Search

The gut–liver axis is defined by dietary and environmental communication between the gut, microbiome and the liver with its redox and immune systems, the overactivation of which can lead to hepatic injury. We used media preconditioning to mimic some aspects of the enterohepatic circulation by treating the human Caco-2 intestinal epithelial cell line with 5, 10 and 20 mM paracetamol (N-acetyl-para-aminophenol; APAP) for 24 h, after which cell culture supernatants were transferred to differentiated human hepatic HepaRG cells for a further 24 h. Cell viability was assessed by mitochondrial function and ATP production, while membrane integrity was monitored by cellular-based impedance. Metabolism by Caco-2 cells was determined by liquid chromatography with tandem mass spectrometry. Caco-2 cell viability was not affected by APAP, while cell membrane integrity and tight junctions were maintained and became tighter with increasing APAP concentrations, suggesting a reduction in the permeability of the intestinal epithelium. During 24 h incubation, Caco-2 cells metabolised 64–68% of APAP, leaving 32–36% of intact starting compound to be transferred to HepaRG cells. When cultured with Caco-2-preconditioned medium, HepaRG cells also showed no loss of cell viability or membrane integrity, completely in contrast to direct treatment with APAP, which resulted in a rapid loss of cell viability and membrane integrity and, ultimately, cell death. Thus, the pre-metabolism of APAP could mitigate previously observed hepatotoxicity to hepatic tight junctions caused by direct exposure to APAP. These observations could have important implications for the direct exposure of hepatic parenchyma to APAP, administered via the intravenous route.

Background While there are postulations that asthma is potentially associated with severe coronavirus disease 2019 (COVID-19), there has been conflicting results from studies on the impact mild-to-moderate COVID-19 on asthma control after recovery. Methods A case control study on the association between mild-to-moderate COVID-19 and asthma control post infection was conducted. The primary outcome was a reduction in Asthma Control Test (ACT) score by ≥ 3 points post-COVID infection. The secondary outcomes included the change in ACT score, the proportion of patient with ACT score who dropped to ≤ 15 on enrolment visit and the need for escalation of asthma maintenance therapy. Results Out of the total of 221 adult patients with asthma recruited, 111 had mild-to-moderate COVID-19 within 30 to 270 days prior to study enrolment. The adjusted odds ratio (aOR) for a reduction in ACT score by ≥ 3 points after COVID-19 was 3.105 (95% CI = 1.385–6.959, p = 0.006). The odds of escalation of asthma maintenance therapy by at least 1 Global Initiative for Asthma (GINA) step was 4.733 (95% CI = 1.151–19.467, p = 0.031) and asthma patient are more likely to become uncontrolled after COVID-19 [aOR = 5.509 (95% CI = 1.061–28.600, p = 0.042)]. Conclusion Mild-to-moderate COVID-19 among asthma patients, upon recovery, was associated with worsening of asthma symptom, lower ACT score, a higher need for escalation of asthma maintenance therapy and more uncontrolled asthma.

Background Intra-hepatocyte triglyceride accumulation is the hallmark of non-alcoholic fatty liver disease (NAFLD). Little is known of the role of lipid droplets in the pathogenesis of this increasingly common condition. Eicosapentaenoic acid (EPA), an omega-3 fatty acid, has been suggested as a potential treatment for NAFLD. Within this study we explore different energy substrates with and without EPA and evaluate biochemical markers alongside lipid droplet characteristics. Methods EPA was added to C3A cells incubated in standard media, oleate (which mimics simple steatosis) or lactate, pyruvate, octanoate and ammonia (LPON), mimicking pathophysiology of NAFLD. At 72 hours, glucose, ketone bodies and various markers of liver function were assessed and triglyceride content was determined using confocal images of cells stained with BODIPY 493/503 and DAPI. Z stacks of confocal images were analysed using Volocity 3D image analysis and Visual Basic software to estimate the physical characteristics of lipid droplets. Liver function was assessed by measuring AST, albumin and LDH. Results Albumin levels increased in control and oleate and LDH showed EPA did not affect viability in concentrations up to 250µm. There was no effect to AST with addition of EPA. Glucose and ketone bodies were reduced in LPON treated cells in a dose dependent manner. EPA reduced triglyceride concentrations and lipid droplets per nucleus in untreated cells compared with control, however, there was no significant difference to total triglyceride concentration of cells treated with oleate + EPA or LPON + EPA. Lipid droplet volume increased in both fat loaded models with the addition of EPA, but untreated control remained the same. Surface area of lipid droplets was higher in oleate model compared to LPON. Conclusion The LPON + EPA model shows reduction of glucose and ketone bodies in a dose dependent manner indicating EPA may be useful as an adjunct to treatment of type 2 diabetes. Accumulation of lipid droplets at different rates and physical characteristics compared to oleate may reflect differences in FFA metabolism or their incorporation into the cell and lipid droplet membrane.

Chlorpromazine (CPZ) is a neuroleptic drug and prototype compound used to study intrahepatic cholestasis. The exact mechanisms of CPZ induced cholestasis remain unclear. Rat hepatocytes, or a sandwich culture of rat and human hepatocytes, have been the most commonly used models for studying CPZ toxicity in vitro. However, to better predict outcomes in pre-clinical trials where cholestasis may be an unwanted consequence, a human in vitro model, based on human HepaRG cells, capable of real-time, non-invasive and label free monitoring, alongside molecular investigations would be beneficial. To address this we used the human hepatic HepaRG cell line, and established concentrations of CPZ ranging from sub-toxic, 25 μM and 50 μM, to toxic 100 μM and compared them with untreated control. To assess the effect of this range of CPZ concentrations we employed electrical cell-substrate impedance sensing (ECIS) to measure viability and cell membrane interactions alongside traditional viability assays, immunocytostaining and qRT-PCR to assess genes of interest within adaptive and inflammatory pathways. Using these methods, we show a concentration dependant response to CPZ involving pro-inflammatory pathway, loss of tight junctions and membrane integrity, and an adaptive response mediated by Cytochrome P450 (CYP) enzyme activation and up-regulation of membrane phospholipid and xenobiotic transporters. In conclusion, structural changes within the membrane caused by sub-toxic and toxic concentrations of CPZ negatively impact the function of the cellular membrane. Damage to efflux transport proteins caused by CPZ induce cholestasis alongside downstream inflammation, which activates compensatory responses for cell survival. Lay summary Chlorpromazine is a drug used to treat patients with schizophrenia, which has a known association with liver damage. Here we show that it causes inflammation and alters the cell membranes in liver and bile duct cells similar to what is seen within a human population. The initiation of the inflammatory response and changes to cellular structure may provide insight into the damage and disease process and inform medical treatment.

Background: Coronavirus Disease 2019 (COVID-19) led to pandemic that affected almost all countries in the world. Many countries have implemented border restriction as a public health measure to limit local outbreak. However, there is inadequate scientific data to support such a practice, especially in the presence of an established local transmission of the disease.Methods: A novel metapopulation Susceptible-Exposed-Infectious-Recovered (SEIR) model with inspected migration was applied to investigate the effect of border restriction between Hong Kong and mainland China on the epidemiological characteristics of COVID-19 in Hong Kong. Isolation facilities occupancy was also studied.Results: At R0 is set to be inversely correlated with temperature of 2.2, the cumulative COVID-19 cases in Hong Kong can be reduced by 13.99% (from 29,163 to 25,084) with complete border closure. At an in-patient mortality of 1.4%, the number of deaths can be reduced from 408 to 351 (57 lives saved). However, border closure alone was insufficient to prevent full occupancy of isolation facilities in Hong Kong; effective public health measures to reduce local R0 to below 1.6 was necessary. Conclusions: As a public health measure to tackle COVID-19, border restriction is effective in reducing cumulative cases and mortality.

Fully differentiated HepaRG™ cells are the hepatic cell line of choice for in vitro study in toxicology and drug trials. They are derived from a hepatoblast-like progenitor (HepaRG-P) that differentiates into a coculture of hepatocyte-like and cholangiocyte-like cells. This process that requires 2 weeks of proliferation followed by 2 weeks of differentiation using dimethyl sulfoxide (DMSO) can be time consuming and costly. Identifying a method to accelerate HepaRG-Ps toward a mature lineage would save both time and money. The ability to do this in the absence of DMSO would remove the possibility of confounding toxicology results caused by DMSO induction of CYP pathways. It has been shown that tissue culture substrates play an important role in the development and maturity of a cell line, and this is particularly important for progenitor cells, which retain some form of plasticity. Oxygen plasma treatment is used extensively to modify cell culture substrates. There is also evidence that patterned rather than planar surfaces have a positive effect on proliferation and differentiation. In this study, we compared the effect of standard tissue culture plastic (TCP), oxygen plasma coated (OPC), and nanopatterned substrates (NPS) on early differentiation and function of HepaRG-P cells. Since NPS were OPC we initially compared the effect of TCP and OPC to enable comparison between all three culture surfaces using OPC as control to asses if patterning further enhanced early differentiation and functionality. The results show that HepaRG-P's grown on OPC substrate exhibited earlier differentiation, proliferation, and function compared with TCP. Culturing HepaRG-P's on OPC with the addition of NPS did not confer any additional advantage. In conclusion, OPC surface appeared to enhance hepatic differentiation and functionality and could replace traditional methods of differentiating HepaRG-P cells into fully differentiated and functional HepaRGs earlier than standard methods. Impact statement We show significantly earlier differentiation and function of HepaRG progenitor cells when grown in dimethyl sulfoxide-free medium on oxygen plasma substrates versus standard tissue culture plastic. Further investigation showed that nanopatterning of oxygen plasma substrates did not confer any additional advantage over smooth oxygen plasma, although one pattern (DSQ120) showed comparable early differentiation and function.

Gene expression analysis by quantitative real-time polymerase chain reaction (RT-qPCR) is routinely used in biomedical studies. The reproducibility and reliability of the data fundamentally depends on experimental design and data interpretation. Despite the wide application of this assay, there is significant variation in the validation process of gene expression data from research laboratories. Since the validity of results depends on appropriate normalisation, it is crucial to select appropriate reference gene(s), where transcription of the selected gene is unaffected by experimental setting. In this study we have applied geNorm technology to investigate the transcription of 12 &lsquo, housekeeping&rsquo, genes for use in the normalisation of RT-qPCR data acquired using a widely accepted HepaRG hepatic cell line in studies examining models of pre-clinical drug testing. geNorm data identified a number of genes unaffected by specific drug treatments and showed that different genes remained invariant in response to different drug treatments, whereas the transcription of &lsquo, classical&rsquo, reference genes such as GAPDH (glyceralde- hyde-3-phosphate dehydrogenase) was altered by drug treatment. Comparing data normalised using the reference genes identified by geNorm with normalisation using classical housekeeping genes demonstrated substantial differences in the final results. In light of cell therapy application, RT-qPCR analyses has to be carefully evaluated to accurately interpret data obtained from dynamic cellular models undergoing sequential stages of phenotypic change.

There are a variety of end-point assays and techniques available to monitor hepatic cell cultures and study toxicity within in vitro models. These commonly focus on one aspect of cell metabolism and are often destructive to cells. Impedance-based cellular assays (IBCAs) assess biological functions of cell populations in real-time by measuring electrical impedance, which is the resistance to alternating current caused by the dielectric properties of proliferating of cells. While the uses of IBCA have been widely reported for a number of tissues, specific uses in the study of hepatic cell cultures have not been reported to date. IBCA monitors cellular behaviour throughout experimentation non-invasively without labelling or damage to cell cultures. The data extrapolated from IBCA can be correlated to biological events happening within the cell and therefore may inform drug toxicity studies or other applications within hepatic research. Because tight junctions comprise the blood/biliary barrier in hepatocytes, there are major consequences when these junctions are disrupted, as many pathologies centre around the bile canaliculi and flow of bile out of the liver. The application of IBCA in hepatology provides a unique opportunity to assess cellular polarity and patency of tight junctions, vital to maintaining normal hepatic function. Here, we describe how IBCAs have been applied to measuring the effect of viral infection, drug toxicity/IC50, cholangiopathies, cancer metastasis and monitoring of the gut-liver axis. We also highlight key areas of research where IBCAs could be used in future applications within the field of hepatology.

Background & Aim SNBTS has been involved in the manufacture of Epstein-Barr virus-specific T cells (EBV-VST) in treatment of post-transplant lymphroliferative disorder (PTLD) for over 20 years. Manufacture of cells for clinical use relies on comprehensive QC analysis to assess product safety, dose, identity, functionality and stability. We have developed an in vitro cytotoxicity assay of EBV-VST against EBV-infected lymphoblastoid cell line (LCL) targets to measure specific lysis compared to controls. Since the LCLs required for this assay may contain live virus, in the advancing regulatory setting this assay is not easily transferable to an accredited QC lab. The aim of this study was to investigate if VST cytotoxicity directly correlated to any other cytotoxic T cell functional assays such as cytokine production or expression of degranulation markers. Methods, Results & Conclusion Cryopreserved EBV-VST products were thawed, counted, and split for use in both a cytotoxicity assay and intracellular cytokine/ degranulation assay. For the cytotoxicity assay, HLA-matched and unmatched LCLs were labeled with PKH67, and then co-cultured alongside EBV-VSTs at increasing VST to LCL ratios against suitable controls. Plates were incubated for 5 hours, and cells subsequently labeled with Annexin V and DRAQ7 to assess target apoptotis and necrosis by flow cytometry. For the intracellular assay, EBV-VSTs were stimulated for 5 hours with EBV consensus peptides and then labeled with surface and intracellular markers in the following flow cytomeric panel: CD8, CD4, CD45RO, CD107a, Perforin, Granzyme B, IFNγ, TNFα, IL-2 and DRAQ7. Both assays were performed from the same start material at same time to ensure a direct comparison. The percentage of IFNγ/TNFα co-expressing cells in response to EBV peptide stimulation positively correlated with specific lysis of HLA-matched target LCLs in EBV-VST co-cultures (R2= 0.5602, p=0.008, n=12). No correlation of LCL specific lysis was seen with any of the other degranulation markers, and interestingly no correlation seen with other product-variable parameters (VST viability, CD8/CD4 ratio, number of HLA matches) suggesting these have less direct impact on overall killing efficiency. Flow cytometric analysis of IFNγ/TNFα expression in response to EBV peptide stimulation is an accurate surrogate marker of EBV-infected LCL killing by VSTs, and therefore presents a valid, safe and compliant alternative functional characterisation method to the conventional LCL cytotoxicity assay.

Background aims Mesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs). Methods MSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs. Results MSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions. Conclusions LPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs.

Introduction Drug induced liver injury accounts for approximately one half of all acute liver failure cases, with Paracetamol (APAP) being one of the most reported hepatotoxins [1]. We used an in vitro approach to model APAP transport from gut to liver where metabolic products (NAPQI) can cause hepatocellular damage. Investigating the mechanisms of a gut-liver axis in vitro may contribute to a better understanding of APAP toxicity. Methods The cellular impedance ECIS Z t platform was used to study the gut-liver axis and effects of APAP. Caco-2 and HepaRGs were seeded on separate 8W10+E ibidi arrays. The Caco-2 array was collagen coated. Step 1: Previously established concentrations of APAP [2] were applied to confluent Caco-2s (day 10), and impedance monitored for 24 hrs. Step 2: This ‘preconditioned’ culture medium was then transferred to HepaRGs (day 11) for 24 hrs. Step 3: preconditioned culture medium was then transferred from HepaRGs to fresh Caco-2s (day 12) for 24 hrs. Results Step 1: using an impedance based cellular assay, we show a tightening of barrier function in Caco-2s treated with 20 mM APAP. Step 2: only the highest concentration (20 mM APAP) shows substantial loss of impedance on hepatocytes. Step 3: we demonstrate that barrier function has become sensitized on Caco-2s with an increase in impedance at 10 mM Discussion and Conclusions This study demonstrates a potential cross-talk between hepatocytes and enterocytes and reveals a homeostatic effect of gut barrier function in presence of APAP. Exposing hepatocytes to toxic levels of APAP taken from Caco-2’s attenuates toxicity at 5 and 10 mM concentrations compared to previous study [2] where a dose-dependent loss of tight junctions was observed (5–20 mM APAP). Further exposure of preconditioned media on fresh Caco-2s (step 3) shows that tightening of barrier function is now achieved with lower APAP concentration (10 mM) to reduce absorption of APAP. It is possible a liver-gut axis regulates APAP absorption through paracrine signals, though more work is needed. This may differ between population phenotypes and potentially be responsible for variability of toxic response in patients with APAP overdose. References Yoon, et al. J Clin Transl Hepatol4(2): 131–142. Gamal A, et al. SREP, 7, 37541.

We congratulate Boeckmans et al., on their comprehensive review of current models of non-alcoholic fatty liver disease (NAFLD) presented ‘Human based systems: Mechanistic NASH modelling just around the corner?’ (1). NAFLD affects a large number of people worldwide and despite advances in our knowledge of the condition, further research is needed to identify biomarkers and target molecules for therapy. We agree that results from animal experiments may not always represent human pathophysiology accurately and we believe that relevant human in vitro models can be informative in understanding NAFLD disease mechanisms and target therapies. In addition, such models should help rationalisation of animal experiments in line with the 3R principles of replacement, reduction and refinement.J. Boeckmans, A. Natale, K. Buyl et al., Human-based systems: Mechanistic NASH modelling just around the corner?, Pharmacol Res. 134 (2018) 257-267, https://doi.org/10.1016/j.phrs.2018.06.029.

Cholangiocarcinoma (CC) is typically diagnosed at an advanced stage and is refractory to surgical intervention and chemotherapy. Despite a global increase in the incidence of CC, little progress has been made toward the development of treatments for this cancer. Here we utilized human tissue; CC cell xenografts; a p53-deficient transgenic mouse model; and a non-transgenic, chemically induced rat model of CC that accurately reflects both the inflammatory and regenerative background associated with human CC pathology. Using these systems, we determined that the WNT pathway is highly activated in CCs and that inflammatory macrophages are required to establish this WNT-high state in vivo. Moreover, depletion of macrophages or inhibition of WNT signaling with one of two small molecule WNT inhibitors in mouse and rat CC models markedly reduced CC proliferation and increased apoptosis, resulting in tumor regression. Together, these results demonstrate that enhanced WNT signaling is a characteristic of CC and suggest that targeting WNT signaling pathways has potential as a therapeutic strategy for CC.

Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes.

It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

Notch signalling has been implicated during haematopoietic development in vivo and in the differentiation of haematopoietic cells from pluripotent cells in vitro. However interpretation of data from many of these studies has been complicated by the heterogeneous nature of cell populations under study and by the fact that the Notch pathway is active during embryogenesis prior to the development of the haematopoietic system. To define the role of Notch signalling in more precise cell populations during the early stages of haematopoietic development within the aorta-gonad-mesonephros (AGM) microenvironment we co-cultured differentiating ESCs on a stromal cell line derived from this region of the embryo. Our co-culture system had no effect on the production of FLK1(+) mesoderm progenitor cells but promoted their subsequent haematopoietic differentiation. We assessed the role of Notch signalling on haematopoietic differentiation of isolated FLK1(+) cells. Notch activity is dynamic and drops to basal levels as FLK1(+) cells commit to a haematopoietic fate. Further reduction of Notch activity by the inducible expression of dominant negative MAML had no functional consequences. In contrast, induction of Notch activity using an inducible NotchIC expression system had an inhibitory effect on haematopoietic differentiation. We used a Cre-mediated recombination strategy whereby NotchIC-expressing cells were marked with the hCD2 receptor and observed a reduction in the number of multi-lineage and myeloid colonies derived from NotchIC(+) compared to NotchIC(-) FLK1(+) cells isolated from the same culture. We believe that our culture system represents a good model for haematopoietic development within the AGM microenvironment and our data suggest that haematopoietic commitment of FLK1(+) cells in this setting occurs when Notch activity is below a specific threshold.

Aims The formation of endothelial cell-colony forming units (EC-CFUs) is increased by vascular injury, although their function in vivo is unclear. We, therefore, examined the constituents of EC-CFUs and the mechanisms of their generation. Methods and results We performed immunohistochemical characterization of EC-CFUs and their mononuclear precursors. Using fluorescent-activated cell sorting, we evaluated the capacity of mononuclear subpopulations to generate EC-CFUs, and monitored their migratory behaviour when co-incubated with EC-CFUs. Time-lapse microscopy was used to observe colony maturation. Cellular proliferation within EC-CFUs was assessed using bromodeoxyuridine (BrdU) and anti-proliferative agents. EC-CFUs exhibited typical endothelial characteristics; however, several endothelial markers were weakly expressed or absent. Macrophage and lymphocyte antigens were intensely expressed. EC-CFUs readily incorporated BrdU, and failed to develop in the presence of anti-proliferative agents ( P < 0.01; n = 12). Time-lapse microscopy demonstrated that the characteristic EC-CFU ‘spindle cells’ are not EC-CFU progeny, but are mononuclear cells migrating towards, and incorporating into colonies. Only CD14+ monocytes were necessary for EC-CFU formation. CD14 expression was progressively down-regulated during colony maturation ( P < 0.001; n = 6). Although unable to generate EC-CFUs directly, CD34+ cells could differentiate into CD14+ cells and potentiate EC-CFU formation. Conclusions EC-CFUs exhibit endothelial characteristics, but are predominantly CD14+ derived macrophages and are a potent stimulus for lymphocyte migration. Proliferation is necessary for EC-CFU generation; however, colony growth also occurs as a result of leucocyte migration. Although confirmatory in vivo studies are required, EC-CFU formation likely reflects leucocyte activation as a reparatory response to vascular denudation or tissue ischaemia.

11β-Hydroxysteroid dehydrogenase type-1 (11β-HSD1) converts inert cortisone into active cortisol, amplifying intracellular glucocorticoid action. 11β-HSD1 deficiency improves cardiovascular risk factors in obesity but exacerbates acute inflammation. To determine the effects of 11β-HSD1 deficiency on atherosclerosis and its inflammation, atherosclerosis-prone apolipoprotein E-knockout (ApoE-KO) mice were treated with a selective 11β-HSD1 inhibitor or crossed with 11β-HSD1-KO mice to generate double knockouts (DKOs) and challenged with an atherogenic Western diet. 11β-HSD1 inhibition or deficiency attenuated atherosclerosis (74–76%) without deleterious effects on plaque structure. This occurred without affecting plasma lipids or glucose, suggesting independence from classical metabolic risk factors. KO plaques were not more inflamed and indeed had 36% less T-cell infiltration, associated with 38% reduced circulating monocyte chemoattractant protein-1 (MCP-1) and 36% lower lesional vascular cell adhesion molecule-1 (VCAM-1). Bone marrow (BM) cells are key to the atheroprotection, since transplantation of DKO BM to irradiated ApoE-KO mice reduced atherosclerosis by 51%. 11β-HSD1-null macrophages show 76% enhanced cholesterol ester export. Thus, 11β-HSD1 deficiency reduces atherosclerosis without exaggerated lesional inflammation independent of metabolic risk factors. Selective 11β-HSD1 inhibitors promise novel antiatherosclerosis effects over and above their benefits for metabolic risk factors via effects on BM cells, plausibly macrophages.—Kipari, T., Hadoke, P. W. F., Iqbal, J., Man, T. Y., Miller, E., Coutinho, A. E., Zhang, Z., Sullivan, K. M., Mitic, T., Livingstone, D. E. W., Schrecker, C., Samuel, K., White, C. I., Bouhlel, M. A., Chinetti-Gbaguidi, G., Staels, B., Andrew, R., Walker, B. R., Savill, J. S., Chapman, K. E., Seckl, J. R. 11β-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis.

Organotypic liver culture models for hepatotoxicity studies that mimic in vivo hepatic functionality could help facilitate improved strategies for early safety risk assessment during drug development. Interspecies differences in drug sensitivity and mechanistic profiles, low predictive capacity and limitations of conventional monocultures of human hepatocytes, with high attrition rates remain major challenges. Herein, we show stable, cell-type specific phenotype/cellular polarity with differentiated functionality in human hepatocyte-like C3A cells (enhanced CYP3A4 activity/albumin synthesis) when in co-culture with human vascular endothelial cells (HUVECs), thus demonstrating biocompatibility and relevance for evaluating drug metabolism and toxicity. In agreement with in vivo studies, acetaminophen (APAP) toxicity was most profound in HUVEC mono-cultures; whilst in C3A:HUVEC co-culture, cells were less susceptible to the toxic effects of APAP, including parameters of oxidative stress and ATP depletion, altered redox homeostasis and impaired respiration. This resistance to APAP is also observed in a primary human hepatocyte (PHH) based co-culture model, suggesting bidirectional communication/stabilization between different cell types. This simple and easy-to-implement human co-culture model may represent a sustainable and physiologically-relevant alternative cell system to PHHs, complementary to animal testing, for initial hepatotoxicity screening or mechanistic studies of candidate compounds differentially targeting hepatocytes and endothelial cells.

Susceptibility to prion infection involves interplay between the prion strain and host genetics, but expression of the host-encoded cellular prion protein is a known prerequisite. Here we consider human embryonic stem cell (hESC) susceptibility by characterizing the genetics and expression of the normal cellular prion protein and by examining their response to acute prion exposure. Seven hESC lines were tested for their prion protein gene codon 129 genotype and this was found to broadly reflect that of the normal population. hESCs expressed prion protein mRNA, but only low levels of prion protein accumulated in self-renewing populations. Following undirected differentiation, up-regulation of prion protein expression occurred in each of the major embryonic lineages. Self-renewing populations of hESCs were challenged with infectious human and animal prions. The exposed cells rapidly and extensively took up this material, but when the infectious source was removed the level and extent of intracellular disease-associated prion protein fell rapidly. In the absence of a sufficiently sensitive test for prions to screen therapeutic cells, and given the continued use of poorly characterized human and animal bioproducts during hESC derivation and cultivation, the finding that hESCs rapidly take up and process abnormal prion protein is provocative and merits further investigation.

Background: Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte-like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver. Aims: In this study, we assess the therapeutic value of HLCs in long-term cell-based therapies in vivo. Methods: hESC-derived HLCs were injected into the spleen of acutely injured NODscidIL-2Rγnull mice and analysed at various time points post-transplantation up to 3 months. Results: Large clusters of human cells engrafted in the spleen after 3 days and had expanded considerably by 31 days. At these time points, we identified human cells expressing parenchymal hepatocyte markers, exhibiting biliary duct-like structures and expressing myofibroblast markers. Three months after transplantation, we could detect human HLCs that were positive for albumin and CK18 by immunostaining and human DNA by fluorescent in situ hybridisation. Moreover, we could detect secretion of human serum albumin by enzyme-linked immunoabsorbant assay. Conclusions: We observed the persistence, engraftment and function of HLCs in vivo up to 3 months post-translation; however, all murine recipients developed large splenic and liver tumours that contained endodermal and mesodermal cell types. Although our studies demonstrate that hESC-derived HLCs have the potential to play an important role in cell-based therapies, current methodologies and transplantation strategies require substantial refinement before they can be deployed safely.

Summary. Background: The most common source of hematopoietic progenitor cells (HPCs) for hematopoietic reconstitution comprises granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs). It has been proposed that endothelial progenitor cells (EPCs) share precursors with HPCs, and that EPC release may accompany HPC mobilization to the circulation following G-CSF administration. Objective: To investigate EPC activity following HPC mobilization, and the direct effects of exogenous G-CSF administration on human umbilical vein endothelial cells (HUVECs) and endothelial outgrowth cells (EOCs), using in vitro and in vivo correlates of angiogenesis. Patients/Methods: Heparinized venous blood samples were collected from healthy volunteers and from cord blood at parturition. G-CSF-mobilized samples were collected before administration, at apheresis harvest, and at follow-up. PBSCs were phenotyped by flow cytometry, and cultured in standard colony-forming unit (CFU)-EPC and EOC assays. The effect of exogenous G-CSF was investigated by addition of it to HUVECs and EOCs in standard tubule formation and aortic ring assays, and in an in vivo sponge implantation model. Results: Our data show that G-CSF mobilization of PBSCs produces a profound, reversible depression of circulating CFU-EPCs. Furthermore, G-CSF administration did not mobilize CD34+CD133− cells, which include precursors of EOCs. No EOCs were cultured from any mobilized PBSCs studied. Exogenous G-CSF inhibited CFU-EPC generation, HUVEC and EOC tubule formation, microvessel outgrowth, and implanted sponge vascularization in mice. Conclusions: G-CSF administration depresses both endothelial cell angiogenesis and monocyte proangiogenic activity, and we suggest that any angiogenic benefit observed following implantation of cells mobilized by G-CSF may come only from a paracrine effect from HPCs.

Side population (SP) cells have recently been identified in a number of tissues although their phenotype and functional abilities are poorly understood. Surface marker characterisation and functional assessment of developing liver SP cells might allow for their isolation and manipulation using clinically relevant techniques. It was hypothesised that SP cells are present early during human liver development and contribute to haematopoietic and epithelial lineage generation. Whilst the SP population remained positive for CD34 during the 1st and 2nd trimester, 1st trimester SP cells were more highly enriched for haematopoietic and epithelial progenitor activity than those from the 2nd trimester in vitro. Marker expression and functional similarities indicate that SP cells in developing human liver may share a temporal relationship with oval/progenitor cells, responsible for liver regeneration after massive or chronic hepatic injury. Furthermore, modification of SP integrin expression during development suggests a potential adaptive interaction with niche components such as fibronectin. Improved understanding of developing human liver SP cells will contribute to the generation of novel cell-based therapies for liver disease.

beta-Defensins comprise a family of cationic, antimicrobial and chemoattractant peptides. The six cysteine canonical motif is retained throughout evolution and the disulphide connectivities stabilise the conserved monomer structure. A murine beta-defensin gene (Defr1) present in the main defensin cluster of C57B1/6 mice, encodes a peptide with only five of the canonical six cysteine residues. in other inbred strains of mice, the allele encodes Defb8, which has the six cysteine motif. We show here that in common with six cysteine beta-defensins, defensin-related peptide 1 (Defr1) displays chemoattractant activity for CD4(+) T cells and immature DC (iDC), but not mature DC cells or neutrophils. Murine Defb2 replicates this pattern of attraction. Defb8 is also able to attract iDC but not mature DC. Synthetic analogues of Defr1 with the six cysteines; restored (Defr1 Y5C) or with only a single cysteine (Defr1-1c(V)) chemoattract CD4(+) T cells with reduced activity, but do not chemoattract DC. beta-Defensins have previously been shown to attract iDC through CC receptor 6 (CCR6) but neither Defr1 or its related peptides nor Defb8, chemoattract cells overexpressing CCR6. Thus, we demonstrate that the canonical six cysteines of beta-defensins are not required for the chemoattractant activity of Defr1 and that neither Defr1 nor the six cysteine polymorphic variant allele Defb8, act through CCR6.

Pluripotent stem cells are derived from the inner cell mass of preimplantation embryos, and display the ability of the embryonic founder cells by forming all three germ lineages in vitro. It is well established that the cellular niche plays an important role in stem cell maintenance and differentiation. Stem cells generally have limited function without the specialized microenvironment of the niche that provides key cell-cell contact, soluble mediators, and extracellular matrices. We were interested in the role that Wnt signaling, in particular Wnt3a, played in human embryonic stem cell (hESC) differentiation to hepatic endoderm in vitro. hESC differentiation to hepatic endoderm was efficient in pure stem cell populations. However, in younger hESC lines, generating stromal cell mesenchyme, our model was very inefficient. The negative effect of stroma could be reversed by pretreating hESCs with Wnt3a prior to the onset of hepatocyte differentiation. Wnt3a pretreatment reinstated efficient hESC differentiation to hepatic endoderm. These studies represent an important step in understanding hepatocyte differentiation from hESCs and the role played by the cellular niche in vitro.

The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However, clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis, a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC, maintained in an optimised cytokine cocktail, to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) in a feeder-free system, without any requirement for small molecules, we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×10(5) seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34(+) cells, we obtained 148 iPS colonies from 10(5) seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function.

Twenty-two patients with acute myeloid leukaemia were recruited into a phase I/II clinical trial investigating the vaccination of patients in complete remission (CR) with autologous dendritic-like leukaemia cells (DLLC). At trial entry, leukaemia cells were harvested and tested for their ability to undergo cytokine-induced dendritic cell differentiation. Patients were then treated with intensive chemotherapy. Five patients achieved both CR and had leukaemia cells that successfully underwent differentiation and therefore proceeded to vaccination. Four escalating doses of DLLC were administered weekly by subcutaneous injection. Vaccination was generally well tolerated although one patient developed extensive eczema and an increased antinuclear factor titre possibly indicating induction of autoimmunity. Development of anti-leukaemic T-cell responses was assessed by enzyme-linked immunospot analysis of gamma-interferon secreting T lymphocytes and by human leucocyte antigen tetramer analysis for WT1-specific T cells. Increases in anti-leukaemic T-cell responses were demonstrated in four patients, but only two of the five remained in remission more than 12 months postvaccination. The study has demonstrated that generation of DLLC is feasible in only a subgroup of patients and is currently neither broadly applicable or clinically effective.

We investigated whether the in vitro differentiation of ES cells into haematopoietic progenitors could be enhanced by exposure to the aorta-gonadal-mesonephros (AGM) microenvironment that is involved in the generation of haematopoietic stem cells (HSC) during embryonic development. We established a co-culture system that combines the requirements for primary organ culture and differentiating ES cells and showed that exposure of differentiating ES cells to the primary AGM region results in a significant increase in the number of ES-derived haematopoietic progenitors. Co-culture of ES cells on the AM20-1B4 stromal cell line derived from the AGM region also increases haematopoietic activity. We conclude that factors promoting the haematopoietic activity of differentiating ES cells present in primary AGM explants are partially retained in the AM20.1B4 stromal cell line and that these factors are likely to be different to those required for adult HSC maintenance.

Recent work has indicated that adult bone marrow-derived cells have the ability to contribute to both the haematopoietic system and other organs. Haematopoietic reconstitution by whole bone marrow and selected but not fully characterised cell populations have resulted in reports indicating high-level repopulation of lung epithelia. The well-characterised cells from the side population have a robust ability for haematopoietic reconstitution. We have used freshly isolated side population cells derived from ROSA26 adult bone marrow and demonstrate that despite being unable to contribute to embryos following blastocyst injection, or air liquid interface cultures or denuded tracheal xenografts, they could contribute to the tracheal epithelium in vivo. Epithelial damage is reported to be important in encouraging the recruitment of marrow-derived stem cells into non-haematopoietic organs. Here we demonstrate that mice engrafted with side population cells have donor-derived cells present in the epithelial lining of the trachea following damage and repair. Donor-derived cells were found at a frequency of 0.83%. Widefield and confocal microscopy revealed donor cells that expressed cytokeratins, indicative of cells of an epithelial nature. These results imply that SP haematopoietic stem cells from the bone marrow do not have the ability to contribute to airway epithelia themselves but require factors present in vivo to allow them to acquire characteristics of this tissue.

Background & Aims: Studies have indicated that stem cells have unexpected plasticity and can differentiate down a multitude of nonhematopoietic cell lineages in rodents. Our aim was to identify whether human cord blood cells, which are a rich source of stem cells, would be able to differentiate into hepatocytes when infused into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. We also wanted to test whether such differentiated cells were the result of cellular fusion or true stem cell transdifferentiation. Methods: Unsorted mononuclear cell preparations of human cord blood were infused into sublethally irradiated NOD-SCID mice. After death, immunohistologic analysis of murine livers was performed using human specific hepatocyte, biliary, and endothelial markers. Fluorescent in situ hybridization (FISH) for mouse and human DNA was also performed. Results: We show that human cord blood cells have the ability to engraft into NOD-SCID liver and become mature hepatocytes. We were unable to identify any biliary or endothelial differentiation. Furthermore, we do not detect any evidence of cell fusion in any of the human cells found in the mouse liver, suggesting that human cord blood cells are capable of true transdifferentiation into hepatocytes in vivo. Conclusions: We conclude that hepatocytes can derive from human cord blood cells when infused into NOD-SCID mice in the absence of fusion. The demonstration that human stem cell differentiation can occur in this murine model permits comprehensive study of human stem cell plasticity in vivo.