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Purpose: Transplanting photoreceptors from human pluripotent stem cell-derived retinal organoids have the potential to reverse vision loss in affected individuals. However, transplantable photoreceptors are only a subset of all cells in the organoids. Hence, the goal of our current study was to accelerate and synchronize photoreceptor differentiation in retinal organoids by inhibiting the Notch signaling pathway at different developmental time-points using a small molecule, PF-03084014 (PF). Methods: Human induced pluripotent stem cell- and human embryonic stem cells-derived retinal organoids were treated with 10 µM PF for 3 days starting at day 45 (D45), D60, D90, and D120 of differentiation. Organoids were collected at post-treatment days 14, 28, and 42 and analyzed for progenitor and photoreceptor markers and Notch pathway inhibition by immunohistochemistry (IHC), quantitative PCR, and bulk RNA sequencing (n = 3-5 organoids from three independent experiments). Results: Retinal organoids collected after treatment showed a decrease in progenitor markers (KI67, VSX2, PAX6, and LHX2) and an increase in differentiated pan-photoreceptor markers (OTX2, CRX, and RCVRN) at all organoid stages except D120. PF-treated organoids at D45 and D60 exhibited an increase in cone photoreceptor markers (RXRG and ARR3). PF treatment at D90 revealed an increase in cone and rod photoreceptors markers (ARR3, NRL, and NR2E3). Bulk RNA sequencing analysis mirrored the immunohistochemistry data and quantitative PCR confirmed Notch effector inhibition. Conclusions: Timing the Notch pathway inhibition in human retinal organoids to align with progenitor competency stages can yield an enriched population of early cone or rod photoreceptors.
Assuntos
Células-Tronco Pluripotentes Induzidas , Organoides , Diferenciação Celular/fisiologia , Humanos , Antígeno Ki-67/metabolismo , Proteínas com Homeodomínio LIM , Organoides/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismoRESUMO
Cases of Leber congenital amaurosis caused by mutations in CRX (LCA7) exhibit an early form of the disease and show signs of significant photoreceptor dysfunction and eventual loss. To establish a translational in vitro model system to study gene-editing-based therapies, we generated LCA7 retinal organoids harboring a dominant disease-causing mutation in CRX. Our LCA7 retinal organoids develop signs of immature and dysfunctional photoreceptor cells, providing us with a reliable in vitro model to recapitulate LCA7. Furthermore, we performed a proof-of-concept study in which we utilize allele-specific CRISPR/Cas9-based gene editing to knock out mutant CRX and saw moderate rescue of photoreceptor phenotypes in our organoids. This work provides early evidence for an effective approach to treat LCA7, which can be applied more broadly to other dominant genetic diseases.
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Edição de Genes/métodos , Predisposição Genética para Doença/genética , Proteínas de Homeodomínio/genética , Amaurose Congênita de Leber/genética , Mutação , Transativadores/genética , Alelos , Sequência de Bases , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Genes Dominantes , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Organoides/citologia , Organoides/metabolismo , Organoides/ultraestrutura , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA-Seq/métodos , Retina/metabolismo , Transativadores/metabolismoRESUMO
Ageing is a major risk factor for vision loss, and inflammation is an important contributor to retinal disease in the elderly. Regenerative medicine based on cell replacement strategies has emerged in recent years as a promising approach to restore vision. However, how the ageing process affects retinal homeostasis and inflammation in the retina and how this may impose a limitation to the success of such interventions remains unknown. Here we report that, in mice and humans, retinal ageing is associated with a reduction in MANF protein levels, specifically in the choroid, where increased densities of activated macrophages can be detected. We further show that the retina of old wild type mice, in the absence of any other genetic alteration, has limited homeostatic capacity after damage imposed by light exposure and reduced engraftment efficiency of exogenously supplied photoreceptors. Finally, we show that supplementation of MANF recombinant protein can improve retinal homeostasis and repair capacity in both settings, correlating with reduced numbers of activated macrophages in the old retina. Our work identifies age-related alterations in retinal homeostasis, independent of genetic alterations, leading to age-related retinal inflammation and damage susceptibility. We suggest that MANF therapy is a potential intervention to maintain retinal homeostasis in the elderly and improve the success of retinal regenerative therapies applied to aged individuals.
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Uncovering the gene regulatory networks that control cone photoreceptor formation has been hindered because cones only make up a few percent of the retina and form asynchronously during development. To overcome these limitations, we used a γ-secretase inhibitor, DAPT, to disrupt Notch signaling and force proliferating retinal progenitor cells to rapidly adopt neuronal identity. We treated mouse retinal explants at the peak of cone genesis with DAPT and examined tissues at several time-points by histology and bulk RNA-sequencing. We found that this treatment caused supernumerary cone formation in an overwhelmingly synchronized fashion. This analysis revealed several categorical patterns of gene expression changes over time relative to DMSO treated control explants. These were placed in the temporal context of the activation of Otx2, a transcription factor that is expressed at the onset of photoreceptor development and that is required for both rod and cone formation. One group of interest had genes, such as Mybl1, Ascl1, Neurog2, and Olig2, that became upregulated by DAPT treatment before Otx2. Two other groups showed upregulated gene expression shortly after Otx2, either transiently or permanently. This included genes such as Mybl1, Meis2, and Podxl. Our data provide a developmental timeline of the gene expression events that underlie the initial steps of cone genesis and maturation. Applying this strategy to human retinal organoid cultures was also sufficient to induce a massive increase in cone genesis. Taken together, our results provide a temporal framework that can be used to elucidate the gene regulatory logic controlling cone photoreceptor development.
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Diferenciação Celular/genética , Perfilação da Expressão Gênica , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Camundongos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
BACKGROUND AIMS: We have previously reported the generation of a current Good Manufacture Practice (cGMP)-compliant induced pluripotent stem cell (iPSC) line for clinical applications. Here we show that multiple cellular products currently being considered for therapy can be generated from a single master cell bank of this or any other clinically compliant iPSC line METHODS: Using a stock at passage 20 prepared from the cGMP-compliant working cell bank (WCB), we tested differentiation into therapeutically relevant cell types of the three germ layers using standardized but generic protocols. Cells that we generated include (i) neural stem cells, dopaminergic neurons and astrocytes; (ii) retinal cells (retinal pigment epithelium and photoreceptors); and (iii) hepatocyte, endothelial and mesenchymal cells. To confirm that these generic protocols can also be used for other iPSC lines, we tested the reproducibility of our methodology with a second clinically compliant line RESULTS: Our results confirmed that well-characterized iPSC lines have broad potency, and, despite allelic variability, the same protocols could be used with minimal modifications with multiple qualified lines. In addition, we introduced a constitutively expressed GFP cassette in Chr13 safe harbor site using a standardized previously described method and observed no significant difference in growth and differentiation between the engineered line and the control line indicating that engineered products can be made using a standardized methodology CONCLUSIONS: We believe that our demonstration that multiple products can be made from the same WCB and that the same protocols can be used with multiple lines offers a path to a cost-effective strategy for developing cellular products from iPSC lines.
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Engenharia Celular/métodos , Engenharia Celular/normas , Linhagem da Célula , Fidelidade a Diretrizes , Células-Tronco Pluripotentes Induzidas/citologia , Astrócitos/citologia , Astrócitos/fisiologia , Diferenciação Celular , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/fisiologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Fidelidade a Diretrizes/normas , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Mesoderma/citologia , Mesoderma/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Guias de Prática Clínica como Assunto/normas , Padrões de Referência , Reprodutibilidade dos Testes , Retina/citologia , Bancos de Tecidos/normasRESUMO
Retinal degeneration often results in the loss of light-sensing photoreceptors, which leads to permanent vision loss. Generating transplantable retinal photoreceptors using human somatic cell-derived induced pluripotent stem cells (iPSCs) holds promise to treat a variety of retinal degenerative diseases by replacing the damaged or dysfunctional native photoreceptors with healthy and functional ones. Establishment of effective methods to produce retinal cells including photoreceptors in chemically defined conditions using current Good Manufacturing Practice (cGMP)-manufactured human iPSC lines is critical for advancing cell replacement therapy to the clinic. In this study, we used a human iPSC line (NCL-1) derived under cGMP-compliant conditions from CD34+ cord blood cells. The cells were differentiated into retinal cells using a small molecule-based retinal induction protocol. We show that retinal cells including photoreceptors, retinal pigmented epithelial cells and optic cup-like retinal organoids can be generated from the NCL-1 iPSC line. Additionally, we show that following subretinal transplantation into immunodeficient host mouse eyes, retinal cells successfully integrated into the photoreceptor layer and developed into mature photoreceptors. This study provides strong evidence that transplantable photoreceptors can be generated from a cGMP-manufactured human iPSC line for clinical applications. Stem Cells Translational Medicine 2018;7:210-219.
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Células-Tronco Pluripotentes Induzidas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Camundongos , Organoides/metabolismo , Degeneração Retiniana/metabolismoRESUMO
Regenerative therapies are limited by unfavorable environments in aging and diseased tissues. A promising strategy to improve success is to balance inflammatory and anti-inflammatory signals and enhance endogenous tissue repair mechanisms. Here, we identified a conserved immune modulatory mechanism that governs the interaction between damaged retinal cells and immune cells to promote tissue repair. In damaged retina of flies and mice, platelet-derived growth factor (PDGF)-like signaling induced mesencephalic astrocyte-derived neurotrophic factor (MANF) in innate immune cells. MANF promoted alternative activation of innate immune cells, enhanced neuroprotection and tissue repair, and improved the success of photoreceptor replacement therapies. Thus, immune modulation is required during tissue repair and regeneration. This approach may improve the efficacy of stem-cell-based regenerative therapies.
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Imunomodulação , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/farmacologia , Fármacos Neuroprotetores/farmacologia , Retina/fisiologia , Cicatrização/imunologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/imunologia , Proteínas do Ovo/metabolismo , Evolução Molecular , Perfilação da Expressão Gênica , Hemócitos/imunologia , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fatores de Crescimento Neural/genética , Fármacos Neuroprotetores/imunologia , Células Fotorreceptoras de Invertebrados/efeitos dos fármacos , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Retina/efeitos dos fármacos , Retina/lesões , Degeneração Retiniana/terapia , Transdução de Sinais , Cicatrização/efeitos dos fármacosRESUMO
The mammalian target of rapamycin (mTOR) pathway regulates stem cell regeneration and differentiation in response to growth factors, nutrients, cellular energetics, and various extrinsic stressors. Inhibition of mTOR activity has been shown to enhance the regenerative potential of pluripotent stem cells. DEPTOR is the only known endogenous inhibitor of all known cellular mTOR functions. We show that DEPTOR plays a key role in maintaining stem cell pluripotency by limiting mTOR activity in undifferentiated embryonic stem cells (ESCs). DEPTOR levels dramatically decrease with differentiation of mouse ESCs, and knockdown of DEPTOR is sufficient to promote ESC differentiation. A strong decrease in DEPTOR expression is also observed during human ESCs differentiation. Furthermore, reduction in DEPTOR level during differentiation is accompanied by a corresponding increase in mTOR complex 1 activity in mouse ESCs. Our data provide evidence that DEPTOR is a novel stemness factor that promotes pluripotency and self-renewal in ESCs by inhibiting mTOR signaling.