Cell therapy for type 1 diabetes.

Cell therapy in the form of human islet transplantation has been a successful form of treatment for patients with type 1 diabetes for over 10 years, but is significantly limited by lack of suitable donor material. A replenishable supply of insulin-producing cells has the potential to address this problem; however to date success has been limited to a few preclinical studies. Two of the most promising strategies include differentiation of embryonic stem cells and induced pluripotent stem cells towards insulin-producing cells and transdifferentiation of acinar or other closely related cell types towards ß-cells. Here, we discuss recent progress and challenges that need to be overcome in taking cell therapy to the clinic.

Inhibition of activin/nodal signalling is necessary for pancreatic differentiation of human pluripotent stem cells.

AIMS/HYPOTHESIS: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) offer unique opportunities for regenerative medicine and for the study of mammalian development. However, developing methods to differentiate hESCs/hIPSCs into specific cell types following a natural pathway of development remains a major challenge. METHODS: We used defined culture media to identify signalling pathways controlling the differentiation of hESCs/hIPSCs into pancreatic or hepatic progenitors. This approach avoids the use of feeders, stroma cells or serum, all of which can interfere with experimental outcomes and could preclude future clinical applications. RESULTS: This study reveals, for the first time, that activin/TGF-ß signalling blocks pancreatic specification induced by retinoic acid while promoting hepatic specification in combination with bone morphogenetic protein and fibroblast growth factor. Using this knowledge, we developed culture systems to differentiate human pluripotent stem cells into near homogenous population of pancreatic and hepatic progenitors displaying functional characteristics specific to their natural counterparts. Finally, functional experiments showed that activin/TGF-ß signalling achieves this essential function by controlling the levels of transcription factors necessary for liver and pancreatic development, such as HEX and HLXB9. CONCLUSION/INTERPRETATION: Our methods of differentiation provide an advantageous system to model early human endoderm development in vitro, and also represent an important step towards the generation of pancreatic and hepatic cells for clinical applications.

Resummed thermodynamic perturbation theory for central force associating potential. Multi-patch models.

A resummed thermodynamic perturbation theory for associating fluids with multiply bondable central force associating potential is extended for the fluid with multiple number of multiply bondable associating sites. We consider a multi-patch hard-sphere model for associating fluids. The model is represented by the hard-sphere fluid system with several spherical attractive patches on the surface of each hard sphere. Resummation is carried out to account for blocking effects, i.e., when the bonding of a particle restricts (blocks) its ability to bond with other particles. Closed form analytical expressions for thermodynamical properties (Helmholtz free energy, pressure, internal energy, and chemical potential) of the models with arbitrary number of doubly bondable patches at all degrees of the blockage are presented. In the limiting case of total blockage, when the patches become only singly bondable, our theory reduces to Wertheim's thermodynamic perturbation theory (TPT) for polymerizing fluids. To validate the accuracy of the theory we compare to exact values, for the thermodynamical properties of the system, as determined by Monte Carlo computer simulations. In addition we compare the fraction of multiply bonded particles at different values of the density and temperature. In general, predictions of the present theory are in good agreement with values for the model calculated using Monte Carlo simulations, i.e., the accuracy of our theory in the case of the models with multiply bondable sites is similar to that of Wertheim's TPT in the case of the models with singly bondable sites.

Resummed thermodynamic perturbation theory for central force associating potential: One-patch model.

A resummed thermodynamic perturbation theory for associating fluids with multiply bondable central force associating potential is proposed. We consider a simple one-patch model for associating fluids. The model is represented by the hard-sphere system with a circular attractive patch on the surface of each hard-sphere. Resummation is carried out to account for the blocking effects, i.e., when the bonding of a particle restricts (blocks) its ability to bond with other particles. Closed form analytical expressions for thermodynamical properties (Helmholtz free energy, pressure, internal energy, and chemical potential) of the model with a doubly bondable patch at all degrees of the blockage are presented. In the limiting case of total blockage, when the particles become only singly bondable, our theory reduces to Wertheim's thermodynamic perturbation theory for dimerizing fluids. To validate the accuracy of the theory we compare to exact values, for the thermodynamical properties of the system, as determined by Monte Carlo computer simulations. In addition we compare the fraction of multiply bonded particles at different values of the density and temperature. Very good agreement between predictions of the theory, corrected for ring formation, and Monte Carlo computer simulation values was found in all cases studied. Less accurate are the original versions of the theory and Wertheim's thermodynamic perturbation theory for dimerization, especially at lower temperatures and larger sizes of the attractive patch.

Investigation of the salting out of methane from aqueous electrolyte solutions using computer simulations.

We calculate the excess chemical potential of methane in aqueous electrolyte solutions of NaCl using Monte Carlo computer simulations. In a recent work [Docherty et al. J. Chem. Phys. 2006, 125, 074510], we presented a new potential model for methane in water which is capable of describing accurately the excess chemical potential of methane in pure water over a range of temperatures, a quantity that can be related to the solubility and which is commonly used to study the hydrophobic effect. Here, we use the same potential model for the water-methane interactions and investigate the effect of added salt on the chemical potential of methane in the solution. The methane molecules are modeled as single Lennard-Jones (LJ) interaction sites, and the water molecules are modeled with the TIP4P/2005 model. A correcting factor of chi = 1.07 for the energetic Berthelot (geometric) combining rule of the methane-water interaction is also used, which mimics the polarization of methane in water. We consider NaCl as the salt and treat the ions with the Smith and Dang model (i.e., as charged LJ interaction sites). Ion-water, ion-ion, and ion-methane interactions are treated using Lorentz-Berthelot combining rules. In addition, the Coulombic potential is used to model charge-charge interactions which are calculated using the Ewald sum. We have carried out isobaric-isothermal (NpT) simulations to determine the equilibrium densities of the solutions. The simulation data is in excellent agreement with experimental densities of aqueous NaCl solutions of different concentration. Hydration numbers are also obtained and found to be in agreement with reported data. Canonical (NVT) simulations at the averaged densities are then performed using the Widom test-particle insertion method to obtain the excess chemical potential of methane in the saline solutions. An increase in the chemical potential of methane, corresponding to a salting out effect, is observed when salt is added to the solution. We investigate different concentrations and ion sizes. An overprediction of the salting out effect as compared with experimental data is observed, which we believe is due to the polarizing effect of the ions in the solution, which is not taken into account by the model. We also find a direct correlation between the increase in the chemical potential and the packing fraction of the solution and argue that the main cause of the observed salting out effect (as represented by an increase in the excess chemical potential) is the increase in the packing fraction of the solutions due to the added salt. Together, with this, we put forward an argument toward explaining the anomalous Hofmeister effect of Li(+).

A potential model for methane in water describing correctly the solubility of the gas and the properties of the methane hydrate.

We have obtained the excess chemical potential of methane in water, over a broad range of temperatures, from computer simulation. The methane molecules are described as simple Lennard-Jones interaction sites, while water is modeled by the recently proposed TIP4P/2005 model. We have observed that the experimental values of the chemical potential are not reproduced when using the Lorentz-Berthelot combining rules. However, we also noticed that the deviation is systematic, suggesting that this may be corrected. In fact, by introducing positive deviations from the energetic Lorentz-Berthelot rule to account indirectly for the polarization methane-water energy, we are able to describe accurately the excess chemical potential of methane in water. Thus, by using a model capable of describing accurately the density of pure water in a wide range of temperatures and by deviating from the Lorentz-Berthelot combining rules, it is possible to reproduce the properties of methane in water at infinite dilution. In addition, we have applied this methane-water potential to the study of the solid methane hydrate structure, commonly denoted as sI, and find that the model describes the experimental value of the unit cell of the hydrate with an error of about 0.2%. Moreover, we have considered the effect of the amount of methane contained in the hydrate. In doing so, we determine that the presence of methane increases slightly the value of the unit cell and decreases slightly the compressibility of the structure. We also note that the presence of methane increases greatly the range of pressures where the sI hydrate is mechanically stable.

Secretion of bioactive human insulin following plasmid-mediated gene transfer to non-neuroendocrine cell lines, primary cultures and rat skeletal muscle in vivo.

The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.

Engineering a glucose-responsive human insulin-secreting cell line from islets of Langerhans isolated from a patient with persistent hyperinsulinemic hypoglycemia of infancy.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a neonatal disease characterized by dysregulation of insulin secretion accompanied by profound hypoglycemia. We have discovered that islet cells, isolated from the pancreas of a PHHI patient, proliferate in culture while maintaining a beta cell-like phenotype. The PHHI-derived cell line (NES2Y) exhibits insulin secretory characteristics typical of islet cells derived from these patients, i.e. they have no K(ATP) channel activity and as a consequence secrete insulin at constitutively high levels in the absence of glucose. In addition, they exhibit impaired expression of the homeodomain transcription factor PDX1, which is a key component of the signaling pathway linking nutrient metabolism to the regulation of insulin gene expression. To repair these defects NES2Y cells were triple-transfected with cDNAs encoding the two components of the K(ATP) channel (SUR1 and Kir6.2) and PDX1. One selected clonal cell line (NISK9) had normal K(ATP) channel activity, and as a result of changes in intracellular Ca(2+) homeostasis ([Ca(2+)](i)) secreted insulin within the physiological range of glucose concentrations. This approach to engineering PHHI-derived islet cells may be of use in gene therapy for PHHI and in cell engineering techniques for administering insulin for the treatment of diabetes mellitus.

Impaired expression of transcription factor IUF1 in a pancreatic beta-cell line derived from a patient with persistent hyperinsulinaemic hypoglycaemia of infancy (nesidioblastosis).

Persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI), or nesidioblastosis, is a rare disorder which may be familial or sporadic, and which is characterized by unregulated secretion of insulin and profound hypoglycaemia in the neonate. The defect has been linked in some patients to mutations in the sulphonyl urea receptor gene (SUR). The present study investigated potential defects in the regulation of the insulin gene by glucose in a beta-cell line (NES 2Y) derived from a patient with PHHI. The results show that the insulin promoter is unresponsive to glucose in PHHI, and that this defect can be attributed to impaired expression of the transcription factor IUF1. Because IUF1 is involved not only in linking glucose metabolism to the control of the insulin, but is also a major regulator of beta-cell differentiation during embryogenesis, we propose that impaired expression of IUF1 contributes to beta-cell dysfunction in PHHI by leading to abnormal beta-cell differentiation.

Expression of parathyroid hormone-related protein in abnormal human parathyroids.

The expression of parathyroid hormone-related protein (PTHrP) in abnormal human parathyroids was investigated. Northern blot analysis of RNA extracted from human benign parathyroid adenomata (n = 4) revealed multiple PTHrP mRNA species ranging in size from 1.8 to 4 kb. The relative abundance of PTHrP mRNA expressed in two of the adenomata was similar to that of a tumour (DAF) associated with humoral hypercalcaemia of malignancy, whereas PTHrP mRNA was of low abundance in a third and was undetectable in the fourth. PTHrP-like immunoreactivity was detected in extracts of abnormal parathyroid tissue (benign adenoma (n = 7), hyperplasia (n = 5) and parathyroid carcinoma (n = 2] using a sensitive specific two-site immunoradiometric assay for human (h) PTHrP(1-86) and a radioimmunoassay for hPTHrP(1-34). Ratios of hPTHrP(1-86)- and hPTHrP(1-34)-like immunoreactivities relative to hPTH(1-84)-like immunoreactivity in the parathyroid tissue extracts were, on average, less than 1%. PTHrP bioactivity in the extracts could not be distinguished from that of PTH, by an osteosarcoma cell bioassay. We conclude that, despite reports of over-expression of PTHrP mRNA in parathyroid adenomata, the potential contribution of PTHrP to the total PTH-like activity of adenomata and other abnormal parathyroid tissue may be insignificant relative to PTH.

Monoclonal antibodies to ROS 17/2.8 cells recognize antigens, some of which are restricted to osteoblasts and chondrocytes.

We have raised a panel of 15 monoclonal antibodies (MAbs) recognizing cell surface antigens of the rat osteoblast-like cell line ROS 17/2.8. The MAbs were selected on the basis of preferential binding to ROS 17/2.8 cells compared to ROS 25/1 cells. Immunohistochemical studies of antigen localization on cryostat sections of rat calvaria, long bone, and soft tissues demonstrated that five of these MAbs, UBIM 1, 2, 3, 12, and 17, recognize antigens that are restricted to normal rat osteoblasts and chondrocytes. The antigens appear to be localized to the cell surface of the osteoblast, with no apparent staining of bone matrix in either undecalcified or decalcified sections. In vitro, these MAbs recognize cell surface antigens present on two additional cell lines, ROS 24/1 and Rat 2 cells, and on the adherent cell population cultured from rat long bone marrow. Of these MAbs, three (UBIM 1, 2, and 3) recognize high-molecular-weight antigens of Mr 200,000-225,000. This study has also identified cell surface antigens of ROS 17/2.8 cells that are not expressed by osteoblasts in vivo. MAbs UBIM 9 and 21 bind to marrow cells in long bone sections, to the 7-day-old nonadherent cell population from cultured marrow, and to lymphoid tissue in sections of spleen. Another four MAbs (UBIM 10, 11, 14, and 22) bind to a variety of cells and tissues both in vitro and in vivo. Studies of the interactions of this panel of MAbs with osteogenic tissues and cell lines may have an important impact on the understanding of osteoblast physiology.

Parathyroid hormone-related proteins in cultured epithelial cells.

The distribution and molecular characteristics of parathyroid hormone-related protein (PTH-rP) in conditioned media and cell extracts of cultured human keratinocytes, and in media from a variety of both normal and transformed epithelial and non-epithelial cell cultures were studied. PTH-rP of Mr 20,000 was observed in keratinocyte-conditioned media, and a larger form, Mr 29,000, in the keratinocyte cell extract. PTH-like bioactivity was also detected in media from 12 out of 17 epithelial cell cultures, but was not present in media from 14 cell cultures of non-epithelial origin. The molecular size of the PTH-like protein present in the epithelial cell media was approximately 20,000, corresponding with the PTH-rP in keratinocyte-conditioned medium. These observations may explain why hypercalcaemia is most commonly associated with tumours of epithelial origin.

Multiple forms of parathyroid hormone-like proteins in a human tumour.

The extensive chromatographic characterization of four parathyroid hormone (PTH)-like proteins in a human bronchial carcinoid tumour associated with humoral hypercalcaemia and severe osteitis fibrosa is described. PTH-like bioactivity was detected in acetic acid extracts of the tumour using an in-vitro osteo-sarcoma cell bioassay. The active tumour proteins were positively charged at physiological pH and had apparent Mr of approximately 29,000, 16,000, 4000-9000 and less than 4000. The proteins were immunologically distinct from PTH, but each stimulated PTH-sensitive adenylate cyclase in cultured osteoblastic cells. There was no evidence of PTH gene expression by the tumour. These proteins represent different molecular forms of PTH-related protein.

Plasma somatostatin 28 increases in response to feeding in man.

Tissue somatostatin-like immunoreactivity (SLI) consists of a number of molecular species including the cyclic tetradecapeptide or SRIF, an N-terminally extended form of SRIF termed somatostatin-28, as well as larger precursor peptides. The function and nature of circulating SLI is not well understood. In this report, we describe techniques for the definition of the components of plasma SLI in normal human plasma. Plasma SLI measured after gel filtration on Bio-gel P-6 columns was found to consist of from 1-3 peaks. The void volume peak was present in greatest concentration (34.2 +/- 8.9 pg/ml) and did not increase in response to a mixed meal. Very low levels of two additional peaks of SLI activity were found. To further characterize these peaks, 10-ml plasma samples were extracted and concentrated on octadecylsilyl silica (C-18) cartridges with subsequent fractionation on Bio-gel P-6 columns. The two peaks that coeluted with synthetic SRIF and S-28 markers, respectively, were present in concentrations of 5.4 +/- 1.4 and 4.8 +/- 1.9 pg/ml in fasting plasma. In response to a mixed meal, the SLI14 peak doubled (12.9 +/- 2.4 pg/ml) while the SLI28 peak increased to 29.9 +/- 7.2 pg/ml at 120 min. These results provide evidence that S-28 circulates in human plasma and its increase after feeding is consistent with a possible biological role for this peptide.

Evidence for the presence of somatostatin 28 in plasma.

Somatostatin-like immunoreactivity (SLI) from dog and rat plasma eluted from Biogel P-6 columns as three distinct peaks. A large-molecular-weight peak was present in the void volume of the column, an intermediate-sized peak (SLI28) coeluted with synthetic somatostatin 28 (S-28), and a small-molecular-weight peak (SLI14) coeluted with SRIF. Material from the SLI28 peak diluted in parallel to the S-28 standard in the radioimmunoassay and behaved identically to S-28 on high pressure liquid chromatography (HPLC). Levels of SLI28 in the portal vein were consistently greater than the simultaneously measured peripheral levels (portal peripheral ratio 2.2 +/- 0.2). Venous samples drawn from multiple sites suggested that SLI28 is segregated by the duodenum and/or pancreas and the intestine. This data is consistent with the possibility that S-28 is a hormone distinct from SRIF.