Human embryonic stem cells for retinal repair : preclinical in vitro and in vivo studies for the treatment of age-related macular degeneration with human embryonic stem cell-derived retinal pigment epithelial cells

Age-related macular degeneration (AMD) is the major cause of vision loss in the industrialized countries in people above sixty years of age. The dry advanced form of the disease, also termed as geographic atrophy (GA), is characterized by the progressive death of retinal pigment epithelial cells (RPE) and consequent loss of the adjacent photoreceptor (PR) layer, leading to an impaired visual function. Since AMD has a multifactorial cause, including both genetic and epigenetic factors, a potential treatment for retinal regeneration relies on the generation of either autologous or allogeneic RPE and PR cells from human pluripotent stem cells (hPSC) in vitro. The overall aim of this thesis was to develop both in vitro and in vivo methods and models to move forward a stem-cell based replacement therapy for patients suffering from dry advanced forms of AMD. Specifically, we first developed a spontaneous, xeno-free and defined protocol to derive RPE from human embryonic stem cells (hESC-RPE) that acquired specific morphological and functional characteristics of native RPE. Additionally, we developed a large-eyed model (rabbit eye) with relevant pre-clinical imaging and surgical advantages when compared to other more commonly used rodent models. In fact, both the subretinal injections of PBS or the chemical NaIO3 created a retinal degeneration phenotype very similar to the lesion present in GA patients with RPE damage and PR loss. A next logical step was to evaluate the behavior of the hESC-RPE in such models of degeneration. From these studies, we first showed that hESC-RPE can rescue the neuroretina from further damage induced at the moment of subretinal injection, and second, that hESC-RPE are not able to integrate in areas of profound retinal degeneration caused by a 7-day pre-injection of either PBS or NaIO3, therefore supporting the idea of an early treatment. The use of allogeneic hESC as a transplantable source comes together with the forthcoming rejection of the donor cells. We then sought to create universal cells that lack HLA-I (hESC-RPEB2M-/- using CRISPR-Cas9 technology) able to evade the host adaptive immune system. Upon co-culture with T-cells under stimulatory conditions, the engineered hESC-RPEB2M-/- dampened CD8+ T-cell proliferation and when mixed with natural killer (NK) cells, a cytotoxic response was triggered. Furthermore, after transplantation of the hESC-RPEB2M-/- in the rabbit xenogeneic model, early stage rejection was reduced and the appearance of anti-human antibodies rejection associated with late rejection was delayed. Altogether, the studies described in this thesis show evidence that allogeneic replacement therapy using subretinal injection of hESC-RPE in suspension can be a successful treatment if (i) the derived cells retain native RPE cell properties; (ii) the cells are transplanted early enough so the subretinal milieu supports their integration; and (iii) the cells can be engineered so that they can evade the host immune system and consequent graft rejection

The Role of Digital Literacy and Self Efficacy in Enhancing Students' Critical Thinking in Learning in the Digital Era

This research is motivated by the low critical thinking skills of students. The purpose of this research is to examine and analyze the role of digital literacy and self-efficacy in increasing students' critical thinking in the digital era. This research uses a quantitative approach with a survey method. The sampling technique used purposive sampling of 100 students. The instruments used are tests and questionnaires. The data analysis technique used is the classical assumption, correlation analysis, and multiple linear regression analysis including hypothesis testing T test, F test and R2 test. The results of this study show 1) Digital literacy has a positive and significant effect on students' critical thinking in the digital era, 2) Self-Efficacy has a positive and significant effect on students' critical thinking in the digital era, and 3) Digital literacy and self-efficacy have a positive and significant effect and are significant to students' critical thinking in the digital era. The findings of this study hint at the importance of digital literacy and self-efficacy in increasing students' critical thinking in the digital era

Immunological considerations and challenges for regenerative cellular therapies.

Funder: Wellcome TrustThe central goal of regenerative medicine is to replace damaged or diseased tissue with cells that integrate and function optimally. The capacity of pluripotent stem cells to produce unlimited numbers of differentiated cells is of considerable therapeutic interest, with several clinical trials underway. However, the host immune response represents an important barrier to clinical translation. Here we describe the role of the host innate and adaptive immune responses as triggers of allogeneic graft rejection. We discuss how the immune response is determined by the cellular therapy. Additionally, we describe the range of available in vitro and in vivo experimental approaches to examine the immunogenicity of cellular therapies, and finally we review potential strategies to ameliorate immune rejection. In conclusion, we advocate establishment of platforms that bring together the multidisciplinary expertise and infrastructure necessary to comprehensively investigate the immunogenicity of cellular therapies to ensure their clinical safety and efficacy

Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

SummaryHuman embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model

Circular Economy for Sustainable Development

As a new paradigm for economic development, the circular economy has significant environmental, economic and social benefits at the global scale. The circular economy concept highlights the notion of replacing the ‘end-of-life’ in current production and consumption practices by reducing, reusing, and recycling products and materials in production, distribution and consumption processes. Promoting circularity aims to accomplish sustainable development, and the circular economy has links to many of the 17 Sustainable Development Goals (SDGs) approved by the United Nations in 2015. This report is a background contribution asked by the Independent group of scientists writing the Global Sustainable Development Report (GSDR) 2019. The GSDR 2019 is the first in a series of comprehensive, in-depth Reports that will be produced every four years to inform the High-level Political Forum on Sustainable Development convened under the auspices of the General Assembly. Thus, this background report seeks to provide a condensed package on the circular economy; the concept, its history, potentials, business opportunities, management and measurement. Some of the key messages entail that moving towards a circular economy presents vast opportunities for businesses of various kind, and that increasing the material circularity of economy can also be a way to alleviate poverty. Yet, the systemic and disruptive changes required for a circular economy transition will not take place without significant changes to existing regulatory structures

OCT Signs of Early Atrophy in Age-Related Macular Degeneration: Interreader Agreement: Classification of Atrophy Meetings Report 6.

PURPOSE: To determine the interreader agreement for incomplete retinal pigment epithelium (RPE) and outer retinal atrophy (iRORA) and complete RPE and outer retinal atrophy (cRORA) and their related features in age-related macular degeneration (AMD). DESIGN: Interreader agreement study. PARTICIPANTS: Twelve readers from 6 reading centers. METHODS: After formal training, readers qualitatively assessed 60 OCT B-scans from 60 eyes with AMD for 9 individual features associated with early atrophy and performed 7 different annotations to quantify the spatial extent of OCT features within regions of interest. The qualitative and quantitative features were used to derive the presence of iRORA and cRORA and also in an exploratory analysis to examine if agreement could be improved using different combinations of features to define OCT atrophy. MAIN OUTCOME MEASURES: Interreader agreement based on Gwet's first-order agreement coefficient (AC1) for qualitatively graded OCT features and classification of iRORA and cRORA, and smallest real difference (SRD) for quantitatively graded OCT features. RESULTS: Substantial or better interreader agreement was observed for all qualitatively graded OCT features associated with atrophy (AC1 = 0.63-0.87), except for RPE attenuation (AC1 = 0.46) and disruption (AC1 = 0.26). The lowest SRD for the quantitatively graded horizontal features was observed for the zone of choroidal hypertransmission (± 190.8 μm). Moderate agreement was found for a 3-category classification of no atrophy, iRORA, and cRORA (AC1 = 0.53). Exploratory analyses suggested a significantly higher level of agreement for a 3-category classification using (1) no atrophy; (2) presence of inner nuclear layer and outer plexiform layer subsidence, or a hyporeflective wedge-shaped band, as a less severe atrophic grade; and (3) the latter plus an additional requirement of choroidal hypertransmission of 250 μm or more for a more severe atrophic grade (AC1 = 0.68; P = 0.013). CONCLUSIONS: Assessment of iRORA and cRORA, and most of their associated features, can be performed relatively consistently and robustly. A refined combination of features to define early atrophy could further improve interreader agreement

H'mong mustard seed production calendar

H’mong mustard seed production calendars for awareness creation and capacity building activities in vegetable and seed production and storage among ethnic minority vegetable farming households in Northern Vietnam. Particularly designed with and for the H’Mong and Dao people in Lao Cai province and Thai people in Son La province in English and Vietnamese

French bean seed production calendar

French bean seed production calendars for awareness creation and capacity building activities in vegetable and seed production and storage among ethnic minority vegetable farming households in Northern Vietnam. Particularly designed with and for the H’Mong and Dao people in Lao Cai province and Thai people in Son La province in English and Vietnamese

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors