From RNA-derived iPS Cells to Retinal Cells
Human induced pluripotent stem (iPS) cells and cells differentiated from iPS cells have widely been used for in vivo models human disease progression. Jason Meyer, of Indiana University Purdue University Indianapolis, uses iPS cell-derived models to study retinogenesis and retinal disease. Two recent papers from his lab highlight the benefits of using Stemgent’s RNA reprogramming technology to enable robust differentiation of iPS cells to the retinal lineage (1, 2). RNA reprogramming technology was chosen in order for these studies to ensure that no vestiges of the reprogramming vectors were retained by the cells or integrated into the genome.
In the first study, published in Stem Cells Translational Medicine, mRNA and retrovirus methods were compared side-by-side for their reprogramming effectiveness and ability to differentiate into cells from the retinal lineage. Both methods generated morphologically distinct colonies which were shown to express the pluripotency marker TRA-1-60 using Stemgent’s LiveStain TRA-1-60 antibody (Cat. Nr. 09-0068). iPS cell lines generated using both methods expressed surface and nuclear markers for pluripotency (Figure 1), and the cells spontaneously differentiated into embryoid bodies that expressed cells from all three germ layers, indicating no difference in pluripotency between iPS cells generated using mRNA and retrovirus.
These iPS cells were differentiated to retinal ganglion cells over about 10 weeks using a differentiation program that closely mimics the in vivo development of the eye. Embryoid bodies were directed toward a neural fate using supplement and heparin, upon which the cells exhibited an eye field fate. The cells were then differentiated toward an optic vesicle fate. After manually isolating the optic vesicle-like structures, the cells were leading to a retinal lineage and ultimately to retinal pigment epithelial (RPE) cells and other cell types (Figure 2).
In the second study, published online in Stem Cells, iPS cells were generated from fibroblasts from healthy volunteers and from a familial glaucoma patient with a mutation in the OPTN gene (which codes for optineurin). iPS cells were generated with Stemgent’s RNA Reprogramming Kit and differentiated to retinal ganglion cells (RGCs) over 10 weeks. The RGCs were characterized by immunocytochemistry (Figure 3) and electrophysiologically. These RGCs exhibited a hyperpolarized resting potential, the ability to fire action potentials, and sodium and potassium channels that could be inhibited by neurotoxins. All of these activities indicate the formation of physiologically active RGCs from mRNA-derived human iPS cells. With the patient-derived RGCs, but not in healthy controls, significant apoptosis was observed, and this apoptosis could be blocked using neuroprotective agents such as BDNF or PEDF.
Together, these studies show that RNA Reprogramming using Stemgent’s RNA Reprogramming technology yields high-quality iPS cells that can be used to develop physiologically relevant models for eye disease. These studies are the most comprehensive analysis to date of the derivation of functional retinal cells using iPS cells, and demonstrate the utility of human iPS cells from retinal disease modeling in general.
Which products did the researchers use?
|RNA Reprogramming Kit||1 Kit||00-0071|
|microRNA Booster Kit||1 Kit||00-0073|
|StemRNA™-SR Reprogramming Kit||1 Kit||00-0075|
|StainAlive™ TRA-1-60 Antibody (DyLight™ 488), Mouse anti-Human||100 µL||09-0068|
|Oct4 Antibody, Rabbit anti-Mouse/Human||100 µL||09-0023|
|Pluriton™ Reprogramming Medium||500 mLt||00-0070|
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1. Sridhar A; Ohlemacher SK; Langer KB; Meyer JS. “Robust differentiation of mRNA-reprogrammed human induced pluripotent stem cells toward a retinal lineage.” Stem Cell Translational Medicine 5:417 (2016)
2. Ohlemacher SK; Sridhar A; Xiao Y; Hochstetler AE; Sarfarazi M; Cummins TR; Meyer JS. “Stepwise differentiation of retinal ganglion cells from human pluripotent stem cells enables analysis of glaucomatous neurodegeneration.” Stem Cells doi: 10.1002/stem.2356 (2016)