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We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore,the results of the transcriptomic profile,coupled with immunostaining,and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells,hepatocytes like cells,and endothelial like cells. However,the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless,the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented. View Publication

Routine techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) include density centrifugation with Ficoll-Paque and isolation by cell preparation tubes (CPTs) and SepMate tubes with Lymphoprep. In a series of experiments,these three PBMC isolation techniques were compared for cell recovery and viability,PBMC population composition,and cell functionality,aiming to provide a starting basis for the selection of the most appropriate method of PBMC isolation for a specific downstream application. PBMCs were freshly isolated from venous blood of healthy male donors,applying the different techniques in parallel. Cell recovery and viability were assessed using a hemacytometer and trypan blue. Immunophenotyping was performed by flow cytometry. Cell functionality was assessed in stimulated (100 ng/mL staphylococcal enterotoxin B [SEB]) and unstimulated 24 hours PBMC cultures,with cytokine production and lactate dehydrogenase (LDH) release as readout measures. PBMC isolation by SepMate and CPT resulted in a 70% higher recovery than Ficoll isolation. CPT-isolated populations contained more erythrocyte contamination. Cell viability,assessed by trypan blue exclusion,was 100% for all three isolation techniques. SepMate and CPT isolation gave higher SEB-induced cytokine responses in cell cultures,for IFNγ and for secondary cytokines. IL-6 and IL-8 release in unstimulated cultures was higher for CPT-isolated PBMCs compared to Ficoll- and SepMate-isolated PBMCs. LDH release did not differ between cell isolation techniques. In addition to criteria such as cost and application practicalities,these data may support selection of a specific PBMC isolation technique for downstream analysis. View Publication

IntroductionHumanization is typically adopted to reduce the immunogenicity of murine antibodies generated by hybridoma technology when used in humans.MethodsTwo different strategies of antibody humanization are popularly employed,including “complementarity determining region (CDR) grafting” and “framework (FR) shuffling” to humanize a murine antibody against human programmed death-1 (PD-1),XM PD1. In CDR-grafting humanization,the CDRs of XM PD-1,were grafted into the human FR regions with high homology to the murine FR counterparts,and back mutations of key residues were performed to retain the antigen-binding affinities. While in FR-shuffling humanization,a combinatorial library of the six murine CDRs in-frame of XM PD-1 was constructed to a pool of human germline FRs for high-throughput screening for the most favorable variants. We evaluated many aspects which were important during antibody development of the molecules obtained by the two methods,including antibody purity,thermal stability,binding efficacy,predicted humanness,and immunogenicity,along with T cell epitope prediction for the humanized antibodies.ResultsWhile the ideal molecule was not achieved through CDR grafting in this particular instance,FR-shuffling proved successful in identifying a suitable candidate. The study highlights FR-shuffling as an effective complementary approach that potentially increases the success rate of antibody humanization. It is particularly noted for its accessibility to those with a biological rather than a computational background. DiscussionThe insights from this comparison are intended to assist other researchers in selecting appropriate humanization strategies for drug development,contributing to broader application and understanding in the field. View Publication

SummaryAntibodies represent a primary mediator of protection against respiratory viruses. Serum neutralizing antibodies (NAbs) are often considered a primary correlate of protection. However,detailed antibody profiles including characterization of antibody functions in different anatomic compartments are poorly understood. Here we show that antibody correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge are different in systemic versus mucosal compartments in rhesus macaques. In serum,NAbs were the strongest correlate of protection and linked to spike-specific binding antibodies and other extra-NAb functions that create a larger protective network. In bronchiolar lavage (BAL),antibody-dependent cellular phagocytosis (ADCP) proved the strongest correlate of protection rather than NAbs. Within BAL,ADCP was linked to mucosal spike-specific immunoglobulin (Ig)G,IgA/secretory IgA,and Fcγ-receptor binding antibodies. Our results support a model in which antibodies with different functions mediate protection at different anatomic sites. Graphical abstract Highlights•Correlates of protection to SARS-CoV-2 Omicron are highly compartment specific•Antibody effector functions are primary correlates of protection at infection site•Mucosal boosting enhances IgA and functionally levered IgG in lower respiratory tract Health sciences; Biological sciences View Publication

Caspase-directed apoptosis usually fragments cells,releasing nonfunctional,prothrombogenic,membrane-bound apoptotic bodies marked for rapid engulfment by macrophages. Blood platelets are functional anucleate cells generated by specialized fragmentation of their progenitors,megakaryocytes (MKs),but committed to a constitutive caspase-independent death. Constitutive formation of the proplatelet-bearing MK was recently reported to be caspase-dependent,apparently involving mitochondrial release of cytochrome c,a known pro-apoptogenic factor. We extend those studies and report that activation of caspases in MKs,either constitutively or after Fas ligation,yields platelets that are functionally responsive and evade immediate phagocytic clearance,and retain mitochondrial transmembrane potential until constitutive platelet death ensues. Furthermore,the exclusion from the platelet progeny of caspase-9 present in the progenitor accounts for failure of mitochondrial release of cytochrome c to activate caspase-3 during platelet death. Thus,progenitor cell death by apoptosis can result in birth of multiple functional anucleate daughter cells. View Publication

Mammary cancer stem cells (MaCSCs) have been identified as a rare population of cells capable of self-renewal to drive mammary tumorigenesis and metastasis. Nevertheless,relatively little is known about the intracellular signaling pathways regulating self-renewal and metastatic activities of MaCSCs in vivo. Using a recently developed breast cancer mouse model with focal adhesion kinase (FAK) deletion in mammary tumor cells (MFCKO-MT mice),here we present evidence suggesting a compensatory function of Pyk2,a FAK-related kinase,in the regulation of MaCSCs and metastasis in these mice. Increased expression of Pyk2 was found selectively in pulmonary metastatic nodules of MFCKO-MT mice,and its inhibition significantly reduced mammary tumor development and metastasis in these mice. Consistent with the idea of metastasis driven by MaCSCs,we detected selective up-regulation of Pyk2 in MaCSCs,but not bulk mammary tumor cells,of primary tumors developed in MFCKO-MT mice. We further showed that inhibition of Pyk2 in FAK-null MaCSCs significantly decreased their tumorsphere formation and migration in vitro as well as self-renewal,tumorigenicity,and metastatic activity in vivo. Last,we identified PI3K/Akt signaling as a major mediator of FAK regulation of MaCSCs as well as a target for the compensatory function of Pyk2 in FAK-null MaCSCs. Together,these results further advance our understanding of FAK and its related tyrosine kinase Pyk2 in regulation of MaCSCs in breast cancer and suggest that pharmaceutically targeting these kinases may hold promise as a novel treatment for the disease by targeting and eradicating MaCSCs. View Publication

Cells die by necrosis due to excessive chemical or thermal stress,leading to plasma membrane rupture,release of intracellular components and severe inflammation. The clearance of necrotic cell debris is crucial for tissue recovery and injury resolution,however,the underlying mechanisms are still poorly understood,especially in vivo. This study examined the role of complement proteins in promoting clearance of necrotic cell debris by leukocytes and their influence on liver regeneration. We found that independently of the type of necrotic liver injury,either acetaminophen (APAP) overdose or thermal injury,complement proteins C1q and (i)C3b were deposited specifically on necrotic lesions via the activation of the classical pathway. Importantly,C3 deficiency led to a significant accumulation of necrotic debris and impairment of liver recovery in mice,which was attributed to decreased phagocytosis of debris by recruited neutrophils in vivo. Monocytes and macrophages also took part in debris clearance,although the necessity of C3 and CD11b was dependent on the specific type of necrotic liver injury. Using human neutrophils,we showed that absence of C3 or C1q caused a reduction in the volume of necrotic debris that is phagocytosed,indicating that complement promotes effective debris uptake in mice and humans. Moreover,internalization of opsonized debris induced the expression of pro-resolving genes in a C3-dependent manner,supporting the notion that debris clearance favors the resolution of inflammation. In summary,complement activation at injury sites is a pivotal event for necrotic debris clearance by phagocytes and determinant for efficient recovery from tissue injury. Graphical Abstract View Publication

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971,rs17615,rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls,P=0.016,OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10,directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC),and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact. View Publication

Complement receptor 2-negative (CR2/CD21(-)) B cells have been found enriched in patients with autoimmune diseases and in common variable immunodeficiency (CVID) patients who are prone to autoimmunity. However,the physiology of CD21(-/lo) B cells remains poorly characterized. We found that some rheumatoid arthritis (RA) patients also display an increased frequency of CD21(-/lo) B cells in their blood. A majority of CD21(-/lo) B cells from RA and CVID patients expressed germline autoreactive antibodies,which recognized nuclear and cytoplasmic structures. In addition,these B cells were unable to induce calcium flux,become activated,or proliferate in response to B-cell receptor and/or CD40 triggering,suggesting that these autoreactive B cells may be anergic. Moreover,gene array analyses of CD21(-/lo) B cells revealed molecules specifically expressed in these B cells and that are likely to induce their unresponsive stage. Thus,CD21(-/lo) B cells contain mostly autoreactive unresponsive clones,which express a specific set of molecules that may represent new biomarkers to identify anergic B cells in humans. View Publication

In the adult bone marrow (BM),endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche,which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM,distinct vascular arteriole,transitional,and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However,the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover,constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this,we describe two tamoxifen-inducible cre -expressing lines,Vegfr3-creER T2 and Cx40-creER T2,that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow,without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre -dependent recombination constitutively-activates MAPK signaling within adult endothelium,we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creER T2 -expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM,providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis. The online version contains supplementary material available at 10.1007/s12015-024-10703-9. View Publication

Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development,but the soundness of this model has been challenged by others,who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7+ within 48 h,with minimal expression of mesoderm markers,including T (Brachyury). Instead,they begin to express a series of trophoblast markers,including HLA-G,demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium,and,over time,produce extensive amounts of human chorionic gonadotropin,progesterone,placental growth factor,and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling,which at day 2 provide colonies that are entirely KRT7+ and in which the majority of cells are transiently CDX2+. Colonies grown on two chemically defined media,including the one in which BMP4 was reported to drive mesoderm formation,also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts. View Publication

ObjectiveGene discovery studies in individuals with diabetes diagnosed within 6 months of life (neonatal diabetes,NDM) can provide unique insights into the development and function of human pancreatic beta-cells.MethodsWe performed genome sequencing in a cohort of 43 consanguineous individuals with NDM in whom all the known genetic causes had previously been excluded. We used quantitative PCR and RNA-sequencing in CRISPR-edited human induced pluripotent stem cells (iPSCs),and CUT&RUN-sequencing in EndoC-?H1 cells to investigate the effect of PAX4 loss on human pancreatic development.ResultsWe describe the identification of homozygous PAX4 loss-of-function variants in 2 individuals with transient NDM: a p.(Arg126?) stop-gain variant and a c.-352_104del deletion affecting the first 4 PAX4 exons. We confirmed the p.(Arg126?) variant causes nonsense mediated decay in CRISPR-edited iPSC-derived pancreatic endoderm cells. Integrated analysis of CUT&RUN-sequencing in EndoC-?H1 cells and RNA-sequencing in PAX4-depleted islet stem cell models identified genes directly regulated by PAX4 involved in both pancreatic islet development and glucose-stimulated insulin secretion.ConclusionWe report the first human cases of complete loss of PAX4,establishing it as a novel cause of NDM and highlighting its role in human beta cell development. Both probands had transient NDM which remitted in early infancy but relapsed at the ages of 2.4 and 6.7 years,demonstrating that in contrast to mouse models,PAX4 is not essential for the development of human pancreatic beta-cells. Highlights•Homozygous loss-of-function variants in PAX4 are a novel genetic cause of transient neonatal diabetes.•PAX4 directly regulates genes involved in pancreatic beta cell development and glucose-sensitive insulin secretion.•The role of PAX4 in humans differs to that observed in mouse and is not essential for beta cell development. View Publication