Behavioral assessment of neuropathic pain, fatigue, and anxiety in experimental autoimmune encephalomyelitis (EAE) and attenuation by interleukin-10 gene therapy.

Relapsing-remitting multiple sclerosis is commonly associated with motor impairments, neuropathic pain, fatigue, mood disorders, and decreased life expectancy. However, preclinical pharmacological studies predominantly rely on clinical scoring of motor deficit as the sole behavioral endpoint. Thus, the translational potential of these studies is limited. Here, we have assessed the therapeutic potential of a novel anti-inflammatory interleukin-10 (IL-10) non-viral gene therapy formulation (XT-101-R) in a rat relapsing remitting experimental autoimmune encephalomyelitis (EAE) model. EAE induced motor deficits and neuropathic pain as reflected by induction of low-threshold mechanical allodynia, suppressed voluntary wheel running, decreased social exploration, and was associated with markedly enhanced mortality. We also noted that voluntary wheel running was depressed prior to the onset of motor deficit, and may therefore serve as a predictor of clinical symptoms onset. XT-101-R was intrathecally dosed only once at the onset of motor deficits, and attenuated each of the EAE-induced symptoms and improved survival, relative to vehicle control. This is the first pharmacological assessment of such a broad range of EAE symptoms, and provides support for IL-10 gene therapy as a clinical strategy for the treatment of multiple sclerosis.

Spinal interleukin-10 therapy to treat peripheral neuropathic pain.

INTRODUCTION: Current research indicates that chronic peripheral neuropathic pain includes a role for glia and the actions of proinflammatory factors. This review briefly discusses the glial and cytokine responses that occur following peripheral nerve damage in support of utilizing anti-inflammatory cytokine interleukin-10 (IL-10) therapy to suppress chronic peripheral neuropathic pain. SPINAL NONVIRAL INTERLEUKIN-10 GENE THERAPY: IL-10 is one of the most powerful endogenous counter-regulators of proinflammatory cytokine function that acts in the nervous system. Subarachnoid (intrathecal) spinal injection of the gene encoding IL-10 delivered by nonviral vectors has several advantages over virally mediated gene transfer methods and leads to profound pain relief in several animal models. NONVIRAL GENE DELIVERY: Lastly, data are reviewed that nonviral deoxyribonucleic acid (DNA) encapsulated by a biologically safe copolymer, poly(lactic-co-glycolic) acid (PLGA), thought to protect DNA, leads to significantly improved therapeutic gene transfer in animal models, which additionally and significantly extends pain relief. CONCLUSIONS: The impact of these early studies exploring anti-inflammatory genes emphasizes the exceptional therapeutic potential of new biocompatible intrathecal nonviral gene delivery approaches such as PLGA microparticles. Ultimately, ongoing expression of therapeutic genes is a viable option to treat chronic neuropathic pain in the clinic.

Trophic factors GDNF and BDNF improve function of retinal sheet transplants.

The aim of this study was to compare glial-derived neurotrophic factor (GDNF) treatment with brain-derived neurotrophic factor (BDNF) treatment of retinal transplants on restoration of visual responses in the superior colliculus (SC) of the S334ter line 3 rat model of rapid retinal degeneration (RD). RD rats (age 4-6 weeks) received subretinal transplants of intact sheets of fetal retina expressing the marker human placental alkaline phosphatase (hPAP). Experimental groups included: (1) untreated retinal sheet transplants, (2) GDNF-treated transplants, (3) BDNF-treated transplants, (4) none surgical, age-matched RD rats, (5) sham surgery RD controls, (6) progenitor cortex transplant RD controls, and (7) normal pigmented rat controls. At 2-8 months after transplantation, multi-unit visual responses were recorded from the SC using a 40 ms full-field stimulus (-5.9 to +1 log cd/m(2)) after overnight dark-adaptation. Responses were analyzed for light thresholds, spike counts, response latencies, and location within the SC. Transplants were grouped into laminated or rosetted (more disorganized) transplants based on histological analysis. Visual stimulation of control RD rats evoked no responses. In RD rats with retinal transplants, a small area of the SC corresponding to the position of the transplant in the host retina, responded to light stimulation between -4.5 and -0.08 log cd/m(2), whereas the light threshold of normal rats was at or below -5 log cd/m(2) all over the SC. Overall, responses in the SC in rats with laminated transplants had lower response thresholds and were distributed over a wider area than rats with rosetted transplants. BDNF treatment improved responses (spike counts, light thresholds and responsive areas) of rats with laminated transplants whereas GDNF treatment improved responses from rats with both laminated and rosetted (more disorganized) transplants. In conclusion, treatment of retinal transplants with GDNF and BDNF improved the restoration of visual responses in RD rats; and GDNF appears to exert greater overall restoration than BDNF.

Release of plasmid DNA-encoding IL-10 from PLGA microparticles facilitates long-term reversal of neuropathic pain following a single intrathecal administration.

PURPOSE: Interleukin-10 (IL-10) is an anti-inflammatory molecule that has achieved interest as a therapeutic for neuropathic pain. In this work, the potential of plasmid DNA-encoding IL-10 (pDNA-IL-10) slowly released from biodegradable microparticles to provide long-term pain relief in an animal model of neuropathic pain was investigated. METHODS: PLGA microparticles encapsulating pDNA-IL-10 were developed and assessed both in vitro and in vivo. RESULTS: In vitro, pDNA containing microparticles activated macrophages, enhanced the production of nitric oxide, and increased the production of IL-10 protein relative to levels achieved with unencapsulated pDNA-IL-10. In vivo, intrathecally administered microparticles embedded in meningeal tissue, induced phagocytic cell recruitment to the cerebrospinal fluid, and relieved neuropathic pain for greater than 74 days following a single intrathecal administration, a feat not achieved with unencapsulated pDNA. Therapeutic effects of microparticle-delivered pDNA-IL-10 were blocked in the presence of IL-10-neutralizing antibody, and elevated levels of plasmid-derived IL-10 were detected in tissues for a prolonged time period post-injection (>28 days), demonstrating that therapeutic effects are dependent on IL-10 protein production. CONCLUSIONS: These studies demonstrate that microparticle encapsulation significantly enhances the potency of intrathecally administered pDNA, which may be extended to treat other disorders that require intrathecal gene therapy.

Impact of lactic acid on cell proliferation and free radical-induced cell death in monolayer cultures of neural precursor cells.

Biomaterials prepared from polyesters of lactic acid and glycolic acid, or a mixture of the two, degrade in the presence of water into the naturally occurring metabolites, lactic acid and glycolic acid. While the lactic acid degradation product that is released from biomaterials is well tolerated by the body, lactic acid can influence the metabolic function of cells; it can serve as an energy substrate for cells, and has been shown to have antioxidant properties. Neural precursor cells, a cell population of considerable interest as a source of cells for neural tissue regeneration strategies, generate a high amount of reactive oxygen species, and when associated with a degradable biomaterial, may be impacted by released lactic acid. In this work, the effect of lactic acid on a neural cell population containing proliferative neural precursor cells was examined in monolayer culture. Lactic acid was found to scavenge exogenously added free radicals produced in the presence of either hydrogen peroxide or a photoinitiator (I2959) commonly utilized in the preparation of photopolymerizable biomaterials. In addition to its effect on exogenously added free radicals, lactic acid reduced intracellular redox state, increased the proliferation of the cell population, and modified the cell composition. The findings of this study provide insight into the role that lactic acid plays naturally on developing neural cells and are also of interest to biomaterials scientists that are focused on the development of degradable lactic-acid-based polymers for cell culture devices. The effect of lactic acid on other cell populations may differ and should be characterized to best understand how cells function in degradable cell culture devices.

Contrasting effects of collagen and bFGF-2 on neural cell function in degradable synthetic PEG hydrogels.

Injectable biodegradable cell carriers provide a potential means to improve transplanted cell viability in the nervous system by providing physical protection from compaction, shear forces, and the acute inflammatory response that occurs following transplantation into the host brain environment. Synthetic polyethylene glycol (PEG) hydrogels are ideal candidates for this purpose, as the degradation profile and mechanical properties of the gel can be controlled. Here we introduce biological components into the synthetic gel with the goal of improving neural cell function in the inert PEG environment. In this study, it was found that (1) bFGF-2 is a survival/mitogenic factor for neural precursor cells in degradable hydrogel cultures, (2) collagen has no measurable effect on cell survival, metabolic activity, or proliferation, and (3) co-application of collagen and bFGF-2 to hydrogel cultures targets cell survival and metabolic activity, an effect that is different than either applied individually. Because collagen and bFGF-2 support the survival and growth of neural cells and other cell types, the co-encapsulation approach and functional characterization described in this study can be extended to the development of an array of tissue engineering applications. These findings suggest the importance of understanding and developing strategies to control the chemical microenvironment surrounding cells in three-dimensional biomaterials.

Three-dimensional growth and function of neural tissue in degradable polyethylene glycol hydrogels.

Graft survival and integration are major factors that limit the efficacy of cell therapies for the treatment of disease and injury in the central nervous system. Efforts to improve cell survival and integration have focused in part on the development of biocompatible scaffolds that support neural cell growth and function. Here we photoencapsulate neural cells within degradable hydrogels and use confocal microscopy to non-invasively monitor these key cell functions over time. By directly imaging fluorescently labeled cells we show that neural cells cultured within three-dimensional polymer networks create their own cellular microenvironment to survive, proliferate and differentiate and form neurons and glia that are electrophysiologically responsive to neurotransmitter. By changing the degradation rate of the polymer network, the time-scale over which neural cells extend processes throughout the hydrogel could be tuned on a time-scale that ranged from 1-3 weeks. These studies were carried out in the absence of serum and extracellular matrix molecules that can be immunogenic and identify degradable PEG hydrogels as suitable synthetic cell carriers for neural transplantation.

Impact of cell type and density on nerve growth factor distribution and bioactivity in 3-dimensional collagen gel cultures.

Local delivery of protein agents is potentially important in many tissue engineering systems. In this report, we evaluate an experimental system for measuring the rate of nerve growth factor (NGF) transport and biological activity within a 3-dimensional, tissue-like environment. Fetal brain cells or PC12 cells were suspended throughout collagen gel cultures; controlled-release matrices were used to control the spatial and temporal pattern of NGF release. Experimentally measured concentration profiles were compared to profiles predicted by a mathematical model encompassing diffusion and first-order elimination. Our results suggest that NGF moves through gels by diffusion while being eliminated at a rate that depends on cell density. Since diffusion and elimination also govern protein transport in brain tissue, the collagen gel serves as a model system that replicates the main features of transport in the brain and, therefore, can be used to identify new strategies that enhance NGF distribution in the central nervous system. As an example of the utility of this biophysical model, we demonstrate that implantation of multiple controlled-release matrices can broaden NGF distribution in gel cultures; this broadening was accompanied by a significant increase in cellular biological activity. This approach may be useful in customizing NGF distribution throughout degenerating or damaged central nervous system tissue while minimizing toxicity to surrounding healthy tissue.

The influence of microchannels on neurite growth and architecture.

Microchannels were produced using a photolithographic technique to pattern polyimide walls (11 microm in height and 20-60 microm in width) onto a planar glass substrate. PC12 cells were seeded onto patterned surfaces. After 3 days of culture in NGF supplemented medium cells were viable and extended neurites. Culture in microchannels influenced the direction of neurite growth (theta Orientation) and the complexity of PC12 cell architecture including neurite length (L(Neurite)), the number of neurites emerging per cell (N(Neurites)), and the angle at which neurites emerged from the cell soma (theta Soma). In microchannels neurites oriented parallel to channel walls and the complexity of neuronal architecture was reduced. Both of these effects were strongest for cells located in channels 20-30 microm wide. Within each channel the magnitude of the effect on orientation and architecture was inversely proportional to the distance of the soma from the channel wall. Microtubule and actin filament mobility within the cytoplasm may underly effect on neurite orientation and cell architecture. By manipulating channel width the overall direction of neurite growth and the complexity of neuronal architecture was controlled. Results from these studies will be applied towards the development of biomaterials for microfluidic platforms and drug discovery studies and in neural regeneration research-two applications that would be significantly improved given the ability to control neurite orientation and the complexity of neuronal architecture.

Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates.

This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15)µm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced under both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (∼ 2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1(+)/Nkx6.1(+) cells, key endocrine markers required for ß-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates.

Tissue engineering approaches to cell-based type 1 diabetes therapy.

Type 1 diabetes mellitus is an autoimmune disease resulting from the destruction of insulin-producing pancreatic ß-cells. Cell-based therapies, involving the transplantation of functional ß-cells into diabetic patients, have been explored as a potential long-term treatment for this condition; however, success is limited. A tissue engineering approach of culturing insulin-producing cells with extracellular matrix (ECM) molecules in three-dimensional (3D) constructs has the potential to enhance the efficacy of cell-based therapies for diabetes. When cultured in 3D environments, insulin-producing cells are often more viable and secrete more insulin than those in two dimensions. The addition of ECM molecules to the culture environments, depending on the specific type of molecule, can further enhance the viability and insulin secretion. This review addresses the different cell sources that can be utilized as ß-cell replacements, the essential ECM molecules for the survival of these cells, and the 3D culture techniques that have been used to benefit cell function.

The administration of BDNF and GDNF to the brain via PLGA microparticles patterned within a degradable PEG-based hydrogel: Protein distribution and the glial response.

Tailored delivery of neurotrophic factors (NFs) is a critical challenge that continues to inhibit strategies for guidance of axonal growth in vivo. Of particular importance is the ability to recreate innervation of distant brain regions by transplant tissue, for instance rebuilding the nigrostriatal track, one focus in Parkinson's disease research. Many strategies have utilized polymer drug delivery to target NF release in space and time, but combinatorial approaches are needed to deliver multiple NFs at relevant therapeutic times and locations without toxic side effects. Here we engineered a paradigm of PLGA microparticles entrapped within a degradable PEG-based hydrogel device to locally release two different types of NFs with two different release profiles. Hydrogel/microparticle devices were developed and analyzed for their ability to release GDNF in the caudal area of the brain, near the substantia nigra, or BDNF in the rostral area, near the striatum. The devices delivered their respective NFs in a region localized to within 100 µm of the bridge, but not exclusively to the targeted rostral or caudal ends. BDNF was slowly released over a 56-day period, whereas a bolus of GDNF was released around 28 days. The timed delivery of NFs from implanted devices significantly reduced the microglial response relative to sham surgeries. Given the coordinated drug delivery ability and reduced localized inflammatory response, this multifaceted PEG hydrogel/PLGA microparticle strategy may be a useful tool for further development in combining tissue engineering and drug delivery, and recreating the nigrostriatal track.

Central nervous system delivery of large molecules: challenges and new frontiers for intrathecally administered therapeutics.

IMPORTANCE OF THE FIELD: Therapeutic proteins and DNA constructs offer promise for the treatment of central nervous system disorders, yet significant biological barriers limit the ability of these molecules to reach the central nervous system from the bloodstream. Direct administrations to the cerebrospinal fluid (intrathecal administration) comprise an emerging field to facilitate the efficient delivery of these biological macromolecules to central nervous system tissues. AREAS COVERED IN THIS REVIEW: Previous reports from 1990 to the present time describing the interactions and turnover of the cerebrospinal fluid within the intrathecal space, characterizations of the effects that therapeutic proteins and DNA have shown after intrathecal delivery through a lumbar route, and reports of emerging technologies to address the limitations of intrathecally administered macromolecules are reviewed. WHAT THE READER WILL GAIN: This review provides an overview of the limitations that must be overcome for intrathecally administered biological macromolecules and the recent advances and promising approaches for surmounting these limitations. TAKE HOME MESSAGE: Emerging approaches that stabilize and sustain the delivery of intrathecally administered biological macromolecules may enhance substantially the clinical relevance of promising therapeutic proteins and DNA constructs for the treatment of various central nervous system disorders.

Inhibition of gamma-secretase activity promotes differentiation of embryonic pancreatic precursor cells into functional islet-like clusters in poly(ethylene glycol) hydrogel culture.

We assessed the ability of a gamma-secretase inhibitor to promote the in vitro differentiation of induced embryonic pancreatic precursor cell aggregates into functional islet-like clusters when encapsulated within a three-dimensional hydrogel. Undifferentiated pancreatic precursor cells were isolated from E.15 rat embryos, dissociated into single cells, and aggregated in suspension-rotation culture. Aggregates were photoencapsulated into poly(ethylene glycol) hydrogels with entrapped collagen type 1 and cultured for 14 days with or without a gamma-secretase inhibitor. Gene expression, proinsulin content, and C-peptide release were measured to determine differentiation and maturation of encapsulated precursor cell aggregates. In the control medium, scattered breakthrough beta cell differentiation was observed; however, cells remained largely insulin negative. Upon addition of a gamma-secretase inhibitor the majority of cells in clusters became insulin positive, and insulin per DNA and glucose-stimulated insulin release measurements for these cultures were comparable with those for adult rat islets. Cluster counts after culture day 14 were 88% of those initially encapsulated, demonstrating excellent cluster survival in hydrogel culture. These results indicate that concerted differentiation of pancreatic precursor cell aggregates into functionally mature islet-like clusters can be achieved in poly(ethylene glycol)-based hydrogel cultures by blocking cell contact-mediated Notch signaling with a gamma-secretase inhibitor.

A novel composite construct increases the vascularization potential of PEG hydrogels through the incorporation of large fibrin ribbons.

Developing a mechanism to vascularize tissue-engineered constructs is imperative for transplant function and integration, particularly when delivering hypoxia-sensitive tissues, such as pancreatic islets. Previous efforts have focused on bulk modifications of scaffold materials rendering the entire construct permissive to vessel penetration or the formation of a porous structure where vessels can infiltrate the empty spaces. Here, we describe a novel construct composed of large fibrin ribbons encapsulated within a poly(ethylene glycol) (PEG) hydrogel. The PEG/fibrin ribbon composite scaffold facilitates coculture of adhesive and nonadhesive cell types, thus providing closely neighboring environments with distinct material properties specific to the needs of two clinically relevant cell populations. This advantage is demonstrated here by the successful coculture of pancreatic islets in the PEG component and vessel-forming endothelial cells in entrapped fibrin ribbons. Transplanted endothelial cells can form anastomosies with host vasculature, suggesting that our cocultures may lead to more rapid scaffold vascularization. Additionally, we show that surface-seeded endothelial cells form multicellular projections that migrate into nonadhesive PEG hydrogels along permissive fibrin ribbons, further demonstrating composite construct vascularization potential. Distribution of large fibrin ribbons throughout PEG hydrogels provide a potential mechanism for vascularization of a well-established biomaterial without inherently changing its desirable properties.

Impact of degradable macromer content in a poly(ethylene glycol) hydrogel on neural cell metabolic activity, redox state, proliferation, and differentiation.

Hydrogels that degrade at different rates were prepared by copolymerizing slowly degrading macromer poly(ethylene glycol) (PEG) dimethacrylate with a faster degrading macromer poly(lactic acid)-b-PEG-b-poly(lactic acid) dimethacrylate. A clinically relevant population of neural cells composed of differentiated neurons and multipotent precursor cells was cultured within hydrogels. Within 2 h after encapsulation, metabolic activity was higher in hydrogels prepared with increasing levels of degradable content. This improvement was accompanied by a reduction in intracellular redox state and an increase in the fraction of glutathione in the reduced state, both of which persisted throughout 7 days of culture and which may be the result of radical scavenging by lactic acid. Importantly, an increase in cellular proliferation was observed in gels prepared with increasing degradable macromer content after 7 days of growth without a shift in the cellular composition of the culture toward the glial cell phenotype. The findings of this study provide additional insight into the growth of neural cells in PEG-based hydrogels. Results suggest that lactic acid released during gel degradation may impact the function of encapsulated cells, a finding of general interest to biomaterials scientists who focus on the development of degradable polymers for cell culture and drug delivery devices.

Effect of macromer weight percent on neural cell growth in 2D and 3D nondegradable PEG hydrogel culture.

Neural precursor cells (NPCs) are a renewable cell source that may be useful for neural cell transplant therapies. Their expansion and differentiation potential have traditionally been explored by culturing them on stiff tissue culture polystyrene. Here we describe advantages of an alternative culture system: bio-inert poly(ethylene glycol) (PEG) hydrogels. Specifically this work reports the effect that macromer weight percent has on the metabolic and apoptotic activity, proliferation, and cellular composition of a mixed population of neurons and multipotent NPCs grown both on 2D and within 3D PEG hydrogels. In 2D culture, hydrogel properties did not affect metabolic or apoptotic activity but did impact cell proliferation and composition leading to an increase in glial cell reactivity as stiffness increased. In 3D culture, low weight percent hydrogels led to greater metabolic activity and lower apoptotic activity with significant proliferation observed only in hydrogels that closely matched the stiffness of native brain tissue. PEG hydrogels therefore provide a versatile in vitro culture system that can be used to culture and expand a variety of neural and glial cell types simply by altering the material properties of the hydrogel.

PEGylation of interleukin-10 for the mitigation of enhanced pain states.

The anti-inflammatory cytokine interleukin-10 (IL-10) shows promise for the treatment of neuropathic pain, but for IL-10 to be clinically useful as a short-term therapeutic its duration needs to be improved. In this study, IL-10 was covalently modified with polyethylene glycol (PEG) with the goal of stabilizing and increasing protein levels in the CSF to improve the efficacy of IL-10 for treating neuropathic pain. Two different PEGylation methods were explored in vitro to identify suitable PEGylated IL-10 products for subsequent in vivo testing. PEGylation of IL-10 by acylation yielded a highly PEGylated product with a 35-fold in vitro biological activity reduction. PEGylation of IL-10 by reductive amination yielded products with a minimal number of PEG molecules attached and in vitro biological activity reductions of approximately 3-fold. In vivo collections of cerebrospinal fluid after intrathecal administration demonstrated that 20 kDa PEG attachment to IL-10 increased the concentration of IL-10 in the cerebrospinal fluid over time. Relative to unmodified IL-10, the 20 kDa PEG-IL-10 product exhibited an increased therapeutic duration and magnitude in an animal model of neuropathic pain. This suggests that PEGylation is a viable strategy for the short-term treatment or, in conjunction with other approaches, the long-term treatment of enhanced pain states.

Selective beta-cell differentiation of dissociated embryonic pancreatic precursor cells cultured in synthetic polyethylene glycol hydrogels.

Continuing advances in islet cell transplantation have been promising; however, several limitations, including severe shortage of transplantable islets, hinder the widespread use of this therapy. Pancreatic precursor cells are one alternative to cadaveric donor islets. These cells found in the developing pancreatic buds are capable of self-renewal and also have the innate ability to become insulin-producing beta-cells. For this work, bioinert polyethylene glycol (PEG) hydrogels were chosen as the supportive three-dimensional matrix for encapsulation of dissociated pancreatic precursor cells obtained from the dorsal pancreatic bud of day-15 rat embryos. This culture system was selected in order to eliminate cell-extracellular matrix and cell-cell signal heterogeneity present when intact pancreatic buds are embedded in protein-based gels, the typical in vitro culture conditions used to study this cell population. In this study it was found that (1) dissociated precursor cells maintain a robust viability for 7 days in PEG hydrogel culture, (2) encapsulated cells selectively differentiate into insulin-expressing beta-cells, and (3) differentiated beta-cells have releasable insulin stores, but are not achieving a mature, glucose responsive phenotype. These findings suggest that encapsulating dissociated pancreatic precursor cells in an environment designed to minimize the heterogeneous signaling cues present during development or in standard culture conditions generates a population highly enriched in pancreatic beta-cells; however, future efforts must focus on achieving glucose responsiveness in this cell population. Further, these results indicate that differentiation down a beta-cell lineage may be the default pathway in pancreatic development.

Entrapped collagen type 1 promotes differentiation of embryonic pancreatic precursor cells into glucose-responsive beta-cells when cultured in three-dimensional PEG hydrogels.

Development of an alternative source of functional, transplantable beta-cells to replace or supplement cadaveric tissue is critical to the future success of islet cell transplantation therapy. Embryonic pancreatic precursor cells are desirable as a renewable source of beta-cells as they are both proliferative and inherently capable of pancreatic cell differentiation. We have previously shown that precursor cells undergo selective beta-cell differentiation when dissociated and photoencapsulated in a polyethylene glycol (PEG) hydrogel network; however, these cells remained immature and were not glucose responsive. Collagen type 1 supports mature cell viability and function in many cell types and we hypothesized that incorporating it within our gels may support differentiating beta-cells and facilitate beta-cell maturation. For these studies, collagen-1 was entrapped with dissociated pancreatic precursor cells in a PEG hydrogel matrix (PEGCol) with the following key findings: (1) mature, glucose-responsive, islet-like structures differentiated from spontaneously forming precursor cell clusters in PEGCol, but not unmodified PEG, hydrogels; (2) a balance existed between providing sufficient collagen-1 signaling to support precursor cell development and providing an overabundance of adhesive sites allowing contaminating mesenchymal cells to thrive' and (3) mechanical stability provided by the PEG hydrogel platform is important for successful precursor cell culture, as PEGCol hydrogels encourage glucose responsiveness and high-insulin gene expression, while pure collagen gel cultures, with the same collagen concentration, have negligible insulin gene expression. These results indicate that PEGCol hydrogels are a useful culture platform to promote differentiation of a glucose-responsive beta-cell population from dissociated precursor cells.