Protective role of osteocalcin in diabetes pathogenesis.

In diabetes, metabolic, inflammatory, and stress-associated alterations conduce to ß-cell failure and tissue damage. Osteocalcin is a bone protein with several endocrine functions in different tissues. In this review, we gathered scientific evidence of how osteocalcin could modulate functional disorders that are altered in diabetes in an integrative way. We include adipose tissue, pancreatic function, and oxidative stress aspects. In the first section, we focus on the role of inflammatory mediators and adiponectin in energy homeostasis and insulin sensitivity. In the following section, we discuss the effect of osteocalcin in metabolic and pancreatic function and its association in insulin signaling and in ß-cell proliferation. Finally, we focus on osteocalcin action in oxidative and endoplasmic reticulum stress, and in antioxidant regulation, since ß-cells are well known by its vulnerability to stress damage. These evidences support the notion that osteocalcin could have an important role in diabetes treatment.

Challenges and Improvements of Developing an Ischemia Mouse Model Through Bilateral Common Carotid Artery Occlusion.

Brain ischemia is one of the principal causes of death and disability worldwide in which prevention or an effective treatment does not exist. In order to develop successful treatments, an adequate and useful ischemia model is essential. Transient global cerebral ischemia is one of the most interesting pathological conditions in stroke studies because of the observed degeneration of forebrain and delayed neuronal cell death in selective vulnerable regions such as hippocampus. Transient occlusion of both common carotid arteries is the most convenient model to induce tGCI. Although there are effective rat and gerbil models using this method, the induction of a reproducible and reliable injury after global ischemia in mouse has presented higher variations, mainly because of its size and the necessary monitoring skills in order to accomplish homogeneous and reproducible results. Further, great variability among cerebral vasculature and susceptibility of the different strains and sub-strains is observed. In recent years, some modifications have been made to the model in order to normalize the heterogenic effects. Analysis of posterior communicating artery patency has been proposed as an exclusion parameter due to the direct relationship reported with the reduction of cerebral blood flow. Another method used to significantly reduce blood flow is the induction of hypotension with isoflurane. Each protocol produces distinct injury outcomes. Further improvements are needed to attain a general, simpler, reproducible and globally accepted model that allows comparisons between research groups, progress in understanding ischemia and the consequent development of therapeutic alternatives for ischemic injury.

Participation of ATP-sensitive K+ channels and µ-opioid receptors in the antinociceptive synergism of the paracetamol-tapentadol co-administration in the formalin-induced pain assay in mice.

Preclinical Research & Development The purpose of this study was to assess the interaction and mechanisms of action of the paracetamol-tapentadol combination in the formalin-induced pain model in mice. Paracetamol (56.23-562.3 mg/kg, i.p.) or tapentadol (1-10 mg/kg, i.p.) were administered 15 min prior the intraplantar injection of formalin. The ED50 value of each drug was determined through the dose-response curves. The ED50 values were used to calculate the combinations in three fixed proportions (1:1, 1:3, and 3:1). Naloxone (1 and 5 mg/kg, i.p.), L-NAME (3 mg/kg, i.p.), or glibenclamide (10 mg/kg, i.p.) were administered before the combination of drugs to evaluate the antinociceptive mechanisms of action. The results showed that the combination 1:1 and paracetamol3-tapenadol1 ratios produced additive effects, whereas the paracetamol1-tapentadol3 proportion showed an antinociceptive synergistic interaction. Moreover, naloxone and glibenclamide reversed the antinociceptive activity of the paracetamol-tapentadol mixture. Our results indicate that the paracetamol-tapentadol combination produces an antinociceptive synergistic interaction with the possible participation of ATP-sensitive K+ channels and µ-opioid receptors in the second phase of the formalin-induced pain model in mice.

SARS-CoV-2: Air pollution highly correlated to the increase in mortality. The case of Guadalajara, Jalisco, México.

Objectives: To determine whether air pollution or changes in SARS-CoV-2 lineages lead to an increase in mortality. Methods: Descriptive statistics were used to calculate rates of infection (2020-2021). RT-PCR was used to compare viral loads from October 2020 to February 2021. Next-generation sequencing (NGS) (n = 92) was used to examine and phylogenetically map SARS-CoV-2 lineages. A correlative "air pollution/temperature" index (I) was developed using regression analysis. PM2.5, PM10, O3, NO2, SO2, and CO concentrations were analyzed and compared to the mortality. Results: The mortality rate during the last year was ∼32%. Relative SARS-CoV-2 viral loads increased in December 2020 and January 2021. NGS revealed that approximately 80% of SARS-CoV-2 linages were B.1.243 (33.7%), B1.1.222 (11.2%), B.1.1 (9%), B.1 (7%), B.1.1.159 (7%), and B.1.2 (7%). Two periods were analyzed, the prehigh- and high-mortality periods and no significant lineage differences or new lineages were found. Positive correlations of air pollution/temperature index values with mortality were found for IPM2.5 and IPM10. INO2. ISO2, and ICO but not for O3. Using ICO, we developed a model to predict mortality with an estimated variation of ∼±5 deaths per day. Conclusion: The mortality rate in the MZG was highly correlated with air pollution indices and not with SARS-CoV-2 lineage.

Antinociception and less gastric injury with the dexketoprofen-tapentadol combination in mice.

The purpose of this study was to evaluate the antinociceptive interaction between dexketoprofen and tapentadol in three different dose ratios, as well as the ulcerogenic activity of this combination. Dose-response curves were carried out for dexketoprofen, tapentadol, and dexketoprofen-tapentadol combinations in the acetic acid-induced writhing test in mice. On the other hand, the gastric damage of all treatments was assessed after the surgical extraction of the stomachs. Intraperitoneal administration of dexketoprofen and tapentadol induced a dose-dependent antinociceptive effect, reaching a maximal effect of about 58% and 99%, respectively. Isobolographic analysis and the interaction index showed that the three proportions produced an analgesic potentiation (synergistic interaction). Interestingly, the 1:1 and 1:3 ratios of the drugs combination produced minor gastric injury in comparison with the 3:1 proportion. Our data suggest that all proportions of the dexketoprofen-tapentadol combination produced a synergistic interaction in the acetic acid-induced visceral pain model in mice with a low incidence of gastric injury.

Selective estrogen receptor modulators in the prevention of breast cancer in premenopausal women: a review.

The detection of premenopausal women at high risk of breast cancer is key to chemoprevention. Therapy with selective estrogen receptor modulators (SERMs) induces a significant antiproliferative effect in estrogen receptor (ER) positive breast cancer. This review was designed according the guidelines of the 2009 PRISMA statement. Searching different databases, including PubMed, MedlinePlus, PLoS One, Cochrane Breast Cancer Specialized Register, Clinical Trials.gov and American Society of Clinical Oncology. From 168 records screened, 15 full text articles were assessed for eligibility and only 7 studies met the inclusion criteria. Three of the studies included analyzed changes in Ki-67 expression, revealing weaker expression after treatment with acolbifene and raloxifene (P<0.001). Three studies also analyzed the breast volume by magnetic resonance imagining (MRI) and demonstrate a significant difference after 1 year with raloxifene treatment (P=0.0017). Moreover, a 20% reduction in breast density was observed after a 2-year treatment with tamoxifen in premenopausal women. SERMs reduce the risk of developing breast cancer. The studies reviewed here demonstrate the modulation of Ki-67 expression and changes in breast density, suggesting an important preventive role for this group of drugs in prevention for premenopausal women at high risk of developing breast cancer.

Kinematic Changes in a Mouse Model of Penetrating Hippocampal Injury and Their Recovery After Intranasal Administration of Endometrial Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Locomotion speed changes appear following hippocampal injury. We used a hippocampal penetrating brain injury mouse model to analyze other kinematic changes. We found a significant decrease in locomotion speed in both open-field and tunnel walk tests. We described a new quantitative method that allows us to analyze and compare the displacement curves between mice steps. In the tunnel walk, we marked mice with indelible ink on the knee, ankle, and metatarsus of the left and right hindlimbs to evaluate both in every step. Animals with hippocampal damage exhibit slower locomotion speed in both hindlimbs. In contrast, in the cortical injured group, we observed significant speed decrease only in the right hindlimb. We found changes in the displacement patterns after hippocampal injury. Mesenchymal stem cell-derived extracellular vesicles had been used for the treatment of several diseases in animal models. Here, we evaluated the effects of intranasal administration of endometrial mesenchymal stem cell-derived extracellular vesicles on the outcome after the hippocampal injury. We report the presence of vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, and interleukin 6 in these vesicles. We observed locomotion speed and displacement pattern preservation in mice after vesicle treatment. These mice had lower pyknotic cells percentage and a smaller damaged area in comparison with the nontreated group, probably due to angiogenesis, wound repair, and inflammation decrease. Our results build up on the evidence of the hippocampal role in walk control and suggest that the extracellular vesicles could confer neuroprotection to the damaged hippocampus.

Establishment of Murine Model of Kidney Failure Induced by Severe Ischemia-Reperfusion Injury Useful to Evaluate Transplantation and Regenerative Therapies.

BACKGROUND: Severe ischemia-reperfusion injury (SIRI) seems to be the key factor that can significantly affect the function of both native kidneys and renal allografts. Therefore, the development of a successful strategy is of a paramount importance in both basic and clinical research. METHODS: To determine the effects of SIRI on the native kidney function, a murine model was planned as follows: group 1 (n = 6) mice underwent to nephrectomy plus ischemia-reperfusion injury for 30 minutes; group 2 (n = 6) mice underwent to nephrectomy without ischemia-reperfusion injury and thus served as sham controls for SIRI. The results of serum creatinine (SCr) were analyzed using Mann-Whitney U tests to calculate the significance between mean values. Survival between groups was measured by Kaplan-Meier test. RESULTS: To reliably achieve an elevation of SCr levels animals were exposed to a SIRI. The values of SCr increased from 0.35 (SD, 0.09) mg/dL to about 2-fold within 2 days and 3-fold within the following 5 days. Under these given conditions the mice displayed signs and histologic findings of severe kidney damage. The survival rate was about 83% of the animals within a week, and they showed no capacity of complete spontaneous self-regeneration. CONCLUSIONS: In this study, we aim to establish a murine model with extensive structural kidney damage and significant elevation of SCr levels, which could be used in basic and translational research of transplantation and regenerative therapies.

Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes.

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter-based cell sorting. ES cell-derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell-derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell-derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.

Reestablishment of microenvironment is necessary to maintain in vitro and in vivo human islet function.

Islet transplantation is associated with an elevated rate of early graft failure. The isolation process leads to structural and functional abnormalities. The reestablishment of the cell-matrix relationship is important to modulate the survival and function of islets. Thus, we evaluated the effect of human fibronectin (hFN) and self-assembling peptide nanofiber (SAPNF) in the ability to support islet function in vitro and after transplantation into streptozotocin (STZ)-induced diabetic severe combined immunodeficiency (SCID) mice. Human isolated islets were cultured with hFN or SAPNF for 7 days. Their ability to maintain insulin production/glucose responsiveness over time was evaluated. Islets embedded in hFN, SAPNF, or alone were transplanted into STZ-induced diabetic SCID mice. Islet grafts were removed after 14 days to evaluate insulin content, insulin expression, and apoptosis. SAPNF-entrapped islets maintained satisfactory morphology/viability and capability of glucose-dependent insulin secretion for over 7 days, whereas islets cultured in hFN underwent widespread deterioration. In vivo grafts containing human islets in SAPNF showed remarkably higher insulin content and expression when compared with human islets in hFn or alone. RT-PCR revealed lower caspase-3 expression in SAPNF islets grafts. These studies indicate that the reestablishment of the cell-matrix interactions by a synthetic matrix in the immediate postisolation period is a useful tool to maintain islet functions in vitro and in vivo.

Laparoscopy-assisted creation of a liver failure model in pigs.

We created a hepatic failure pig model that was suitable for the assessment of cell therapies, such as hepatocyte transplantation and bioartificial livers, using a laparoscopic surgical technique. In our model, all of three hepatic arteries were resected, 5, 7.5, or 10 ml of carbon tetrachloride (CCL4) was injected into the liver through the portal vein, and subsequently the portal vein was mechanically occluded for 30 min. After the portal occlusion was released, a liver biopsy was performed, and then the surgery was completed. Blood samples were regularly taken during the surgery in order to perform biochemical examinations. All of five pigs in which 5 ml of CCL4 was infused recovered spontaneously and survived; in contrast, all of five pigs that received 10 ml CCL4 died within 1.5 h after surgery. The pigs in which 7.5 ml CCL4 was administered developed liver failure and survived for 6.4 h on average (+/-1.4 SD). Induction of liver failure with the use of 7.5 ml CCL4 and 30-min hepatic ischemia fulfilled five of the six criteria that were proposed by Terblanche and Hickman: reversibility, reproducibility, death from liver failure, a therapeutic window, and a large-animal model. We believe that our model is the first report on creation of a reliable model for liver failure in pigs to assess the efficacy of liver-targeted cell therapies.

A human beta-cell line for transplantation therapy to control type 1 diabetes.

A human pancreatic beta-cell line that is functionally equivalent to primary beta-cells has not been available. We established a reversibly immortalized human beta-cell clone (NAKT-15) by transfection of primary human beta-cells with a retroviral vector containing simian virus 40 large T-antigen (SV40T) and human telomerase reverse transcriptase (hTERT) cDNAs flanked by paired loxP recombination targets, which allow deletion of SV40T and TERT by Cre recombinase. Reverted NAKT-15 cells expressed beta-cell transcription factors (Isl-1, Pax 6, Nkx 6.1, Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic severe combined immunodeficiency mice resulted in perfect control of blood glucose within 2 weeks; mice remained normoglycemic for longer than 30 weeks. The establishment of this cell line is one step toward a potential cure of diabetes by transplantation.

Pancreas development and beta-cell differentiation of embryonic stem cells.

Embryonic stem (ES) cells may offer an unlimited cell source for the treatment of diabetes. However, a successful derivation of ES cells into islet-cells has proven to be more difficult than it was initially expected. Considering that the pancreas coordinates the global use of energy in the organism by secreting digestive enzymes and hormones, it is understandable that a sophisticated and tight regulation that lies on the pancreas itself to orchestrate its own tissue development and maturation. The complex process of endocrine cell differentiation can be better understood by analyzing the normal development of the pancreas. The proper detection of the signals provided in the pancreatic environment gives us a clue as to how the stem cells give rise to the whole pancreas. Careful and extensive screening of the natural or synthetic cytokines and growth factors and biochemical compounds that are essential in pancreatic development is required to properly mimic the process in vitro. Such a study would allow the researchers to achieve selective control of the differentiation and proliferation of the stem cells. The development and identification of the key molecules can provide us new insights into the pancreatic differentiation of the stem cells. We herein discuss the role of the microenvironment and transcriptional factors and cytokines, which have been recognized as important molecules during the major steps of the development of the pancreas. Finally, a more complete comprehension of the mechanisms that drive the pancreatic regeneration will provide us with new perspectives for future prophylactic and therapeutic interventions.

Construction and transplantation of an engineered hepatic tissue using a polyaminourethane-coated nonwoven polytetrafluoroethylene fabric.

BACKGROUND: Acute liver failure (ALF) is a serious condition that has a high mortality rate. Construction of an efficient culture and transplantation engineering system of hepatic tissue is an important approach to treat patients suffering from ALF to provide short-term hepatic support until the damaged liver spontaneously recovers or a donor liver becomes available for transplantation. Here, we evaluate the construction and transplantation of an engineered hepatic tissue (EHT) using primary isolated hepatocytes cultured onto polyaminourethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric. METHODS: The isolated hepatocytes cultured onto PAU-coated PTFE fabric were able to adhere and spread over the individual fibers of the net and formed hepatic clusters after 3 days, such clusters revealed Gap junctions and well-developed bile canaliculi. RESULTS: When PAU-coated PTFE was utilized, ammonia-, and diazepam- metabolizing capacities and albumin production ability were significantly increased compared with collagen control. To test the function of this hepatic tissue in vivo, we transplanted a nonwoven PAU-coated PTFE fabric inoculated with one million hepatocytes on the surface of the spleen of Balb/c mice suffering from ALF induced by 90% hepatectomy, and found that this EHT prolonged the survival of liver failure-induced mice without adverse effects. Ultrastructure analyses showed good attachment of the cells on the surface of PTFE fabric and strong albumin expression seven days after the newly formed hepatic tissue was transplanted. CONCLUSION: We have here demonstrated the efficient construction and transplantation of hepatic tissue using primary hepatocytes and PAU-coated PTFE fabric.

Locomotion in intact and in brain cortex-ablated cats.

The current decerebration procedures discard the role of the thalamus in the motor control and decortication only rules out the brain cortex part, leaving a gap between the brain cortex and the subthalamic motor regions. In here we define a new preparation denominated Brain Cortex-Ablated Cat (BCAC), in which the frontal and parietal brain cortices as well as the central white matter beneath them were removed, this decerebration process may be considered as suprathalamic, since the thalamus remained intact. To characterize this preparation cat hindlimb electromyograms (EMG), kinematics and cutaneous reflexes (CR) produced by electrical stimulation of sural (SU) or saphenous (SAPH) nerves were analyzed during locomotion in intact and in BCAC. In cortex-ablated cats compared to intact cats, the hindlimb EMG amplitude was increased in the flexors, whereas in most extensors the amplitude was decreased. Bifunctional muscle EMGs presented complex and speed-dependent amplitude changes. In intact cats CR produced an inhibition of extensors, as well as excitation and inhibition of flexors, and a complex pattern of withdrawal responses in bifunctional muscles. The same stimuli applied to BCAC produced no detectable responses, but in some cats cutaneous reflexes produced by electrical stimulation of saphenous nerve reappeared when the locomotion speed increased. In BCAC, EMG and kinematic changes, as well as the absence of CR, imply that for this cat preparation there is a partial compensation due to the subcortical locomotor apparatus generating close to normal locomotion.

Serum levels of undercarboxylated osteocalcin are related to cardiovascular risk factors in patients with type 2 diabetes mellitus and healthy subjects.

AIM: To determine a potential relationship between serum undercarboxylated (ucOC) concentration and cardiovascular risk factors in type 2 diabetes (T2D) patients and healthy subjects (HS). METHODS: A cross-sectional study was conducted on 140 subjects classified into two groups, 70 with T2D and 70 HS. Medical history and physical examination with anthropometric measurements were obtained from all subjects. Body fat percentage was determined by bioelectrical impendency analysis. Serum ucOC concentration was determined by enzyme immunoassay, while serum levels of insulin and hsCRP were obtained using high sensitivity enzyme-linked immunosorbent assay. Insulin resistance was determined using the homeostasis model assessment-IR. Lipid profile [triglycerides, total cholesterol (TC), high-density lipoproteins (HDL-c), low density lipoproteins (LDL-c), very low-density lipoproteins] was determined by spectrophotometry and standard formulas when applicable. RESULTS: The T2D patient group showed significantly higher values of waist circumference, waist-to-hip ratio, systolic blood pressure (SBP), diastolic blood pressure (DBP), current smoking, and alcohol use when compared to the HS group (P < 0.05). We observed a significantly lower serum ucOC concentration in T2D than in HS (1.5 ± 1.4 vs 2.3 ± 1.8, P < 0.05). In the whole study population, ucOC concentration was inversely correlated with body mass index (BMI) (r = -0.236, P < 0.05), fasting plasma glucose (r = -0.283, P < 0.01) and HDL-c (r = -0.255, P < 0.05); and positively correlated with LDL-c/HDL-c ratio (r = 0.306, P < 0.05) and TC/HDL-c ratio (r = 0.284, P < 0.05). In the T2D group, serum ucOC concentration was inversely correlated with BMI (r = -0.310, P < 0.05) and body-fat percentage (r = -0.311, P < 0.05), and positively correlated with DBP (r = 0.450, P < 0.01). In HS group a positive correlation between serum levels of ucOC and SBP (r = 0.277, P < 0.05) was observed. CONCLUSION: Serum ucOC is a potential marker for cardiovascular risk in Mexicans because it is related to adiposity parameters, blood pressure and lipid profile.

Prolonged survival of mice with acute liver failure with transplantation of monkey hepatocytes cultured with an antiapoptotic pentapeptide V5.

BACKGROUND: Because hepatocyte transplantation has been considered to be an attractive method to treat acute liver failure (ALF), efficient recovery of hepatocytes and maintenance of differentiated hepatocyte functions is of extreme importance. We here report the usefulness of an antiapoptotic pentapeptide V5, composed of Val-Pro-Met-Leu-Lys, in the monkey hepatocyte cultures. METHODS: We evaluated albumin production, metabolizing abilities of ammonia, lidocaine, and diazepam of monkey hepatocytes cultured with V5. The protein expression of apoptosis-associated molecules was analyzed using power blot analysis. An unwoven cloth inoculated with V5-treated monkey hepatocytes was transplanted on the surface of the spleen of both SCID mice and Balb/c mice suffering from ALF induced by 90% hepatectomy. RESULTS: When 100 microM V5 was utilized, ammonia-, lidocaine- and diazepam- metabolizing capacities and albumin production ability were significantly increased in V5-treated monkey hepatocytes. Such hepatocytes showed decreased Annexin V binding and increased the expression of anti-apoptotic and/or cytoprotective molecules, including Ku70, NF-kappaB, IKAP, hILP/XIAP, IkappaB, and CAS. Transplantation of the cloth containing the monkey hepatocytes significantly improved blood levels of glucose and ammonia and encephalopathy score and prolonged the survival of the mice with ALF. CONCLUSIONS: The present work clearly demonstrates the usefulness of V5 for maintaining the functions of monkey hepatocytes in tissue culture.

A newly developed bioartificial pancreas successfully controls blood glucose in totally pancreatectomized diabetic pigs.

Construction of a safe and functional bioartificial pancreas (BAP) that provides an adequate environment for islet cells may be an important approach to treating diabetic patients. Various types of BAP devices have been developed, but most of them involve extravascular implantation of islets in microcapsules or diffusion chambers. These devices have poor diffusive exchange between the islets and blood, and often rupture. To overcome these problems, we developed a new type of BAP composed of polyethylene-vinyl alcohol (EVAL) hollow fibers that are permeable to glucose and insulin and a poly-amino-urethane-coated, non-woven polytetrafluoroethylene (PTFE) fabric that allows cell adhesion. Porcine islets attached to the surface of the PTFE fabric, but not to the surface of the EVAL hollow fibers, allowing nutrient and oxygen exchange between blood flowing inside the fibers and cells outside. We inoculated this BAP with porcine islets and connected it to the circulation of totally pancreatectomized diabetic pigs. We found that blood glucose levels were reduced to a normal range and general health was improved, resulting in longer survival times. In addition, regulation of insulin secretion from the BAP was properly controlled in response to glucose both in vitro and in vivo. These results indicate that our newly developed BAP may be a potential therapy for the treatment of diabetes in humans.

Functional hepatocyte culture and its application to cell therapies.

Since Berry and Friend developed methods to isolate hepatocytes from the liver by a collagenase digestion technique in 1969, studies in laboratory animals have demonstrated that hepatocyte transplantation could potentially be used for the treatment of liver failure and inborn errors of liver-based metabolism. Healthy human hepatocytes are an ideal source for hepatocyte transplantation; however, their relative scarcity is one of the major drawbacks, further compounded by the competing demands of liver transplantation. Notably, most of the hepatocytes are isolated from discarded livers that are not suitable for organ transplantation for a variety of reasons, including excessive fat content. Importantly, the hepatocyte isolation procedure itself exerts major stress on hepatocytes by the disruption of cell-to-cell and cell-to-matrix contacts, resulting in hepatocytic apoptosis. Prevention of apoptosis would maximize yield of healthy cells and maintain hepatocyte differentiated function in culture. In this review, we describe methods to prevent apoptosis by utilizing both antiapoptotic molecules and matrices. We also introduce a new type of liver tissue engineering, hepatocyte sheet transplantation, which utilizes unwoven cloth having a cellular adhesive property.

Differentiation of human embryonic stem cells to hepatocytes using deleted variant of HGF and poly-amino-urethane-coated nonwoven polytetrafluoroethylene fabric.

Human embryonic stem (hES) cells have recently been studied as an attractive source for the development of a bioartificial liver (BAL). Here we evaluate the differentiation capacity of hES cells into hepatocytes. hES cells were subjected to suspension culture for 5 days, and then cultured onto poly-amino-urethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric in the presence of fibroblast growth factor-2 (bFGF) (100 ng/ml) for 3 days, then with deleted variant of hepatocyte growth factor (dHGF) (100 ng/ml) and 1% dimethyl sulfoxide (DMSO) for 8 days, and finally with dexamethasone (10(-7) M) for 3 days. The hES cells showed gene expression of albumin in a time-dependent manner of the hepatic differentiation process. The resultant hES-derived hepatocytes metabolized the loaded ammonia and lidocaine at 7.8% and 23.6%, respectively. A million of such hepatocytes produced albumin and urea at 351.2 ng and urea at 7.0 microg. Scanning electron microscopy showed good attachment of the cells on the surface of the PTFE fabric and well-developed glycogen rosettes and Gap junction. In the present work we have demonstrated the efficient differentiation of hES cells to functional hepatocytes. The findings are useful to develop a BAL.