Towards the use of hydrogels in the treatment of limbal stem cell deficiency

Corneal blindness caused by limbal stem cell deficiency (LSCD) is a prevailing disorder worldwide. Clinical outcomes for LSCD therapy using amniotic membrane (AM) are unpredictable. Hydrogels can eliminate limitations of standard therapy for LSCD, because they present all the advantages of AM (i.e. biocompatibility, inertness and a biodegradable structure) but unlike AM, they are structurally uniform and can be easily manipulated to alter mechanical and physical properties. Hydrogels can be delivered with minimum trauma to the ocular surface and do not require extensive serological screening before clinical application. The hydrogel structure is also amenable to modifications which direct stem cell fate. In this focussed review we highlight hydrogels as biomaterial substrates which may replace and/or complement AM in the treatment of LSCD

Encapsulation as a 3D model for human embryonic stem cell propagation and differentiation

Type 1 diabetes is a chronic disease caused by the destruction of the beta cells of the pancreas. Current treatments for are whole pancreas or islet transplantation, which is limited by scarcity of cadaveric pancreata. Therefore, developing a cell replacement therapy with renewable sources of surrogate beta cells is essential. Human embryonic stem cells (hESCs) have an indefinite replication capacity and can be directed to differentiate into many types of specialized cells. Currently, efficient protocols to generate beta cells rely on a step-wise ontogenic approach which mimics the in vivo pancreatic development. This approach requires a three dimensional (3D) culture system that can induce cell-cell and cell-matrix interactions which are essential for functional maturation. This project investigated the use of cell encapsulation technology to maintain hESC propagation and enhance their differentiation into beta cells in a 3D system. This system also offers potential for immune-isolation and prevention of teratoma formation by hESCs during transplantation. For the first time conditions were optimized for encapsulation of single cell population of the hESCs in alginate microcapsules with an extended proliferation. A 5-stage ontogeny-based protocol for differentiation to beta cells was then applied to these cells. Encapsulated hESCs were successfully differentiated into definitive endoderm (DE); however, this 3D model was not efficient to derive functional beta cells due to low cell viability maintained during the latter differentiation stages. To enhance this differentiation process, further investigation was performed by using glucagon-like peptide-1 (GLP-1) which has been shown to stimulate differentiation of stem and progenitor cells to an endocrine phenotype, induce insulin expression and prevent beta cell apoptosis. The present study demonstrated that undifferentiated and differentiated hESCs expressed GLP-1 receptor (GLP-1R). Attempts were made by supplementing the GLP-1R agonist, exendin-4 (Ex-4) to the culture media during each stage of differentiation. However, Ex-4 supplementation had no additive effect on cell viability. In contrast, further investigation demonstrated a significant reduction of the DE gene expression levels. Examination of miRNA profiles of hESC-derived DE revealed that Ex-4 supplementation resulted in the increase in pluripotency-associated miRNAs. Furthermore, in apoptosis-inducing medium, Ex-4 in combination with bFGF resulted in a significant downregulation of pro-apoptotic markers in hESCs. While these data revealed the possible role of Ex-4 in the maintenance of pluripotency and prevention of apoptosis in hESCs, future work is necessary to clarify the mechanism of GLP-1R-ligand interactions and their relevance to hESC development. Findings from this thesis demonstrate the establishment of a novel 3D model for hESC propagation and differentiation. Encapsulated hESCs successfully proliferated and differentiated into DE. Further optimization of the differentiation protocol and modification of biomaterial surface are required to generate functional beta cells. However, this study provides the possibility of using the alginate microcapsules for hESC 3D propagation and differentiation into other lineages and may be useful for cell replacement therapy upon transplantation

Pluripotent stem cells and diabetes : basic science to clinical applications

Type 1 diabetes is caused by the loss of insulin-producing (β) cells in pancreas. A significant advance in cell therapy for diabetes has been the development of a protocol for islet transplantation from Dr. James Shapiro and colleagues at the University of Alberta in Edmonton, Canada (the Edmonton protocol). However, lack of suitable organ donors for transplantation is a critical factor that limits this therapy from majority of individuals suffering from diabetes. Research in the last decade has therefore been largely focused on generating insulin-producing cells that can be easily obtained (derived) and used in transplantation setting for replacement therapy in diabetes. Although insulinproducing cells have been obtained from various sources including human ES cells, bone marrowderived mesenchymal cells, umbilical cord-blood derived mesenchymal cells, transdifferentiation of liver / gallbladder cells, pancreatic duct cells, exocrine cells as well as islet-derived mesenchymal cells, the amount of insulin produced by most of these cell types is significantly less as compared to the amount of insulin produced by a normal adult pancreas. Present research is therefore focused on understanding signalling molecules and processes that would enhance differentiation of these cells. Although embryonic pluripotent/stem cells may be limited due to ethical issues, adult tissue-derived progenitor cells are believed to possess inherent traits that result in "commitment" to a particular phenotype, demonstrated by their relatively restricted differentiation capacity. In this chapter, we discuss the cell types that have been studied for replacement therapy in diabetes with specific reference to their possibility for use in a clinical setting

Alginate microcapsule as a 3D platform for propagation and differentiation of human embryonic stem cells (hESC) to different lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche(1-3). The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment(4) such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures(2). These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded(5). Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions(2) to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes(1). We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, >100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future

An Intelligent Neural Stem Cell Delivery System for Neurodegenerative Diseases Treatment

Transplanted stem cells constitute a new therapeutic strategy for the treatment of neurological disorders. Emerging evidence indicates that a negative microenvironment, particularly one characterized by the acute inflammation/immune response caused by physical injuries or transplanted stem cells, severely impacts the survival of transplanted stem cells. In this study, to avoid the influence of the increased inflammation following physical injuries, an intelligent, double‐layer, alginate hydrogel system is designed. This system fosters the matrix metalloproeinases (MMP) secreted by transplanted stem cell reactions with MMP peptide grafted on the inner layer and destroys the structure of the inner hydrogel layer during the inflammatory storm. Meanwhile, the optimum concentration of the arginine‐glycine‐aspartate (RGD) peptide is also immobilized to the inner hydrogels to obtain more stem cells before arriving to the outer hydrogel layer. It is found that blocking Cripto‐1, which promotes embryonic stem cell differentiation to dopamine neurons, also accelerates this process in neural stem cells. More interesting is the fact that neural stem cell differentiation can be conducted in astrocyte‐differentiation medium without other treatments. In addition, the system can be adjusted according to the different parameters of transplanted stem cells and can expand on the clinical application of stem cells in the treatment of this neurological disorder