Purchase this article with an account.
Takahiro Suzuki, Yasuyuki Suzuki; Establishment of lysosomal disease sialidosis patient-derived iPS cells and characterization of induced retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):117.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Sialidosis is an autosomal recessive hereditary disorder caused by the primary defect of lysosomal enzyme, neuraminidase 1 (neu1). Sialidosis patients develop symptoms of myoclonus, cherry red spot, a result of lysosomal storage, and progressive visual loss. We have previously reported that inhibition of neu1 in retinal pigment epithelium (RPE)-derived cell line ARPE-19 resulted in the imparied autophagy flux. In order to investigate the disease, we have established sialidosis patient-derived iPS cells, from which RPE cells were induced and examined.
Mononuclear cells collected from peripheral blood samples were cultured with anti-CD3/CD28 coated beads in the presence of rIL-2. After several days culture, reprogramming 4 factors were transfected via SeVdp vector (M.Nakanishi, JBC 2011). For induction of RPE cells, iPS cells were cultured in nonadherent plate for 21 days in the absence of bFGF and then transferred to laminin-coated plate with 10ng/ml bFGF and 0.5 mM SB15432 for 40 to 60 days. RPE-like cell sheet was isolated and cultured. Autophagy flux was examined by the culture with/without chloroquine under amino acid starved or normal condition. Cell lysates were prepared and applied to Western blotting with anti-LC3 and anti-p62 antibodies.
Frequency of iPS cell establishment from sialidosis patient was quite low compared to that from healthy donor controls. Similarly, during the inducing culture into RPE cells, patient-derived cells showed vulnerable feature although induced RPE cells stably proliferated. Patient-derived RPE cells showed impaired autophagy flux.
Impaired autophagy was observed in sialidosis patient derived RPE, suggesting that autophagy is implicated in the pathology of cherry red spot.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
This PDF is available to Subscribers Only