Repopulation of decellularized retinas with hiPSC-derived retinal pigment epithelial and ocular progenitor cells shows cell engraftment, organization and differentiation

Decellularization; Ocular progenitors; RetinaDescelularización; Progenitores oculares; RetinaDescel·lularització; Progenitors oculars; RetinaThe retinal extracellular matrix (ECM) provides architectural support, adhesion and signal guidance that controls retinal development. Decellularization of the ECM affords great potential to tissue engineering; however, how structural retinal ECM affects in vitro development, differentiation and maturation of ocular cells remains to be elucidated. Here, mouse and porcine retinas were decellularized and the protein profile analyzed. Acellular retinal ECM (arECM) scaffolds were then repopulated with human iPSC-derived retinal pigment epithelial (RPE) cells or ocular progenitor cells (OPC) to assess their integration, proliferation and organization. 3837 and 2612 unique proteins were identified in mouse and porcine arECM, respectively, of which 93 and 116 proteins belong to the matrisome. GO analysis shows that matrisome-related proteins were associated with the extracellular region and cell junction and KEGG pathways related to signalling transduction, nervous and endocrine systems and cell junctions were enriched. Interestingly, mouse and porcine arECMs were successfully repopulated with both RPE and OPC, the latter exhibiting cell lineage-specific clusters. Retinal cells organized into different layers containing well-defined areas with pigmented cells, photoreceptors, Müller glia, astrocytes, and ganglion cells, whereas in other areas, conjunctival/limbal, corneal and lens cells re-arranged in cell-specific self-organized areas. In conclusion, our results demonstrated that decellularization of both mouse and porcine retinas retains common native ECM components that upon cell repopulation could guide similar ocular cell adhesion, migration and organization.This work was supported by La Marató de TV3 Foundation (484/C/2012); ERA-NET EuroNanoMed III-AC19/00080/ISCIII (CELLUX); Instituto de Salud Carlos III (CA18/00045 and PI18/00219); and the European Social Fund, the Ministerio de Ciencia, Innovación y Universidades, which is part of the Agencia Estatal de Investigación (PTA2018-016371-I). A.D. was supported by PT13/0001/0041 PRB2-ISCIII-SGEFI-FEDER-PE I+D+i 2013–2016 and ISCIII-FEDER RETICS (Oftared; RD16/0008). We thank the CERCA Programme/Generalitat de Catalunya for institutional support

All-trans retinoic acid modulates pigmentation, neuroretinal maturation, and corneal transparency in human multiocular organoids

Ácido transretinoico; Retina; Células madreÀcid transreitnoic; Retina; Cèl·lules mareAll-trans retinoic acid; Retina; Stem cellsBackground All-trans retinoic acid (ATRA) plays an essential role during human eye development, being temporally and spatially adjusted to create gradient concentrations that guide embryonic anterior and posterior axis formation of the eye. Perturbations in ATRA signaling can result in severe ocular developmental diseases. Although it is known that ATRA is essential for correct eye formation, how ATRA influences the different ocular tissues during the embryonic development of the human eye is still not well studied. Here, we investigated the effects of ATRA on the differentiation and the maturation of human ocular tissues using an in vitro model of human-induced pluripotent stem cells-derived multiocular organoids. Methods Multiocular organoids, consisting of the retina, retinal pigment epithelium (RPE), and cornea, were cultured in a medium containing low (500 nM) or high (10 µM) ATRA concentrations for 60 or 90 days. Furthermore, retinal organoids were cultured with taurine and T3 to further study photoreceptor modulation during maturation. Histology, immunochemistry, qPCR, and western blot were used to study gene and protein differential expression between groups. Results High ATRA levels promote the transparency of corneal organoids and the neuroretinal development in retinal organoids. However, the same high ATRA levels decreased the pigmentation levels of RPE organoids and, in long-term cultures, inhibited the maturation of photoreceptors. By contrast, low ATRA levels enhanced the pigmentation of RPE organoids, induced the opacity of corneal organoids—due to an increase in collagen type IV in the stroma— and allowed the maturation of photoreceptors in retinal organoids. Moreover, T3 promoted rod photoreceptor maturation, whereas taurine promoted red/green cone photoreceptors. Conclusion ATRA can modulate corneal epithelial integrity and transparency, photoreceptor development and maturation, and the pigmentation of RPE cells in a dose-dependent manner. These experiments revealed the high relevance of ATRA during ocular tissue development and its use as a potential new strategy to better modulate the development and maturation of ocular tissue through temporal and spatial control of ATRA signaling.This research project was funded by a grant from ERA-NET EuroNanoMed III-ISCIII (AC19/00080) (CELLUX), and Instituto de Salud Carlos III (PI18/00219). A.D. was supported by ISCIII-FEDER RETICS (Oftared; RD16/0008). H.IM. was supported by EuroNanoMed III-ISCIII (AC19/00080)

Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy

Modelo animal; Medicina regenerativa; RetinaModel animal; Medicina regenerativa; RetinaAnimal model; Regenerative medicine; RetinaBackground: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). Methods: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. Results: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. Conclusions: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA.This work was supported by Spanish Ministry of Science and Innovation (MICINN, RTI2018-095377-B-100), Instituto de Salud Carlos III ISCIII/FEDER (PRB2 PT13/0001/0041; TerCel RD16/0011/0024 and Oftared-RETICS RD16/0008), ERA-NET EuroNanoMed III/ISCIII (AC19/00080), the Catalan Government, AGAUR (2017-SGR-899), CERCA Programme/Generalitat de Catalunya, and by Fundació Carmen i Mª José Godó (Fundació CMJ Godó 2017)

Avenços en la fisiopatologia de la Leucoencefalopatia Megalencefàlica amb Quists subcorticals

La Leucoencefalopatia Megalencefàlica amb quists subcorticals (MLC) és un tipus de leucodistròfia espongiforme d'herència autosòmica recessiva. Està considerada una malaltia rara que es caracteritza per macrocefàlia els primers anys de vida, deteriorament de les funcions motores, atàxia i espasticitat, lleuger retràs mental i en alguns casos atacs epilèptics. El diagnòstic es fa per ressonància magnètica d'imatge ja que els pacients presenten la substància blanca anormalment difusa i quists subcorticals a la regió anterio-temporal i fronto-parietal. Es va descriure el primer gen causant de la malaltia, MLC1 mapat al cromosoma 22qtel, del qual s'han descrit més de 60 mutacions diferents. Però existeix un 20% dels pacients de MLC que no presenten mutacions en aquest gen, suggerint més d'un gen implicat a la malaltia. En l'actualitat encara no es coneix la funció de la proteïna MLC1. L'expressió de MLC1 és bàsicament en el sistema nerviós en ratolí, a astròcits i neurones. En la present tesi s'han generat tres anticossos contra la proteïna humana MLC1 per tal de caracteritzar l'expressió i localització de MLC1 en humans. MLC1 s'expressa a sistema nerviós central i perifèric en adult, principalment a astròcits perivasculars i concretament en la membrana en contacte entre els astròcits, amb petites diferències en l'etapa fetal on és predominantment expressió neuronal. Trobem una sobreexpressió de MLC1 en la zona de penombra en condicions d'infart cerebral. S'ha desenvolupat dos models cel·lulars per estudiar la fisiopatologia de MLC1. En el model primari d'astròcits, MLC1 es localitza als processos astrocitaris entre astròcits en unions complexes, formades per proteïnes d'unions tight, gap i adherents, colocalitzant i co¬immunoprecipitant amb ZO-1 i GlialCAM. La seva localització depèn dels microfilaments d'actina. En el model primari de neurones, MLC1 està localitzat al tracte axonal. L'estudi de les mutacions de MLC1 en el model astrocitari provoquen un defecte de plegament amb la retenció de la proteïna a reticle endoplasmàtic, sense poder arribar a membrana. MLC1 s'expressa a monòcits de sang perifèrica, una nova eina pel diagnòstic dels pacients. L'estudi de pacients de MLC amb diferents mutacions revelen una manca total de la proteïna MLC1. El model astrocitari knock-down de MLC1, utilitzant la tecnologia de RNA d'interferència i adenovirus, presenta canvis morfològics en els astròcits. La manca de MLC1 provoca una reducció de la mida cel·lular i l'aparició de vacuoles intracel·lulars. La seva expressió i localització es veu alterada per condicions hipoosmòtiques del medi. La sobreexpressió de MLC1 provoca un augment en la permeabilitat de l'aigua en els astròcits i una disminució de la capacitat de recuperar el volum cel·lular RVD (regulatory colume decrese) en condicions hipoosmòtiques, i en condicions fisiològiques, produeix una major sortida d'aspartat dels astròcits degut a l'activació dels VRACs (volume.regulated anion channels). La nostre hipòtesi és que MLC1 podria actuar com un canal d'aigua, un canal/transporador de Cl-o K+, o un VRAC.Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a type of spongiform leukodystrophy with an autosomal recessive inheritance. It is considered a rare disease characterized by early life macrocephaly, impaired motor functions, ataxia and spasticity, slight mental delay and some cases of seizures. Magnetic resonance imaging is used as diagnostic of patients and shows abnormally diffuse white matter and subcortical cysts in the anterio-temporal and fronto-parietal regions. We describe the first gene causing the disease; MLC1 is mapped to chromosome 22qtel, which have been described more than 60 different mutations. But there is 20% of MLC patients who have no mutations in this gene, suggesting more than one gene involved in disease. The function of MLC1 protein is unknown. The expression of MLC1 is basically in the nervous system in mice, in neurons and astrocytes. In this thesis, we have generated three antibodies against MLC1 human protein to characterize the expression and localization of human MLC1. MLC1 is expressed in central and peripheral nervous system in adults, mainly in perivasculars astrocytes and specifically in the membrane contact between cells, with small differences in the fetal stage where it is predominantly neuronal expression. We found an overexpression of MLC1 in the penumbra zone of a cerebral stroke. We have developed two cell models to study the pathophysiology of MLC1. In the primary astrocytes model, MLC1 is localized in the astrocytic processes between astrocytes, in a complex junctions formed by proteins of tight, gap and adherent junctions, interacting with ZO-1 and GlialCAM. Its location depends on the actin microfilaments. In the model of primary neurons, MLC1 is located in axonal tracts. In the astrocytes model, mutations in MLC1 cause protein folding defects and an endoplasmic reticulum retention, unable to reach membrane. MLC1 is expressed in peripheral blood monocytes, a tool for the diagnosis of patients. Studies of different MLC1 mutations in patients with MLC reveal a total lack of MLC1 protein. The astrocyte knock-down model of MLC1, using RNA interference and adenovirus technology, produces cell morphological changes. The lack of MLC1 causes a reduction in cell size and the appearance of intracellular vacuoles. The expression and localization is altered by hypoosmotical extracellular conditions. In this conditions, the overexpression of MLC1 causes an increase in the permeability of water in astrocytes and a reduced ability to recover the cellular volume RVD (regulatory volume decrease), and under physiological conditions, it triggers a greater efflux of aspartate due to the activation of VRACs (volume-regulated anions channels). Our hypothesis is that MLC1 could act as a water channel, a channel or transporter of Cl-or K+, or a VRAC

Avenços en la fisiopatologia de la leucoencefalopatia megalencefàlica amb quists subcorticals

Descripció del recurs: el 24 març 2011BibliografiaLa Leucoencefalopatia Megalencefàlica amb quists subcorticals (MLC) és un tipus de leucodistròfia espongiforme d'herència autosòmica recessiva. Està considerada una malaltia rara que es caracteritza per macrocefàlia els primers anys de vida, deteriorament de les funcions motores, atàxia i espasticitat, lleuger retràs mental i en alguns casos atacs epilèptics. El diagnòstic es fa per ressonància magnètica d'imatge ja que els pacients presenten la substància blanca anormalment difusa i quists subcorticals a la regió anterio-temporal i fronto-parietal. Es va descriure el primer gen causant de la malaltia, MLC1 mapat al cromosoma 22qtel, del qual s'han descrit més de 60 mutacions diferents. Però existeix un 20% dels pacients de MLC que no presenten mutacions en aquest gen, suggerint més d'un gen implicat a la malaltia. En l'actualitat encara no es coneix la funció de la proteïna MLC1. L'expressió de MLC1 és bàsicament en el sistema nerviós en ratolí, a astròcits i neurones. En la present tesi s'han generat tres anticossos contra la proteïna humana MLC1 per tal de caracteritzar l'expressió i localització de MLC1 en humans. MLC1 s'expressa a sistema nerviós central i perifèric en adult, principalment a astròcits perivasculars i concretament en la membrana en contacte entre els astròcits, amb petites diferències en l'etapa fetal on és predominantment expressió neuronal. Trobem una sobreexpressió de MLC1 en la zona de penombra en condicions d'infart cerebral. S'ha desenvolupat dos models cel·lulars per estudiar la fisiopatologia de MLC1. En el model primari d'astròcits, MLC1 es localitza als processos astrocitaris entre astròcits en unions complexes, formades per proteïnes d'unions tight, gap i adherents, colocalitzant i co¬immunoprecipitant amb ZO-1 i GlialCAM. La seva localització depèn dels microfilaments d'actina. En el model primari de neurones, MLC1 està localitzat al tracte axonal. L'estudi de les mutacions de MLC1 en el model astrocitari provoquen un defecte de plegament amb la retenció de la proteïna a reticle endoplasmàtic, sense poder arribar a membrana. MLC1 s'expressa a monòcits de sang perifèrica, una nova eina pel diagnòstic dels pacients. L'estudi de pacients de MLC amb diferents mutacions revelen una manca total de la proteïna MLC1. El model astrocitari knock-down de MLC1, utilitzant la tecnologia de RNA d'interferència i adenovirus, presenta canvis morfològics en els astròcits. La manca de MLC1 provoca una reducció de la mida cel·lular i l'aparició de vacuoles intracel·lulars. La seva expressió i localització es veu alterada per condicions hipoosmòtiques del medi. La sobreexpressió de MLC1 provoca un augment en la permeabilitat de l'aigua en els astròcits i una disminució de la capacitat de recuperar el volum cel·lular RVD (regulatory colume decrese) en condicions hipoosmòtiques, i en condicions fisiològiques, produeix una major sortida d'aspartat dels astròcits degut a l'activació dels VRACs (volume.regulated anion channels). La nostre hipòtesi és que MLC1 podria actuar com un canal d'aigua, un canal/transporador de Cl-o K+, o un VRAC.Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a type of spongiform leukodystrophy with an autosomal recessive inheritance. It is considered a rare disease characterized by early life macrocephaly, impaired motor functions, ataxia and spasticity, slight mental delay and some cases of seizures. Magnetic resonance imaging is used as diagnostic of patients and shows abnormally diffuse white matter and subcortical cysts in the anterio-temporal and fronto-parietal regions. We describe the first gene causing the disease; MLC1 is mapped to chromosome 22qtel, which have been described more than 60 different mutations. But there is 20% of MLC patients who have no mutations in this gene, suggesting more than one gene involved in disease. The function of MLC1 protein is unknown. The expression of MLC1 is basically in the nervous system in mice, in neurons and astrocytes. In this thesis, we have generated three antibodies against MLC1 human protein to characterize the expression and localization of human MLC1. MLC1 is expressed in central and peripheral nervous system in adults, mainly in perivasculars astrocytes and specifically in the membrane contact between cells, with small differences in the fetal stage where it is predominantly neuronal expression. We found an overexpression of MLC1 in the penumbra zone of a cerebral stroke. We have developed two cell models to study the pathophysiology of MLC1. In the primary astrocytes model, MLC1 is localized in the astrocytic processes between astrocytes, in a complex junctions formed by proteins of tight, gap and adherent junctions, interacting with ZO-1 and GlialCAM. Its location depends on the actin microfilaments. In the model of primary neurons, MLC1 is located in axonal tracts. In the astrocytes model, mutations in MLC1 cause protein folding defects and an endoplasmic reticulum retention, unable to reach membrane. MLC1 is expressed in peripheral blood monocytes, a tool for the diagnosis of patients. Studies of different MLC1 mutations in patients with MLC reveal a total lack of MLC1 protein. The astrocyte knock-down model of MLC1, using RNA interference and adenovirus technology, produces cell morphological changes. The lack of MLC1 causes a reduction in cell size and the appearance of intracellular vacuoles. The expression and localization is altered by hypoosmotical extracellular conditions. In this conditions, the overexpression of MLC1 causes an increase in the permeability of water in astrocytes and a reduced ability to recover the cellular volume RVD (regulatory volume decrease), and under physiological conditions, it triggers a greater efflux of aspartate due to the activation of VRACs (volume-regulated anions channels). Our hypothesis is that MLC1 could act as a water channel, a channel or transporter of Cl-or K+, or a VRAC