RESUMEN
BACKGROUND AIMS: Age-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease. METHODS: Replacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location. RESULTS: We found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2-5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium-derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology. CONCLUSIONS: The possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation.
Asunto(s)
Degeneración Macular , Células Madre Pluripotentes , Humanos , Anciano , Diferenciación Celular , Degeneración Macular/terapia , Criopreservación , Células Epiteliales , Pigmentos RetinianosRESUMEN
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.
Asunto(s)
Envejecimiento/genética , Metilación de ADN , ADN/genética , Células Madre/citología , Adolescente , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Células Cultivadas , Niño , Preescolar , Cromatina/genética , Epigénesis Genética , Histonas/genética , Humanos , Análisis por Micromatrices , Persona de Mediana Edad , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Análisis de Secuencia de ADN , Gemelos Monocigóticos , Adulto JovenRESUMEN
OBJECTIVE: Endothelial cells (ECs), pericytes, and vascular smooth muscle cells (vSMCs) are essential for vascular development, and their dysfunction causes multiple cardiovascular diseases. Primary vascular cells for research are, however, difficult to obtain. Human-induced pluripotent stem cells (hiPSCs) derived from somatic tissue are a renewable source of ECs and vSMCs; however, their use as disease models has been limited by low and inconsistent efficiencies of differentiation and the lack of phenotypic bioassays. APPROACH AND RESULTS: Here, we developed defined conditions for simultaneous derivation of ECs and pericytes with high efficiency from hiPSCs of different tissue origin. The protocol was equally efficient for all lines and human embryonic stem cells (hESCs). The ECs could undergo sequential passage and were phenotypically indistinguishable, exhibiting features of arterial-like embryonic ECs. Moreover, hiPSC-derived ECs formed an authentic vascular plexus when cocultured with hiPSC-derived pericytes. The coculture system recapitulated (1) major steps of vascular development including EC proliferation and primary plexus remodeling, and (2) EC-mediated maturation and acquisition of contractile vSMC phenotype by pericytes. In addition, hiPSC-derived ECs integrated into developing vasculature as xenografts in zebrafish. This contrasts with more widely used ECs from human umbilical vein, which form only unstable vasculature and were completely unable to integrate into zebrafish blood vessels. CONCLUSIONS: We demonstrate that vascular derivatives of hiPSC, such as ECs and pericytes, are fully functional and can be used to study defective endothelia-pericyte interactions in vitro for disease modeling and studies on tumor angiogenesis.
Asunto(s)
Diferenciación Celular , Células Endoteliales/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Neovascularización Fisiológica , Pericitos/fisiología , Animales , Biomarcadores/metabolismo , Comunicación Celular , Línea Celular , Linaje de la Célula , Proliferación Celular , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Células Endoteliales/trasplante , Regulación de la Expresión Génica , Xenoinjertos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Pericitos/metabolismo , Pericitos/trasplante , Fenotipo , Vasoconstricción , Pez CebraRESUMEN
Production of large quantities of hepatocytes remains a major challenge for a number of clinical applications in the biomedical field. Directed differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells (HLCs) provides an advantageous solution and a number of protocols have been developed for this purpose. However, these methods usually follow different steps of liver development in vitro, which is time consuming and requires complex culture conditions. In addition, HLCs lack the full repertoire of functionalities characterising primary hepatocytes. Here, we explore the interest of forward programming to generate hepatocytes from hPSCs and to bypass these limitations. This approach relies on the overexpression of three hepatocyte nuclear factors (HNF1A, HNF6, and FOXA3) in combination with different nuclear receptors expressed in the adult liver using the OPTi-OX platform. Forward programming allows for the rapid production of hepatocytes (FoP-Heps) with functional characteristics using a simplified process. We also uncovered that the overexpression of nuclear receptors such as RORc can enhance specific functionalities of FoP-Heps thereby validating its role in lipid/glucose metabolism. Together, our results show that forward programming could offer a versatile alternative to direct differentiation for generating hepatocytes in vitro.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Diferenciación Celular , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Hígado , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.
Asunto(s)
Células Madre Embrionarias Humanas , Degeneración Macular , Animales , Diferenciación Celular/genética , Humanos , Degeneración Macular/genética , Degeneración Macular/terapia , Epitelio Pigmentado de la Retina , Pigmentos RetinianosRESUMEN
The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.
Asunto(s)
Hepatocitos , Hígado , Humanos , Hígado/metabolismo , Hepatocitos/metabolismo , Diferenciación Celular , Organoides , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Medicina Regenerativa , Ingeniería Genética , Rechazo de Injerto/inmunología , Prueba de Histocompatibilidad , Humanos , Tolerancia Inmunológica , Inmunidad Innata , Activación de Linfocitos , Trasplante de Células Madre , Linfocitos T/inmunologíaRESUMEN
As pluripotent stem cell (PSC)-based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in development, and treatment for age-related macular degeneration is one of the first that has reached patients. We have previously established a xeno-free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)-derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole-genome sequencing (WGS) to assess genome stability, single-cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC-based therapies and the data support safety of the hESC-RPE cells generated through our in vitro differentiation methodology.
Asunto(s)
Células Madre Embrionarias Humanas/metabolismo , Degeneración Macular/terapia , Células Madre Pluripotentes/metabolismo , Anciano , Diferenciación Celular , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes/citologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and -521 without the need for manual isolation.
Asunto(s)
Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Células Epiteliales/metabolismo , Neuronas/metabolismo , Pigmentos Retinianos/metabolismo , Animales , Antígeno CD56 , Células Madre Embrionarias , Humanos , Laminina/genética , Degeneración Macular/metabolismo , Conejos , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.
Asunto(s)
Células Epiteliales/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Madre Embrionarias Humanas/citología , Epitelio Pigmentado de la Retina/citología , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Citotoxicidad Inmunológica , Xenoinjertos , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inmunomodulación , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple/genética , Linfocitos T/metabolismo , Transactivadores/metabolismo , Microglobulina beta-2/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.
Asunto(s)
Atrofia Geográfica/diagnóstico , Células Madre Embrionarias Humanas/trasplante , Degeneración Macular/terapia , Retina/trasplante , Epitelio Pigmentado de la Retina/trasplante , Animales , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Atrofia Geográfica/patología , Humanos , Conejos , Retina/citología , Epitelio Pigmentado de la Retina/citología , Trasplante HeterólogoRESUMEN
Purpose: Subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the capacity to form functional monolayers in naive eyes. We explore hESC-RPE integration when transplanted in suspension to a large-eyed model of geographic atrophy (GA). Methods: Derivation of hESC-RPE was performed in a xeno-free and defined manner. Subretinal bleb injection of PBS or sodium iodate (NaIO3) was used to induce a GA-like phenotype. Suspensions of hESC-RPE were transplanted to the subretinal space of naive or PBS-/NaIO3-treated rabbits using a transvitreal pars plana technique. Integration of hESC-RPE was monitored by multimodal real-time imaging and by immunohistochemistry. Results: Subretinal blebs of PBS or NaIO3 caused different degrees of outer neuroretinal degeneration, RPE hyperautofluorescence, focal RPE loss, and choroidal atrophy; that is, hallmark characteristics of GA. In nonpretreated naive eyes, hESC-RPE integrated as subretinal monolayers with preserved overlying photoreceptors, yet not in areas with outer neuroretinal degeneration and native RPE loss. When transplanted to eyes with PBS-/NaIO3-induced degeneration, hESC-RPE failed to integrate. Conclusions: In a large-eyed preclinical model, subretinal suspension transplants of hESC-RPE did not integrate in areas with GA-like degeneration.
Asunto(s)
Células Epiteliales/trasplante , Atrofia Geográfica/terapia , Células Madre Embrionarias Humanas/citología , Epitelio Pigmentado de la Retina/citología , Animales , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Humanos , Inyecciones Intraoculares , ConejosRESUMEN
Human 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.
Asunto(s)
Diferenciación Celular/fisiología , Atrofia Geográfica/fisiopatología , Células Madre Embrionarias Humanas/fisiología , Epitelio Pigmentado de la Retina/fisiología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Línea Celular , Medios de Cultivo/química , Medios de Cultivo/farmacología , Modelos Animales de Enfermedad , Atrofia Geográfica/terapia , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/trasplante , Humanos , Laminina/metabolismo , Microscopía Confocal , Conejos , Epitelio Pigmentado de la Retina/citología , Trasplante de Células Madre/métodos , Imagen de Lapso de Tiempo , Trasplante Heterólogo , Xenobióticos/química , Xenobióticos/farmacologíaRESUMEN
PURPOSE: To analyze the morphologic effects of subretinal blebs in rabbits using real-time imaging by spectral-domain optical coherence tomography (SD-OCT), infrared-confocal scanning laser ophthalmoscopy (IR-cSLO), and blue-light fundus autofluorescence (BAF). METHODS: Subretinal blebs of PBS or balanced salt solution (BSS) were induced in albino or pigmented rabbits using a transvitreal pars plana technique. Spectral-domain optical coherence tomography, IR-cSLO, and BAF were done at multiple intervals for up to 12 weeks after subretinal bleb injection. The morphologic effects were compared with histologic analysis on hematoxylin-eosin-stained sections of the neurosensory retina and on flat-mounts of phalloidin-labeled RPE. RESULTS: Scans of SD-OCT of the normal rabbit posterior segment revealed 11 bands including six layers of the photoreceptors. Subretinal blebs of PBS or BSS caused acute swelling of the neurosensory retina followed by gradual atrophy. Outer retinal thickness was significantly reduced with pronounced degeneration of all the photoreceptor OCT layers. En face IR-cSLO showed a hyperreflective area corresponding to the progressive photoreceptor degeneration, whereas BAF revealed both hyper- and hypofluorescent changes in the RPE layer. The in vivo results were confirmed by histology and on subretinal flatmounts demonstrating extensive photoreceptor loss and disruption of the RPE mosaic. CONCLUSIONS: Subretinal blebs induce pronounced photoreceptor degeneration and RPE changes in the rabbit as demonstrated by in vivo imaging using SD-OCT, IR-cSLO, and BAF.
Asunto(s)
Diagnóstico por Imagen , Angiografía con Fluoresceína/métodos , Oftalmoscopía/métodos , Células Fotorreceptoras de Vertebrados/patología , Segmento Posterior del Ojo/patología , Degeneración Retiniana/diagnóstico , Tomografía de Coherencia Óptica/métodos , Animales , Modelos Animales de Enfermedad , Fondo de Ojo , Conejos , Reproducibilidad de los Resultados , Degeneración Retiniana/etiologíaRESUMEN
Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease, as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture, differentiation, isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 d), isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes, respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs.