Pitfalls and promises of bile duct alternatives:There is plenty of room in the regenerative surgery

Current abdominal surgery has several approaches for biliary reconstruction.However,the creation of functional and clinically applicable bile duct substitutes still represents an unmet need.In the paper by Miyazawa and colleagues,approaches to the creation of bile duct alternatives were summarized,and the reasons for the lack of development in this area were explained.The history of bile duct surgery since the nineteenth century was also traced,leading to the conclusion that the use of bioabsorbable materials holds promise for the creation of bile duct substitutes in the future.We suggest three ideas that may stimulate progress in the field of bile duct substitute creation.First,a systematic analysis of the causative factors leading to failure or success in the creation of bile duct substitutes may help to develop more effective approaches.Second,the regeneration of a bile duct is delicately balanced between epithelialization and subsequent submucosal maturation within limited time frames,which may be more apparent when using quantitative models to estimate outcomes.Third,the utilization of the organism’s endogenous regeneration abilities may enhance the creation of bile duct substitutes.We are convinced that an interdisciplinary approach,including quantitative methods,machine learning,and deep retrospective analysis of the causes that led to success and failure in studies on the creation of bile duct substitutes,holds great value.Additionally,more attention should be directed towards the balance of epithelialization and submucosal maturation rates,as well as induced angiogenesis.These ideas deserve further investigation to pave the way for bile duct restoration with physiologically relevant outcomes.

Clinical safety and efficacy of allogenic human adipose mesenchymal stromal cells-derived exosomes in patients with mild to moderate Alzheimer’s disease:a phaseⅠ/Ⅱclinical trial

Background There have been no effective treatments for slowing or reversing Alzheimer’s disease(AD)until now.Growing preclinical evidence,including this study,suggests that mesenchymal stem cells-secreted exosomes(MSCs-Exos)have the potential to cure AD.Aims The first three-arm,drug-intervention,phase I/II clinical trial was conducted to explore the safety and efficacy of allogenic human adipose MSCs-Exos(ahaMSCs-Exos)in patients with mild to moderate AD.Methods The eligible subjects were assigned to one of three dosage groups,intranasally administrated with ahaMSCs-Exos two times per week for 12 weeks,and underwent follow-up visits at weeks 16,24,36 and 48.Results No adverse events were reported.In the medium-dose arm,Alzheimer’s Disease Assessment Scale–Cognitive section(ADAS-cog)scores decreased by 2.33(1.19)and the basic version of Montreal Cognitive Assessment scores increased by 2.38(0.58)at week 12 compared with baseline levels,indicating improved cognitive function.Moreover,the ADAS-cog scores in the medium-dose arm decreased continuously by 3.98 points until week 36.There were no significant differences in altered amyloid or tau deposition among the three arms,but hippocampal volume shrank less in the medium-dose arm to some extent.Conclusions Intranasal administration of ahaMSCs-Exos was safe and well tolerated,and a dose of at least 4×10^(8)particles could be selected for further clinical trials.

(EDTA)^(4-) supplements as superior modifier of the in-vitro-degradation properties of the magnesium alloy coated through discharge-assisted process

The(EDTA)^(4-)organic substituents were supplemented with alkaline silicates electrolyte,however in varying concentration to explore the microstructure,chemical composition and subsequent effects on the biodegradation of the PEO coated Mg AZ91 alloy.From surface analysis-,carried out through SEM,samples with zero-(EDTA)^(4-)were found-,lacking pancake structural features;however,supplementing electrolyte with(EDTA)^(4-),bulky and layered pancakes were obtained with minimum cracks and minimum porosity for EDT-1.From the phase and inner layer analysis,it was found that EDTA can significantly alter the surface layers composition and could act as a passivation agent-;however,individual polycrystalline phases were not found to form.The hardness value obtained for EDT-1 was~909.69 HV,the highest value found here which occurs with the shallowest indents found in this study.In-vitro degradation analysis was carried out using potentiodynamic polarization at room temperature,cyclic polarization at RT and high fever conditions(HFT~40°C).The corrosion potential for coated AZ91 was obtained as a positive value of~0.1 V,for the first time in the PEO research;however,the results were not supported by the cyclic polarization tests at room temperature and at HFT.The post corrosion microstructural analysis confirmed the effective role of EDTA up to 1.0 g·L^(-1)and revealed the formation of thin films during the forward polarization at HFT.

Expression of Rab25 correlates with the invasion and metastasis of gastric cancer

The objective of this study was to determine the expression of the important vesicle trafficking- regulating factor Rab25 in human gastric cancer tissues, to analyze the correlation between Rab25 protein expression with gastric cancer occurrence and development, and to discuss the correlation of Rab25 protein expression with gastric cancer cell metastasis. The overall aim was to provide experimental evidence that can be used to design future biological treatments of human gastric cancer. Human gastric cancer tissue and the adjacent normal gastric tissue were surgically removed, and immunohistochemistry and Western blotting were used to detect Rab25 protein expression. The correlation between Rab25 protein expression with the development and pathological characteristics of gastric cancer was analyzed. Using RNAi, Rab25 expression was reduced in the gastric cancer cell line MGC80-3, and the changes in MGC80-3 cell invasiveness were then monitored. Immunohistochemistry showed that the Rab25 protein expression rates were 78.21% and 23.08% in gastric carcinoma and the adjacent normal gastric tissue, respectively. Immunohistochemistry and Western blot results showed that Rab25 protein expression in gastric cancer was significantly higher than in adjacent normal gastric tissues (P<0.01). Less differentiated gastric cancer cells had higher expression of Rab25 protein (P<0.01). Gastric carcinomas from patients with a late pathological stage (III-IV) had significantly higher Rab25 protein expression than early stage (I-II) patients (P<0.01). Gastric carcinomas from patients with lymph node metastasis had significantly higher Rab25 protein expression than lymph node metastasis- free patients (P<0.01). Gastric carcinomas from patients with distant metastases had significantly higher Rab25 protein expression than the distant metastasis-negative patients (P<0.01). Rab25 protein expression in gastric cancer was not affected by the patients , sex, age, or tumor size (P>0.05). MGC80-3 cells transfected with Rab25 siRNA had significantly lower Rab25 protein expression (P<0.01) and a significantly lower number of cells that passed through a Transwell chamber compared with non-transfected controls and the transfected control group (P<0.01). Rab25 protein expression is associated with the development of gastric cancer. siRNA knockdown of Rab25 protein expression in MGC80-3 gastric cancer cells reduced MGC80-3 cell invasiveness and provided experimental evidence for potential future biological treatment strategies of human gastric cancer.

Quantitative evaluation of long-term liver repopulation and the reconstitution of bile ductules after hepatocellular transplantation

AIM: The treatment of liver disease is severely limited by a shortage of donor livers. In trying to address this growing problem, hepatocellular transplantation (HTx) has received much attention as an alternative to whole organ transplant.However, the expansion of transplanted cells is at low level,and the reconstitution of functional liver tissue is limited by this cellular property. We set up an animal model to better understand cell dose effect and the kinetics of liver repopulation following HTx.METHODS: Dipeptidyl peptidase Ⅳ (DPPIV)-deficient rats treated with retrorsine and subjected to partial hepatectomy were infused with DPPIV-positive hepatocytes. Rats were injected with varying numbers of donor hepatocytes down to 100 cells low, and liver repopulation was examined at different time points up to 20 mo long. Repopulation was assessed by computer-aided quantitative detection.RESULTS: Transplanted hepatocytes underwent multiple rounds of proliferation and stably repopulated the injured livers after 20 mo and at all cell doses. Transplanted cells divided 14 times within the 3-mo time period following infusion, and the liver repopulation reached a plateau between 3 and 20 mo. Approximately 90% replacement occurred. Donor-derived cells also reconstituted the bile ductules of the recipients.CONCLUSION: The ability of transplanted hepatocytes to fully reconstitute injured livers strongly supports further investigation into the clinical potential of HTx. Additionally,the observation that transplanted hepatocytes also form components of the biliary system suggests that these cells may have bi-potential property of the stem cells.

Methylation of RAR-β2, RASSF1A, and CDKN2A Genes Induced by Nickel Subsulfide and Nickel-carcinogenesis in Rats

Objective To investigate the expression variation of RAR‐β2, RASSF1A, and CDKN2A gene in the process of nickel‐induced carcinogenesis. Methods Nickel subsulfide (Ni 3 S 2 ) at dose of 10 mg was given to Wistar rats by intramuscular injection. The mRNA expression of the three genes in induced tumors and their lung metastasis were examined by Real‐time PCR. The methylation status of the 5' region of these genes were detected by Quantitative Real‐time methylation specific PCR. Results The mRNA expressions of the three genes both in muscle and lung tumor were decreased distinctly in comparison with normal tissue. But hypermethylation was found only in muscle tumor. Conclusion These findings suggest that loss of function or decrease of RAR‐β2, RASSF1A, and CDKN2A, as well as the hypermethylation of 5' region of these genes, are related with nickel exposure.

Anticancer effect of linalool via cancer-specific hydroxyl radical generation in human colon cancer

AIM To investigate the anticancer mechanisms of the monoterpenoid alcohol linalool in human colon cancer cells.METHODS The cytotoxic effect of linalool on the human colon cancer cell lines and a human fibroblast cell line was examined using the WST-8 assay. The apoptosisinducing effect of linalool was measured using the terminal deoxynucleotidyl transferase d UTP nickend labeling assay and flow cytometry with Annexin V. Oxidative stress was investigated by staining for diphenyl-1-pyrenylphosphine, which is a cellular lipid peroxidation marker, and electron spin resonance spectroscopy. Sixteen SCID mice xenografted with human cancer cells were randomized into 3 groups for in vivo analysis: control and low-dose and high-dose linalool groups. The control group was administered tap water orally every 3 d. The linalool treatment groups were administered 100 or 200 μg/kg linalool solution orally for the same period. All mice were sacrificed under anesthesia 21 d after tumor inoculation, and tumors and organs were collected for immunohistochemistry using an anti-4-hydroxynonenal antibody. Tumor weights were measured and compared between groups. RESULTS Linalool induced apoptosis of cancer cells in vitro, following the cancer-specific induction of oxidative stress, which was measured based on spontaneous hydroxyl radical production and delayed lipid peroxidation. Mice in the high-dose linalool group exhibited a 55% reduction in mean xenograft tumor weight compared with mice in the control group(P < 0.05). In addition, tumor-specific lipid peroxidation was observed in the in vivo model.CONCLUSION Linalool exhibited an anticancer effect via cancerspecific oxidative stress, and this agent has potential for application in colon cancer therapy.

Cytokines in adipose-derived mesenchymal stem cells promote the healing of liver disease

Adipose-derived mesenchymal stem cells(ADSCs) are a treatment cell source for patients with chronic liver injury. ADSCs are characterized by being harvested from the patient's own subcutaneous adipose tissue, a high cell yield(i.e., reduced immune rejection response), accumulation at a disease nidus, suppression of excessive immune response, production of various growth factors and cytokines, angiogenic effects, antiapoptotic effects, and control of immune cells via cellcell interaction. We previously showed that conditioned medium of ADSCs promoted hepatocyte proliferation and improved the liver function in a mouse model of acute liver failure. Furthermore, as found by many other groups, the administration of ADSCs improved liver tissue fibrosis in a mouse model of liver cirrhosis. A comprehensive protein expression analysis by liquid chromatography with tandem mass spectrometry showed that the various cytokines and chemokines produced by ADSCs promote the healing of liver disease. In this review, we examine the ability of expressed protein components of ADSCs to promote healing in cell therapy for liver disease. Previous studies demonstrated that ADSCs are a treatment cell source for patients with chronic liver injury. This review describes the various cytokines and chemokines produced by ADSCs that promote the healing of liver disease.

Erythropoietin Receptor Positive Circulating Progenitor Cells and Endothelial Progenitor Cells in Patients with Different Stages of Diabetic Retinopathy

Objective To investigate the possible involvement of erythropoietin (EPO)/erythropoietin receptor (EPOR) system in neovascularization and vascular regeneration in diabetic retinopathy (DR). Methods EPOR positive circulating progenitor cells (CPCs: CD34+) and endothelial pro-genitor cells (EPCs: CD34+KDR+) were assessed by flow cytometry in type 2 diabetic patients with different stages of DR. The cohort consisted of age- and sex-matched control patients without diabetes (n=7), non-proliferative DR (NPDR, n=7), proliferative DR (PDR, n=8), and PDR complicated with diabetic nephropathy (PDR-DN, n=7). Results The numbers of EPOR+ CPCs and EPOR+ EPCs were reduced remarkably in NPDR compared with the control group (both P<0.01), whereas rebounded in PDR and PDR-DN groups in varying degrees. Similar changes were observed in respect of the proportion of EPOR+ CPCs in CPCs (NPDR vs. control, P<0.01) and that of EPOR+ EPCs in EPCs (NPDR vs. control, P<0.05). Conclusion Exogenous EPO, mediated via the EPO/EPOR system of EPCs, may alleviate the impaired vascular regeneration in NPDR, whereas it might aggravate retinal neovasculariza-tion in PDR due to a rebound of EPOR+ EPCs associated with ischemia.

Topographic patterns of vascular disease: HOX proteins as determining factors?

Steadily increasing evidence supports the idea that genetic diversities in the vascular bed are, in addition to hemodynamic influences, a major contributing factor in determining region-specific cardiovascular disease susceptibility. Members of the phylogenetically highly conserved Hox gene family of developmental regulators have to be viewed as prime candidates for determining these regional genetic differences in the vasculature. During embryonic patterning, the regionally distinct and precisely choreographed expression patterns of HOX transcription factors are essential for the correct specification of positional identities. Apparently, these topographic patterns are to some degree retained in certain adult tissues, including the circulatory system. While an understanding of the functional significance of these localized Hox activities in adult blood vessels is only beginning to emerge, an argument can be made for a role of Hox genes in the maintenance of vessel wall homeostasis and functional integrity on the one hand, and in regulating the development and progression of regionally restricted vascular pathologies, on the other. Initial functional studies in animal models, as well as data from clinical studies provide some level of support for this view. The data suggest that putative genetic regulatory networks of Hox-dependent cardiovascular disease processes include genes of diverse functional categories(extracellular matrix remodeling, transmembrane signaling, cell cycle control, inflammatory response, transcriptional control, etc.), as potential targets in both vascular smooth muscle and endothelial cells, as well as cell populations residing in the adventitia.

Construction of Ad-EGFP-BDNF vector and its expression in neural stem cells

BACKGROUND:Brain-derived neurotrophic factor(BDNF) provides nourishment to injured neurons.Neural stem cells can differentiate into neurons to repair neuronal injury in vivo.It has been hypothesized that continuous secretion of BDNF from neural stem cells could benefit brain injury repair.OBJECTIVE:To transfect BDNF and enhanced green fluorescent protein(EGFP) into neural stem cells with adenovirus vector and to observe expression of BDNF and EGFP in transfected neural stem cells.DESIGN,TIME AND SETTING:Observational,cellular,molecular study was performed at the Biochemistry Laboratory,Tongji University School of Medicine,China from July 2004 to September 2006.MATERIALS:Neural stem cells were provided by the Anatomy and Histoembryology Laboratory of Fudan University Medical School,China.METHODS:BDNF cDNA was extracted by reverse transcription polymerase chain reaction from the rat hippocampus.Following gene cloning and packaging by HEK293.BDNF,the EGFP gene was transfected into cultured neural stem cells with the Ad-EGFP-BDNF vector.BDNF-expressing neural stem cell clones were selected by G418 selection.MAIN OUTCOME MEASURES:EGFP expression and cell morphology were observed by fluorescent microscopy;neural stem cell expressing BDNF mRNA was examined by reverse transcription polymerase chain reaction;BDNF expression was detected by enzyme-linked immunosorbent assay from supernatant of infected neural stem cells.RESULTS:High transfection efficiency was obtained using 5 × 108 virus titers to transfect neural stem cells.G418-resistant neural stem cell clones integrated BDNF mRNA fragments.Enzyme-linked immunosorbent assay results showed that BDNF expression in the supernatant increased with increasing culture time and peaked at 72 hours.CONCLUSION:Adenovirus-mediated BDNF and EGFP genes were successfully transfected into neural stem cells and were expressed in neural stem cells for a long period of time.

Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment

BACKGROUND To solve the problem of liver transplantation donor insufficiency,an alternative cell transplantation therapy was investigated.We focused on amniotic epithelial cells(AECs)as a cell source because,unlike induced pluripotent stem cells,they are cost-effective and non-tumorigenic.The utilization of AECs in regenerative medicine,however,is in its infancy.A general profile for AECs has not been comprehensively analyzed.Moreover,no hepatic differentiation protocol for AECs has yet been established.To this end,we independently compiled human AEC libraries,purified amniotic stem cells(ASCs),and co-cultured them with mesenchymal stem cells(MSCs)and human umbilical vein endothelial cell(HUVECs)in a 3D system which induces functional hepatic organoids.AIM To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells METHODS AECs,MSCs,and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients.Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry,Alkaline phosphatase(AP)staining,and flow cytometry.An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay.AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics.The 2D and 3D culture were compared by relative gene expression using several differentiation protocols.ASCs,MSCs,and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging,periodic acid Schiff,and an indocyanine green(ICG)test.RESULTS AECs have certain stemness markers such as EPCAM,SSEA4,and E-cadherin.One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers.Moreover,it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage.Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells.TJP1,associated with epidermal growth factor receptor,and MET,associated with hepatocyte growth factor receptor,were upregulated and may be important for hepatic differentiation.In conventional flat culture,the cells turned unviable and did not readily differentiate into hepatocytes.In 3D culture,however,hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol.Furthermore,the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity,hepatic-like glycogen storage,and ICG absorption/elimination.CONCLUSION Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness.Under a 3D co-culture system,functional hepatic organoids were generated in a multicellular microenvironment.

Hepatoprotective effect of nitric oxide in experimental model of acute hepatic failure

AIM:To evaluate the effect of nitric oxide(NO)on the development and degree of liver failure in an animal model of acute hepatic failure(AHF).METHODS:An experimental rat model of galactosamine-induced AHF was used.An inhibitor of NO synthase,nitroarginine methyl ester,or an NO donor,arginine,were administered at various doses prior to or after the induction of AHF.RESULTS:All tested groups developed AHF.Following inhibition of the endogenous NO pathway,most liver parameters improved,regardless of the inhibitor dose before the induction of liver damage,and depending on the inhibitor dose after liver damage.Prophylactic administration of the inhibitor was more effective in improving liver function parameters than administration of the inhibitor after liver damage.An attempt to activate the endogenous NO pathway prior to the induction of liver damage did not change the observed liver function parameters.Stimulation of the endogenous NO pathway after liver damage,regardless of the NO donor dose used,improved most liver function parameters.CONCLUSION:The endogenous NO pathway plays an important role in the development of experimental galactosamine-induced AHF.

Characterization of Side Cell Populations Obtained from Human Amnion Mesenchymal Cells

Human amnion mesenchymal cells (AMCs) contain multipotent cells. To enrich such multipotent stem cells, we applied to AMCs the new method for the isolation of side population (SP) cells used for the enrichment of multipotent stem cells from many tissues. We succeeded in obtaining SP cells from AMCs (AMC-SP cells). AMC-SP cells were found in 0.2% of AMCs, irrespective of the length of pregnant period, ranging from 37 to 40 weeks. Cell cycle analyses suggested that AMC-SP cells belonged to a cell population that proliferated very slowly and/or was in a quiescent state in the amniotic membrane. Upon culturing, they proliferated with 40 to 80 cell doublings. However, they did not form colonies in a soft agarose culture, whereas HepG2 cells, representative human hepatoma cells formed many large colonies. These results suggest that AMC-SP cells that have considerable value for the use of regenerative medicine can be managed safely in vitro.

Development of the nervous system in mouse liver

BACKGROUND The role of the hepatic nervous system in liver development remains unclear.We previously created functional human micro-hepatic tissue in mice by co-culturing human hepatic endodermal cells with endothelial and mesenchymal cells.However,they lacked Glisson’s sheath[the portal tract(PT)].The PT consists of branches of the hepatic artery(HA),portal vein,and intrahepatic bile duct(IHBD),collectively called the portal triad,together with autonomic nerves.AIM To evaluate the development of the mouse hepatic nervous network in the PT using immunohistochemistry.METHODS Liver samples from C57BL/6J mice were harvested at different developmental time periods,from embryonic day(E)10.5 to postnatal day(P)56.Thin sections of the surface cut through the hepatic hilus were examined using protein gene product 9.5(PGP9.5)and cytokeratin 19(CK19)antibodies,markers of nerve fibers(NFs),and biliary epithelial cells(BECs),respectively.The numbers of NFs and IHBDs were separately counted in a PT around the hepatic hilus(center)and the peripheral area(periphery)of the liver,comparing the average values between the center and the periphery at each developmental stage.NF-IHBD and NF-HA contacts in a PT were counted,and their relationship was quantified.SRYrelated high mobility group-box gene 9(SOX9),another BEC marker;hepatocyte nuclear factor 4α(HNF4α),a marker of hepatocytes;and Jagged-1,a Notch ligand,were also immunostained to observe the PT development.RESULTS HNF4αwas expressed in the nucleus,and Jagged-1 was diffusely positive in the primitive liver at E10.5;however,the PGP9.5 and CK19 were negative in the fetal liver.SOX9-positive cells were scattered in the periportal area in the liver at E12.5.The Jagged-1 was mainly expressed in the periportal tissue,and the number of SOX9-positive cells increased at E16.5.SOX9-positive cells constructed the ductal plate and primitive IHBDs mainly at the center,and SOX-9-positive IHBDs partly acquired CK19 positivity at the same period.PGP9.5-positive bodies were first found at E16.5 and HAs were first found at P0 in the periportal tissue of the center.Therefore,primitive PT structures were first constructed at P0 in the center.Along with remodeling of the periportal tissue,the number of CK19-positive IHBDs and PGP9.5-positive NFs gradually increased,and PTs were also formed in the periphery until P5.The numbers of NFs and IHBDs were significantly higher in the center than in the periphery from E16.5 to P5.The numbers of NFs and IHBDs reached the adult level at P28,with decreased differences between the center and periphery.NFs associated more frequently with HAs than IHBDs in PTs at the early phase after birth,after which the number of NF-IHBD contacts gradually increased.CONCLUSION Mouse hepatic NFs first emerge at the center just before birth and extend toward the periphery.The interaction between NFs and IHBDs or HAs plays important roles in the morphogenesis of PT structure.

Evaluation of Steroid Receptors mRNA Fingerprints in Two Groups of Normozoospermic Patients: Men from Unexplained Infertility Couples vs. Men from Couples with Tubal Factor Infertility

The study of sperm cellular components at molecular level is crucial for the diagnosis of male unexplained infertility. The aim of the study was to compare the molecular profile of steroid receptors and aromatase in spermatozoa obtained from two normozoospermic groups of patients issued from couples treated for infertility. We investigated 46 male patients from unexplained infertility couples and from men, 38 where female partners presented with tubal infertility. Sperm ERs (estrogen receptors: alpha and beta), GPER (G protein-coupled estrogen receptor), AR (androgen receptor) and aromatase mRNA expression levels by TaqMan qPCR were analyzed. AR transcript level was significantly lower in sperm of men from unexplained infertility couples vs. men from couples with tubal factor infertility (P = 0.04). Although the AR mRNA expression level did not had any effect on embryo development and its implantation, a significant correlation between AR mRNA levels and clinical pregnancy in unexplained infertility patients was observed. Taken together, AR transcript presence in ejaculated spermatozoa could be a potential marker for unexplained infertility.

Biofabrication of size-controlled liver microtissues incorporated with ECM-derived microparticles to prolong hepatocyte function

Multicellular microtissues of primary human hepatocytes(PHHs)co-cultured with other supporting cell types are a promis-ing model for drug screening and toxicological studies.However,these liver microtissues(LMs)rapidly lose their functions during ex vivo culture.Here,in order to mimic the cellular and structural hepatic microenvironment,we co-cultured PHHs with human mesenchymal stromal cells(MSCs)and human umbilical vein endothelial cells(HUVECs)in the presence of cell-sized microparticles(MPs)derived from liver extracellular matrix(LEMPs).The microwell culture platform enabled biofabrication of size-controlled multicellular microtissues(PHH:HUVEC:MSC=3:2:1)with efficient LEMP incorporation(about 70%at a 2:1 ratio of cells:MP).The biofabricated liver microtissues(BLMs)were cultured ex vivo for 14 days and compared to the cell-only LM in terms of gene and protein expression,functional activity,cytochrome P450(CYP450)enzyme inducibility,and drug sensitivity.The results supported superior hepatic-related gene expression,functional activity,and polarity for PHH in BLM compared to LM.CYP450 enzyme inducibility and dose-responsive sensitivity to toxic drugs were significantly higher in the BLM group.In conclusion,microtissue engineering by incorporation of tissue-specific microparticles within a multicellular microtissue can offer some advantages for drug discovery studies and cell transplantation applications.In the near future,this approach could generate a scalable platform of several functional biofabricated microtissues representing different organs.

Inflammation in diabetic retinopathy: possible roles in pathogenesis and potential implications for therapy

Diabetic retinopathy, characterized as a microangiopathy and neurodegenerative disease, is the leading cause of visual impairment in diabetic patients. Many clinical features observed in diabetic retinopathy, such as capillary occlusion, acellular capillaries and retinal non-perfusion, aggregate retinal ischemia and represent relatively late events in diabetic retinopathy. In fact, retinal microvascular injury is an early event in diabetic retinopathy involving multiple biochemical alterations, and is manifested by changes to the retinal neurovascular unit and its cellular components. Currently, intravitreal anti-vascular endothelial growth factor therapy is the firstline treatment for diabetic macular edema, and benefits the patient by decreasing the edema and improving visual acuity. However, a significant proportion of patients respond poorly to anti-vascular endothelial growth factor treatments, indicating that factors other than vascular endothelial growth factor are involved in the pathogenesis of diabetic macular edema. Accumulating evidence confirms that low-grade inflammation plays a critical role in the pathogenesis and development of diabetic retinopathy as multiple inflammatory factors, such as interleukin-1β, monocyte chemotactic protein-1 and tumor necrosis factor-α, are increased in the vitreous and retina of diabetic retinopathy patients. These inflammatory factors, together with growth factors such as vascular endothelial growth factor, contribute to blood-retinal barrier breakdown, vascular damage and neuroinflammation, as well as pathological angiogenesis in diabetic retinopathy, complicated by diabetic macular edema and proliferative diabetic retinopathy. In addition, retinal cell types including microglia, Müller glia, astrocytes, retinal pigment epithelial cells, and others are activated, to secrete inflammatory mediators, aggravating cell apoptosis and subsequent vascular leakage. New therapies, targeting these inflammatory molecules or related signaling pathways, have the potential to inhibit retinal inflammation and prevent diabetic retinopathy progression. Here, we review the relevant literature to date, summarize the inflammatory mechanisms underlying the pathogenesis of diabetic retinopathy, and propose inflammation-based treatments for diabetic retinopathy and diabetic macular edema.

Epigenetic modifications and metabolic memory in diabetic retinopathy:beyond the surface

Epigenetics focuses on DNA methylation,histone modification,chromatin remodeling,noncoding RNAs,and other gene regulation mechanisms beyond the DNA sequence.In the past decade,epigenetic modifications have drawn more attention as they participate in the development and progression of diabetic retinopathy despite tight control of glucose levels.The underlying mechanisms of epigenetic modifications in diabetic retinopathy still urgently need to be elucidated.The diabetic condition facilitates epigenetic changes and influences target gene expression.In this review,we summarize the involvement of epigenetic modifications and metabolic memory in the development and progression of diabetic retinopathy and propose novel insights into the treatment of diabetic retinopathy.