Hypoxia increases the yield of photoreceptors differentiating from mouse embryonic stem cells and improves the modeling of retinogenesis in vitro.

Retinitis pigmentosa (RP), a genetically heterogeneous group of diseases together with age-related macular degeneration (AMD), are the leading causes of permanent blindness and are characterized by the progressive dysfunction and death of the light sensing photoreceptors of the retina. Due to the limited regeneration capacity of the mammalian retina, the scientific community has invested significantly in trying to obtain retinal progenitor cells from embryonic stem cells (ESC). These represent an unlimited source of retinal cells, but it has not yet been possible to achieve specific populations, such as photoreceptors, efficiently enough to allow them to be used safely in the future as cell therapy of RP or AMD. In this study, we generated a high yield of photoreceptors from directed differentiation of mouse ESC (mESC) by recapitulating crucial phases of retinal development. We present a new protocol of differentiation, involving hypoxia and taking into account extrinsic and intrinsic cues. These include niche-specific conditions as well as the manipulation of the signaling pathways involved in retinal development. Our results show that hypoxia promotes and improves the differentiation of mESC toward photoreceptors. Different populations of retinal cells are increased in number under the hypoxic conditions applied, such as Crx-positive cells, S-Opsin-positive cells, and double positive cells for Rhodopsin and Recoverin, as shown by immunofluorescence analysis. For the first time, this manuscript reports the high efficiency of differentiation in vivo and the expression of mature rod photoreceptor markers in a large number of differentiated cells, transplanted in the subretinal space of wild-type mice. STEM CELLS 2013;31:966¿978This work was supported by funds for research from Junta de Andalucía PI-0113-2010 (S.E.) and ‘‘Miguel Servet’’ contract of Instituto de Salud Carlos III of Spanish Ministry of Science and Innovation (S.E.).Peer Reviewe

Modeling AIPL1-defect using iPS-derived retinal progenitors from a patient Leber Congenital Amaurosis

Póster presentado al Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), celebrado en Seattle, Washington (US) del 1 al 5 de mayo de 2016.Patient-derived cellular models can provide a specific tool to test new therapeutic approaches for retinal degenerative diseases. Mutations in AIPL1 are associated with Leber Congenital Amaurosis (LCA). Considering that AIPL1 protein is only expressed in photoreceptors we decided to establish a cellular model of AIPL1-LCA by differentiation of induced pluripotent stem cells (iPS) towards photoreceptor lineage.Grants: Junta de Andalucía; Fundación Progreso y Salud; Ministerio de Economía y Competitividad; Instituto de Salud Carlos III; EU H2020.Peer Reviewe

Highly efficient neural conversion of human pluripotent stem cells in adherent and animal-free conditions

Lukovic, Dunja et al.Neural differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can produce a valuable and robust source of human neural cell subtypes, holding great promise for the study of neurogenesis and development, and for treating neurological diseases. However, current hESCs and hiPSCs neural differentiation protocols require either animal factors or embryoid body formation, which decreases efficiency and yield, and strongly limits medical applications. Here we develop a simple, animal-free protocol for neural conversion of both hESCs and hiPSCs in adherent culture conditions. A simple medium formula including insulin induces the direct conversion of >98% of hESCs and hiPSCs into expandable, transplantable, and functional neural progenitors with neural rosette characteristics. Further differentiation of neural progenitors into dopaminergic and spinal motoneurons as well as astrocytes and oligodendrocytes indicates that these neural progenitors retain responsiveness to instructive cues revealing the robust applicability of the protocol in the treatment of different neurodegenerative diseases. The fact that this protocol includes animal-free medium and human extracellular matrix components avoiding embryoid bodies makes this protocol suitable for the use in clinic.This work was supported by Wings for Life Foundation, funds for research from the “Miguel Servet” contract of Institute of Health Carlos III of Spanish Ministry of Science and Innovation (CP10/00579) (S.E.), Fund for Health of Spain PI14‐02209 (S.E.), Platform of Biomolecular and Bioinformatics Resources of the Institute of Health Carlos III PT13/0001/0042, Spain (D.L. and S.E.), Czech National Foundation GA CR P304/12/G069 (E.S.), and by the project „BIOCEV “(CZ.1.05/1.1.00/02.0109)” (P.J. and S.E.) and by the European infrastructure for translational medicine (EATRIS‐CZ LM2015064) (E.S.).Peer Reviewe

Generation and characterization of the human iPSC line CABi001-A from a patient with retinitis pigmentosa caused by a novel mutation in PRPF31 gene

PRPF31 gene codes for a ubiquitously expressed splicing factor but mutations affect exclusively the retina, producing the progressive death of photoreceptor cells. We have identified a novel PRPF31 mutation in a patient with autosomal dominant retinitis pigmentosa. A blood sample was obtained and mononuclear cells were reprogrammed using the non-integrative Sendai virus to generate the cell line CABi001-A. The iPSC line has been characterized for pluripotency and differentiation capacity and will be differentiated toward photoreceptors and retinal pigment epithelium cells to study the molecular mechanism of the disease and test possible therapeutic strategies

Sirt1 activators induced neuroprotection of photoreceptors in rd10 mice

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.[Purpose]: It has been reported that Sirt1 nuclear expression decreases in the outer nuclear layer (ONL) of retinal degeneration 10 (rd10) mice at postnatal day 15 (P15), and this abnormal pattern is correlated with the beginning of photoreceptor degeneration. Sirt1 is a class III histone deacetylase, and over-expression of these proteins have shown neuroprotective effect in several experimental models of neurodegenerative diseases. However, it is still unknown whether Sirt1 modulation could induce neuroprotection of photoreceptors. We tested the hypothesis that subretinal injection of new Sirt1 activators would slow degeneration of photoreceptors using an experimental mouse model of autosomal recessive retinitis pigmentosa (RP).[Methods]: A library of resveratrol derivatives were newly synthesized in order to improve the solubility and bioavailability of this lead compound. The new optimized Sirt1 activators were tested for preclinical drug development. Rd10 mice (P13) were subretinal injected with 1 µL of vehicle, resveratrol or several new Sirt1 activators. Electroretinogram (ERG), fundus, and histological evaluation were performed 15 days after injections. Amplitude of a- and b-waves were quantified in ERG recordings, and the statistical analyses were calculated by one-way ANOVA and post hoc DMS. A p value < 0.05 was considered significant.[Results]: Fundus evaluation showed less amount of pigment patches in both JC19 and JC21 treated mouse retinas when compared with non-treated and vehicle treated rd10 mice (controls). In addition, the number of photoreceptors nuclei in the ONL and the immunostaining of rhodopsin were preserved in JC19 and JC21 treated mice. b-Wave amplitude in dark-adapted ERG (flash intensities of 0.2, 1, 3 and 10 cd x s/m2) significantly increased in JC21 treated mice when compared with controls. b-Wave amplitude also increased in resveratrol and JC19 treated groups but in a lesser extent. The amplitude of b-wave in dark-adapted ERG-10 was: non-treated=68.4 ± 6.7 µV, vehicle=79.3 ± 10.3 µV, resveratrol= 103.8 ± 12.1 µV (p < 0.05), JC19=110.3 ± 12.5 µV (p < 0.05), JC21= 129.8 ± 27.7 µV (p < 0.01). a-Wave amplitude also showed statistically significant differences in Sirt1 activators treated mice.[Conclusions]: Our results are consistent with our hypothesis that Sirt1 activators induce neuroprotection of photoreceptors in a mouse model of RP. Other retinal diseases could be potentially treated with these drugs.Peer reviewe

Sirt1 activators induced neuroprotection of photoreceptors in rd10 mice

Póster presentado al Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), celebrado en Seattle, Washington (US) del 1 al 5 de mayo de 2016.It has been report ed that Sirt1 nuclear expression decreases in the outer nuclear layer (ONL) of retinal degeneration 10 (rd10 ) mice at postnatal day 15 (P15), and this abnormal pattern is correlated with the beginning of photoreceptor degeneration. Sirt1 is a class III histone deacetylase, and over-­expression of these p roteins have shown neuroprotective effect in several experimental models of neurodegenerative diseases. However, it is still unknown whether Sirt1 modulation could induce neuroprotection of ph otoreceptors. We tested the hypothesis that subretinal injection of new Sirt1 activators would slow degeneration of photoreceptors using an experimental mouse model of autosomal recessive retinitis pigmentosa (RP).Support: ISCIII grants (Miguel Servet-­I, CP15/00071) and co-­funded by the European Regional Development Fund (ERDF). Andalusia Regional Government (FQM-­7316).Peer Reviewe

Subretinal Transplant of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium on Nanostructured Fibrin-Agarose

Damage to the retinal pigment epithelium (RPE) in age-related macular degeneration and other diseases results in photoreceptor cell death and blindness. Replacement of RPE is therefore being explored as a therapy for several retinal diseases. To move toward a future personalized autologous transplant approach, we have prepared a biocompatible implant using RPE derived from induced pluripotent stem cells (iPSCs) reprogrammed from a healthy donor's monocytes. The correct positioning of the polarized RPE is essential to fulfill its role and protect photoreceptors from degeneration. Hence, we have used a biocompatible hydrogel matrix of fibrin and agarose that allows the surgical placement of an RPE sheet in the subretinal space, keeping its functional orientation. Our aim was to demonstrate safety and viability of the transplant in preclinical models. Pigs were used to test the feasibility of a regular vitreoretinal surgery. Our results show that this implant is suitable for subretinal transplantation allowing human RPE cells to survive and maintain their phenotype and orientation without any local or systemic adverse events. The ability to transplant the iPSC-derived RPE sheet in its natural orientation will surely increase the chance to obtain a therapeutic effect in future translational studies. In the promising field of cellular therapy for retinal degenerative diseases, a new biomaterial is proposed as a scaffold to grow and surgically introduce a monolayer of retinal pigment epithelial cells into the subretinal space, keeping the orientation of the cells for a proper functional integration of the transplant. The use of induced pluripotent stem cells as the starting material for retinal pigment epithelial cells is intended to advance toward a personalized medicine approach.Study supported by Consejería de Salud, Junta de Andalucía and by ISCIII (Miguel Servet-I, 2015) cofinanced by European Regional Development Fund (ERDF) (CP/00071)