Dataset Information

Early retinal differentiation of human pluripotent stem cells in microwell suspension cultures.

ABSTRACT:

Objective

To develop a microwell suspension platform for the adaption of attached stem cell differentiation protocols into mixed suspension culture.

Results

We adapted an adherent protocol for the retinal differentiation of human induced pluripotent stem cells (hiPSCs) using a two-step protocol. Establishing the optimum embryoid body (EB) starting size and shaking speed resulted in the translation of the original adherent process into suspension culture. Embryoid bodies expanded in size as the culture progressed resulting in the expression of characteristic markers of early (Rx, Six and Otx2) and late (Crx, Nrl and Rhodopsin) retinal differentiation. The new process also eliminated the use of matrigel, an animal-derived extracellular matrix coating.

Conclusions

Shaking microwells offer a fast and cost-effective method for proof-of-concept studies to establish whether pluripotent stem cell differentiation processes can be translated into mixed suspension culture.

SUBMITTER: Sharma VS

PROVIDER: S-EPMC5247545 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Publications

Early retinal differentiation of human pluripotent stem cells in microwell suspension cultures.

Sharma Vishal S VS Khalife Rana R Tostoes Rui R Leung Leonard L Kinsella Rose R Ruban Ludmilla L Veraitch Farlan S FS

Biotechnology letters 20161103 2

<h4>Objective</h4>To develop a microwell suspension platform for the adaption of attached stem cell differentiation protocols into mixed suspension culture.<h4>Results</h4>We adapted an adherent protocol for the retinal differentiation of human induced pluripotent stem cells (hiPSCs) using a two-step protocol. Establishing the optimum embryoid body (EB) starting size and shaking speed resulted in the translation of the original adherent process into suspension culture. Embryoid bodies expanded i ...[more]

PMID: 27812821

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Project description:UnlabelledThe derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications.SignificanceIn the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hiPSCs), followed by the differentiation of these cells toward a retinal lineage, including photoreceptors, retinal ganglion cells, and retinal pigment epithelium, has been demonstrated. The use of mRNA reprogramming to yield pluripotency represents a unique ability to derive pluripotent stem cells without the use of DNA vectors, ensuring the lack of genomic integration and constitutive expression. The studies reported in the present article serve to establish a more reproducible system with which to derive retinal cell types from hiPSCs through the prevention of genomic integration of delivered genes and should also eliminate the risk of constitutive expression of these genes. Such ability has important implications for the study of, and development of potential treatments for, retinal degenerative disorders and the development of novel therapeutic approaches to the treatment of these diseases.

Dataset Information

Early retinal differentiation of human pluripotent stem cells in microwell suspension cultures.

Objective

Results

Conclusions

Publications

Early retinal differentiation of human pluripotent stem cells in microwell suspension cultures.

Similar Datasets

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