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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. PM 2.5 , PM 10 , O 3 , NO 2 , SO 2 , 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 IPM 2.5 and IPM 10 . INO 2 . ISO 2 , and ICO but not for O 3 . 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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

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., (© 2020 Société Française de Pharmacologie et de Thérapeutique.)

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.

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., (Copyright © 2020 León-Moreno, Castañeda-Arellano, Aguilar-García, Desentis-Desentis, Torres-Anguiano, Gutiérrez-Almeida, Najar-Acosta, Mendizabal-Ruiz, Ascencio-Piña, Dueñas-Jiménez, Rivas-Carrillo and Dueñas-Jiménez.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 ED 50 value of each drug was determined through the dose-response curves. The ED 50 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., (© 2018 Wiley Periodicals, Inc.)

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., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest to report.

Background: Diabetes is complex disease, which involves primary metabolic changes followed by immunological and vascular pathophysiological adjustments. However, it is mostly characterized by an unbalanced decreased number of the β-cells unable to maintain the metabolic requirements and failure to further regenerate newly functional pancreatic islets. The objective of this study was to analyze the properties of the endothelial cells to facilitate the islet cells engraftment after islet transplantation., Methods: We devised a co-cultured engineer system to coat isolated islets with vascular endothelial cells. To assess the cell integration of cell-engineered islets, we stained them for endothelial marker CD31 and nuclei counterstained with DAPI dye. We comparatively performed islet transplantations into streptozotocin-induced diabetic mice and recovered the islet grafts for morphometric analyses on days 3, 7, 10, and 30. Blood glucose levels were measured continuously after islet transplantation to monitor the functional engraftment and capacity to achieve metabolic control., Results: Cell-engineered islets showed a well-defined rounded shape after co-culture when compared with native isolated islets. Furthermore, the number of CD31-positive cells layered on the islet surface showed a direct proportion with engraftment capacities and less TUNEL-positive cells on days 3 and 7 after transplantation., Conclusions: We observed that vascular endothelial cells could be functional integrated into isolated islets. We also found that islets that are coated with vascular endothelial cells increased their capacity to engraft. These findings indicate that islets coated with endothelial cells have a greater capacity of engraftment and thus establish a definitely vascular network to support the metabolic requirements., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Islet transplantation has become a widely accepted therapeutic option for selected patients with type 1 diabetes mellitus. However, in order to achieve insulin independence a great number of islets are often pooled from 2 to 4 pancreata donors. Mostly, it is due to the massive loss of islets immediately after transplant. The endothelium plays a key role in the function of native islets and during the revascularization process after islet transplantation. However, if a delayed revascularization occurs, even the remaining islets will also undergo to cell death and late graft dysfunction. Therefore, it is essential to understand how the signals are released from endothelial cells, which might regulate both differentiation of pancreatic progenitors and thereby maintenance of the graft function. New strategies to facilitate islet engraftment and a prompt revascularization could be designed to intervene and might lead to improve future results of islet transplantation.

Objectives: Diabetes is the clinical consequence of the loss of the majority of the β-cell population and failure to regenerate new pancreatic β cells. The current therapies based on β-cell replacement have failed to achieve β-cell renewal and thus, long-term insulin freedom. We have hypothesized that early rejection of endothelial elements within the islet grafts may seriously hamper islet regeneration in both native and islet grafts., Methods: In the present study, we analyzed the role of endothelial cells to activate pancreatic stem cells during islet regeneration. Mice were pretreated with or without endothelial pharmacological ablation of endothelial cells, followed by an acute β-cell injury using a single intraperitoneal injection of streptozotocin. We performed comparative morphometric analyses of recovered pancreata on days 3, 7, 10, and 30 after streptozotocin injury, staining with bromodeoxyuridine (BrdU) for representative cell types, β cells, endothelial elements, and stem cells. Blood glucose levels were measured continuously after the injury to monitor the capacity for metabolic control., Results: Morphometric analyses revealed an increasing number of cells over time to be stained with a stem cell and BrdU markers among animals only injured with streptozotocin but not with endothelial ablation. Notably, on day 10, stem cell markers were dramatically decrease nearly to basal levels, with appearance of numerous insulin-positive cells. Intact vessels with cobblestone-shaped endothelial elements were observed in direct proportion to the better outcomes, both by morphometric and by metabolic parameters. In contrast, fewer insulin-positive cells were observed in pancreata that had been ablated of endothelial cells showing extensive collapse of endocrine functions., Conclusions: We observed that endothelial elements promoted stem cell proliferation and islet regeneration after a β-cell insult. We believe that preservation of endothelial cells positively affects the process of pancreatic regeneration., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The development of a bioartificial pancreas (BAP) with immunoisolating fashion has been gaining attention as a new method for treating diabetes. We have been proceeding with the development of a bag-type BAP that can be easily implanted and that allows for the optional injection or rejection of cells at any time. If fibrosis develops around a BAP device, then the permeability of substances transmitted through a semipermeable membrane will decrease, thereby reducing the reactivity with glucose, so it is necessary for the material of the device to have an excellent histocompatibility. Furthermore, in order to improve the efficacy of BAP treatment, it is important to maintain an environment of ample blood flow around the device. We have created a bag-type device for BAP that is 20 x 20 mm in size and comprises two layers of membranes. We have used an EVAL membrane for the outer membrane of the two layers. The EVAL membrane is a semipermeable membrane with good insulin permeability, which functions as an immunoisolation membrane. The inner membrane consists of PAU-coated HD-PE (nonwoven material processed with polyaminourethan) and it is designed to function as a scaffold for cells. We used Lewis rats to determine whether the effectiveness of fibroblast growth factor 2 (bFGF) can be improved by concomitantly using bFGF with a capacity for blood vessel regeneration as well as bFGF immersed in a sheet of gelatin. We placed the BAP in the abdominal cavity and covered it with the greater omentum. We were able to significantly increase the blood flow and the number of new blood vessels in the tissue surrounding the BAP device by using gelatinized bFGF. There were only a few instances of fibrosis as a biological reaction to the EVAL membrane, and the infiltration of inflammatory cells was mild. There were no adverse effects related to implantation of the device. We confirmed in this study that the use of an implantable BAP device and bFGF allowed for a better blood flow around the BAP device. There were only minor instances of fibrosis and inflammation reaction around the BAP, thus indicating the BAP that we are currently developing to have an excellent histocompatibility.

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.

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.

Background/aims: Hepatocyte transplantation and bioartificial liver treatment are attractive alternatives to liver transplantation. The availability of well-characterized human hepatocyte lines facilitates such cell therapies., Methods: Human hepatocytes were immortalized with a retroviral vector SSR#197 expressing catalytic subunit of human telomerase reverse transcriptase (hTERT) and enhanced green fluorescent protein (EGFP) cDNAs flanked by a pair of loxP recombination targets. Then, Tamoxifen-dependent Cre recombinase was expressed in SSR#197-immortalized hepatocytes. Cre/LoxP recombination was performed in the established cells by simple exposure to 500 nM Tamoxifen for a week. Then, the reverted population of the cells was recovered by EGFP-negative cell sorting and characterized in vitro and in vivo using a pig model of acute liver failure (ALF) induced by d-galactosamine (0.5 g/kg) injection., Results: A human hepatocyte cell line 16T-3 was established. Reverted 16-T3 cells showed the increased expression of hepatic markers in association with enhanced levels of transcriptional factors. Compared to normal human hepatocytes, albumin production and lidocaine-metabolizing activities of reverted 16-T3 cells were 0.32 and 0.50-fold, respectively. Transplantation of reverted 16T-3 cells significantly prolonged the survival of ALF pigs., Conclusions: Here we demonstrate the usefulness of Cre/LoxP -mediated reversible immortalization of human hepatocytes with Tamoxifen-mediated self-recombination.

Objective: Treatment of diabetic patients by pancreatic islet transplantation often requires the use of islets from two to four donors to produce insulin independence in a single recipient. Following isolation and transplantation, islets are susceptible to apoptosis, which limits their function and probably long-term islet graft survival., Research Design and Methods: To address this issue, we examined the effect of the cell-permeable apoptosis inhibitor pentapeptide Val-Pro-Met-Leu-Lys, V5, on pancreatic islets in a mouse model., Results: V5 treatment upregulated expression of anti-apoptotic proteins Bcl-2 and XIAP (X-linked inhibitor of apoptosis protein) by more than 3- and 11-fold and downregulated expression of apoptosis-inducing proteins Bax, Bad, and nuclear factor-kappaB-p65 by 10, 30, and nearly 50%, respectively. Treatment improved the recovered islet mass following collagenase digestion and isolation by 44% and in vitro glucose-responsive insulin secretion nearly fourfold. Following transplantation in streptozotocin-induced diabetic mice, 150 V5-treated islet equivalents functioned as well as 450 control untreated islet equivalents in normalizing blood glucose., Conclusions: These studies indicate that inhibition of apoptosis by V5 significantly improves islet function following isolation and improves islet graft function following transplantation. Use of this reagent in clinical islet transplantation could have a dramatic impact on the number of patients that might benefit from this therapy and could affect long-term graft survival.

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.

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.

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 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.

Development of an efficient preculture system of islets is ideal. Toward that goal, we constructed a human pancreatic islet-derived fibroblast cell line MNNK-1 for a source as a coculture system for freshly isolated islets to maintain islet functions. Human pancreatic islet cells were nucleofected with a plasmid vector pYK-1 expressing simian virus 40 large T antigen gene (SV40T) and hygromycin resistance gene (HygroR). One of the transduced cell lines, MNNK-1, was established and served as a feeder cell in the coculture for freshly isolated mouse, rat, and pig islets. Morphology, viability, and glucose-responding insulin secretion were analyzed in the coculture system. MNNK-1 cells were morphologically spindle shaped and were negative for pancreatic endocrine markers. MNNK-1 cells were positive for α-smooth muscle actin and collagen type I and produced fibroblast growth factor. Coculture of the mouse, rat, and pig islets with MNNK-1 cells maintained their viability and insulin secretion with glucose responsiveness. A human pancreatic islet-derived fibroblast cell line MNNK-1 was established. MNNK-1 cells were a useful means for maintaining morphology and insulin secretion of islets in the coculture system.

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.

Human embryonic stem (hES) cells have the ability to differentiate into a variety of different cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. Here we investigated an efficient method of hepatic differentiation from hES cells. A human ES cell line, KhES-1, was used and maintained by a nonfeeder method. KhES-1 cells were cultured for 5 days in the presence of human activin A (50 ng/ml) and then treated with a deleted variant of hepatocyte growth factor (dHGF) at 0, 100, or 500 ng/ml for 7 days. The resultant cells were biologically analyzed. The expression of the endodermal genes SOX17 and FOXA2 increased in KhES-1 cells after activin A treatment. In contrast, Oct4, a self-renewal undifferentiated marker, decreased in a time-dependent manner in KhES-1 cells. Following a 7-day treatment of the resultant cells with dHGF, especially at 500 ng/ml, KhES-1 cells showed an expression of the hepatic makers albumin, AFP, and CK18. Transitional electron microscopy showed well-developed glycogen rosettes and a gap junction in KhES-1 cells treated with 500 ng/ml of dHGF. We developed an efficient method to differentiate KhES-1 cells into hepatocyte-like cells in vitro using 50 ng/ml of activin A and 500 ng/ml of dHGF.

Development of a subcutaneously implantable bioartificial pancreas (BAP) with immunoisolatory function could have a great impact on the treatment of diabetes mellitus. We have developed an implantable BAP device with an ethylene vinyl alcohol (EVAL) membrane. In the present study, we used basic fibroblast growth factors (bFGF), which was incorporated in a carrier for sustained release, in order to induce neovascularization when the device was implanted subcutaneously. To maintain the vasculature thus formed, a cell infusion port was attached to the BAP device, through which the device was filled with human liver vascular endothelial cell line TMNK-1, and the vasculature could be adequately maintained. Mice were divided into the following three groups. In group 1, a bFGF-free BAP device was implanted subcutaneously. In group 2, a sustained-release bFGF-impregnated BAP device was implanted. In group 3, a sustained-release bFGF-impregnated BAP device was implanted, and 3 x 10(6) TMNK-1 cells were infused into the implanted device every week. Neovascularization induced in the subcutaneous tissue around the implanted BAP device was macroscopically examined and histologically evaluated. In addition, the tissue blood flow was measured using a laser blood flow meter. In mice in group 3, neovascularization was significantly induced and maintained until week 8 postimplantation. It was confirmed by scanning electron microscopy that infused TMNK-1 cells adhered to the inner polyethylene surface of the device. It was demonstrated that the use of bFGF and vascular endothelial TMNK-1 cells induced and maintained adequate vasculature and tissue blood flow surrounding the implantable bag-type BAP device. We believe that the present study will contribute to BAP development for the treatment of diabetes.

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.

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.

Artificial bones have often used for bone regeneration due to their strength, but they cannot provide an adequate environment for cell penetration and settlement. We therefore attempted to explore various materials that may allow the cells to penetrate and engraft in bone defects. PuraMatrix is a self-assembling peptide scaffold that produces a nanoscale environment allowing both cellular penetration and engraftment. The objective of this study was to investigate the effect of PuraMatrix on bone regeneration in a mouse bone defect model of the calvaria. Matrigel was used as a control. The expression of bone-related genes (alkaline phosphatase, Runx2, and Osterix) in the PuraMatrix-injected bone defects was stronger than that in the Matrigel-injected defects. Soft X-ray radiographs revealed that bony bridges were clearly observed in the defects treated with PuraMatrix, but not in the Matrigel-treated defects. Notably, PuraMatrix treatment induced mature bone tissue while showing cortical bone medullary cavities. The area of newly formed bones at the site of the bone defects was 1.38-fold larger for PuraMatrix than Matrigel. The strength of the regenerated bone was 1.72-fold higher for PuraMatrix (146.0 g) than for Matrigel (84.7 g). The present study demonstrated that PuraMatrix injection favorably induced functional bone regeneration.

Fibroblast growth factor (FGF)-2 has been recognized to be a key element involved in angiogenesis and a putative factor involved in stem cell-mediated islet regeneration. However, the usefulness of FGF-2 in an islet transplantation setting has not yet been explored. We therefore evaluated the effect of FGF-2 on both islet culture and islet transplantation. Isolated islets were cultured in the presence of 100 ng/ml FGF-2 for a week and then the glucose-responding insulin secretion and insulin contents were measured. Gelatinized FGF-2 (100 ng), which allowed the controlled release of FGF-2, was used for islet transplantation of streptozotocin-induced diabetic mice. Islets (150 IEQ), obtained from a single donor, mixed with gelatinized FGF-2, were transplanted into the subrenal capsule of the mice and the animals were observed for 30 days. Revascularization around the islet grafts was examined. The blood glucose levels were measured and the intraperitoneal glucose tolerance test (IPGTT) was performed. The supplementation of FGF-2 maintained proper insulin secretion and insulin contents in an in vitro culture. The use of gelatinized FGF-2 facilitated revascularization and favorable islet engraftment, thus resulting in an amelioration of the blood glucose levels in diabetic mice. The utilization of FGF-2 showed increased contents of insulin in the islet grafts and revealed a similar pattern as that of normal healthy mice in IPGTT. In contrast, the transplantation of islets without FGF-2 supplementation showed poor revascularization and failed to control the blood glucose levels in the diabetic mice.

Freshly isolated porcine hepatocytes are a very attractive cell source in the cell-based therapies to treat liver failure because of unlimited availability. However, due to the loss of hepatocyte functions in vitro, there is a need to develop a functional culture system to keep the cells metabolically active. Here we compared the effect of a self-assembling peptide nanofiber (SAPNF) as an extracellular matrix (ECM) with collagen type I on hepatocyte metabolic and secretion activities following hepatocyte isolation. Isolated porcine hepatocytes were cultured in SAPNF and collagen type I. Morphological assessment at different time points was performed by using SEM and phase contrast microscope. Metabolic and secretion activities were comparatively performed in the groups, by means of ammonia, lidocaine, and diazepam as well as albumin. Hepatocytes cultured on SAPNF revealed a three-dimensional spheroidal formation, thus maintaining cell differentiation status during 2 weeks of culture. On the other hand, hepatocytes in collagen revealed a spread shape, and by day 14 no hepatocyte-like cells were observed, but cells with long shape were present, thus revealing a degree of dedifferentiation in collagen culture. Hepatocytes in SAPNF were capable of drug-metabolizing activities and albumin secretion in higher ratio than those cultured on collagen. The present work clearly demonstrates the usefulness of SAPNF for maintaining differentiated functions of porcine hepatocytes in culture.

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.

Background: Considering the scarcity of donor livers, it is extremely important to establish a functional culture method for isolated hepatocytes. As a tool for maintaining hepatocyte functions in vitro, dHGF, a variant of HGF (hepatocyte growth factor) with a deletion of five amino acids, attracted our attention because it is less cytotoxic compared with HGF., Methods: We evaluated growth, albumin production, metabolizing abilities of ammonia, lidocaine, and diazepam of human hepatocytes in the presence of dHGF (10-1000 ng/ml). The gene expression of liver markers was comparatively analyzed. The effect of intrasplenic transplantation of dHGF-treated human hepatocytes into severe combined immunodeficient (SCID) mice was evaluated in an acute liver failure (ALF) model induced by D-galactosamine (D-gal)., Results: When 100 ng/ml of dHGF was utilized, metabolism rates of ammonia, lidocaine, and diazepam and albumin production per unit cell significantly increased. The gene expression analysis demonstrated the enhanced expression of albumin, HNF-4alpha, and C/EBPalpha in the hepatocytes treated with 100 ng/ml of dHGF. Transplantation of such hepatocytes prolonged the survival of the SCID mice with ALF induced by D-gal., Conclusions: The present work clearly demonstrates the usefulness of dHGF (100 ng/ml) for maintaining the differentiated functions of human hepatocytes in tissue culture.

Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer).