Diabetes mellitus: a potential target for stem cell therapy

Type 1 diabetes mellitus has received much attention recently as a potential target for the emerging science of stem cell medicine. In this autoimmune disease, the insulin-secreting beta-cells of the pancreas are selectively and irreversibly destroyed by autoimmune assault. Advances in islet transplantation procedures now mean that patients with the disease can be cured by transplantation of primary human islets of Langerhans. A major drawback in this therapy is the availability of donor islets, and the search for substitute transplant tissues has intensified in the last few years. This review will describe the essential requirements of a material designed as a replacement beta-cell and will look at the potential sources of such replacements. These include embryonic stem (ES) cells and multipotent adult stem/progenitor cells from a range of tissues including the pancreas, intestine, liver, bone marrow and brain. These stem cell populations will be evaluated and the different experimental approaches that have been employed to derive functional insulin-expressing cells will be discussed. The review will also look at the capability of human ES (hES) cells generated by somatic cell nuclear transfer and some adult stem cell populations such as bone marrow-derived stem cells, to offer autologous transplant material that would remove the need for immunosuppression. In patients with Type 1 diabetes, auto-reactive T-cells are programmed to recognise the insulin-producing beta-cells. As a result, for therapeutic replacement tissues, it may be more sensible to derive cells that behave like beta-cells but are immunologically distinct. Thus, the potential of cells derived from non-beta-cell origin to avoid the autoimmune response will also be discussed. Finally, the review will summarise the future prospects for stem cell therapies for diabetes and will highlight some of the problems that may be faced by researchers working in this area, such as malignancy, irreproducible differentiation strategies, immune-system rejection and social and ethical concerns over the use of hES cells.     

Generation of insulin-expressing cells from mouse embryonic stem cells

The therapeutic potential of transplantation of insulin-secreting pancreatic beta-cells has stimulated interest in using pluripotent embryonic stem (ES) cells as a starting material from which to generate insulin secreting cells in vitro. Mature beta-cells are endodermal in origin so most reported differentiation protocols rely on the identification of endoderm-specific markers. However, endoderm development is an early event in embryogenesis that produces cells destined for the gut and associated organs in the embryo, and for the development of extra-embryonic structures such as the yolk sac. We have demonstrated that mouse ES cells readily differentiate into extra-embryonic endoderm in vitro, and that these cell populations express the insulin gene and other functional elements associated with beta-cells. We suggest that the insulin-expressing cells generated in this and other studies are not authentic pancreatic beta-cells, but may be of extra-embryonic endodermal origin.     

Activation of protein kinase C is essential for sustained insulin secretion in response to cholinergic stimulation

Insulin secretion from isolated rat islets of Langerhans is enhanced by cholinergic agonists, such as carbachol (CCh), in the presence of a stimulatory concentration of glucose. Depletion of islet protein kinase C activity by prolonged exposure to a tumour-promoting phorbol ester did not prevent the initial secretory response to CCh, but markedly reduced the duration of CCh-induced elevated secretory rates. These results suggest that the major action of PKC is in maintaining rather than initiating the insulin secretory response to cholinergic agonists.     

Binding proteins for sweet compounds from gustatory papillae of the cow, pig and rat

The intensely sweet proteins thaumatin and monellin were covalently attached to affinity column supports. Lingual tissue extracts were incubated with the affinity columns which were then eluted with glycine-HCl pH 3.4, the sweet peptide aspartame, or gymnemic acid, which is a sweet taste modifier. SDS-PAGE analysis of eluates from the columns showed that 156 kDa and 47 kDa proteins were the main components from cow fungiform papillae which were specifically bound to thaumatin and monellin. These proteins could be displaced from the column with 0.5 mM aspartame or 0.5 mg/ml gymnemic acid. With circumvallate papillae small amounts of 47 kDa protein were also found. The 47 kDa protein was also the major component bound to a gymnemic acid affinity column and could be displaced from the column with 0.5 mg/ml gymnemic acid. Control experiments with other lingual tissue components indicated that these proteins are localised in the gustatory papillae. Similar protein patterns were also found in extracts of pig fungiform papillae and rat lingual preparations.     

Ion transport across the frog olfactory mucosa: the basal and odorant-stimulated states

The Ussing method was adapted to study the basal electrolyte transfer as well as the events that occur upon odorant stimulation in frog olfactory mucosa. The unstimulated short-circuit current was due mainly to a furosemide-sensitive ion transport system on the apical side of the olfactory mucosa. This current was not amiloride sensitive. The current-voltage relationship of the unstimulated state was linear. That of the odorant-evoked current was non-linear and amiloride-sensitive. Ouabain caused collapse of both the unstimulated and odorant-stimulated short-circuit current. In this case, voltage-clamping the tissue to non-zero values restored the odorant-evoked current with polarity depending on that of the clamping voltage. This suggested that the direction of the current is determined by that of the sodium electrochemical potential difference. Our results indicate that the unstimulated short-circuit current occurs through an apical sodium cotransport system, while the odorant-evoked current is due to odorant-activated, passive sodium channels that are amiloride sensitive.     

Evaluation of cytotoxic T-lymphocyte responses in human and nonhuman primate subjects infected with human immunodeficiency virus type 1 or simian/human immunodeficiency virus

Cytotoxic T-lymphocyte (CTL) responses have been implicated as playing an important role in control of human immunodeficiency virus (HIV) infection. However, it is technically difficult to demonstrate CTL responses consistently in nonhuman primate and human subjects using traditional cytotoxicity assay methods. In this study, we systematically evaluated culture conditions that may affect the proliferation and expansion of CTL effector cells and presented a sensitive method for detection of cytotoxicity responses with bulk CTL cultures. We confirmed the sensitivity and specificity of this method by demonstration of vigorous CTL responses in a simian-HIV (SHIV)-infected rhesus macaque. The expansion of epitope-specific CTL effector cells was also measured quantitatively by CTL epitope-major histocompatibility complex tetramer complex staining. In addition, two new T-cell determinants in the SIV gag region are identified. Last, we showed the utility of this method for studying CTL responses in chimpanzee and human subjects.     

The perception of visual images encoded in musical form: a study in cross-modality information transfer

This study demonstrates the ability of blind (previously sighted) and blindfolded (sighted) subjects in reconstructing and identifying a number of visual targets transformed into equivalent musical representations. Visual images are deconstructed through a process which selectively segregates different features of the image into separate packages. These are then encoded in sound and presented as a polyphonic musical melody which resembles a Baroque fugue with many voices, allowing subjects to analyse the component voices selectively in combination, or separately in sequence, in a manner which allows a subject to patch together and bind the different features of the object mentally into a mental percept of a single recognizable entity. The visual targets used in this study included a variety of geometrical figures, simple high-contrast line drawings of man-made objects, natural and urban scenes, etc., translated into sound and presented to the subject in polyphonic musical form.