Surface Antigen Profile Research Articles

Serovar-level identification of bacterial foodborne pathogens from full-length 16S rRNA gene sequencing.

The resolution of variation within species is critical for interpreting and acting on many microbial measurements. In the key foodborne pathogens Salmonella and Escherichia coli, the primary subspecies classification scheme used is serotyping: differentiating variants within these species by surface antigen profiles. Serotype prediction from whole-genome sequencing (WGS) of isolates is now seen as comparable or preferable to traditional laboratory methods where WGS is available. However, laboratory and WGS methods depend on an isolation step that is time-consuming and incompletely represents the sample when multiple strains are present. Community sequencing approaches that skip the isolation step are, therefore, of interest for pathogen surveillance. Here, we evaluated the viability of amplicon sequencing of the full-length 16S rRNA gene for serotyping Salmonella enterica and E. coli. We developed a novel algorithm for serotype prediction, implemented as an R package (Seroplacer), which takes as input full-length 16S rRNA gene sequences and outputs serovar predictions after phylogenetic placement into a reference phylogeny. We achieved over 89% accuracy in predicting Salmonella serotypes on in silico test data and identified key pathogenic serovars of Salmonella and E. coli in isolate and environmental test samples. Although serotype prediction from 16S rRNA gene sequences is not as accurate as serotype prediction from WGS of isolates, the potential to identify dangerous serovars directly from amplicon sequencing of environmental samples is intriguing for pathogen surveillance. The capabilities developed here are also broadly relevant to other applications where intraspecies variation and direct sequencing from environmental samples could be valuable.IMPORTANCEIn order to prevent and stop outbreaks of foodborne pathogens, it is important that we can detect when pathogenic bacteria are present in a food or food-associated site and identify connections between specific pathogenic bacteria present in different samples. In this work, we develop a new computational technology that allows the important foodborne pathogens Escherichia coli and Salmonella enterica to be serotyped (a subspecies level classification) from sequencing of a single-marker gene, and the 16S rRNA gene often used to surveil bacterial communities. Our results suggest current limitations to serotyping from 16S rRNA gene sequencing alone but set the stage for further progress that we consider likely given the rapid advance in the long-read sequencing technologies and genomic databases our work leverages. If this research direction succeeds, it could enable better detection of foodborne pathogens before they reach the public and speed the resolution of foodborne pathogen outbreaks.

MCT1 gene silencing enhances the immune effect of dendritic cells on cervical cancer cells.

Dendritic cells (DCs) are a key class of immune cells that migrate to the draining lymph nodes and present processed antigenic peptides to lymphocytes after being activated by external stimuli, thereby establishing adaptive immunity. Moreover, DCs play an important role in tumor immunity. The aim of the study was to investigate whether MCT1 gene silencing in DCs affects their ability to mount an immune response against cervical cancer cells. We silenced the expression of MCT1 in DCs from mouse bone marrow (BM) by infection with adenovirus. The surface antigen profile of DCs was analyzed by flow cytometry and cytokine secretion was evaluated using enzyme-linked immunosorbent assay (ELISA) following sodium lactate (sLA) exposure and lipopolysaccharide (LPS) stimulation. Then, various groups of DC-induced cytotoxic T lymphocytes (CTLs) were prepared and their cytotoxicity against U14 was tested. Without sLA exposure, silencing MCT1 did not affect the expression of CD1a, CD80, CD83, CD86, and major histocompatibility complex class II (MHCII) in DCs after LPS challenge. Similar results were found for interleukin (IL)-6, IL-12 p70 and tumor necrosis factor alpha (TNF-α). After sLA exposure, silencing MCT1 significantly decreased the expression of CD1a, CD80, CD83, CD86, and MHCII in DCs after the LPS challenge, as well as the secretion of IL-6, IL-12 p70 and TNF-α. In addition, sLA exposure significantly reduced the toxicity and inhibited the proliferation of DC-induced CTLs compared to U14 cells in vitro and in vivo. However, silencing MCT1 significantly attenuated the changes caused by sLA exposure. At the same time, in the absence of sLA, silencing MCT1 did not affect the toxicity nor inhibit the proliferation of DC-induced CTLs on U14 cells. Lactate exposure reduces the immune effect of DCs on cervical cancer cells, but MCT1 gene silencing attenuates these alterations.

Effect of low-intensity pulsed ultrasound on osteogenic differentiation of human induced membrane-derived cells in Masquelet technique

IntroductionThe Masquelet technique is a relatively new method for large bone defect treatment. In this technique, grafted bone tissue is used, and after the cement is removed, the induced membrane (IM; that form around the cement spacers placed in the bone defect region) is thought to play an important role in promoting bone formation. On the other hand, low-intensity pulsed ultrasound (LIPUS) is known to promote fracture healing and angiogenesis through mechanical stimulation. This study aimed to investigate the in vitro effects of LIPUS on the osteogenic differentiation of human induced membrane-derived cells (IMCs). MethodsSeven patients who had been treated using the Masquelet technique were enrolled. The IM was harvested during the second stage of the technique. IMCs were isolated, cultured in growth medium, and then divided into two groups: (1) control group, IMCs cultured in osteogenic medium without LIPUS, and (2) LIPUS group, IMCs cultured in osteogenic medium with LIPUS treatment. Adherent cells from the IM samples were harvested after the first passage and evaluated for cell surface protein expression using immunostaining. A cell proliferation assay was used to count the number of IMCs using a hemocytometer. Osteogenic differentiation capability was assessed using an alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, and real-time reverse transcription-polymerase chain reaction. ResultsCell surface antigen profiling revealed that the IMCs contained cells positive for the mesenchymal stem cell–related markers CD73, CD90, and CD105. No significant difference in cell numbers was found between the control and LIPUS groups. The ALP activity of IMCs in the LIPUS group was significantly higher than that in the control group on days 7 and 14. Alizarin red S staining intensity was significantly higher in the LIPUS group than in the control group on day 21. Runx2 and VEGF expression was significantly upregulated on days 7 and 14, respectively, compared with levels in the control group. ConclusionWe demonstrated the significant effect of LIPUS on the osteogenic differentiation of human IMCs. This study indicates that LIPUS can be used as an additional tool for the enhancement of the healing process of the Masquelet technique.

Bone marrow-derived dedifferentiated fat cells exhibit similar phenotype as bone marrow mesenchymal stem cells with high osteogenic differentiation and bone regeneration ability

BackgroundMesenchymal stem cells (MSCs) are known to have different differentiation potential depending on the tissue of origin. Dedifferentiated fat cells (DFATs) are MSC-like multipotent cells that can be prepared from mature adipocytes by ceiling culture method. It is still unknown whether DFATs derived from adipocytes in different tissue showed different phenotype and functional properties. In the present study, we prepared bone marrow (BM)-derived DFATs (BM-DFATs), BM-MSCs, subcutaneous (SC) adipose tissue-derived DFATs (SC-DFATs), and adipose tissue-derived stem cells (ASCs) from donor-matched tissue samples. Then, we compared their phenotypes and multilineage differentiation potential in vitro. We also evaluated in vivo bone regeneration ability of these cells using a mouse femoral fracture model.MethodsBM-DFATs, SC-DFATs, BM-MSCs, and ASCs were prepared from tissue samples of knee osteoarthritis patients who received total knee arthroplasty. Cell surface antigens, gene expression profile, and in vitro differentiation capacity of these cells were determined. In vivo bone regenerative ability of these cells was evaluated by micro-computed tomography imaging at 28 days after local injection of the cells with peptide hydrogel (PHG) in the femoral fracture model in severe combined immunodeficiency mice.ResultsBM-DFATs were successfully generated at similar efficiency as SC-DFATs. Cell surface antigen and gene expression profiles of BM-DFATs were similar to those of BM-MSCs, whereas these profiles of SC-DFATs were similar to those of ASCs. In vitro differentiation analysis revealed that BM-DFATs and BM-MSCs had higher differentiation tendency toward osteoblasts and lower differentiation tendency toward adipocytes compared to SC-DFATs and ASCs. Transplantation of BM-DFATs and BM-MSCs with PHG enhanced bone mineral density at the injection sites compared to PHG alone in the mouse femoral fracture model.ConclusionsWe showed that phenotypic characteristics of BM-DFATs were similar to those of BM-MSCs. BM-DFATs exhibited higher osteogenic differentiation potential and bone regenerative ability compared to SC-DFATs and ASCs. These results suggest that BM-DFATs may be suitable sources of cell-based therapies for patients with nonunion bone fracture.

Peripheral immune cell profiling of double-hit lymphoma by mass cytometry

BackgroundDouble-hit or Triple-hit lymphoma (DHL/THL) is a subset of high-grade B cell lymphoma harboring rearrangements of MYC and BCL2 and/or BCL6, and usually associate with aggressive profile, while current therapies tend to provide poor clinical outcomes and eventually relapsed. Further explorations of DHL at cellular and molecular levels are in demand to offer guidance for clinical activity.MethodsWe collected the peripheral blood of DHL patients and diffused large B cell lymphoma (DLBCL) patients from single institute and converted them into PBMC samples. Mass cytometry was then performed to characterize these samples by 42 antibody markers with samples of healthy people as control. We divided the immune cell subtypes based on the expression profile of surface antigens, and the proportion of each cell subtype was also analyzed. By comparing the data of the DLBCL group and the healthy group, we figured out the distinguished immune cell subtypes of DHL patients according to their abundance and marker expression level. We further analyzed the heterogeneity of DHL samples by pairwise comparison based on clinical characteristics.ResultsWe found double-positive T cells (DPT) cells were in a significantly high percentage in DHL patients, whereas the ratio of double-negative T cells (DNT) was largely reduced in patients. Besides, CD38 was uniquely expressed at a high level on some naïve B cells of DHL patients, which could be a marker for the diagnosis of DHL (distinguishing from DLBCL), or even be a drug target for the treatment of DHL. In addition, we illustrated the heterogeneity of DHL patients in terms of immune cell landscape, and highlighted TP53 as a major factor that contributes to the heterogeneity of the T cells profile.ConclusionOur study demonstrated the distinct peripheral immune cell profile of DHL patients by contrast to DLBCL patients and healthy people, as well as the heterogeneity within the DHL group, which could provide valuable guidance for the diagnosis and treatment of DHL.

Elucidation of adipogenic differentiation regulatory mechanism in human maxillary/mandibular bone marrow-derived stem cells

ObjectiveThis study aims to investigate the underlying molecular mechanisms that regulate the adipogenic differentiation of maxillary/mandibular bone marrow-derived mesenchymal stem cells (MBMSCs). DesignMBMSCs and iliac bone marrow-derived MSCs (IBMSCs) were compared for osteogenic, chondrogenic, and adipogenic differentiation. Cell surface antigen expression was examined using flow cytometry, and stem cell marker expression was assessed using real-time polymerase chain reaction (PCR). Various adipogenic regulatory factors’ expression was evaluated using real-time PCR and western blotting. ResultsNo significant differences in cell surface antigen profiles or stem cell marker expression in MBMSCs and IBMSCs were observed. MBMSCs and IBMSCs displayed similar osteogenic and chondrogenic potentials, whereas MBMSCs showed significantly lower adipogenic potentials than those shown by IBMSCs. Expression of CCAAT/enhancer binding protein β (C/EBPβ), C/EBPδ, early B-cell factor 1 (Ebf-1), and Krüppel-like factor 5 (KLF5), which are early adipogenic differentiation factors, was suppressed in MBMSCs compared to that in IBMSCs. Peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα, which play important roles in the terminal differentiation of adipocytes, was lower in MBMSCs than that in IBMSCs. Furthermore, the level of zinc finger protein 423 (Zfp423), which is involved in the commitment of undifferentiated MSCs to the adipocyte lineage, was significantly lower in MBMSCs than that in IBMSCs. ConclusionsMBMSCs are negatively regulated in the commitment of undifferentiated MSCs to the adipocyte lineage (preadipocytes) as well as in the terminal differentiation of preadipocytes into mature adipocytes. These results may elucidate the site-specific characteristics of MBMSCs.

Comparison of the yields and properties of dedifferentiated fat cells and mesenchymal stem cells derived from infrapatellar fat pads.

Infrapatellar fat pad (IFP)-derived mesenchymal stem cells (MSCs) have high chondrogenic potential and are attractive cell sources for cartilage regeneration. During ceiling culture to acquire the characteristics of MSCs, mature adipocytes from fat tissue are known to undergo dedifferentiation, generating dedifferentiated fat (DFAT) cells. The purpose of the present study was to compare the yields and biological properties of IFP-derived MSCs and IFP-derived DFAT cells. IFPs were harvested from the knees of 8 osteoarthritis (OA) patients. DFAT cells were obtained using a ceiling culture of adipocytes isolated from the floating top layer of IFP digestion. MSCs were obtained by culturing precipitated stromal vascular fraction cells. We compared the P0 cell yields, surface antigen profile, colony formation ability, and multipotency of DFAT cells and MSCs. The P0 cell yields per flask and the estimated total cell yields from 1g of IFP were much greater for MSCs than for DFAT cells. Both MSCs and DFAT cells were positive for MSC markers. No obvious difference was observed in colony formation ability. In differentiation assays, DFAT cells produced greater amounts of lipid droplets, calcified tissue, and glycosaminoglycan than MSCs did. Adipogenic and chondrogenic gene expressions were upregulated in DFAT cells. IFP-derived DFAT cells showed higher adipogenic and chondrogenic potentials than IFP-derived MSCs, but they had a poor cell yield.

Clinical Safety of NK Cell in the Prevention of Leukemia Relapse Post-transplantation and in Treatment of the Elderly Leukemia Patients

To observe the safety of donor NK cell infusions in the settings of hematopoietic stem cell transplantation and after consolidation chemotherapy in elderly patients with acute myeloid leukemia (AML). Forty patients with AML were included, in which 21 patients aged over 60 years were at the stage of complete remission (CR) and 19 patients that received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mononucleated cells were isolated from peripheral blood from the donors (for allo-HSCT) or healthy immediate family members (elderly AML). The cells were seeded into the flasks pre-coated with NK cell specific activators, and expanded in media containing recombinant human IL-15 and IL-2 for 14 days. The cells were transfused intravenously after the identification of quality control. Trypan blue exclusion test was used for the determination of cell viability and counting. Flow cytometry analysis was performed to assess the surface antigenic profile. Seventy-eight infusions of the cell products were received by the elderly patients with AML after consolidation chemotherapy, 11 infusions were received by the patients during allo-HSCT and 32 infusions 3 moths after transplantation. The safety of cell therapy, body temperature, blood pressure and other indexes were observe during and 48 hours after cell transfusion. Meanwhile, the occurrence and severity of acute graft-versus-host disease (GVHD) were documented. Flow cytometry analysis showed that the proportion of NK cells (CD3-CD56+) in the mononucleated cells before culture was (14.10±4.22)% (n=121), and the proportion increased dramatically up to (87.29±8.75)% (n=121) after culture for 14 days, the number of NK cells increased to 753.47±140.13 times (n=121). The doses of the infused NK cells was (7.58±2.50)×107/kg per infusion. Moderate fever occurred in three cases after multiple infusions, and the temperature restored to normal on the same day after treatment. Fever was observed in one patient after every infusion of four times in total. The temperature reached to 38.5-39.0 ℃ and returned to normal within 1-2 hours after adequate antipyretic treatment, and then there was no discomfort. No GVHD was observed in the elderly AML patients, while 6 cases that received allo-HSCT developed moderate acute GVHD, among them grade I in 5 cases and grade II in 1 case. No other severe toxicities were observed. NK cell products with a high-purity could be obtained by ex vivo expansion with this protocol. The transfusion of these expanded cells is generally safe in the elderly patients with AML that have received chemotherapy or patients that received hematopoietic stem cell transplantation.

Characterization of adipose tissue-derived stromal cells of mice with nonalcoholic fatty liver disease and their use for liver repair.

IntroductionFreshly isolated uncultured adipose tissue-derived stromal cells (u-ADSCs), containing miscellaneous cells like the relatively abundant mesenchymal stem cells, are attractive for repair and regenerative therapy. However, the detailed characteristics and therapeutic efficacy of u-ADSCs obtained from disease-affected hosts are unknown. We compared the properties of u-ADSCs obtained from wild-type mice and from a mouse model of non-alcoholic steatohepatitis (NASH).MethodsThe NASH model was established by feeding C57BL/6J mice an atherogenic high-fat diet for 4 (NASH (4w)) or 12 weeks (NASH (12w)), followed by the isolation and characterization of u-ADSCs. Wild-type u-ADSCs or NASH-derived u-ADSCs were administered to mice with NASH cirrhosis, followed by analyses of hepatic inflammatory cells, antigen profiles, fibrosis, and gene expression.ResultsWild-type u-ADSCs and NASH-derived u-ADSCs did not show marked differences in surface antigen profiles. In NASH (4w) u-ADSCs, but not NASH (12w) u-ADSCs, the frequencies of the leukocyte markers CD11b, CD45, and CD44 were elevated; furthermore, we observed an increase in the M1/M2 macrophage ratio only in NASH (12w) u-ADSCs. Only in NASH-4w u-ADSCs, the expression levels cell cycle-related genes were higher than those in u-ADSCs. Wild-type u-ADSCs administered to mice with NASH-related cirrhosis decreased the infiltration of CD11b+, F4/80+, and Gr-1+ inflammatory cells, ameliorated fibrosis, and had a restorative effect on liver tissues, as determined by gene expression profiles and the NAFLD activity score. The therapeutic effects of NASH (4w) u-ADSCs and NASH (12w) u-ADSCs on NASH-related cirrhosis were highly similar to the effect of wild-type u-ADSCs, including reductions in inflammation and fibrosis.ConclusionsNASH-derived u-ADSCs, similar to wild-type u-ADSCs, are applicable for reparative and regenerative therapy in mice with NASH.

Mitochondrial DNA Mutations Distinguish Individual Donor- and Recipient-Derived Immune Cells Following Matched Unrelated Allogeneic Stem Cell Transplantation

▪Reconstitution of donor hematopoiesis after allogeneic hematopoietic stem cell transplantation (HSCT) forms the basis for effective graft-versus-leukemia responses, but mixed chimerism is not an infrequent outcome. How the donor and host hematopoietic system interact under conditions of mixed chimerism remains incompletely understood. Multi-modal single cell sequencing platforms are increasingly available and provide information regarding cell identities and interactions at high resolution. However, the analysis of post-transplant immune reconstitution requires consistent distinguishing of donor- and recipient-derived cells, which for sparse single cell sequencing data until now has remained a challenge.Recently, mitochondrial DNA (mtDNA) mutations have been recognized for their potential as personal genetic barcodes that can be detected with mitochondrial single cell assay for transposase-accessible chromatin with sequencing (mtscATAC-seq). We hypothesized that individual-specific mtDNA mutations could provide a sensitive and robust approach for distinguishing donor- from recipient-derived cells, and therefore tested this approach on bone marrow (BM) samples from patients with relapsed acute myeloid leukemia (AML) post-HSCT. We employed ATAC with select cell surface antigen profiling by sequencing (ASAP-seq), which enables the detection of mtDNA mutations within distinct surface marker-defined cell populations alongside chromatin accessibility. We selected serial samples collected from the ETCTN 10026 study, which tested combined decitabine (days 1-5, every 4 weeks, start cycle 0) and ipilimumab (day 1, every 4 weeks, start cycle 1) in relapsed AML post-HSCT. We focused on 13 samples (study entry, on treatment and disease progression) from 3 patients: AML1012 (HSCT from a matched related donor [MRD]), and AML1010 and AML1026 (matched unrelated donor [MUD]-HSCT).In total, we obtained 33,943 ASAP-seq profiles, including 3,283 single T cells. While clustering using single cell chromatin profiles alone only allowed identification of either CD4 + or CD8 + T cells, integration with surface marker expression enabled more detailed annotations of 8 T cell subpopulations and NK cells. Further, phenotypically distinct subpopulations such as CD57 + CD4 + and CD8 + T cells shared highly similar chromatin profiles, and 11.1% of CD4 + and 33.7% of CD8 + T cells would have been mislabeled based on clustering of chromatin profiles alone. Thus, ASAP-seq identified T cell subsets with markedly improved accuracy and resolution than scATAC-seq alone.Upon evaluation of mtDNA mutations to discriminate donor- and recipient-derived single T cells, we found that this was unreliable for MRD-HSCT (AML1012), but highly robust in the setting of MUD-HSCT (AML1010, AML1026), consistent with maternal inheritance of mitochondrial genomes. For the latter two patients, we identified 48 donor- and 26 recipient-specific mtDNA mutations, all with high heteroplasmy (range 82 - 99%). Presence of donor- and recipient-derived mtDNA mutations was mutually exclusive, and recipient-specific mtDNA mutations were also detectable in AML cells. Clinical bulk and mtDNA mutation-based single T cell chimerisms were highly correlated (r = 0.97).AML1010 had sustained complete T cell chimerism (>97%) during study treatment. In AML1026, the mtDNA mutation-based T cell chimerism rose from 55% to 71% after 1 cycle of decitabine and then remained stable until disease progression 3 months later. This was associated with increased percentage of donor-derived CD4 + T cells (45% [study entry] vs. 71% [after 1 cycle of decitabine], p < 0.01), while donor-derived CD8 + T cells remained unchanged at 76%. Across all studied timepoints in AML1026, donor versus recipient skewing was also highest in CD4 + T cell subsets, with fewer naïve (20% vs. 31%, p < 0.01) but more donor-derived CD57 + CD4 + T cells (13% vs. 3%, p < 0.01).We demonstrate that mtDNA mutations can discriminate between donor- and recipient-derived single cells, enabling detection and in-depth characterization of chimeric immune cell dynamics after MUD HSCT. This approach will allow to systematically dissect conditions of mixed chimerism in the post-transplant setting with larger studies. DisclosuresDeAngelo: Abbvie: Research Funding; Takeda: Consultancy; Servier: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Incyte: Consultancy; Forty-Seven: Consultancy; Autolus: Consultancy; Amgen: Consultancy; Agios: Consultancy; Blueprint: Research Funding; Glycomimetrics: Research Funding. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Sankaran: Cellarity: Consultancy; Forma: Consultancy; Novartis: Consultancy; Branch Biosciences: Consultancy; Ensoma: Consultancy. Soiffer: Jasper: Consultancy; Jazz Pharmaceuticals, USA: Consultancy; Precision Biosciences, USA: Consultancy; Juno Therapeutics, USA: Other: Data Safety Monitoring Board; Kiadis, Netherlands: Membership on an entity's Board of Directors or advisory committees; Rheos Therapeutics, USA: Consultancy; Gilead, USA: Other: Career Development Award Committee; NMPD - Be the Match, USA: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy. Garcia: Genentech: Research Funding; Prelude: Research Funding; Pfizer: Research Funding; AstraZeneca: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company. OffLabel Disclosure:ipilimumab to modulate anti-leukemia immunity in the post-transplant and transplant-naive context

Circulating extracellular vesicles are endowed with enhanced procoagulant activity in SARS-CoV-2 infection.

BackgroundCoronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents.MethodsWe analyzed serum-derived EV from 67 participants who underwent nasopharyngeal swabs molecular test for suspected SARS-CoV-2 infection (34 positives and 33 negatives) and from 16 healthy controls (HC), as referral. A sub-analysis was performed on subjects who developed pneumonia (n = 28). Serum-derived EV were characterized for their surface antigen profile and tested for their procoagulant activity. A validation experiment was performed pre-treating EV with anti-CD142 antibody or with recombinant FVIIa. Serum TNF-α levels were measured by ELISA.FindingsProfiling of EV antigens revealed a surface marker signature that defines circulating EV in COVID-19. A combination of seven surface molecules (CD49e, CD209, CD86, CD133/1, CD69, CD142, and CD20) clustered COVID (+) versus COVID (-) patients and HC. CD142 showed the highest discriminating performance at both multivariate models and ROC curve analysis. Noteworthy, we found that CD142 exposed onto surface of EV was biologically active. CD142 activity was higher in COVID (+) patients and correlated with TNF-α serum levels.InterpretationIn SARS-CoV-2 infection the systemic inflammatory response results in cell-release of substantial amounts of procoagulant EV that may act as clotting initiation agents, contributing to disease severity.FundingCardiocentro Ticino Institute, Ente ospedaliero Cantonale, Lugano-Switzerland.

Patient-derived organoids of bladder cancer recapitulate antigen expression profiles and serve as a personal evaluation model for CAR-T cells in vitro.

ObjectivesRecent advances in patient‐derived cancer organoids have opened a new avenue for personalised medicine. We aimed to establish an in vitro technological platform to evaluate chimeric antigen receptor (CAR)‐T cell‐mediated cytotoxicity against bladder cancer.MethodsPatient‐derived bladder cancer organoids (BCOs) were derived using classic medium containing R‐spondin 1 and noggin. The features of BCOs were characterised via H&E, whole‐exome sequencing and immunofluorescence of specific markers. Surface antigen expression profiles of the recently identified CAR‐recognisable targets were determined with a panel of antibodies via immunohistochemistry. A co‐cultivation system consisting of BCOs and engineered T cells targeting a specific antigen was utilised to test its efficacy to model immunotherapy by cytotoxic assays and ELISA.ResultsBladder cancer organoid lines of basal and luminal subtypes were established. The histopathological morphology, genomic alteration, and specific marker expression profiles showed that the BCO lines retained the characteristics of the original tumors. Among all tested CAR‐recognisable antigens in other solid tumors, MUC1 was simultaneously expressed in organoids and parental tumor tissues. Given the surface antigen profiles, second‐generation CAR‐T cells targeting MUC1 were prepared for modelling in vitro immunotherapy responses in BCOs. Specific immune cytotoxicity occurred only in the MUC1+ organoids but not in the MUC1‐ organoids or control CAR‐T cells.ConclusionPatient‐derived BCOs recapitulate the heterogeneity and key features of parental cancer tissues, and these BCOs could be useful for preclinical testing of CAR‐T cells in vitro.

Comprehensive Genetic Analysis Revealed Myeloid/Natural Killer (NK) Cell Precursor Acute Leukemia As a Novel Distinctive Leukemia Entity

Introduction: Myeloid/Natural killer (NK) cell precursor acute leukemia (MNKPL) is a rare hematologic malignancy prevalent in East Asia. MNKPL is characterized by extramedullary involvement, immature lymphoblastoid morphology without myeloperoxidase (MPO) reactivity, the CD7+/CD33+/CD34+/CD56+/HLA−DR+ phenotype. MNKPL is classified as mixed phenotype acute leukemia, and not otherwise specified rare types (MPAL NOS rare types) in WHO classification. However, its characteristic clinical feature and undetermined genetic feature suggests that MNPKL leaves open the possibility of a new independent disease concept. Here, we report clinical features and genetic alterations in patients with MNKPL. Methods: The Leukemia and Lymphoma Committee of the Japanese Society of Pediatric Hematology and Oncology (JSPHO) sent out questionnaires to 110 JSPHO affiliated hospitals and collected cases of MNPKL diagnosed during the period 2000-2013. Besides, the cases published as literature were recruited. The data of clinical features, cell surface antigen profiling, overall survival (OS), and event-free survival (EFS) defined as relapse or death were also collected as a secondary survey. The protocol of this retrospective study was approved by the review boards of JSPHO and Ehime Prefectural Central Hospital. Comprehensive genetic analysis including 13 whole-exome sequences (WES), 2 target sequence, 6 RNA sequence (RNA-seq), and 8 DNA methylation analysis was performed. We also performed single-cell RNA-seq using 1 sample of MNKPL patients and a normal bone marrow sample as the reference. The research protocol was approved by the review board of TMDU. Results: Sixteen children or young adults (&amp;lt; 39 years old) and 2 older adults with MNKPL were identified. The median age of MNKPL patients was 11 (0.5-75) years old. There are 12 males and 6 females. The extramedullary involvement was observed in 7 patients. Complete remission after induction therapy was achieved in 8/14 (57%) patients treated with acute myeloid leukemia (AML) type chemotherapy and 2/4 (50%) patients treated with acute lymphoblastic leukemia (ALL)/non-Hodgkin lymphoma type chemotherapy, respectively. Fifteen patients underwent hematopoietic cell transplantation (HCT). The median follow-up period was 3.8 (0.1-16.0) years. 5-year OS and 5-year EFS was 49.5% and 40.7%, respectively. In genetic analysis, median 388 somatic mutations in MNKPL were identified by WES. The recurrent mutations were observed in NOTCH1 (n=5), MAML3 (n=4), NRAS, MAP3K4, RECQL4, CREBBP, ASXL2, and KMT2D (n=3, respectively), and MAML2, MAP3K1, FLT3, CARD11, MSH4, FANCI, WT1, ZNF384, and ERG (n=2, respectively). The distinct expression pattern, higher expression of RUNX3 and NOTCH1, and lower expression of BCL11B were identified in MNKPL samples which were compared to MPAL, AML, and T cell ALL in RNA-seq. The distinct methylation profile, hypomethylation of RUNX3 regulatory region, and hypermethylation of BCL11B regulatory region were identified in DNA methylation analysis. Single-cell RNA-seq analysis also showed distinct 4 subsets of MNKPL. Discussion and Conclusions: NK cells are the founding member of a family of innate lymphoid cells (ILC). Genetic abnormality of NOTCH1 pathway is a hallmark of MNPKL. RUNX3 is required for NK cell survival and proliferation in response to IL-15 signaling. RUNX3 high expression and hypomethylation of RUNX3 regulatory region also characterize MNKPL. Currently, MNKPL is classified as MPAL NOS, our genetic analysis revealed that MNKPL is a distinct group from MPAL. The prognosis of MNKPL was not satisfactory even though HCT was performed. The development of new therapeutic approaches based on these genetic analyses is highly expected. Disclosures Saito: Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.

OR31-03 Single-Cell Profiling of Adult Human Ovarian Cortex Reveals Six Main Cell Types but No Germline Stem Cells

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been suggested that oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Our study aimed at comprehensive characterization of all cell types present in the ovarian cortex, including the previously reported oogonial stem cells. We developed methods to dissociate human ovarian cortex to a viable single cell solution allowing subsequent analysis by single cell transcriptomic profiling and cell surface antigen screening. In all analyses, cells captured by DDX4 antibodies (DDX4 Ab+) were included as a reference. High quality ovarian cortex tissue from gender reassignement and caesarean section patients was used in the analyses. Our single cell transcriptomic analyses based on >24,000 cells revealed the presence of six main cell types in ovarian cortex; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Surface marker screening showed robust expression of 43 cell surface antigens in ovarian cortex cells. With the help of transcriptomic and cell surface antigen profiles, the DDX4 Ab+ cells were identified as perivascular cells. This finding was validated by immunostaining of ovarian tissue showing DDX4 Ab+ cells lining CD31 positive endothelial cells of blood vessels. To search for germline stem cells on a broader front, we compared our data with human fetal ovary cells including pre-meiotic germ cells (Li et al. 2017) and found no evidence for the presence of germ line stem cells of any kind in adult human ovarian cortex. In summary, we provide the first cell map of human ovarian cortex. Our results demonstrate six main cell types, but cannot provide support to the existence of oogonial stem cells. This dataset will be a valuable tool for studying the role of specific cell populations in ovarian biology, dissecting causes of infertility, and developing novel assisted reproductive technologies or even contraceptives.

Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.

Conversion of mesenchymal stem cells into a canine hepatocyte-like cells by Foxa1 and Hnf4a.

IntroductionHepatocytes, which account for the majority of liver tissue, are derived from the endoderm and become hepatocytes via differentiation of hepatic progenitor cells. Induced hepatocyte-like (iHep) cells and artificial liver tissues are expected to become useful, efficient therapies for severe and refractory liver diseases and to contribute to drug discovery research. The establishment of iHep cell lines are needed to carry out liver transplants and assess liver toxicity in the rising number of dogs affected by liver disease. Recently, direct conversion of non-hepatocyte cells into iHep cells was achieved by transfecting mouse adult fibroblasts with the Forkhead box protein A1 (Foxa1) and hepatocyte nuclear factor 4 homeobox alpha (Hnf4α) genes. Here, we applied this conversion process for the differentiation of canine bone marrow stem cells (cBMSCs) into hepatocyte-like cells.MethodsBone marrow specimens were collected from four healthy Beagle dogs and used to culture cBMSCs in Dulbecco's Modified Eagle's Medium (DMEM). The cBMSCs displayed the following characteristic features: plastic adherence; differentiation into adipocytes, osteoblasts and chondrocytes; and a cell surface antigen profile of CD29 (+), CD44 (+), CD90 (+), CD45 (−), CD34 (−) and CD14 (−), or CD11b (−) and CD79a (−), or CD19 (−) and HLA class II(−). The cBMSCs were seeded in a collagen I-coated plate and cultured in DMEM with 10% fetal bovine serum and transfected with retroviruses expressing Foxa1 and Hnf4α the following day. Canine iHep cells were differentiated from cBMSCs in culture on day 10, and were analyzed for morphology, RNA expression, immunocytochemistry, urea production, and low-density lipoprotein (LDL) metabolism.ResultsThe cBMSCs expressed CD29 (98.06 ± 1.14%), CD44 (99.59 ± 0.27%) and CD90 (92.78 ± 4.89%), but did not express CD14 (0.47 ± 0.29%), CD19 (0.44 ± 0.39%), CD34 (0.33 ± 0.25%), CD45 (0.46 ± 0.34%) or MHC class II (0.54 ± 0.40%). The iHep cells exhibited morphology that included circular to equilateral circular shapes, and the formation of colonies that adhered to each other 10 days after Foxa1 and Hnf4α transfection. Quantitative RT-PCR analysis showed that the expression levels of the genes encoding albumin (ALB) and cadherin (CDH) in iHep cells on day 10 were increased approximately 100- and 10,000-fold, respectively, compared with cBMSCs. Corresponding protein expression of ALB and epithelial-CDH was confirmed by immunocytochemistry. Important hepatic functions, including LDL metabolic ability and urea production, were increased in iHep cells on day 10.ConclusionWe successfully induced cBMSCs to differentiate into functional iHep cells. To our knowledge, this is the first report of canine liver tissue differentiation using Foxa1 and Hnf4α gene transfection. Canine iHep cells are expected to provide insights for the construction of liver models for drug discovery research and may serve as potential therapeutics for canine liver disease.

Parallel comparison of fibroblast-like synoviocytes from the surgically removed hyperplastic synovial tissues of rheumatoid arthritis and osteoarthritis patients

BackgroundFibroblast-like synoviocytes (FLS) are essential cellular components in inflammatory joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Despite the growing use of FLS isolated from OA and RA patients, a detailed functional and parallel comparison of FLS from these two types of arthritis has not been performed.MethodsIn the present study, FLS were isolated from surgically removed synovial tissues from twenty-two patients with OA and RA to evaluate their basic cellular functions.ResultsPure populations of FLS were isolated by a sorting strategy based on stringent marker expression (CD45−CD31−CD146−CD235a−CD90+PDPN+). OA FLS and RA FLS at the same passage (P2-P4) exhibited uniform fibroblast morphology. OA FLS and RA FLS expressed a similar profile of cell surface antigens, including the fibroblast markers VCAM1 and ICAM1. RA FLS showed a more sensitive inflammatory status than OA FLS with regard to proliferation, migration, apoptosis, inflammatory gene expression and pro-inflammatory cytokine secretion. In addition, the responses of OA FLS and RA FLS to both the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and the anti-inflammatory drug methotrexate (MTX) were also evaluated here.ConclusionThe parallel comparison of OA FLS and RA FLS lays a foundation in preparation for when FLS are considered a potential therapeutic anti-inflammatory target for OA and RA.

Identification of Cell Surface Targets for CAR T Cell Immunotherapy.

Immunotherapy has become a prominent approach for the treatment of cancer. Targeted killing of malignant cells by adoptive transfer of chimeric antigen receptor (CAR) T cells is a promising immunotherapy technique in oncology. However, the identification of cell surface antigens unique to tumor cells against which CAR T cells can be engineered has historically been challenging and not well documented in solid tumors. Here, we describe a generalized method to construct a cell subtype-specific surface antigen profile (i.e., surfaceome) from cell lines and identify high-confidence antigens as effective targets for CAR T cell therapy by integrating transcriptomics and cell surface proteomics. This method is widely applicable to all therapies utilizing CAR T cells, such as cancer, as well as infectious and autoimmune diseases.

Generation of bioartificial hearts using decellularized scaffolds and mixed cells

BackgroundPatients with end-stage heart failure must receive treatment to recover cardiac function, and the current primary therapy, heart transplantation, is plagued by the limited supply of donor hearts. Bioengineered artificial hearts generated by seeding of cells on decellularized scaffolds have been suggested as an alternative source for transplantation. This study aimed to develop a tissue-engineered heart with lower immunogenicity and functional similarity to a physiological heart that can be used for heart transplantation.Materials and methodsWe used sodium dodecyl sulfate (SDS) to decellularize cardiac tissue to obtain a decellularized scaffold. Mesenchymal stem cells (MSCs) were isolated from rat bone marrow and identified by flow cytometric labeling of their surface markers. At the same time, the multi-directional differentiation of MSCs was analyzed. The MSCs, endothelial cells, and cardiomyocytes were allowed to adhere to the decellularized scaffold during perfusion, and the function of tissue-engineered heart was analyzed by immunohistochemistry and electrocardiogram.ResultsMSCs, isolated from rats differentiated into cardiomyocytes, were seeded along with primary rat cardiomyocytes and endothelial cells onto decellularized rat heart scaffolds. We first confirmed the pluripotency of the MSCs, performed immunostaining against cardiac markers expressed by MSC-derived cardiomyocytes, and completed surface antigen profiling of MSC-derived endothelial cells. After cell seeding and culture, we analyzed the performance of the bioartificial heart by electrocardiography but found that the bioartificial heart exhibited abnormal electrical activity. The results indicated that the tissue-engineered heart lacked some cells necessary for the conduction of electrical current, causing deficient conduction function compared to the normal heart.ConclusionOur study suggests that MSCs derived from rats may be useful in the generation of a bioartificial heart, although technical challenges remain with regard to generating a fully functional bioartificial heart.

GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells

BackgroundHuman mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines.MethodsIn the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium).ResultsThe results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum.ConclusionsThe findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine.Graphical abstract