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A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here,we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform,under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm²,where they attach,migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521,in combination with E-cadherin,allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities. View Publication

BACKGROUND: Induced pluripotent stem cells (iPSCs) can be differentiated into potentially unlimited lineages of cell types for use in autologous cell therapy. However,the efficiency of the differentiation procedure and subsequent function of the iPSC-derived cells may be influenced by epigenetic factors that the iPSCs retain from their tissues of origin; thus,iPSC-derived cells may be more effective for treatment of myocardial injury if the iPSCs were engineered from cardiac-lineage cells,rather than dermal fibroblasts. METHODS AND RESULTS: We show that human cardiac iPSCs (hciPSCs) can be generated from cardiac fibroblasts and subsequently differentiated into exceptionally pure (textgreater92%) sheets of cardiomyocytes (CMs). The hciPSCs passed through all the normal stages of differentiation before assuming a CM identity. When using the fibrin gel-enhanced delivery of hciPSC-CM sheets at the site of injury in infarcted mouse hearts,the engraftment rate was 31.91%+/-5.75% at Day 28 post transplantation. The hciPSC-CM in the sheet also appeared to develop a more mature,structurally aligned phenotype 28 days after transplantation and was associated with significant improvements in cardiac function,vascularity,and reduction in apoptosis. CONCLUSIONS: These data strongly support the potential of hciPSC-CM sheet transplantation for the treatment of heart with acute myocardial infarction. View Publication

Regeneration of human cartilage is inherently inefficient; an abundant autologous source,such as human induced pluripotent stem cells (hiPSCs),is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks,with an overall efficiency textgreater90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression,extracellular matrix production,and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition,2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance,autologous nature,and potential to generate articular-like cartilage rather than fibrocartilage. In addition,hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening. View Publication

Rheumatoid Arthritis (RA) causes chronic inflammation of joints. The cytokines TNFalpha and IFNgamma are central players in RA,however their source has not been fully elucidated. Natural Killer (NK) cells are best known for their role in elimination of viral-infected and transformed cells,and they secrete pro-inflammatory cytokines. NK cells are present in the synovial fluids (SFs) of RA patients and are considered to be important in bone destruction. However,the phenotype and function of NK cells in the SFs of patients with erosive deformative RA (DRA) versus non-deformative RA (NDRA) is poorly characterized. Here we characterize the NK cell populations present in the blood and SFs of DRA and NDRA patients. We demonstrate that a distinct population of activated synovial fluid NK (sfNK) cells constitutes a large proportion of immune cells found in the SFs of DRA patients. We discovered that although sfNK cells in both DRA and NDRA patients have similar phenotypes,they function differently. The DRA sfNK secrete more TNFalpha and IFNgamma upon exposure to IL-2 and IL-15. Consequently,we suggest that sfNK cells may be a marker for more severely destructive RA disease. View Publication

INTRODUCTION The stem cell portion of the dental pulp derived cultures (DPSCs) showed a higher resistance to cytotoxic effect of restorative dental materials compared to pulpal fibroblasts (DPFs). Here,we aimed to compare the expression of some drug resistant genes between these cells. METHODS AND MATERIALS To separate DPSCs from DPFs,we used magnetic cell sorting technique based on CD146 expression. To assess the stem cell properties,the positive and negative portions underwent colony forming assays and were induced to be differentiated into the adipocytes,osteoblasts,hepatocytes,and neural cells. Cell surface antigen panels were checked using immune fluorescence and flow-cytometry techniques. The mRNA expression of 14 ABC transporters including ABCA2,ABCB1,ABCB11,ABCC1,ABCC2,ABCC3,ABCC4,ABCC5-2,ABCC5-4,ABCC5-13,ABCC6,ABCC10,ABCC11,and ABCG2 genes was assessed,using quantitative RT-PCR technique. RESULTS Only the CD146 positive portion could be differentiated into the desired fates,and they formed higher colonies (16.7 ± 3.32 vs. 1.7 ± 1.67,p {\textless} .001). The cell surface antigen panels were the same,except for CD146 and STRO-1 markers which were expressed only in the positive portion. Among the ABC transporter genes studied,the positive portion showed a higher expression (approximately two-fold) of ABCA2,ABCC5-13,and ABCC5-2 genes. CONCLUSION Dental pulp stem cells which can be separated from dental pulp fibroblasts based on CD146 expression,express higher levels of some drug resistance genes which probably accounts for their features of more resistance to cytotoxic effects of some dental materials. This needs to be more validated in future. View Publication

BACKGROUND CD47/SIRP$\alpha$ axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer,particularly in hematological malignancies. In preclinical models,CD47/SIRP$\alpha$ blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here,we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. METHODS The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry,transcriptomic analysis,and in vivo immune-cell depletion experiments. RESULTS We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells,monocytes,dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably,molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation,including IFN$\gamma$ and IL-12b. Moreover,TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701,highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition,NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro,resulting in an increased cross-priming of tumor-specific CD8 T cells. CONCLUSIONS The study described the mechanisms afforded by the CD47xCD19 bispecific antibody,NI-1701,at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035). View Publication

BACKGROUND Specific immune response is a hallmark of cancer immunotherapy and shared tumor-associated antigens (TAAs) are important targets. Recent advances using combined cellular therapy against multiple TAAs renewed the interest in this class of antigens. Our study aims to determine the role of TAAs in esophago-gastric adenocarcinoma (EGA). METHODS RNA expression was assessed by NanoString in tumor samples of 41 treatment-na{\{i}}ve EGA patients. Endogenous T cell and antibody responses against the 10 most relevant TAAs were determined by FluoroSpot and protein-bound bead assays. Digital image analysis was used to evaluate the correlation of TAAs and T-cell abundance. T-cell receptor sequencing in vitro expansion with autologous CD40-activated B cells (CD40Bs) and in vitro cytotoxicity assays were applied to determine specific expansion clonality and cytotoxic activity of expanded T cells. RESULTS 68.3% of patients expressed ??5 TAAs simultaneously with coregulated clusters which were similar to data from The Cancer Genome Atlas (n=505). Endogenous cellular or humoral responses against ??1??TAA were detectable in 75.0% and 53.7% of patients respectively. We found a correlation of T-cell abundance and the expression of TAAs and genes related to antigen presentation. TAA-specific T-cell responses were polyclonal could be induced or enhanced using autologous CD40Bs and were cytotoxic in vitro. Despite the frequent expression of TAAs co-occurrence with immune responses was rare. CONCLUSIONS We identified the most relevant TAAs in EGA for monitoring of clinical trials and as therapeutic targets. Antigen-escape rather than missing immune response should be considered as mechanism underlying immunotherapy resistance of EGA." View Publication

Despite the key role that antibodies play in protection,the cellular processes mediating the acquisition of humoral immunity against malaria are not fully understood. Using an infection model of severe malaria,we find that germinal center (GC) B cells upregulate the transcription factor T-bet during infection. Molecular and cellular analyses reveal that T-bet in B cells is required not only for IgG2c switching but also favors commitment of B cells to the dark zone of the GC. T-bet was found to regulate the expression of Rgs13 and CXCR3,both of which contribute to the impaired GC polarization observed in the absence of T-bet,resulting in reduced IghV gene mutations and lower antibody avidity. These results demonstrate that T-bet modulates GC dynamics,thereby promoting the differentiation of B cells with increased affinity for antigen. View Publication

AbstractEncouraged by the observations of significant B7-H3 protein overexpression in many human solid tumors compared to healthy tissues,we directed our focus towards targeting B7-H3 using chimeric antigen receptor (CAR) T cells. We utilized a nanobody as the B7-H3–targeting domain in our CAR construct to circumvent the stability issues associated with single-chain variable fragment–based domains. In efforts to expand patient access to CAR T-cell therapy,we engineered our nanobody-based CAR into human Epstein-Barr virus–specific T cells (EBVST),offering a readily available off-the-shelf treatment. B7H3.CAR-armored EBVSTs demonstrated potent in vitro and in vivo activities against multiple B7-H3–positive human tumor cell lines and patient-derived xenograft models. Murine T cells expressing a murine equivalent of our B7H3.CAR exhibited no life-threatening toxicities in immunocompetent mice bearing syngeneic tumors. Further in vitro evaluation revealed that while human T,B,and natural killer cells were unaffected by B7H3.CAR EBVSTs,monocytes were targeted because of upregulation of B7-H3. Such targeting of myeloid cells,which are key mediators of cytokine release syndrome (CRS),contributed to a low incidence of CRS in humanized mice after B7H3.CAR EBVST treatment. Notably,we showed that B7H3.CAR EBVSTs can target B7-H3–expressing myeloid-derived suppressor cells (MDSC),thereby mitigating MDSC-driven immune suppression. In summary,our data demonstrate that our nanobody-based B7H3.CAR EBVSTs are effective as an off-the-shelf therapy for B7-H3–positive solid tumors. These cells also offer an avenue to modulate the immunosuppressive tumor microenvironment,highlighting their promising clinical potential in targeting solid tumors.Significance:Clinical application of EBVSTs armored with B7-H3–targeting CARs offer an attractive solution to translate off-the-shelf CAR T cells as therapy for solid tumors. View Publication

Myeloperoxidase (MPO) is found almost exclusively in granulocytes and immature myeloid cells. It plays a key role in the innate immune system,catalysing the formation of reactive oxygen species that are important in anti‐microbial action,but MPO also oxidatively transforms the topoisomerase II (TOP2) poison etoposide to chemical forms that have elevated DNA damaging properties. TOP2 poisons such as etoposide are widely used anti‐cancer drugs,but they are linked to cases of secondary acute myeloid leukaemias through a mechanism that involves DNA damage and presumably erroneous repair leading to leukaemogenic chromosome translocations. This leads to the possibility that myeloperoxidase inhibitors could reduce the rate of therapy‐related leukaemia by protecting haematopoietic cells from TOP2 poison‐mediated genotoxic damage while preserving the anti‐cancer efficacy of the treatment. We show here that myeloperoxidase inhibition reduces etoposide‐induced TOP2B‐DNA covalent complexes and resulting DNA double‐strand break formation in primary ex vivo expanded CD34 + progenitor cells and unfractionated bone marrow mononuclear cells. Since MPO inhibitors are currently being developed as anti‐inflammatory agents this raises the possibility that repurposing of these potential new drugs could provide a means of suppressing secondary acute myeloid leukaemias associated with therapies containing TOP2 poisons. View Publication

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with high death rates that have remained substantially unaltered for decades. Therefore,new treatment approaches are urgently needed. Human papillomavirus-negative tumors harbor areas of terminally differentiated tissue that are characterized by cornification. Dissecting this intrinsic ability of HNSCC cells to irreversibly differentiate into non-malignant cells may have tumor-targeting potential. We modeled the cornification of HNSCC cells in a primary spheroid model and analyzed the mechanisms underlying differentiation by ATAC-seq and RNA-seq. Results were verified by immunofluorescence using human HNSCC tissue of distinct anatomical locations. HNSCC cell differentiation was accompanied by cell adhesion,proliferation stop,diminished tumor-initiating potential in immunodeficient mice,and activation of a wound-healing-associated signaling program. Small promoter accessibility increased despite overall chromatin closure. Differentiating cells upregulated KRT17 and cornification markers. Although KRT17 represents a basal stem cell marker in normal mucosa,we confirm KRT17 to represent an early differentiation marker in HNSCC tissue. Cornification was frequently found surrounding necrotic areas in human tumors,indicating an involvement of pro-inflammatory stimuli. Indeed,inflammatory mediators activated the differentiation program in primary HNSCC cells. In HNSCC tissue,distinct cell differentiation states were found to create a common tissue architecture in normal mucosa and HNSCCs. Our data demonstrate a loss of cell malignancy upon faithful HNSCC cell differentiation,indicating that targeted differentiation approaches may be therapeutically valuable. Moreover,we describe KRT17 to be a candidate biomarker for HNSCC cell differentiation and early tumor detection. Subject terms: Cancer stem cells,Oral cancer View Publication

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells,making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis,Nampt,in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells,but not normal donor as well as MM patients PBMCs. Importantly,FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6,IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly,cytotoxicity of FK866 triggered autophagy,but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally,FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model,associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology,providing the basis for a novel targeted therapeutic approach. View Publication