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

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

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

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

Immunological considerations and challenges for regenerative cellular therapies

Abstract: The 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

Preclinical safety studies of human embryonic stem cell‐derived retinal pigment epithelial cells for the treatment of age‐related macular degeneration

International audienc

Publisher Correction: Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells

An amendment to this paper has been published and can be accessed via a link at the top of the paper