Sharon Mason

Biphasic Induction of Pdx1 in Mouse and Human Embryonic Stem Cells Can Mimic Development of Pancreatic β‐Cells

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes. Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic β‐cell development. Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse and human ES cell. By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage‐dependent requirement for this transcription factors activity. Mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas‐like cell phenotype, in which 30% of the cells were β‐cell‐like. Cell markers consistent with the different β‐cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development. Furthermore, in mouse ES‐derived cultures the differentiated β‐like cells secreted C‐peptide (insulin) in response to KCl and 3‐isobutyl‐1‐methylxanthine, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells. Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non‐β‐cells in the developing endocrine pancreas. Collectively, we show that this method of in vitro differentiation provides a template for inducing and studying ES cell differentiation into insulin‐secreting cells. STEM CELLS 2009;27:341–351

Progenitors for the Corneal Endothelium and Trabecular Meshwork: A Potential Source for Personalized Stem Cell Therapy in Corneal Endothelial Diseases and Glaucoma

Several adult stem cell types have been found in different parts of the eye, including the corneal epithelium, conjunctiva, and retina. In addition to these, there have been accumulating evidence that some stem-like cells reside in the transition area between the peripheral corneal endothelium (CE) and the anterior nonfiltering portion of the trabecular meshwork (TM), which is known as the Schwalbes Ring region. These stem/progenitor cells may supply new cells for the CE and TM. In fact, the CE and TM share certain similarities in terms of their embryonic origin and proliferative capacity in vivo. In this paper, we discuss the putative stem cell source which has the potential for replacement of lost and nonfunctional cells in CE diseases and glaucoma. The future development of personalized stem cell therapies for the CE and TM may reduce the requirement of corneal grafts and surgical treatments in glaucoma.

Plasma polymer coatings to aid retinal pigment epithelial growth for transplantation in the treatment of age related macular degeneration

Subretinal transplantation of functioning retinal pigment epithelial (RPE) cells grown on a synthetic substrate is a potential treatment for age-related macular degeneration (AMD), a common cause of irreversible vision loss in developed countries. Plasma polymers give the opportunity to tailor the surface chemistry of the artificial substrate whilst maintaining the bulk properties. In this study, plasma polymers with different functionalities were investigated in terms of their effect on RPE attachment and growth. Plasma polymers of acrylic acid (AC), allyl amine (AM) and allyl alcohol (AL) were fabricated and characterised using X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. Octadiene (OD) hydrocarbon films and tissue culture polystyrene were used as controls. Wettability varied from hydrophobic OD to relatively hydrophilic AC. XPS demonstrated four very different surfaces with the expected functionalities. Attachment, proliferation and morphological examination of an RPE cell line and primary RPE cells were investigated. Both cell types grew on all surfaces, with the exception of OD, although the proliferation rate of primary cells was low. Good epithelial morphology was also demonstrated. Plasma polymerised films show potential as cell carrier surfaces for RPE cells in the treatment of AMD.

Replacement of the RPE monolayer.

There are numerous scenarios in which replacing the diseased RPE monolayer is an attractive but as yet unrealised goal. The proof of concept that vision can be improved by placing a healthy neuroretina onto a different, healthy, underlying RPE layer is demonstrated in patch graft transplantations. The surgical procedure to relocate the neuroretina is both complex and is hampered by postoperative complications and as such newer replacement procedures are also being investigated including stem cell replacement therapies. Past studies have largely focused on using cell suspensions and have had disappointing outcomes largely due to the lack of control over cellular differentiation, incomplete attachment onto Bruchs membrane and subsequent integration into the existing RPE monolayer. The choice of which cells to transplant is still under investigation and is complicated by factors such as the ease of collection of an adequate sample, rejection following implantation, the age of the cells and ethical issues. In all these situations, however, understanding the mechanisms of cellular differentiation are likely to be prerequisite to future successes.The current research into replacing the RPE monolayer is briefly discussed with reference to our experiences comparing IPE and RPE cells in an in vitro environment.

Ocular epithelial transplantation: current uses and future potential.

Visual loss may be caused by a variety of ocular diseases and places a significant burden on society. Replacing or regenerating epithelial structures in the eye has been demonstrated to recover visual loss in a number of such diseases. Several types of cells (e.g., embryonic stem cells, adult stem/progenitor/differentiated epithelial cells and induced pluripotent cells) have generated much interest and research into their potential in restoring vision in a variety of conditions: from ocular surface disease to age-related macular degeneration. While there has been some success in clinical transplantation of conjunctival and particularly corneal epithelium utilizing ocular stem cells, in particular, from the limbus, the replacement of the retinal pigment epithelium by utilizing stem cell sources has yet to reach the clinic. Advances in our understanding of all of these cell types, their differentiation and subsequent optimization of culture conditions and development of suitable substrates for their transplantation will enable us to overcome current clinical obstacles. This article addresses the current status of knowledge concerning the biology of stem cells, their progeny and the use of differentiated epithelial cells to replace ocular epithelial cells. It will highlight the clinical outcomes to date and their potential for future clinical use.

Transplantation in the treatment of age-related macular degeneration: past, present and future directions

This review aims to cover the transplant procedures that have been developed and investigated as potential treatments for age-related macular degeneration. The choice of transplant materials that will be discussed ranges from isolated cells (including stem cells, iris pigment epithelial cells and retinal pigment epithelial cells) to monolayers of cells on artificial or nonartificial substrates and also to whole patches of tissue (e.g., fetal retina, full-thickness patch grafts and macular relocation). Finally, we will address the current and future technologies and questions that need to be addressed in order that transplant procedures can have an effective role in the treatment of patients with age-related macular degeneration.

Yield and Viability of Human Limbal Stem Cells From Fresh and Stored Tissue

PURPOSE We compared cell number, putative stem cell markers, and clonogenic ability in fresh uncultured human limbal epithelial cells to that obtained from stored organ-cultured tissue. METHODS Cell suspensions were formed from fresh and organ culture-stored human limbal epithelium. Expression of putative stem cell markers ΔNp63 and TrkA was performed using immunofluorescent staining before culture. Colony-forming efficiency (CFE) assays were performed at first passage. The effects of tissue storage, age, and postmortem/culture times were analyzed in a general linear model. RESULTS Limbal tissue from 94 donors (34 fresh and 60 stored) was compared. Three times more cells were obtained per eye from fresh (35.34 × 104; SD, 17.39) than stored (11.24 × 104; SD, 11.57; P < 0.01) tissue. A higher proportion of cells from fresh tissue were viable (91.9%; SD, 5.7 vs. 85%; SD, 10.8) P < 0.01. Higher total cell expression of ΔNp63 (20.19 × 104; SD, 15.5 vs. 3.28 104; SD, 4.33) and TrkA (59.24 × 104; SD, 13.21 vs. 7.65 × 104; SD, 1.05) was observed in fresh than stored tissue (P < 0.01). Colony-forming efficiency was higher for fresh (1.42; SD, 0.12) than stored (0.43; SD, 0.15; P < 0.01) cells. For stored tissue only, there was a significant inverse relationship between donor age and total number of cells isolated (R2 = 0.27, P < 0.001). CONCLUSIONS Storage of corneoscleral discs in organ culture medium leads to significant reduction in limbal epithelial cell number, expression of ΔNp63 and TrkA, and viability compared to fresh tissue. There is a smaller basal stem cell population in stored compared to fresh tissue.

Supercontinuum ultra-high resolution line-field OCT; experimental spectrograph comparison and comparison with current clinical OCT systems by the imaging of a human cornea

Ultra high axial resolution (UHR) was demonstrated early in the development of optical coherence tomography (OCT), but has not yet reached clinical practice. We present the combination of supercontinuum light source and line field (LF-) OCT as a technical and economical route to get UHR-OCT into clinic and other OCT application areas. We directly compare images of a human donor cornea taken with low and high resolution current generation clinical OCT systems with UHR-LF-OCT. These images highlight the massive information increase of UHR-OCT. Application to pharmaceutical pellets, and the functionality and imaging performance of different imaging spectrograph choices for LF- OCT are also demonstrated.

Deformation velocity imaging using optical coherence tomography and its applications to the cornea

Optical coherence tomography (OCT) can monitor human donor corneas non-invasively during the de-swelling process following storage for corneal transplantation, but currently only resultant thickness as a function of time is extracted. To visualize and quantify the mechanism of de-swelling, we present a method exploiting the nanometer sensitivity of the Fourier phase in OCT data to image deformation velocities. The technique was demonstrated by non-invasively showing during de-swelling that osmotic flow through an intact epithelium is negligible and removing the endothelium approximately doubled the initial flow at that interface. The increased functional data further enabled the validation of a mathematical model of the cornea. Included is an efficient method of measuring high temporal resolution (1 minute demonstrated) corneal thickness, using automated collection and semi-automated graph search segmentation. These methods expand OCT capabilities to measure volume change processes for tissues and materials.