RESUMO
Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Epitélio Pigmentado da Retina/citologia , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes/citologia , Reação em Cadeia da PolimeraseRESUMO
Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.
Assuntos
Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Engenharia Genética/métodos , Células Ganglionares da Retina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Expressão Gênica , Humanos , Imuno-Histoquímica , Potenciais da Membrana/genética , Camundongos , Microscopia de Fluorescência , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígenos Thy-1/metabolismo , Fatores de Tempo , Fator de Transcrição Brn-3B/genética , Fator de Transcrição Brn-3B/metabolismoRESUMO
The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.