Age related macular degeneration (AMD) is a leading cause of vision loss in the developed world, with an increasing number of people suffering from blindness or severe visual impairment. Transplantation of retinal pigment epithelium (RPE) cells supported on a synthetic, biomimetic-like Bruch's membrane (BM) is considered a promising treatment. However, the synthetic scaffolds used do not mimic the microenvironment of the RPE cell supporting layers, required for the development of a functional RPE monolayer. This study indicated that porous, laminin coated, 70nm PLLA ENMs supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. These findings indicate the potential clinical use of porous, laminin coated, 70nm PLLA ENMs in fabricating retinal constructs aimed at treating dry AMD.

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Age-related macular degeneration (AMD) is a highly prevalent form of blindness caused by loss death of cells of the retinal pigment epithelium (RPE). Transplantation of pluripotent stem cell (PSC)-derived RPE cells is considered a promising therapy to regenerate cell function and vision. Objective The objective of this study is to develop a rapid directed differentiation method for production of RPE cells from PSC which is rapid, efficient, and fully defined and produces cells suitable for clinical use. Design A protocol for cell growth and differentiation from hESCs was developed to induce differentiation through screening small molecules which regulated a primary stage of differentiation to the eyefield progenitor, and then, a subsequent set of molecules to drive differentiation to RPE cells. Methods for cell plating and maintenance have been optimized to give a homogeneous population of cells in a short 14-day period, followed by a procedure to support maturation of cell function. Results We show here the efficient production of RPE cells from human embryonic stem cells (hESCs) using small molecules in a feeder-free system using xeno-free/defined medium. Flow cytometry at day 14 showed ~ 90% of cells expressed the RPE markers MITF and PMEL17. Temporal gene analysis confirmed differentiation through defined cell intermediates. Mature hESC-RPE cell monolayers exhibited key morphological, molecular, and functional characteristics of the endogenous RPE. Conclusion This study identifies a novel cell differentiation process for rapid and efficient production of retinal RPE cells directly from hESCs. The described protocol has utility for clinical-grade cell production for human therapy to treat AMD.

Cardiovascular disease, especially ischemic heart disease resulting from coronary artery disease (CAD), is one of the major causes of death and disability in the United States. Even though the dead myocardial cells can be replaced by scar tissue in the healing process, the resulting myocardium cannot function as well as the preinfarcted myocardium, because scar tissues cannot contract. This "normal" healing process results in decreased cardiac output, which can lead to heart failure. Moreover, the scar tissue has abnormal electrical properties, which can lead to sometimes fatal arrhythmias. Previous studies demonstrated that when Lac-Z-labeled healing cells were infused into two animal models of myocardial infarction, that these cells were found to be located within the myocardium, the cardiac skeleton, and the vasculature undergoing repair. These results suggested that healing cells have the potential to repair damaged hearts. The current series of studies were undertaken to determine whether healing cells customarily reside in normal non-injured hearts of small and large animals, and whether autologous healing cells could be infused safely into a post-myocardial infarction patient. Adult rats were euthanized following the guidelines of Mercer University's IACUC. Adult pigs were euthanized following the guidelines of Fort Valley State University's IACUC. The human study was performed under the guidance of the Medical Center of Central Georgia's IRB. Animal hearts were harvested, fixed, cryosectioned, and stained with three antibodies: carcinoembryonic antigen-cell adhesion molecule-1 (CEA-CAM-1) for totipotent stem cells, stage-specific embryonic antigen-4 (SSEA-4) for pluripotent stem cells, and smooth muscle alpha-actin (IA4) for smooth muscle in the wall of the accompanying vasculature, thus serving as the positive procedural control. Cells positive for both CEA-CAM-1 and SSEA-4 were found to be located in adult rat and porcine hearts. Infusion of autologous healing cells into a post-myocardial infarcted patient resulted in an increase in their cardiac output after two successive healing cell infusions. Current IRB-approved studies are underway to assess the safety and efficacy of infused healing cells into individuals with cardiovascular disease.

The retinal pigment epithelium (RPE) is a multifunctional monolayer located at the back of the eye required for the survival and function of the light-sensing photoreceptors. In Age-related Macular Degeneration (AMD), the loss of RPE cells leads to photoreceptor death and permanent blindness. RPE cell transplantation aims to halt or reverse vision loss by preventing the death of photoreceptor cells and is considered one of the most viable applications of stem cell therapy in the field of regenerative medicine. Proof-of-concept of RPE cell transplantation for treating retinal degenerative disease, such as AMD, has long been established in animal models and humans using primary RPE cells, while recent research has focused on the transplantation of RPE cells derived from human pluripotent stem cells (hPSC). Early results from clinical trials indicate that transplantation of hPSC-derived RPE cells is safe and can improve vision in AMD patients. Current hPSC-RPE cell production protocols used in clinical trials are nevertheless inefficient. Treatment of large numbers of AMD patients using stem cellderived products may be dependent on the ability to generate functional cells from multiple hPSC lines using robust and clinically-compliant methods. Transplantation outcomes may be improved by delivering RPE cells on a thin porous membrane for better integration into the retina, and by manipulation of the outcome through control of immune rejection and inflammatory responses.

Summary StatementThe generation and transcriptome analysis of the first induced pluripotent stem cells from the platypus reveals SOX2 has been a key driver of the expanded pluripotency regulatory network in placental mammals.ABSTRACTThe mechanisms by which pluripotency has evolved remain unclear. To gain insight into the evolution of mammalian pluripotency we have generated induced pluripotent stem cells (piPSCs) from the platypus. Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors OCT4, SOX2 and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 million years and 166 million years ago primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. We further show that the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1 indicating that there is no upregulation of X-linked genes and that there is no preference for silencing of maternal or paternal alleles (ie imprinting).

Stage-specific antigen-4 (SSEA-4) positive cells and carcinoembryonic antigen-cell adhesion molecule-1 (CEA-CAM-1) positive cells, indicative of pluripotent stem cells and totipotent stem cells, respectively, have been isolated and characterized from the skeletal muscle and blood of adult animals, including humans. The current study was undertaken to determine their location in the dermis and underlying connective tissues of the adult pig. Adult pigs were euthanized following the guidelines of Fort Valley State University's IACUC. The skin (epidermis through hypodermis) was harvested, fixed, cryosectioned, and stained with the two antibodies: SSEA-4 and CEA-CAM-1. SSEA-4 positive cells were located preferentially in the reticular dermis of the skin and to some extent in the underlying hypodermis. In contrast, CEA-CAM-1 positive stem cells were preferentially located within the hypodermis of the pig skin within the loose fibrous connective tissues surrounding adipose tissue. CEA-CAM-1 positive cells were also located, to a lesser extent, in the dermis as well. These results demonstrate the presence of native populations of pluripotent stem cells and totipotent stem cells within the dermis, hypodermis, and adipose tissue of adult pig skin. Studies are ongoing to address the functional significance of these cells in normal injury and repair.

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