Purchase this article with an account.
Jason S Meyer, Akshayalakshmi Sridhar, Clara Iglesias, Sarah Ohlemacher; Comparison of viral and mRNA-reprogrammed human induced pluripotent stem cells for retinal differentiation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1374.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The 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 risk of genomic integration or constitutive transgene expression due to viral delivery. The delivery of reprogramming factors by mRNA eliminates these risks and may provide a safer alternative, but the efficient retinal differentiation of such cells has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs for retinal differentiation.
Human fibroblasts were transfected daily with mRNAs encoding for reprogramming genes, whereas similar cultures of human fibroblasts were infected with retroviruses to deliver these genes in parallel. Pluripotency was confirmed by live cell staining for cell surface antigens and positive colonies were identified and manually isolated, yielding stable lines of hiPSCs. New lines of hiPSCs were differentiated to a retinal fate following established protocols, and the efficiency of retinal specification from hiPSCs was compared between the two systems at various stages of differentiation.
Both mRNA and retroviral methods of reprogramming yielded stable lines of hiPSCs expressing numerous pluripotency-related characteristics as assessed by immunocytochemistry and RT-PCR. Upon differentiation, no overt differences were found in the ability of these cells to adopt a retinal fate as assessed by the expression of retinal progenitor-associated genes. Within two months of differentiation, many types of retinal cells could be derived from both sources, including retinal pigment epithelium, photoreceptors, and retinal ganglion cells.
The data presented demonstrates the feasibility of utilizing mRNA-based reprogramming strategies to derive lines of patient-specific hiPSCs for purposes of retinal differentiation. Differences in the differentiation capacity of these cells from both sources were not readily observed. Given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages because they lack the risks of genomic integration or constitutive expression, such methods likely represent a promising new approach for retinal stem cell research, particularly those for translational purposes.
This PDF is available to Subscribers Only