurons with cell tropism relevant for Huntington’s and Parkinson’s disease. View Publication

This work demonstrates that group 2 KLF family transcription factors are critical for specifying the identity of distinct tissue-resident macrophages. KLF2 directly controls expression of genes previously shown to be necessary in cavity macrophages,while KLF4 may play a similar role in alveolar macrophages. Tissue-resident macrophages adopt distinct gene expression profiles and exhibit functional specialization based on their tissue of residence. Recent studies have begun to define the signals and transcription factors that induce these identities. Here we describe an unexpected and specific role for the broadly expressed transcription factor Krüppel-like factor 2 (KLF2) in the development of embryonically derived large cavity macrophages (LCMs) in the serous cavities. KLF2 not only directly regulates the transcription of genes previously shown to specify LCM identity,such as retinoic acid receptors and GATA6,but also is required for induction of many other transcripts that define the identity of these cells. Our results suggest that KLF4 may similarly regulate the identity of alveolar macrophages in the lung. These data demonstrate that broadly expressed transcription factors,such as group 2 KLFs,can play important roles in the specification of distinct identities of tissue-resident macrophages. View Publication

Genetic variations in the Trpm8 gene that encodes the cold receptor TRPM8 have been linked to protection against polygenic migraine,a disabling condition primarily affecting women. Noteworthy,TRPM8 has been recently found in brain areas related to emotional processing,suggesting an unrecognized role in migraine comorbidities. Here,we use mouse behavioural models to investigate the role of Trpm8 in migraine-related phenotypes. Subsequently,we test the efficacy of rapamycin,a clinically relevant TRPM8 agonist,in these behavioural traits and in human induced pluripotent stem cell (iPSC)-derived sensory neurons. We report that Trpm8 null mice exhibited impulsive and depressive-like behaviours,while also showing frequent pain-like facial expressions detected by an artificial intelligence algorithm. In a nitroglycerin-induced migraine model,Trpm8 knockout mice of both sexes developed anxiety and mechanical hypersensitivity,whereas wild-type females also displayed depressive-like phenotype and hypernociception. Notably,rapamycin alleviated pain-related behaviour through both TRPM8-dependent and independent mechanisms but lacked antidepressant activity,consistent with a peripheral action. The macrolide ionotropically activated TRPM8 signalling in human sensory neurons,emerging as a new candidate for intervention. Together,our findings underscore the potential of TRPM8 for migraine relief and its involvement in affective comorbidities,emphasizing the importance of addressing emotional symptoms to improve clinical outcomes for migraine sufferers,especially in females. The online version contains supplementary material available at 10.1186/s10194-025-02082-4. View Publication

The diagnosis of Parkinson's disease (PD) is initiated after the occurrence of motor symptoms,such as resting tremors,rigidity,and bradykinesia. According to previous reports,non-motor symptoms,notably gastrointestinal dysfunction,could potentially be early biomarkers in PD patients as such symptoms occur earlier than motor symptoms. However,connecting PD to the intestine is methodologically challenging. Thus,we generated in vitro human intestinal organoids from PD patients and ex vivo mouse small intestinal organoids from aged transgenic mice. Both intestinal organoids (IOs) contained the human LRRK2 G2019S mutation,which is the most frequent genetic cause of familial and sporadic PD. By conducting comprehensive genomic comparisons with these two types of IOs,we determined that a particular gene,namely,Iroquois homeobox protein 2 (IRX2),showed PD-related expression patterns not only in human pluripotent stem cell (PSC)-derived neuroectodermal spheres but also in human PSC-derived neuronal cells containing dopaminergic neurons. We expected that our approach of using various cell types presented a novel technical method for studying the effects of multi-organs in PD pathophysiology as well as for the development of diagnostic markers for PD. View Publication

The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However� View Publication

Human pluripotent stem cells (hPSCs) are a promising source of cells for clinical applications,such as transplantation of clinically engineered tissues and organs,and drug discovery programs due to their ability to self-renew and to be differentiated into cells from the three embryonic germ layers. In this study,the differentiation of two hPSC-lines into neural precursors (NPs) was accomplished with more than 80 % efficiency,by means of the dual-SMAD inhibition protocol,based on the use of two small molecules (SB431542 and LDN193189) to generate Pax6 and Nestin-positive neural entities. One of the major hurdles related to the in vitro generation of PSC-derived populations is the tumorigenic potential of cells that remain undifferentiated. These remaining hPSCs have the potential to generate teratomas after being transplanted,and may interfere with the outcome of in vitro differentiation protocols. One strategy to tackle this problem is to deplete these contaminating" cells during the differentiation process. Magnetic activated cell sorting (MACS) was used for the first time for purification of hPSC-derived NPs after the neural commitment stage using anti-Tra-1-60 micro beads for negative selection of the unwanted hPSCs. The depletion had an average efficiency of 80.4 ± 5 % and less than 1.5 % of Tra-1-60 positive cells were present in the purified populations. After re-plating� View Publication

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein,tandem dimer Tomato (tdTomato),are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons,evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations. View Publication

Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms,yet their clinical translation has been compromised by their biosafety concern. Here,we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/ progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immu-nodeficient mice. Moreover,removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplanta-tion,ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable,depending on the oncogenic load,with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus,transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:694–702 SIGNIFICANCE Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products,especially when reprogrammed with integrating vectors. Two major under-lying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzy-matic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in test-ing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature b cell phenotype would lead to safe islet replacement therapy for diabetes. View Publication