Improving the function of islet and beta-cell grafts

Background: Human islet transplantation would offer a less invasive and more physiological alternative than whole pancreas transplantation and insulin injections respectively for the treatment of diabetes mellitus if islet graft survival can be improved. Initial recipient post-transplant insulin independence declines to <10% after 5 years. Factors contributing to graft failure include enzymatic disruption of the islet microenvironment during isolation, diabetogenic effects of immunosuppressants and metabolic stress resulting from slow revascularisation. Aims: To investigate the effect of co-culture in both static (SC) and rotational culture (RC) of BRINBDII beta-cells (Dl1) and human umbilical vein endothelial cells (HUVEC) on Dl1 insulin secretion; and the effect of a thiazolidinedione (TZD) on DII function and HUVEC proliferation. To assess the effect of culture media, SC, RC and a TZD on human islet morphology, insulin secretion and VEGF production. To initiate in vivo protocol development for assessment of revascularisation of human islet grafts. Methods: D11 cells were cultured +/-TZD and co-cultured with HUVEC +/-TZD in SC and RC. Dl1 insulin secretion was induced by static incubation with low glucose (1.67mM), high glucose (l6.7mM: and high glucose with 10mM theophylline (G+T) and determined by ELISA. HUVEC were cultured +/-TZD in SC and RC and proliferation was assessed by ATP luminescence assay and VEGF ELISA. D II and HUVEC morphology was determined by immunocytochemistry. Human islets were cultured in SC and RC in various media +/-TZD. Insulin secretion was determined as above and VEGF production by fluorescence immunocytochemistry (FI) and ELISA. Revascularisation of islet grafts was assessed by vascular corrosion cast and FI. Results: Dll cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and further improved by adding 10mM TZD. Untreated Dll/HUVEC co-cultures displayed significantly increased insulin secretion in response to 16.7mM and G+T over basal, again enhanced by RC and improved with 10mM TZD. 10mM TZD significantly increased HUVEC proliferation over control. Human islets maintained in medium 199 (mI99) in SC and RC exhibited comparable maintenance of morphology and insulin secretory profiles compared to islets maintained in RPMI, endothelial growth media and dedicated islet medium Miami# I. All cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and in certain instances further improved by adding 25mM TZD. TZD increased VEGF production and release as determined by ELISA. Post-implant vascular corrosion casts of mouse kidneys analysed by x-ray micro tomography indicates a possible TZD enhancement of microvessel growth via VEGF upregulation. Conclusions: D II /HUVEC co-culture in SC or RC does not alter the morphology of either cell type and supports D 11 function. TZD improves 0 I I and D I I/HUVEC SC and RC co-culture insulin secretion while increasing HUVEC proliferation. Human islet RC supports islet functional viability and structural integrity compared to SC while the addition of TZD occasionally further improves secretagogue induced insulin secretion. Expensive, 'dedicated' islet media showed no advantage over ml99 in terms of maintaining islet morphology or function. TZD upregulates VEGF in islets as shown by ELISA and suggested by x-ray micro tomography analysis of vascular corrosion casts. Maintenance of islets in RC and treatment with TZD prior to transplant may improve the functional viability and revascularisation rate of islet grafts.

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.

Mesenchymal stem cell-conditioned medium accelerates skin wound healing:an in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-beta1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

Aims/hypothesis - Loss of the trophic support provided by surrounding non-endocrine pancreatic cell populations underlies the decline in beta cell mass and insulin secretory function observed in human islets following isolation and culture. This study sought to determine whether restoration of regulatory influences mediated by ductal epithelial cells promotes sustained beta cell function in vitro. Methods - Human islets were isolated according to existing protocols. Ductal epithelial cells were harvested from the exocrine tissue remaining after islet isolation, expanded in monolayer culture and characterised using fluorescence immunocytochemistry. The two cell types were co-cultured under conventional static culture conditions or within a rotational cell culture system. The effect of co-culture on islet structural integrity, beta cell mass and insulin secretory capacity was observed for 10 days following isolation. Results - Human islets maintained under conventional culture conditions exhibited a characteristic loss in structural integrity and functional viability as indicated by a diminution of glucose responsiveness. By contrast, co-culture of islets with ductal epithelial cells led to preserved islet morphology and sustained beta cell function, most evident in co-cultures held within the rotational cell culture system, which showed a significantly (p<0.05) greater insulin secretory response to elevated glucose compared with control islets. Similarly, insulin/protein ratio data suggested that the presence of ductal epithelial cells is beneficial for the maintenance of beta cell mass. Conclusions/interpretation - The data indicate a supportive role for ductal epithelial cells in islet viability. Further characterisation of the regulatory influences may lead to novel strategies to improve long-term beta cell function both in vitro and following islet transplantation.

Functional astrocyte-neuron lactate shuttle in a human stem cell-derived neuronal network

The NT2.D1 cell line is one of the most well-documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of the neuronal cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time that human stem cell-derived astrocytes produce glycogen and that co-cultures of these cells demonstrate a functional astrocyte-neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake, which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2-derived neurons and astrocytes, we have shown that these cells modulate their glucose uptake in response to glutamate. Additionally, we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown after treatment with glutamate, potassium, isoproterenol, and dbcAMP. Together, these results demonstrate for the first time a functional ANLS in a human stem cell-derived co-culture. © 2013 ISCBFM.

Aβ(1-42) induced metabolic effects in a stem cell derived neuron and astrocyte network.

Alzheimer’s Disease (AD) is the most common form of dementia currently affecting more than 35 million people worldwide. Hypometabolism is a major feature of AD and appears decades before cognitive decline and pathological lesions. This has a detrimental impact on the brain which has a high energy demand. Current models of AD fail to mimic all the features of the disease, which has an impact on the development of new therapies. Human stem cell derived models of the brain have attracted a lot of attention in recent years as a tool to study neurodegenerative diseases. In this thesis, neurons and astrocytes derived from the human embryonal carcinoma cell line (NT2/D1) were utilised to determine the metabolic coupling between neurons and astrocytes with regards to responses to hypoglycaemia, neuromodulators and increase in neuronal activity. This model was then used to investigate the effects of Aß(1-42) on the metabolism of these NT2-derived co-cultures as well as pure astrocytes. Additionally primary cortical mixed neuronal and glial cultures were utilised to compare this model to a widely accepted in vitro model used in Alzheimer’s disease research. Co-cultures were found to respond to Aß(1-42) in similar way to human and in vivo models. Hypometabolism was characterised by changes in glucose metabolism, as well as lactate, pyruvate and glycogen. This led to a significant decrease in ATP and the ratio of NAD+/NADH. These results together with an increase in calcium oscillations and a decrease in GSH/GSSG ratio, suggests Aß-induces metabolic and oxidative stress. This situation could have detrimental effects in the brain which has a high energy demand, especially in terms of memory formation and antioxidant capacity.

A collagen IV matrix is required for guinea pig gastric epithelial cell monolayers to provide an optimal model of the stomach surface for biopharmaceutical screening

The surface epithelial cells of the stomach represent a major component of the gastric barrier. A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. Primary cultures of guinea pig gastric mucous epithelial cells were grown on filter inserts (Transwells®) for 3 days. Tight-junction formation, assessed by transepithelial electrical resistance (TEER) and permeability of mannitol and fluorescein, was enhanced when collagen IV rather than collagen I was used to coat the polycarbonate filter. TEER for cells grown on collagen IV was close to that obtained with intact guinea pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [ 3H]glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on plastic culture plates, but no major difference was found between cells grown on collagens I and IV. The proportion of cells, which stained positively for mucin with periodic acid Schiff reagent, was greater than 95% for all culture conditions. Monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide, and were resistant to acidification of the apical medium to pH 3.0 for 30 min. A screen of nonsteroidal anti-inflammatory drugs revealed a novel effect of diclofenac and niflumic acid in reversibly reducing permeability by the paracellular route. In conclusion, the mucous cell preparation grown on collagen IV represents a good model of the gastric surface epithelium suitable for screening procedures. © 2005 The Society for Biomolecular Screening.

Development of a neurotoxicity test-system, using human post-mitotic, astrocytic and neuronal cell lines in co-culture

Astrocytes are essential for neuronal function and survival, so both cell types were included in a human neurotoxicity test-system to assess the protective effects of astrocytes on neurons, compared with a culture of neurons alone. The human NT2.D1 cell line was differentiated to form either a co-culture of post-mitotic NT2.N neuronal (TUJ1, NF68 and NSE positive) and NT2.A astrocytic (GFAP positive) cells (∼2:1 NT2.A:NT2.N), or an NT2.N mono-culture. Cultures were exposed to human toxins, for 4 h at sub-cytotoxic concentrations, in order to compare levels of compromised cell function and thus evidence of an astrocytic protective effect. Functional endpoints examined included assays for cellular energy (ATP) and glutathione (GSH) levels, generation of hydrogen peroxide (H2O2) and caspase-3 activation. Generally, the NT2.N/A co-culture was more resistant to toxicity, maintaining superior ATP and GSH levels and sustaining smaller significant increases in H2O2 levels compared with neurons alone. However, the pure neuronal culture showed a significantly lower level of caspase activation. These data suggest that besides their support for neurons through maintenance of ATP and GSH and control of H2O2 levels, following exposure to some substances, astrocytes may promote an apoptotic mode of cell death. Thus, it appears the use of astrocytes in an in vitro predictive neurotoxicity test-system may be more relevant to human CNS structure and function than neuronal cells alone. © 2007 Elsevier Ltd. All rights reserved.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.

Human mesenchymal stem cells stimulate EaHy926 endothelial cell migration:combined proteomic and in vitro analysis of the influence of donor-donor variability

Mesenchymal stem cells (MSCs) stimulate angiogenesis within a wound environment and this effect is mediated through paracrine interactions with the endothelial cells present. Here we report that human MSC-conditioned medium (n=3 donors) significantly increased EaHy-926 endothelial cell adhesion and cell migration, but that this stimulatory effect was markedly donor-dependent. MALDI-TOF/TOF mass spectrometry demonstrated that whilst collagen type I and fibronectin were secreted by all of the MSC cultures, the small leucine rich proteoglycan, decorin was secreted only by the MSC culture that was least effective upon EaHy-926 cells. These individual extracellular matrix components were then tested as culture substrata. EaHy-926 cell adherence was greatest on fibronectin-coated surfaces with least adherence on decorin-coated surfaces. Scratch wound assays were used to examine cell migration. EaHy-926 cell scratch wound closure was quickest on substrates of fibronectin and slowest on decorin. However, EaHy-926 cell migration was stimulated by the addition of MSC-conditioned medium irrespective of the types of culture substrates. These data suggest that whilst the MSC secretome may generally be considered angiogenic, the composition of the secretome is variable and this variation probably contributes to donor-donor differences in activity. Hence, screening and optimizing MSC secretomes will improve the clinical effectiveness of pro-angiogenic MSC-based therapies.

Amyloid β 1-42 induces hypometabolism in human stem cell-derived neuron and astrocyte networks

Alzheimer's disease (AD) is the most common form of dementia, affecting more than 35 million people worldwide. Brain hypometabolism is a major feature of AD, appearing decades before cognitive decline and pathologic lesions. To date, the majority of studies on hypometabolism in AD have used transgenic animal models or imaging studies of the human brain. As it is almost impossible to validate these findings using human tissue, alternative models are required. In this study, we show that human stem cell-derived neuron and astrocyte cultures treated with oligomers of amyloid beta 1-42 (Aβ1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose, pyruvate, lactate, and glutamate. In addition, a significant increase in the glycogen content of cells was also observed. These changes were accompanied by changes in NAD+ /NADH, ATP, and glutathione levels, suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aβ-induced hypometabolism. Further research using this model may elucidate the mechanisms associated with Aβ-induced hypometabolism.

Functional astrocyte-neuron lactate shuttle in a human stem cell derived neuronal network

The development of stem cell-derived neuronal networks will promote experimental system development for drug screening, toxicological testing and disease modelling, providing that they mirror closely the functional competencies of their in vivo counterparts. The NT2 cell line is one of the best documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of these cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time in a human stem cell derived co-culture model that these cultures are also metabolically competent and demonstrate a functional astrocyte neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2 derived neurons and astrocytes we have shown that these cells modulate their glucose uptake in response to glutamate, an effect that was blocked by cytochalasin B and ouabain. Additionally we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown following treatment with glutamate, potassium, Isoproterenol and dbcAMP. Together these results demonstrate for the first time a functional ANLS in a human stem cell derived co-culture.