Generation of functional islet-like clusters after monolayer culture and intracapsular aggregation of adult human pancreatic islet tissue.

BACKGROUND: Cellular replacement therapy represents a promising strategy for treating type I diabetes; however, such an approach is limited due to the inadequate availability of human donor tissue. Here we investigated the extent to which human islet tissue can be expanded in monolayer culture and brought back to islet function. METHODS: Adult human pancreatic cells were proliferated with a serum-free media in monolayer cultures through multiple passages. Expanded cells were dispersed and encapsulated in alginate-poly-l-lysine microcapsules wherein the cells spontaneously coalesced into islet-like clusters. Encapsulated cell clusters were subsequently transplanted into the peritoneal cavity of streptozotocin-induced diabetic severe combined immunodeficiency mice. RESULTS: The cultured monolayer cells secreted insulin in response to glucose stimulation and maintained endocrine gene expression. Encapsulated islet-like clusters displayed cellular architecture similar to freshly isolated and encapsulated adult human islets maintained in culture, exhibiting an immunoreactive core of insulin, glucagon, and somatostatin, as well as peripheral cytokeratin-19 staining. Encapsulated aggregates significantly reduced hyperglycemia in transplanted mice within 1 week and normoglycemia was achieved after 5 weeks. Human C-peptide was detected in transplanted mice concomitant with the reduction in hyperglycemia. Capsules recovered 8 weeks posttransplantation exhibited insulin immunoreactivity. CONCLUSIONS: Collectively, these data indicate that adult human pancreatic islet cells can be expanded by three serial passages while maintaining their endocrine properties and can yield functional islet-like cell clusters through intracapsular aggregation that reverse hyperglycemia in diabetic mice. This culture and aggregation process could serve as a platform for proliferation and differentiation studies of endocrine lineage cells.

Synaptic targeting of retrogradely transported trophic factors in motoneurons: comparison of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and cardiotrophin-1 with tetanus toxin.

Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and cardiotrophin-1 (CT-1) are the most potent neurotrophic factors for motoneurons, but their fate after retrograde axonal transport is not known. Internalized trophic factors may be degraded, or they may be recycled and transferred to other neurons, similar to the known route of tetanus toxin. We tested whether neonatal rat hypoglossal motoneurons target retrogradely transported trophic factors to synaptic sites on their dendrites within the brainstem and subsequently transfer these trophins across the synaptic cleft to afferent synapses (transsynaptic transcytosis). Motoneurons retrogradely transport from the tongue radiolabeled GDNF, BDNF, and CT-1 as well as tetanus toxin. Quantitative autoradiographic electron microscopy showed that GDNF and BDNF were transported into motoneuron dendrites with labeling densities similar to those of tetanus toxin. Although tetanus toxin accumulated rapidly (within 8 h) at presynaptic sites, GDNF accumulated at synapses more slowly (within 15 h), and CT-1 never associated with synapses. Thus, some retrogradely transported neurotrophic factors are trafficked similarly but not identically to tetanus toxin. Both GDNF and BDNF accumulate at the external (limiting) membrane of multivesicular bodies within proximal dendrites. We conclude that tetanus toxin, GDNF, and BDNF are released from postsynaptic sites and are internalized by afferent presynaptic terminals, thus demonstrating transsynaptic transcytosis. CT-1, however, follows a strict degradation pathway after retrograde transport to the soma. Synaptic and transcytotic trafficking thus are restricted to particular neurotrophic factors such as GDNF and BDNF.

GDNF increases the survival of developing oculomotor neurons through a target-derived mechanism.

Glial cell line-derived neurotrophic factor (GDNF) is the most potent motoneuron survival factor. We show here that in the chick oculomotor system, endogenous GDNF is derived largely from extraocular muscle but less from glial cells and not from muscle spindles. Increased levels of GDNF exclusively in the target rescued 30% of oculomotor neurons that would normally die during developmental cell death, a rate of rescue similar to that with systemic GDNF application. Thus, GDNF supports motoneuron survival in a retrograde, target-derived fashion, as opposed to a local paracrine route or an indirect route via sensory afferents. Persephin, another member of the GDNF family, did not increase survival with target delivery, despite its retrograde transport from the target. Unlike GDNF, however, persephin increased neurite outgrowth from oculomotor nuclei in vitro. Thus, one GDNF family member acts as a muscle-derived retrograde survival factor, whereas another one has distinct functions on neurite outgrowth.

Differential tissue shrinkage and compression in the z-axis: implications for optical disector counting in vibratome-, plastic- and cryosections.

The optical disector is among the most efficient cell counting methods, but its accuracy depends on an undistorted particle distribution in the z-axis of tissue sections. Because the optical disector samples particle densities exclusively in the center of sections, it is essential for unbiased estimates of particle numbers that differential shrinkage or compression (and resulting differences in particle densities along the z-axis) are known and corrected. Here we examined, quantified, and compared differential shrinkage and compression of vibratome-, celloidin- and cryosections. Vibratome sections showed a significant z-axis distortion, while celloidin- and cryosections were minimally distorted. Results were directly compared with previous data obtained from paraffin and methacrylate sections. We conclude that z-axis distortion varies significantly between embedding and sectioning methods, and that vibratome-, methacrylate- and paraffin sections can result in grossly biased estimates. We describe a simple method for assessing differential z-axis shrinkage or compression, as well as simple strategies to minimize the bias of the optical disector. Minimal bias can be achieved by either adjusting the placement and extent of counting boxes and guard spaces for sampling, or by applying a correction factor in cases when guard spaces are deemed essential for particle recognition.

Anterograde axonal transport of internalized GDNF in sensory and motor neurons.

Endogenous glial cell line-derived neurotrophic factor (GDNF) has been shown to be anterogradely transported in sensory and motor neurons which do not express detectable levels of GDNF mRNA. The source of the anterograde axonal GDNF in these neurons has remained unclear. Here we show that radiolabeled GDNF is internalized by dorsal root ganglion (DRG) and hypoglossal motor neurons at their cell bodies and/or their dendrites and that a mechanism exists to transport the internalized GDNF anterogradely in their axons. Since adjacent glial cells express GDNF mRNA, these data support the concept that Schwann cell- or oligodendrocyte-derived GDNF is taken up by DRG and motor neurons, respectively, and transported anterogradely along the axons for release from terminals, resulting in neuronal transcytosis.

Target-derived cardiotrophin-1 and insulin-like growth factor-I promote neurite growth and survival of developing oculomotor neurons.

Several trophic factors support the survival of developing motoneurons, but it is not known whether these factors act in a retrograde fashion from the motoneuron target muscle or are derived from other sources. Cardiotrophin-1 (CT-1) and the insulin-like growth factors (IGFs) are candidate target-derived motoneuron survival factors as both are expressed in muscle during naturally occurring motoneuron death and, applied systemically, support the survival of developing motoneurons. By using the embryonic chick oculomotor system, we show that CT-1 and IGF-I promote neurite outgrowth from E13-derived oculomotor explants and are retrogradely transported from muscle to nerve cell body in vivo, and injection of CT-1 or IGF-I into eye muscles increases motoneuron survival by 20 and 30%, respectively, as evidenced by calibrated stereological counting techniques. Pharmacological depletion of endogenous target-derived IGF-I in vivo reduces oculomotor neuron survival by up to 30% in a dose-dependent manner. These results significantly extend previous studies using systemic administration of trophic factors and are the first to demonstrate a target-derived retrograde mechanism of developing motoneuron survival factors.