Type 2 diabetes, characterized by dysfunction of pancreatic β-cells and insulin resistance in peripheral organs, accounts for more than 90% of all diabetes. Despite current developments of new drugs and strategies to prevent/treat diabetes, there is no ideal therapy targeting all aspects of the disease. Restoration, however, of insulin-producing β-cells, as well as insulin-responsive cells, would be a logical strategy for the treatment of diabetes. In recent years, generation of transplantable cells derived from stem cells in vitro has emerged as an important research area. Pluripotent stem cells, either embryonic or induced, are alternative and feasible sources of insulin-secreting and glucose-responsive cells. This notwithstanding, consistent generation of robust glucose/insulin-responsive cells remains challenging. In this review, we describe basic concepts of the generation of induced pluripotent stem cells and subsequent differentiation of these into pancreatic β-like cells, myotubes, as well as adipocyte- and hepatocyte-like cells. Use of these for modeling of human disease is now feasible, while development of replacement therapies requires continued efforts. The prevalence of diabetes, a life-threatening disease, is increasing worldwide; 463 million people have diabetes and 374 million exhibit impaired glucose tolerance. It has been estimated that 578 million people by the year 2030, and 700 million by the year 2045, will suffer from diabetes . Type 2 In recent years, generation of transplantable glucose-responsive, insulin-secreting β-like cells, and insulin-responsive cells in vitro is not only theoretically attractive but is also becoming feasible. Pluripotent stem cells (PSC), such as embryonic stem cells (ESC) and induced PSCs (iPSC), are potential alternative sources of insulin- and glucose-responsive cells owing to their ability to differentiate into all major somatic cell lineages and their unlimited renewal capacity . Although both ESCs and iPSCs are PSCs, for several reasons iPSCs may be considered as the first choice for modeling and treatment of diseases. There are ethical concerns regarding the use of human embryos for research purposes. In addition, genetic manipulation will likely be required to introduce specific mutations when using ESCs as a model of a genetic disorder. In contrast, iPSCs can be derived from individuals carrying a specific mutation or genetic variant that may be of pathogenetic relevance (for more detail see ). For thFor these reasons, this review will focus on the generation of iPSCs from somatic cells of healthy individuals or patients suffering from various disorders related to T2D and subsequent differentiation of such cells into pancreatic β-like cells, as well as myotubes, and adipocyte- and hepatocyte-like cells. The limitations and challenges of successful therapeutic application of iPSC-derived cells in diabetes, such as development of methods to substitute these cells for host cells, standardization of the treatment protocols, and quality control will also be discussed.iPSCs are generated through reprogramming of somatic cells into an embryonic-like state. This is achieved by transduction of pluripotency-associated transcription factors. This was first shown by use of four transcription factors, KLF4 (Kruppel-like factor-4), OCT-3/4 (octamer-binding transcription factor-3/4), SOX2 (sex determining region Y-box 2), and c-MYC, which can reprogram mouse fibroblast into PSCs, exhibiting morphological and molecular features resembling those of ESCs . The iniTransposon-based methods have been applied to remove integrated transgenes from iPSCs. A polycistronic plasmid harboring four factors and a piggyBac transposon have been constructed and integrated into the genome in the presence of piggyBac transposase ,23. The To reduce the limitation of integrative methods, novel non-integrative approaches have been developed for reprogramming purposes. These are divided into four main categories, integration-defective viral delivery , episomal delivery, RNA delivery, and protein delivery. Using adenovirus was one of the first attempts to generate integration-free iPSCs. This allows for transient expression of exogenous genes without integration into the genome. Viruses are instead lost by dilution via cell division . The effTo circumvent the problem of non-replication episomal vectors, oriP/EBNA1 vectors have been developed. This approach successfully generates iPSC colonies and one-third of them are free of plasmid DNA . The rep2 with addition of valproic acid in the medium also enhances efficiency [Utilizing mRNA to express reprogramming factors has high efficiency, which can be even further improved by adding Lin28 to the Yamanaka reprogramming factors . Culturificiency . Furtherficiency . This meficiency . The recficiency .Numerous small molecules and soluble factors can increase the reprogramming efficiency through several mechanisms, such as inhibition of transforming growth factor-β (TGF-β)  or Rho-aDepending on the starting cell types and species, the kinetics of reprogramming are different. This can be due in part to the epigenetic memory in iPSCs; during the reprogramming process, the genes responsible for cell specificity may remain under-reprogrammed . In addiIn summary, several methods have been developed to generate iPSCs. Some hurdles still remain, such as undesirable tumor formation and variable efficiency of reprogramming of different somatic cells  ,45. TherDevelopment of the pancreas is, in general, very similar in different vertebrate species. Since ethical considerations limit studies and use of human embryos, knowledge about pancreas development has largely been derived from the study of mouse pancreatic embryogenesis. During gastrulation, the primitive gut tube  is formed from definitive endoderm. The foregut is then split to the anterior and posterior foregut . TransiePtf1a and continued expression of Nkx6.1 lead to the development of ductal/endocrine lineages, while the exocrine lineage depends on the maintenance of Ptf1a and loss of Nkx6.1 [Several transcription factors and signaling molecules initiate and regulate pancreatic development  by the af Nkx6.1 . PDX1 inf Nkx6.1 ,53.Protocols to generate glucose-responsive pancreatic β-cells from iPSCs largely follow strategies established for ESCs . They ar+ definitive endoderm. For a response to activin/Nodal, PI3K signaling must be suppressed. For this purpose, compounds such as wortmannin and LY2994002, which inhibit PI3K signaling, have been found to promote definitive endoderm formation [Formation of definitive endoderm is the rate-determining step in pancreatic differentiation . It is bormation . Anotherormation . Inclusiormation . Indeed,ormation ,58; it cormation .+ cells rate. Transplantation of these cells into streptozotocin (STZ)-induced diabetic mice reduces blood glucose levels over the next 4 weeks [Inhibition of GSK-3β (using CHIR99021) for induction of definitive endoderm and spheroid formation at the final stage are important for the generation of functional iPSC-derived β-like cells . CHIR990 4 weeks . Express 4 weeks .PDX1, while at higher concentrations it promotes more posterior endoderm fates [+ progenitors [+/NKX6.1+ progenitors cannot be developed in the absence of FGF7 [PDX1 expression, most likely through increases in progenitor cell survival [HNF1B and HNF4A is increased [PDX1, NGN3, NKX2.2 and NKX6.1 expression following retinoic acid treatment [SHH inhibitors and FGF10 prime definitive endoderm to form cells characteristic of the primitive gut tube. At low concentrations, FGF promotes posterior foregut fate and expression of rm fates ,62. Membrm fates ,53,57. Agenitors ; therefo of FGF7 . Furthersurvival . SHH actsurvival . Thus, isurvival ,67. At tncreased . There areatment ,67,68.+/NKX6.1+ progenitors differentiate into monohormonal β-cells, while PDX1+/NKX6.1− progenitors differentiate into polyhormonal cells [+/NKX6.1+ progenitors is high under optimized conditions [+/NKX6.1− population is further increased when duration of the posterior foregut stage is prolonged [+/NKX6.1+ progenitors is reasonably stable, using the same protocol on different iPSC lines leads to a variable NKX6.1 induction, ranging from 37% to 84% [−/NKX6.1+ progenitor cells have been found during differentiation of iPSCs to β-like cells [+ cells. PDX1−/NKX6.1+ progenitors demonstrate downregulation of pancreatic epithelial genes and upregulation of neuronal development genes, indicating that they represent a unique source for generating INS+ cells of a non-epithelial origin [Pancreatic progenitor cells express a group of transcription factors, of which PDX1 and NKX6.1 are critical markers for β-cell maturation and functionality . PDXal cells ,72. The nditions ,73; the rolonged . Althoug% to 84% . This inke cells ; these pl origin .+ progenitors and persists upon completion of the differentiation, as ~95% of endocrine progenitors and insulin-expressing β-cells do not express YAP. Inhibition of YAP during the specification of early PDX1+ to late NKX6.1+ progenitors decreases the number of NKX6.1+ progenitors, while its inhibition during endocrine specification, leads to differentiation of pancreatic progenitors into NGN3+ endocrine progenitors and then into NKX6.1+/C-peptide+ β-cells more efficiently than control cells [+/NKX6.1+ progenitor cells into diabetic mice reverses hyperglycemia [Expression of NKX6.1 is promoted by use of nicotinamide and EGF, which increase generation of pancreatic progenitors . Additiool cells . Transplglycemia .+ progenitors [HES1, a downstream target of notch signaling [Fgf10-deficient mice exhibit reduced proliferation of pancreatic epithelial progenitor cells and display pancreatic hypoplasia [Fgf10 leads to pancreatic hyperplasia, decreased differentiation of endocrine progenitor cells, and increased progenitor cell numbers [Forskolin (an adenylate cyclase activator), activin receptor-like kinase (ALK5) inhibitors, ROCK inhibitors, γ-secretase inhibitor XXI, nicotinamide, triiodothyronine (T3), exendin-4, heparin, and dexamethasone also enhance the generation of insulin-expressing cells from PDX1genitors ,54,79. Fignaling . Fgf10-dpoplasia . In cont numbers .+/NKX6.1+ progenitors leads to the generation of NGN3+ endocrine progenitors. The transcription factor NGN3 plays an important role at this stage as impaired Notch receptor activation or signaling upregulates NGN3 expression, resulting in premature endocrine cell differentiation. NGN3 is expressed early in the specification of the pancreatic endocrine lineage [NGN3 are associated with neonatal diabetes and blocking of β-cell differentiation [Ngn3-deficient mice [+ progenitors and then into NGN3-expressing pancreatic endocrine progenitors [of NKX6.1 is increased, demonstrating the specification of endocrine pancreas. A significant portion of endodermal chromogranin A−/PDX1+ cells also expresses NKX6.1 [Inhibition of Notch signaling following induction of the PDX1 lineage . Null muntiation , as wellent mice . Inhibitent mice ; blockinent mice . High cogenitors . Differegenitors . This cas NKX6.1 .MAFA increases expression of glucokinase, glucose transporter GLUT2, PDX1, NKX6.1, glucagon-like peptide-1 receptor (GLP1R), and pyruvate carboxylase [Immature fetal β-cells require several weeks to gain responsiveness to glucose ; this inboxylase , as wellboxylase ).MAFA [NKX6.1, PDX1, insulin, and NEUROD1, likely through increasing MAFA and NGN3 expression. The differentiation of the endocrine progenitors into β-like cells is promoted by vitamin E, T3, betacellulin, heparin, ALK5 inhibitors, AXL receptor tyrosine kinase inhibitors, exendin-4, hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and N-acetyl cysteine, which induce the expression of MAFA ,67,90,91ATP channel) activity [2+ response and increased insulin secretion following glucose stimulation [2+ increase is heterogeneous, demonstrating both oscillatory and biphasic kinetics [2+ increase in iPSC-derived β-cells [ATP channels and voltage-gated Ca2+ channels in these β-like cells are functional.Using step-wise differentiation protocols, several groups have successfully generated glucose-responsive iPSC-derived β-like cells. Glucose sensing and the amount of secreted insulin are, however, not equivalent to that observed in cadaveric human islets in vitro. The insulin secretion capacity of iPSC-derived β-cells is qualitatively similar to that of cadaveric islets in terms of the ability to regulate de- and re-polarization of the plasma membrane in response to KCl or altering ATP-sensitive potassium channel  has been suggested as a master regulator of β-cell maturation . Postnat+ cells at the end of differentiation. Inhibition of ROCKII enhances NKX6.1, INS, UCN3, MAFA and G6PC2 expression levels, improves glucose sensitivity, as well as reduces expression of cell cycle and focal adhesion genes, conferring robustness to functionally mature iPSC-derived β-cells [Pretreatment of mesenchymal stromal cells with epigenetic modifiers has been shown to increase expression of insulin, GLUT2, glucokinase, as well as the transcription factors PDX1, NKX6.1, and MAFA . The dif β-cells .Using the differentiation protocol described by Ameri et al. , our gro+ cells) [Although in vitro iPSC differentiation protocols do not generate pure populations of β-like cells, some cell surface markers can be used to isolate pure and functional iPSC-derived β-cells and re-aggregate them before encapsulation for transplantation. Cell surface markers SUSD2, CD200, and CD318 identify endocrine progenitors (NGN3+ cells) . Glycopr+ cells) ,100,101.+ cells) . Current+ cells) . The mol+ cells) , circula+ cells) ,105, and+ cells)  have, hoPancreatic islet architecture with intra-islet cell-cell communication between β-cells and other endocrine cell types are essential for β-cell functionality and glucose responsiveness. During islet development, for proper β-cell lineage specification, differentiation and maturation, intact cell-cell  and cell-matrix  stiffness, topography, fluid shear forces, and geometry) interactions are crucial for coordinating and supporting cells ,111,112.Success in producing and maintaining functional β-cells in vitro greatly depends on the ability to recapitulate the native islet 3D microenvironment, with its spatial, chemical and mechanical attributes. The 3D microenvironment of the islet niche is composed of multiple layers of interconnected cells, nerve fibers, blood vessels, and ECM molecules . All together, they serve to generate synchronized insulin secretion ,118,119.+ endocrine progenitors, rather than spread out loose cells, reflecting a more ductal phenotype [Alterations of the ECM are sensed by β-cell mechanosensors , which transduce signaling cascades intracellularly. Thus, cytoskeletal dynamics and formation of integrin adhesion complexes are brought into motion ,110. Thehenotype . This phhenotype .2+ fluxes similar to those in primary human islet cells [To provide 3D cell-cell interactions, suspension/aggregate/spinner flask culture systems have been developed to induce formation of islet-like organoids, which further promote formation of glucose-responsive β-cells from stem cells ,123,124.et cells .NEUROG3, and NKX6.1 expression, hereby inducing pancreatic endocrine lineage [Although 3D culture systems are considered superior to 2D systems, recently, differential actin cytoskeleton states (polymerized vs. depolymerized), cell adhesion intensity to the surface of culture plates, and substrate stiffness have been shown to control the timing of  lineage . A 2D pl lineage . This en lineage .Generation of β-cells from PSCs holds great promise in the treatment of diabetes. In addition, diabetes patient-derived iPSCs could serve as experimental models to investigate disease mechanisms. There are more than 400 genetic signals associated with T2D and fasting glucose . Many ofEncapsulation of iPSC-derived β-cells could solve issues with immunity: a flat-sheet macroencapsulation device, to graft the iPSC-derived pancreatic endoderm cells under the skin of immune-compromised mice, increases circulating C-peptide levels in response to glucose administration ; both faInsulin resistance, a key component of T2D pathophysiology is defined as a state with decreased metabolic actions of insulin in target tissues, namely liver, skeletal muscle, and adipose tissue [This notwithstanding, longitudinal studies have shown that insulin resistance is also a heritable trait and develops in people at risk for T2D many years before glucose intolerance ,135. As FOXA proteins regulate almost all liver-specific genes [Hepatocytes, the principal liver cell type, are derived from embryonic endoderm . Liver-sic genes . Cardiogic genes ,137. Theic genes .A combination of growth factors and small molecules is used for differentiating hepatic developmental stages from definitive endoderm to immature hepatocytes (hepatic progenitors) and finally mature hepatocytes  138,139139.For differentiation of definitive endoderm, various signaling pathways controlling the early cell fate decisions of pluripotent stem cells, such as those involving Wnt, Activin, BMP, and FGF are activated. Following generation of endodermal cells from iPSCs, hepatoblasts are formed. To this end, HGF or KGF is added. At this stage, differentiated hepatoblasts display expression of FOXA1, FOXA2, CCAAT enhancer-binding protein alpha (CEBPα), peroxisome proliferator-activated receptor alpha (PPARα), alpha-fetoprotein (AFP), and cytokeratin (KRT)-18 and -19 ,142,143.Oncostatin M (OSM) is important for hepatic maturation . OncostaiPSC-derived hepatocytes secrete albumin ,10,72, sMoreover, genes involved in cell-cycle and cell-proliferation pathways have significantly higher expression in fetal than adult liver. In contrast, genes highly expressed in adult liver are metabolism-related genes, including three major functional categories: metabolism of fatty acids, xenobiotics, and glucose . To mimiiPSC-derived hepatocytes have been used to resolve inherited metabolic disorders of the liver, such as A1AT deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a . Gene edPax3 is then downregulated and the delaminating progenitor cells differentiate into myoblasts [MyF5), myogenic regulatory factor (MrF4), and myogenic differentiation (MyoD) is increased in the myoblasts, which differentiate into myocytes through the action of myogenin (MyoG), MrF4, and MyoD [Nearly all skeletal muscle cells are derived from paraxial mesoderm, forming somites, then dermamyotome and finally the myotome . For myoyoblasts . The expand MyoD . The myoand MyoD . + or PAX3+/PAX7+ dermomyotomal progenitors [Pax7 and downregulation of Pax3 are initiated in a subset of PAX3+ myogenic progenitors. These PAX7+ myogenic precursors form primary fibers and then generate the secondary or fetal fibers expressing specific markers, such as β-enolase or MYL3 [+ progenitors. A portion of the PAX7+ progenitors will also generate satellite cells, the pool of adult muscle stem cells [During the early embryonic/primary phase, primary myofibers are generated, which derive from PAX3genitors . These m or MYL3 ,199. At There are two protocols for derivation of skeletal muscle cells from iPSCs: i) forced overexpression of muscle-specific transcription factors, such as PAX3, PAX7, and MYOD, and ii) a step-wise induction of skeletal muscle by small molecules to inhibit or activate relevant signaling pathways in myogenesis . MYOD in a variety of differentiated cell lines is sufficient to activate a downstream program for terminal muscle differentiation [PAX7 and MYOD1 in iPSCs [MYOD1 overexpression, particularly in undifferentiated cells, drives them along the myogenic lineage with 70–90% efficiency, and myocytes reach maturity within two weeks of differentiation [PAX7 in two well-characterized human iPSC lines, generated from normal donor’s fibroblasts, converts these cells into myotubes [MyoD induces cell cycle arrest [Activation of ntiation . Differein iPSCs ,201. MYOntiation . Overexpmyotubes ,201. Promyotubes . Howevere arrest . As thes+/MYOG+ populations in vitro [Some small molecules secreted as paracrine factors play important roles in muscle development, controlling proliferation, migration, and differentiation from mesodermal cells into somites and dermomyotome . iPSCs ain vitro .FGF2, EGF, IGF-1, HGF, and platelet-derived growth factor (PDGF) have also been used to enhance myogenic differentiation ,207,208.MYOD1, as well as of MYH and the cholinergic receptor CHRNA1 [+ cells during the main part of the expansion period [After 7, 12, and 36 days, cells are differentiated into embryoid bodies (EBs), myoblasts, and myotubes, respectively . From dar CHRNA1 . Under pn period  , and PRD1-BF-1-RIZ1 homologous domain containing protein-16 (PRDM16) [Cebpa is important for terminal differentiation of adipocytes, as its absence leads to insulin resistance; development of BAT is independent of Cebpa [The overall function of white adipose tissue (WAT) is to store energy, whereas brown adipose tissue (BAT) dissipates energy in a heat-producing process called thermogenesis. Postnatally, a third form of adipose tissue has been identified (‘beige’) but its function and origin is enigmatic; it has been reported that beige adipocytes are generated from unique precursor cells , but the(PRDM16) . PPARγ, (PRDM16) . In addi(PRDM16) . Expressof Cebpa . The expof Cebpa . PRDM16 of Cebpa ; its delof Cebpa .Two protocols for differentiation of adipocytes from iPSCs have been reported: (i) forced overexpression of white/brown adipocyte-specific transcription factors, such as PPARγ, PRDM16, and CEBPα, and (ii) step-wise induction of adipocytes by small molecules to inhibit or activate relevant signaling pathways in adipogenesis .AFP or FOX2 (for endoderm), SOX1 or β3-tubulin (for ectoderm), and Brachyury or α-smooth muscle actin (AKTA2) (for mesoderm) [Embryoid bodies are three-dimensional cell aggregates formed in suspension by PSCs, which differentiate into cells of all three germ layers. Formation of EBs is a routine inductive step to generate specific cell lineages from PSCs. For formation of EBs, iPSCs are cultured in a floating condition with medium containing undefined components, such as fetal bovine serum (FBS), knock-out serum replacement (KSR) or albumin products for 2–7 days ,230. Shoesoderm) ,233.PPARG and FABP4), but also expression of brown fat genes (uncoupling protein 1 (UCP1) and PGC1A) [PPARG, FABP4, UCP1, and PGC1A) [PGC1A, PPARG, FABP4, and UCP1 [The second step of differentiation, consists of culturing iPSC-derived EBs under adherent conditions with an adipogenic cocktail, including FBS, dexamethasone, isobutylmethylxanthin (IBMX), pioglitazone, and insulin ,231,234.d PGC1A) . IBMX ald PGC1A) ,235. Dexd PGC1A)  and upreand UCP1 . Using pand UCP1 . CEBPA, PPARG2, LEP, FABP4, and fatty acid-binding protein 4 (aP2) [PPARG2, in combination with adipogenic differentiation medium, is used to enhance differentiation efficiency [PPARG2 transgene expression, and exhibit insulin-stimulated glucose uptake and inducible lipolysis [Human iPSC-derived white adipocyte-like cells exhibit lipid accumulation and transcription of adipogenesis-related genes, such as  4 (aP2) ,233,234.ficiency . Transfeipolysis . Withoutipolysis ; althougipolysis . Patientipolysis . Lipid-cPPARG2, CEBPB, and PRDM16 in human [Overexpression of in human ,238 or min human ,238, higin human ,238 and in human , with elin human , importain human . Transpl+ splanchnic mesoderm toward beige adipocyte-like cells [+ adipogenic precursors within subcutaneous adipose tissue and increases their differentiation into beige adipocytes [UCP1 transcription, as well as beige adipogenic precursor markers (PDGFRA and EBF2) [UCP1, PGC1A, and PRDM16; this characterizes beige and brown adipocytes but not progenitor or mature brown adipocyte markers (MYF5 or ZIC1), suggesting that iPSC-derived cells are not of the brown adipocyte lineage [Adding the Wnt agonist (CHIR99021), recombinant BMP4, VEGFA proteins, and TGF-β inhibitor (SB431542) with an adipogenic cocktail efficiently differentiates FOXF1ke cells . Differeke cells . Cold exipocytes . Pretreand EBF2) . Treatme lineage . Activat lineage . Accordi lineage . Moreove lineage . Treatin lineage .Moving the iPSC-derived insulin/glucose-responsive cells from bench to bedside requires overcoming several obstacles. These range from iPSC reprogramming to transplantation of differentiated cells, as well as maintenance of phenotypic stability, and avoiding tumor formation.Most iPSCs are established utilizing retroviral vectors. There are numerous transgene integrations in the genomes of iPSCs developed by retroviral vectors, which may cause leaky expression that can interrupt the function of endogenous transcription factors or introduce mutations that increase tumorigenic risk after transplantation. Non-viral reprogramming methods reduce toxicity or other adverse effects of the vectors and their delivery systems, but their efficiency of iPSC induction is typically lower than that of retroviral vectors, possibly due to low transduction efficiency and unstable expression. Furthermore, all reprogramming strategies influence and impede cell cycle control genes, such as p53.Variations in the iPSC lines strongly influence iPSC reprogramming and differentiation. Embryonic and tail-tip fibroblasts in mouse and skin fibroblast and peripheral blood cells in human are widely used for reprogramming, mainly due to their availability and relative ease of culture. Mouse tail-tip fibroblast iPSCs demonstrate high tumorigenic propensity, while that of gastric epithelial- and hepatocyte-derived iPSCs is lower . This in+/glucagon+) cells alongside diverse categories of progenitor cells [Despite an increasing number of differentiation protocols, production of mature and functional iPSC-derived insulin/glucose-responsive cells is yet to be convincingly achieved. The final percentage of differentiated β-cells obtained in most studies without purification is at best 30–60%. Most remaining cells are undifferentiated/unwanted cells. iPSC-derived β-like cells demonstrate increased basal insulin levels and insufficient glucose responsiveness. Furthermore, in vitro differentiation results in generation of heterogeneous cell populations, composed of bihormonal , paracrine soluble factors secreted by MSCs or HUVECs, even without cell-cell surface contact, are sufficient to induce hepatic differentiation . HoweverSimilarly, using a 3D culture system and a step-wise differentiation protocol, pancreatic clusters have been produced, containing α- and β-like cells, and enterochromaffin-like cells, as well as non-endocrine cell type . Using eStandardization of iPSCs meets more difficulty when using patient-specific iPSCs. Insulin mutations impair differentiation of β-cells in a patient-derived iPSC model of neonatal diabetes and triggers ER-stress concomitantly with insulin expression . IncreasSuccessful survival post-transplantation is another challenge, which can be attributed to the lack of suitable transplantation approaches and immune rejection in the host. To date, iPSC-derived insulin/glucose-responsive cells in vitro lack many properties of mature cells. Further maturation occurs in vivo after transplantation to immunodeficient mice. Therefore, more work is needed to understand what factors in the in vivo milieu are crucial for functional maturation. Factors may be provided by the in vivo niche at the transplantation site. Maturation develops more efficiently in female mice, indicating the potential role of estrogen in maturation . While hAfter satisfying all these requirements, PSC-based therapies may show superiority to any other antidiabetic treatment strategies. Increasing medical expenditures, as a result of the rising prevalence of diabetes, will become an economic concern in the near future. Thus, prevention and treatment of diabetes are very important from a societal perspective. Indeed, restoring or maintaining the function of residual insulin/glucose-responsive cells prevents the progression of diabetes and reduces medical costs.Generation of iPSC-derived insulin/glucose-responsive cells in vitro offers great opportunities for both in vitro disease modeling and treatment of diabetes in humans. There is continuous improvement of the differentiation protocols for ESCs and iPSCs, allowing for use in scalable in vitro generation of insulin/glucose-responsive cells, as well as in approaches for transplantation. Despite these advances, a more complete understanding of in vitro differentiation processes and maturation in vivo is required to generate properly functional and safe cells for replacement therapy. This is a pre-requisite for generation of large and sufficient quantities of insulin/glucose-responsive cells, and will also refine and improve in vitro disease models derived from iPSCs.


SMC3), miR-122 (SGO1), and miR-206 (AURKA) were involved in ovarian organogenesis and hormone secretion by oocyte meiosis. Furthermore, miR-202-5p (EREG) and miR-485-3p (FLT3) were involved in follicular maturation through the MAPK signaling pathway, and miR-2404 (BMP7 and CDKN1C) played a key role in follicular development through the TGF-β signaling pathway and cell cycle; nevertheless, further research is warranted. To our knowledge, this is the first study to investigate miRNA expression patterns in OS, LF, and SF of Chuanzhong black goats during estrus. Our findings provide a theoretical basis to elucidate the role of miRNAs in follicular maturation. These key miRNAs might provide candidate biomarkers for the diagnosis of follicular maturation and will assist in developing new therapeutic targets for female goat infertility.microRNAs (miRNAs) play a significant role in ovarian follicular maturity, but miRNA expression patterns in ovarian stroma (OS), large follicles (LF), and small follicles (SF) have been rarely explored. We herein aimed to identify miRNAs, their target genes and signaling pathways, as well as their interaction networks in OS, LF, and SF of Chuanzhong black goats at the estrus phase using small RNA-sequencing. We found that the miRNA expression profiles of LF and SF were more similar than those of OS—32, 16, and 29 differentially expressed miRNAs were identified in OS vs. LF, OS vs. SF, and LF vs. SF, respectively. Analyses of functional enrichment and the miRNA-targeted gene interaction network suggested that miR-182 ( Fecundity is a complex quantitative character, which is mainly restricted by genetic factors and has an economic importance in the goat industry . KiddingmicroRNAs (miRNAs), a type of non-coding small RNA with a length of 19–24 nucleotides (nt), regulate gene expression at the post-transcriptional level by interfering with or degrading mRNA expression ,8. For tCapra hircus) is an excellent local goat breed in China [Chuanzhong (CZ) black goat , large follicles , and small follicles  in CZ black goats during estrus, and we studied and compared miRNA expression patterns among them. We also assessed whether miRNA expression profiles of OS, LF, and SF were consistent, and searched for miRNAs that might be related to follicular maturation in each tissue, with the aim of providing a reference for further studies on molecular mechanisms underlying follicular development in goats. The key miRNAs discovered in this study might be applied as activators or inhibitors in the future to regulate follicular maturation and ovulation, thus improving ovulation and kidding rate in goats and providing high-fertility goats for the goat industry.All experimental procedures and sample collection methods complied with the Regulation on the Administration of Laboratory Animals  and were performed in strict accordance with the Institutional Animal Care and Use Committees of South China Agricultural University .We selected CZ black goats from Wens Foodstuff Group Co., Ltd., as the experimental animals . Under the condition of natural light, 10 healthy female goats of the same age (approximately 3.5–4.5 years), who had >3 litters, were given ad libitum access to food and water. After estrus synchronization, we slaughtered them, collected their ovaries, and separated the OS, LF, and SF. The LF and SF that were isolated from follicles were analyzed by small RNA-sequencing , as previously described . We sepa260/280 <2.2, and RNA integrity number >8.5) was used for preparing the cDNA libraries.Total RNA was extracted from OS, LF, and SF using TRIzol . The RNA quality was estimated using an Agilent 2100 Bioanalyzer  and NanoDrop spectrophotometer . The RNA integrity was evaluated by 1% agarose gel; purified RNA was stored at −80 °C. High quality RNA  to extract approximately 2 μg of total RNA from each sample to construct the small RNA libraries. All libraries used for high-throughput sequencing of miRNAs were amplified by PCR, with sequencing connectors and index sections added. Next, 18–36 nt RNAs were purified using a 6% Novex TBE PAGE gel  and quantified with an Agilent 2100 Bioanalyzer. Single-stranded cDNA templates were used for bridge PCR, and Illumina single-end sequencing was performed on a HiSeq 2500 sequencer . http://www.mirbase.org/) with mature and precursor miRNA sequences of the species, and the detected miRNAs were annotated. A novel miRNA prediction analysis was performed based on MIREAP analysis of the unannotated sequences. The read count values of the miRNAs were calculated based on the number of mature miRNA sequences. Herein, we analyzed the expression difference of the known miRNAs.Raw reads were handled by a script consisting of read alignment, index trimming, and read counting. To obtain clean reads, raw reads were further filtered as per the following rules: (1) removing low-quality reads containing a >1 low quality  base or unknown nt (N); (2) removing reads without 3′ adapters; (3) removing reads with 5′ adapters; (4) removing reads containing 3′ and 5′adapters but without small RNA fragments between them; (5) removing reads containing poly-A from small RNA fragments; and (6) removing reads that were <18 nt (excluding adapters). The excised clean reads were mapped to the goat reference genome  using miRDeep2 (v2.0.0.8), in which the mapper.pl program called Bowtie was used to align the anti-repeat sequence with the reference genome sequence. The de-repeat sequences were aligned in miRBase  values were used for inter-sample correction of total reads . The density distribution of the CPM values was estimated using the density function in R (v4.0.1). Principal components analysis (PCA) was performed by the procmp function in R. Differential expression in each group was identified by DESeq (v1.18.0) with R, and DEmiRNAs were identified by |log2FC| > 1 and The ggplots2 package in R was used to generate volcanic plots with the DEmiRNAs, and the heatmap package in R was used to cluster all miRNAs. The Euclidean distance was calculated according to the expression levels of the same miRNA in different samples and the expression patterns of different miRNAs in the same sample. The complete linkage hierarchical clustering method with the largest intercluster distance was used for clustering. Based on possible functional relationships between the DEmiRNAs and genes, a systematic bioinformatic analysis was performed. The target genes of the DEmiRNAs were predicted by miRanda, with the 3′-UTR mRNA sequence serving as the target sequence. The parameters used to predict the miRNA–target interaction were a mapping score >140 and free energy <1.0. Cytoscape (v3.5.1) was then used for visualization of the DEmiRNAs and target genes, and the degree represented the number of target genes of the DEmiRNAs. http://geneontology.org/) and the Kyoto Encyclopedia of Genes and Genomes  in DAVID (https://david.ncifcrf.gov). The degree of enrichment was measured by the Rich factor, false discovery rate, and number of genes enriched in a pathway. A p ≤ 0.05 indicated statistical significance.The target genes of the DEmiRNAs were analyzed by gene ontology  are listed in Based on the heatmap shown in We herein used miRanda to predict the target genes of the DEmiRNAs . In OS vLIMCH1) was predicted to be targeted by 17 DEmiRNAs. In OS vs. SF, both zinc finger protein 827 (ZNF827) and myocardin-related transcription factor B (MRTFB) were predicted to target 11 DEmiRNAs. Finally, in LF vs. SF, it was predicted that the new gene ENCHIG000015853 was targeted by 19 DEmiRNAs. Most miRNAs were predicted to regulate hundreds or even thousands of target genes at the same time. In OS vs. LF, OS vs. SF, and LF vs. SF, it was predicted that the DEmiRNA with the most target genes was miR-543-5p, miR-412-3p, and miR-195-5p, respectively, and the number of target genes was 1026, 919, and 1469, respectively. Many target genes may be regulated by multiple miRNAs. In OS vs. LF, LIM and calponin-homology domains 1 (p < 0.05), such as tissue development, extracellular space, and sequence-specific DNA binding of the transcription regulatory region. In OS vs. SF, 1132 GO terms were significantly enriched (p < 0.05), such as tissue development, cell differentiation regulation, and GTPase regulator activity. In LF vs. SF, 1032 GO terms were significantly enriched (p < 0.05), such as cell proliferation, neuron synapse, and glycosaminoglycan binding. The top 10 GO terms in each category with a significant difference (p < 0.05) are shown in GO functional enrichment analyses  revealedp < 0.05) enriched by the target genes of the DEmiRNAs, including the TGF-β signaling pathway, cell adhesion molecule, MAPK signaling pathway, and oocyte meiosis. In OS vs. SF, there were 31 signaling pathways (p < 0.05) enriched by the target genes of the DEmiRNAs, including the cell adhesion molecule, TGF-β signaling pathway, oocyte meiosis, Notch signaling pathway, and ovarian steroid production. In LF vs. SF, there were 41 signaling pathways (p < 0.05) enriched by the target genes of the DEmiRNAs, including the cell adhesion molecule, TGF-β signaling pathway, cell cycle, and MAPK signaling pathway. The top 20 significantly enriched (p < 0.05) pathways are shown in Based on the KEGG pathway enrichment analysis , in OS vA comprehensive comparison showed that 19 signaling pathways were enriched in the triple comparison ; 2 signaling pathways were enriched in OS vs. LF and OS vs. SF; 4 in OS vs. LF and LF vs. SF; and 4 in OS vs. SF and LF vs. SF. Further, 14, 6, and 14 signaling pathways were only significantly enriched in OS vs. LF, OS vs. SF, and LF vs. SF, respectively D.The ovary is an important reproductive organ that directly affects the estrous cycle and reproductive ability in mammals . Many stOverall, there were 16 miRNAs with CPM > 10,000 in at least one tissue , among which 2 miRNAs (miR-143-3p and miR-21-5p) were significantly different in OS vs. LF. In a study on ovarian cancer, miR-143-3p was noted to be potentially serve as a tumor suppressor, and its expression upregulated in normal ovarian tissue . FurtherINHBB, which plays a key role in the apoptosis of granulosa cells and regulation of the cell cycle [We then screened miRNAs that were only expressed in one tissue. The miRNAs in at least half of the samples with CPM ≥ 1 were considered as expressed and those with CPM < 1 were considered as not expressed. Accordingly, only 8, 21, and 5 miRNAs were expressed in OS, LF, and SF, respectively. Only 7 miRNAs  were significantly differentially expressed, and only miR-187 was found to be related to ovarian function. miR-187 is involved in the regulation of ovarian cancer by targeting disabled homolog-2, which supposedly promotes tumor progression in advanced cancers via epithelial–mesenchymal transition ,43. In all cycle , and is ll cycle . Moreove2(FC) |> 1, and p < 0.05) were used, and 15, 25, and 6 DEmiRNAs were found in OS, LF, and SF, respectively. Specifically, miR-182, miR-122, and miR-206 were significantly upregulated in OS compared with the other two tissues. miR-182, miR-122, and miR-206 have been reported to regulate oocyte maturation and granulosa cell development [SMC3, SGO1, and AURKA, respectively. SMC3 promotes oocyte apoptosis and affects the pathogenesis of polycystic ovary syndrome [AURKA is a rate-limiting factor that promotes microtubule growth as oocytes resume meiosis [SGO1 plays a key role in the centromere cohesion of sister chromatids and movement of chromosomes to the spindle pole [EREG and FLT3, respectively. EREG can be induced by gonadotropin-releasing hormone and regulates ovulation [FLT3 is highly expressed in oocytes and related to follicular growth and maturation [EREG and FLT3, respectively. In addition, miR-2404 was significantly upregulated in SF compared with OS, but there was no significant difference compared with LF. It is noteworthy that miR-2404 has not been found to be related to ovarian development. Through target gene prediction of miRNAs, BMP7 and CDKN1C targeted by miR-2404 were found to participate in the TGF-β signaling pathway and cell cycle, respectively. BMP7 is an active regulator of the transformation from primordial to primary follicle [FSHR in human granulosa cells [CDKN1C is highly expressed in mouse ovaries [BMP7 and CDKN1C.To further screen reliable DEmiRNAs, three screening conditions , the expression of miR-1185-3p, miR-190b, and miR-487a-3p was significantly different (expression levels: LF > SF > OS). The functions of these three have not been reported as yet; we herein speculate their possible functions by analyzing the functions of their target genes. We found that the Hippo signaling pathway, cell adhesion molecules, and TGF-β signal pathway were the key elements that may be involved in the ovarian function and follicular maturation. As the potential role of these miRNAs in ovarian maturation was predicted, further experiments were needed to verify their function. Specifically, as reported in the literature , a serieAREG [WWTR1 [CTGF [AREG, WWTR1, and CTGF were predicted to be the target genes of miR-1185-3p, suggesting that miR-1185-3p regulates them through the Hippo signaling pathway, in turn regulating ovarian function and follicular development. Cell adhesion molecules are essential for the separation and aggregation of different cell types to form different tissues; they maintain the integrity of the germ cells in female gonads [CLDN10 [CADM1 [CLDN10 and CADM1 and mediated the cell adhesion molecules to regulate ovarian function. Further, the TGF-β signaling pathway is widely involved in functions such as cell proliferation, apoptosis, differentiation, and migration, which affect follicular development, embryonic development, and organ formation [BMP7 [BMP5 [FMOD [TGFB2 [BMPR1B [SMAD7 [BMP7, BMP5, and FMOD; miR-190b targeted TGFB2 and BMPR1b; and miR-487a-3p targeted TGFB2 and SMAD7; thus, they seem to mediate the TGF-β signaling pathway to regulate the ovulation rate.The Hippo signaling pathway has a key role in regulating the proliferation and apoptosis of mammalian cells and in maintaining ovarian function as well as promoting follicular growth ,69. AREGAREG ,71, WWTRG [WWTR1 ,73, and R1 [CTGF ,74 are ie gonads ,76. CLDN [CLDN10 ,78 and C0 [CADM1  in this ormation ,81. BMP7on [BMP7 , BMP5 [8P7 [BMP5 , FMOD [8P5 [FMOD , TGFB2 [ [BMPR1B , and SMAB [SMAD7  are the SMC3), miR-122 (SGO1), and miR-206 (AURKA) were involved in ovarian organogenesis and hormone secretion by oocyte meiosis, whereas miR-202-5p (EREG) and miR-485-3p (FLT3) were involved in follicular maturation through the MAPK signaling pathway. Moreover, miR-2404 (BMP7 and CDKN1C) was observed to play a key role in follicular development through the TGF-β signaling pathway and cell cycle. In addition, the expression of miR-1185-3p, miR-190b, and miR-487a-3p may increase with follicular development and maturation, and they seemed to play a pivotal role through the Hippo signaling pathway, cell adhesion molecules, and TGF-β signaling pathway. In a future study, we aim to verify the relationship between candidate miRNAs and their target genes and study the mechanism of their influence on follicular maturation so as to reveal the mechanisms underlying miRNAs in goat ovarian follicular maturation. Overall, based on the expression profiling of miRNAs, we speculated that the miRNA expression patterns of OS, LF and SF were similar. Most miRNAs were not differentially expressed in OS, LF, and SF; only a few miRNAs were differentially expressed according to the miRNA expression abundance. Our data suggested that miR-182 (


Similarly, the clinical immunology laboratory could play an important role in elucidating diverse immunological abnormalities associated with the disease. In particular, immunological testing could better categorise the severe and critical forms of COVID-19, and subsequently assist treating physicians during the entire course of therapy. The purpose of this opinion article is to highlight the position of the immunology laboratory in the management of severe and critical COVID-19 cases, specifically in countries with limited resources.The contribution of the medical laboratory to the categorisation of COVID-19 clinical forms is not yet well defined.4 Other biological abnormalities may predict the severity or progression of the disease, such as abnormal coagulation activation, leukopenia, high levels of C-reactive protein, ferritin, D-dimers, aminotransferase, lactate dehydrogenase and creatin kinase.5 Elevated levels of pro-inflammatory cytokines have been shown to characterise severe lung infection, which manifests as respiratory distress, multiple organ failure and adverse outcomes in SARS-CoV-2 infection.7 In addition, IL-6, IL-10, and TNF-α levels significantly increase during infection and drop during recovery,8 suggesting that the intensity of the cytokine release correlates with disease activity. Interestingly, IL-6 may be a marker for monitoring serious COVID-19 cases.9COVID-19 is marked by an overproduction of pro-inflammatory cytokines and chemokines, mainly interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, IL-1β, IL-2, IL-10, interferon-γ and monocyte chemoattractant protein-1.3Moreover, simultaneous high levels of IL-6 and D-dimers have been shown to be narrowly associated with severe forms of the disease in adult patients. Therefore, measuring them in combination allows for greater specificity and sensitivity for predicting severe cases of COVID-19.10; direct infection of lymphocytes by SARS-CoV-2 has been proposed as a cause for acute lymphocyte decline.11 Lymphocytes express the receptor angiotensin-converting enzyme 2, which has been suggested to be the primary receptor targeted by SARS-CoV-2.12Deregulation of the immune response, particularly T lymphocytes, appears to be strongly linked to the pathology of COVID-1911Moreover, altered T lymphocytes could be an important factor in worsening symptoms in patients, which makes lymphopenia a relevant marker of disease severity, hence the need for intensive care unit admission.13 On the other hand, COVID-19 patients who require intensive care unit hospitalisation have significantly higher levels of IL-6, IL-10 and TNF-α, with lower levels of CD4 and CD8 T cell counts,8 with which levels of cytokines and T cells are inversely correlated.8 Moreover, because T lymphocytes are generally crucial for amortising exaggerated natural immune reactions against viral infection, T cell defects can lead to worsening inflammatory responses during COVID-19, whereas restoring the number of these lymphocytes can improve them.6It has been demonstrated that the severe respiratory syndrome that manifests due to COVID-19 is characterised by cluster of differentiation (CD)4 and CD8 T lymphopenia, correlating with disease severity.6 Owing to the cytokine storm, and other immunological predictors of severity of SARS-CoV2 infection, these may also be helpful in choosing anti-inflammatory drugs, especially corticosteroids for their potential benefit in the reduction of inflammation-induced lung damage in severe COVID-19 patients.1In accordance with this hypothesis, it has been shown that 4–6 days following the onset of infection, T lymphocyte counts drop to their lowest level, whereas cytokine levels reach their maximum. Conversely, the restoration of the number of T lymphocytes is associated with a decrease in serum levels of various cytokines, such as IL-6, IL-10 and TNF-α.10 Cytokine levels, especially that of IL-6, can be individually measured on immunoassay analysers, whereas other cytokine profiles can be explored using either enzyme-linked immunosorbent assays or other specific biotechnologies like Luminex®, 200™ (or multiplex)  and flow cytometry systems, which allow for a large-scale quantitative measurement.14 The assessment of the main T cell subsets, such as CD3+, CD4+ and CD8+, requires only a simple phenotyping procedure conductible on a mini-cytometer, which is largely available worldwide even in limited-resource context laboratories, thanks to HIV management programs. More developed flow cytometry platforms allow for comprehensive phenotyping assays, enabling the investigation of naive, memory and regulatory T cells; in severe COVID-19 patients, early data on T cell subpopulation abnormalities show increased naive helper T cells and decreased memory helper T cells, associated with a lower number of regulatory T cells.10Having shown that the most relevant immunological parameters include inflammatory cytokines and T lymphocyte subpopulations, the clinical immunology laboratory is positioned to be important for assessing COVID-19 patients, especially for categorising, or even predicting severe cases.2 considered 559 cells/µL, 235 cells/µL, and 104 cells/µL of CD3+, CD4+T, and CD8+ T cell subsets as warning values, below which there was a significantly higher risk of in-hospital death.2 Indeed, there is a need for regional data analyses before determining cut-off values of these T cell subsets. However, T cell lymphopenia might be accentuated by possible primary or acquired immune deficiency conditions, potentially revealed by COVID-19, and must then be considered first when interpreting cell phenotyping values and when managing patients as well.15Using a receiver operating characteristic curve to compare fatal and recovered COVID-19 cases, Xu et al.For SARS-CoV-2 infection, alongside other clinical and biological parameters, the measurement of inflammatory cytokines, mainly IL-6, as well as CD4+ and CD8+ T cell assessment, should be systematically planned during management of the disease. These markers could be useful for identifying severe cases requiring prompt admission to intensive care units, and for monitoring patient disease progression. These investigations are within the reach of almost all clinical immunology laboratories in the world. Close collaboration between immunologists and physicians is essential for effective global efforts against this highly threatening pandemic.


However, N-acetylcysteine (NAC), a ROS scavenger, significantly attenuated the apoptosis-inducing effect of coptisine. It is worth noting that coptisine significantly upregulated phosphorylation of ROS-dependent c-Jun N-terminal kinase (JNK), whereas treatment with JNK inhibitor could suppress an apoptosis-related series event. Taken together, our results suggest that coptisine has an anticancer effect in Hep3B cells through ROS-mediated activation of the JNK signaling pathway.Hepatocellular carcinoma (HCC) has a high mortality rate worldwide, and treatment is very limited due to its high recurrence and low diagnosis rate, and therefore there is an increasing need to develop more effective drugs to treat HCC. Coptisine is one of the isoquinoline alkaloids, and it has various pharmacological effects. However, the evidence for the molecular mechanism of the anticancer efficacy is still insufficient. Therefore, this study investigated the antiproliferative effect of coptisine on human HCC Hep3B cells and identified the action mechanism. Our results showed that coptisine markedly increased DNA damage and apoptotic cell death, which was associated with induction of death receptor proteins. Coptisine also significantly upregulated expression of proapoptotic Bax protein, downregulated expression of anti-apoptotic Bcl-2 protein, and activated caspase-3, -8, and -9. In addition, coptisine remarkably increased the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP), and release of cytochrome  Hepatocellular carcinoma (HCC) is a major form of liver malignancy, which is the second highest cause of cancer death worldwide ,2. In thRhizoma coptidis (roots of Coptis chinensis Franch) [Over recent decades, natural plant products, including medicinal herbs and phytochemicals, have been reported to have many advantages, due to their cost effectiveness, few adverse effects, and higher safety, and they have been used as potent therapeutic and chemopreventive agents for various chronic diseases ,11. Accu Franch) ,13, and  Franch) , bacteri Franch) , and met Franch) ,17 are w Franch) ,20,21,22 Franch)  demonstr Franch) ,24. AlthTo assess the cytotoxic effect of coptisine in Hep3B cells, we measured cell viability using a 3--2-,5-diphenyltetrazolium bromide (MTT) assay. As shown in In order to evaluate the effect of coptisine on apoptotic DNA damage, comet assay, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and immunofluorescence analysis of phospho-H2A histone family member X (p-γH2AX) were performed. Next, we investigated whether either extrinsic or intrinsic pathways were involved in coptisine-mediated apoptotic cell death. Our results demonstrated that coptisine precisely increased the expression of death receptor 4 (DR4) and DR5, but the expression of Fas remained unchanged A. CoptisOn the basis of the above result that coptisine regulated the expression of Bcl-2 family proteins, we investigated the effect of coptisine on the function of mitochondria in Hep3B cells. Consequently, the population of depolarized cell was substantially enhanced by coptisine A,B. AddiN-acetylcysteine (NAC), a ROS scavenger  signaling pathway including the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. The results demonstrated that coptisine upregulated the phosphorylation of JNK (c-JUN) with increasing concentration, while phosphorylated ERK (p-ERK) was decreased without affecting p38 MAPK A. AdditiRhizoma coptidis [Coptisine is one of the active compounds discovered in coptidis . Althougcoptidis ,19,20, tApoptosis is a very tightly programmed cell death process that plays a crucial role in several biological processes in normal tissues. Defects in apoptosis can promote tumor development, and also make cancer cells resistant to treatment. In this respect, the evasion of apoptosis is a noticeable hallmark of cancer ,26. ActuDNA damage is a primary response with the biological goal of protecting a multicellular organism against damaged cells . Faulty c, were released into the cytoplasm. Cytochrome c released into the cytoplasm activated caspase-9, which, in turn, activated executioner caspases and led to the induction of apoptosis through the cleavage of various cellular substrates, such as PARP [c. Meanwhile, coptisine-induced apoptotic cell death was suppressed by Z-VAD-FMK, a pan caspase inhibitor. Therefore, based on those observations, we speculated that the proapoptotic effect of coptisine in Hep3B cells can be induced by simultaneously activating caspase-dependent extrinsic and intrinsic pathways.It is well known that apoptosis is regulated by intrinsic and extrinsic signaling pathways that are triggered by multiple factors ,35. On t as PARP ,39. Our Many previous studies have found that a number of anticancer drugs induce apoptosis through their oxidative properties, such as destroying cellular antioxidant systems or increasing ROS generation ,41. AlthIt is well know that the MAPKs signaling pathway regulates various cellular responses including apoptosis. Numerous studies have established that JNK and p38 MAPK signaling pathways are involved in cell death, whereas the ERK signaling pathway is associated with the survival of cells ,43. As rIn summary, the present study has demonstrated that coptisine exerts antitumor effects in Hep3B cells through induction of extrinsic and intrinsic apoptosis pathways. Coptisine induces ROS-dependent apoptosis by targeting JNK signaling pathway, DNA damage, and mitochondrial dysfunction . Taken tl-glutamine, and 1% antibiotics  at 37 °C in a humidified 5% CO2 atmosphere. Coptisine  was purchased from Sigma-Aldrich Chemical Co. , dissolved in dimethyl sulfoxide . Prior to use, the stock solution of coptisine was diluted to the required concentrations in culture medium.The Hep3B cells were purchased from the American Type Culture Collection . Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM 3 cells per well. After 24 h of incubation, the cells were treated with different concentrations of coptisine, for 24 h, in the presence or absence of NAC . Subsequently, the cells were incubated with 0.5 mg/mL MTT (AMRESCO) solution, at 37 °C. After incubation for 2 h, the medium was removed, and then 200 µL of DMSO was added to the well to dissolve the formazan. The cell viability was determined by measuring the optical density at 540 nm with a microplate reader .Cell viability was measured using an MTT assay, as previously described . Hep3B cThe quantification of apoptosis was determined by flow cytometry using an annexin V-FITC/PI double staining kit , according to the manufacturer’s instruction. Briefly, cells were treated with the desired concentrations of coptisine, for 24 h, in the presence or absence of Z-VAD-FMK . The collected cells were re-suspended in binding buffer, and then stained with PI solution and FITC-conjugated annexin V, for 15 min, in dark, as previously described . The fluThe comet assay was assessed to detect the DNA damage in individual cells, as previously describe . In brieThe extent of the DNA breaks in apoptotic cells was evaluated using the TUNEL staining kit  . In brieCells were seeded in 4-well plates and treated as indicated in each experiment. After 24 h, cells were fixed with methanol at −20 °C for 10 min, and then washed three times using phosphate-buffered saline (PBS). The cells were permeabilized using 0.1% Triton X-100  and blocked with 5% bovine serum albumin  in PBS, for 1 h. The cells were incubated with anti-p-γH2AX  antibody in 5% BSA overnight at 4 °C, and then washed with Tris-buffered saline and Tween 20 (TBST). Following washing for 1 h with anti-rabbit Alexa-conjugated secondary antibody  and staining with DAPI, they were observed by fluorescence microscopy.The cells were collected, washed with PBS, and then lysed with lysis buffer, at 4 °C, for 30 min, to extract whole cell proteins . The cytCaspase-3, -8, and -9 activities were determined using colorimetric assay kits , according to the manufacturer’s instruction. The cells were treated with coptisine for 24 h, and then cells were collected and lysed in the lysis buffer. After centrifugation, the supernatants were collected and incubated with the supplied reaction buffer, which contained the caspases substrates and dithiothreitol  for caspase-3, Ile–Glu–Thr–Asp (IETD) for caspase-8, and Leu–Glu–His–Asp (LEHD) for caspase-9), at 37 °C, for 2 h in the dark. The activities of caspases were determined by a microplate reader using wavelength of 405 nm.The MMP was measured by JC-1 , which is a mitochondria-specific fluorescent dye. The cells were treated with the indicated concentrations of coptisine. After 24 h, the cells were subjected to 10 μM JC-1 dye, for 20 min, at 37 °C. After washing to remove unbound dye, the green (JC-1 monomers) and red (JC-1 aggregates) fluorescence was immediately acquired using flow cytometry.The production of intracellular and mitochondrial ROS was evaluated using DCF-DA  and MitoSOX™ red dye (Thermo Fisher Scientific), respectively. The cells were treated with coptisine for 30 min, and then incubated with 10 μM DCF-DA or 1 μM MitoSOX™ red, for 20 min, at 37 °C, without light. The fluorescence intensity was detected by flow cytometry. For image analysis of the ROS production, the cells were collected using a Thermo Shandon Cytospin 3 cytocentrifuge , and stained with DAPI, which is a nuclear-specific fluorescence dye. Subsequently, the cells were mounted using a mounting solution , and the images were visualized using an EVOS Cell Imaging System .All results were expressed as the mean ± SD. Statistical analysis was by a one-way analysis of variance (ANOVA) and Tukey’s post hoc test using GraphPad Prism software . The p-value was less than 0.05, and the differences between groups were considered statistically significant.Overall, our results suggest that coptisine inhibited cell viability and simultaneously increased apoptosis that concerned both extrinsic and intrinsic pathways in Hep3B cells. In addition, coptisine induced DNA damage and mitochondrial dysfunction by the ROS-mediated JNK signaling pathway. Consequently, coptisine exerts an anticancer effect in Hep3B cells via the regulation of the ROS-induced JNK pathway, and this result provides further information for the treatment of HCC.


Interestingly, those assembled materials exhibit interesting external stimuli responsiveness to e.g., variable concentrations, changed pH values, different temperature, as well as the addition/removal of competition guests and ions. Thus, they could further be used for diverse applications such as detection, sorption, and separation of significant multi-analytes including metal cations, anions, and amino acids.Pillararenes trimer with particularly designed structural geometry and excellent capacity of recognizing guest molecules is a very efficient and attractive building block for the fabrication of advanced self-assembled materials. Pillararenes trimers could be prepared via both covalent and noncovalent bonds. The classic organic synthesis reactions such as click reaction, palladium-catalyzed coupling reaction, amidation, esterification, and aminolysis are employed to build covalent bonds and integrate three pieces of pillararenes subunits together into the “star-shaped” trimers and linear foldamers. Alternatively, pillararenes trimers could also be assembled in the form of host-guest inclusions and mechanically interlocked molecules via noncovalent interactions, and during those procedures, pillararenes units contribute the cavity for recognizing guest molecules and act as a “wheel” subunit, respectively. By fully utilizing the driving forces such as host-guest interactions, charge transfer, hydrophobic, hydrogen bonding, and C–H Self-assembly has attracted much more attention in fundamental researches of material science and interesting applications to practical engineering areas ,2, e.g.,Macrocycles is a kind of particular cyclic oligomers with the hollow cavity for recognizing guest molecules ,7, proviTo enlarge the family of assemblies with controllable multi-dimensional morphologies such as two-dimensional materials, pillararenes trimers have been designed and have attracted much more attentions recently. Different from pillararenes monomers and dimers, pillararenes trimers integrate three pieces of pillararenes units together via a “core” bridge, contribute a planar geometry as building blocks for advanced self-assemblies, and generate unique physiochemical properties such as aggregation-induced absorption .PT1–PT10 according to the timeline, G1–G9, In this review, we briefly summarized the recent progress about pillararenes trimers palladium(II) were used in coupling alkynes together for the synthesis of PT2  20]..PT2 amine in the toluene/methanol mixture could produce PT8  NMR measurements, ESI-MS and gel permeation chromatography (GPC) spectra.Mechanically interlocked molecules are fabricated by noncovalent bonds, but only can be destroyed by damaging the covalent bonds . The desX11  performsX11 amine , quite different from the behaviors shown by inclusions between pillararenes monomers/dimers and guests.In another example, benzene-1,3,5-trispillar[5]arene PT4  could efPT4 G3,  and trismine G4,  via hostG2 amine subunit on PT8 and N-phenyl-3-(phenylimino)-3H-phenothiazin-7-amine  indicates the binding strength of inclusions, and is able to be detected by e.g., the Benesi–Hildebrand equation arenes , which a


Background: Several studies suggested cross talk among components of hemostasis, inflammation, and immunity pathways in the pathogenesis, neurodegeneration, and occurrence of cerebral microbleeds (CMBs) in multiple sclerosis (MS).Objectives: This study aimed to evaluate the combined contribution of the hemostasis inhibitor protein C (PC) and chemokine C-C motif ligand 18 (CCL18) levels to brain atrophy in MS and to identify disease-relevant correlations among circulating levels of hemostasis inhibitors, chemokines, and adhesion molecules, particularly in CMB occurrence in MS.Methods: Plasma levels of hemostasis inhibitors , CCL18, and soluble adhesion molecules  were evaluated by multiplex in 138 MS patients [85 relapsing-remitting (RR-MS) and 53 progressive (P-MS)] and 42 healthy individuals (HI) who underwent 3-T MRI exams. Association of protein levels with MRI outcomes was performed by regression analysis. Correlations among protein levels were assessed by partial correlation and Pearson's correlation.Results: In all patients, regression analysis showed that higher PC levels were associated with lower brain volumes, including the brain parenchyma (p = 0.002), gray matter (p < 0.001), cortex (p = 0.001), deep gray matter (p = 0.001), and thalamus (p = 0.001). These associations were detectable in RR-MS but not in P-MS patients. Higher CCL18 levels were associated with higher T2-lesion volumes in all MS patients (p = 0.03) and in the P-MS (p = 0.003). In the P-MS, higher CCL18 levels were also associated with lower volumes of the gray matter (p = 0.024), cortex (p = 0.043), deep gray matter (p = 0.029), and thalamus (p = 0.022). PC-CCL18 and CCL18-PAI1 levels were positively correlated in both MS and HI, PC–sVAP1 and PAI1–sVCAM1 only in MS, and PC–sICAM1 and PC–sNCAM only in HI. In MS patients with CMBs (n = 12), CCL18–PAI1 and PAI1–sVCAM1 levels were better correlated than those in MS patients without CMBs, and a novel ADAMTS13–sVAP1 level correlation  was observed.Conclusions: Differences between clinical phenotype groups in association of PC and CCL18 circulating levels with MRI outcomes might be related to different aspects of neurodegeneration. Disease-related pathway dysregulation is supported by several protein level correlation differences between MS patients and HI. The integrated analysis of plasma proteins and MRI measures provide evidence for new relationships among hemostasis, inflammation, and immunity pathways, relevant for MS and for the occurrence of CMBs. In multiple sclerosis (MS) pathogenesis, blood–brain barrier (BBB) disruption and vascular changes interact in a vicious cycle with altered immune trafficking and the inflammatory processes, supported by adhesion molecules and chemokines –3. SeverPlasma levels of protein C (PC) have been associated with neurodegenerative magnetic resonance imaging (MRI) outcomes in MS patients . Among hNeurodegenerative outcomes in MS patients have been also associated with higher plasma levels of C-C motif ligand 18 (CCL18), a chemokine involved in immune cell chemotaxis . To noteAmong the main regulators of hemostasis, the disintegrin-like and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) has been suggested to support vascular integrity –16, and The progressive failure of BBB integrity might have the pathological features of cerebral microbleeds (CMBs) , revealeTaking advantage of main findings reported in our previous studies, focused on different biological pathways and performed on the same MS cohort, we hypothesized that circulating concentration of hemostasis inhibitors could participate in the immunity and inflammation MS-related network. To assess this hypothesis, in the current study, (i) we evaluated the combined contribution of the main coagulation inhibitor PC and the CCL18 chemokine levels to MS brain atrophy, (ii) we compared PC and CCL18 plasma concentrations for their ability to explain the observed neurodegeneration, and (iii) we investigated the correlations among circulating levels of hemostasis inhibitors , CCL18, and adhesion molecules , particularly in relation to CMBs.The population used for this analysis included 138 MS patients and 42 healthy individuals (HI) derived from the CEG-MS study. Details on the collection, diagnosis, and demographics of this cohort have been previously described . The stuMultiplex magnetic-bead technology  was used to measure the following panel of hemostasis inhibitors, chemokines, and adhesion molecules: ADAMTS13, PC, PAI1, CCL18, sICAM1, sNCAM, sVAP1, and sVCAM1 , 20, 21.Brain MRI was performed by 3-T GE Signa Excite HD 12.0 scanner  with an eight-channel head and neck coil. Details of the acquisition protocol and MRI analyses were previously provided , 14 and 2 field of view (FOV), resulting in a nominal in-plane resolution of 1 × 1 mm2. For the whole-brain coverage, 48 gap-less 3-mm-thick slices were acquired. The sequence-specific parameters were as follows: dual fast spin echo (FSE) proton density and T2-WI , 4:31 min long; FLAIR , 4:16 min long; and spin-echo T1-WI (TE/TR = 16/600 ms), 4:07 min long. Last, a 3D high-resolution T1-WI fast spoiled gradient echo sequence with a magnetization-prepared inversion recovery pulse was obtained , 4:39 min long, with 184 slices of 1 mm.Acquisition of two-dimensional (2D) T2/PD-weighted images (WI), fluid-attenuated inversion recovery (FLAIR), spin-echo T1-WI with and without gadolinium contrast, and a three-dimensional (3D) high-resolution T1-WI was performed. 2D sequences were collected using a 256 × 192 matrix and 256 × 192 mmFor the image analysis, a semi-automated edge detection thresholding technique was used to assess T2- and T1-LV, as previously reported . Prior tThe CMB analysis was performed on susceptibility-weighted imaging (SWI) minimum-intensity projection images and susceptibility maps. CMBs were classified as focal, small, and round to ovoid punctuate areas of signal hypointensity on SWI minimum-intensity projection images, as previously reported . Signal 2, Student's t-test, or Mann–Whitney U-test.Analyses were performed using SPSS . Demographic and clinical variables were compared using χTo evaluate the contribution of PC and CCL18 to MS brain atrophy, multiple regression analysis was conducted with each MRI characteristic used as the dependent variable while age, sex, body mass index (BMI), and the plasma protein levels were predictor variables. The first block included the forced entry of age, sex, and BMI; and the second block included the stepwise entry of PC and CCL18 natural logarithmic values. To further determine the findings' validity, multivariate regression analysis by enter method with 1000-sample bootstrapping procedure was performed.Association analysis of logarithmic values of protein levels in MS patients and HI was performed by partial correlation with 1000-sample bootstrapping procedure, using age and sex as covariates. Due to the low number of MS patients with CMBs, Pearson's correlation with 1000-sample bootstrapping procedure was used to assess associations among logarithmic values of protein levels.p-values are based on two-tailed statistical tests, with a significance level of 0.05.All reported p < 0.001), and the RR-MS and P-MS groups differed in clinical characteristics and brain MRI measures (The progressive (P)-MS group comprised 46 secondary-progressive MS patients and seven primary-progressive MS patients. As expected, P-MS patients were older than relapsing-remitting (RR)-MS patients .In the whole MS population, higher CCL18 levels were associated with higher T2-lesion volume (LV). CCL18 alone was able to predict the 10% of total variation of T2-LV, and for one logarithmic unit (~2.7 ng/ml) increase in CCL18 was associated with 8.16-ml increase in T2-LV. This result showed a trend for significance in bootstrap analysis  increase in PC was associated with decrease in GMV (41.2 ml), in CV (30 ml), in DGMV (5.04 ml), and in thalamic volume (1.7 ml).In P-MS patients, one logarithmic unit (~2.7 ng/ml) increase in CCL18 was associated with decrease in GMV (44.4 ml), in CV (32.4 ml), in DGMV (5.8 ml), and in thalamic volume (2.1 ml) and with increase in T2-LV (25 ml).In MS patients and in HI, PC levels were positively associated with CCL18 levels .p = 0.008, CI 95% = 0.08, 0.48) but not in progressive patients .A focused sub-analysis in clinical phenotype groups showed that PC-CCL18 levels were correlated in RR-MS patients  was even stronger than in MS patients without CMBs . Similarly, in MS with CMBs, the correlation between PAI1 and sVCAM1  was better than in MS patients without CMBs .In MS patients with CMBs, the correlation between CCL18 and PAI1 . Correlation between ADAMTS13 and sVAP1 was detectable neither/nor in MS without CMBs  nor in HI .Levels of ADAMTS13 were correlated with those of sVAP1  the differences between PC and CCL18 levels in the ability to predict neurodegeneration in MS patients and (ii) the positive correlation between PC and CCL18 levels present in diseased and healthy conditions. Both observations support the hypothesis of a relation between the hemostasis and chemokine pathways, which might act in the disease pathophysiology. Our findings in patients are coherent with previous data: (i) PC and CCL18 levels have been positively associated with neurodegeneration , 10, ii, which bThe different results of the regression analysis between the clinical phenotype groups suggest that the circulating levels of PC and CCL18 might be related to different aspects of neurodegeneration. The relation of PC levels with GM-related volumes in whole MS population and RR-MS could be interpreted as an increase of PC expression-associated with inflammation. This might represent the response to diffuse neuronal loss associated with inflammatory and oxidative injuries, which might occur independently of focal lesions . Our datThese hypotheses are strengthened by the analysis of level correlation among proteins related to MRI findings in MS patients, which pointed out the noticeable correlation between levels of PC and CCL18, both associated with neurodegeneration. This correlation would suggest factors able to upregulate expression of both PC and CCL18 by mechanisms that are only partially known –35. On tThe hemostasis inhibitors–immunity link was even stronger in the low number of patients with CMBs, who displayed high correlation between CCL18 and PAI1 levels. The high association of PAI1 with sVCAM1 extends this molecular relationship to adhesion molecules. Several differences between MS patients and HI in PC correlation with sICAM1, sNCAM, and sVAP1 concentrations support dysregulation, associated with the MS disease, of the adhesion molecules and PC pathways. The absence of correlation between PC and sICAM and sNCAM in MS patients could be reflected in a decreased inhibition of leukocyte adhesion and transmigration , 8.Based on the high correlation between ADAMTS13 with sVAP1 detected only in MS patients with CMBs, and on low ADAMTS13  and highIn conclusion, with the limitations of a cross-sectional study and the low number of MS patients with CMBs, the integrated analysis of plasma proteins and MRI measures, here reported, provides evidence for new MS disease-relevant relationships among hemostasis, inflammation, and immunity pathways.The datasets presented in this article are not readily available because they are restricted to the Buffalo Neuroimaging Analysis Center. Requests to access the datasets should be directed to Robert Zivadinov, rzivadinov@bnac.net.The studies involving human participants were reviewed and approved by The study protocols were approved by the institutional review boards of the University at Buffalo (USA) (ID:MODCR00000352) and the University/Hospital of Ferrara  (ID:170585). The patients/participants provided their written informed consent to participate in this study.NZ, RZ, MR, GM, and FB substantially contributed to the concept and design of the study. MB contributed to assay setup. NZ contributed to data acquisition, analysis, and interpretation. DJ, MB, NB, DR, BW-G, and RZ contributed to data acquisition. RZ, DJ, NB, and BW-G contributed to data interpretation, discussion, and manuscript preparation. NZ, GM, and FB wrote the manuscript. All authors critically revised the manuscript for important intellectual content and contributed to the article and approved the submitted version.RZ reports personal fees from EMD Serono, Genzyme-Sanofi, Bristol Myers Squibb, and Novartis for speaking and consultant fees, and grants from Genzyme-Sanofi, Novartis, Bristol Myers Squibb, Mapi Pharma, Keystone Heart, Boston Scientific, Protembis, and V-Vawe Medical unrelated to the submitted work. BW-G reports grants and personal fees from Biogen, EMD Serono, Novartis, and Genentech, and personal fees from Mallinckrodt, outside the submitted work. MR reports grants from Otsuka Pharmaceutical Research and Development and grants from National Institute of Neurological Diseases and Stroke, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.