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YUQIANG HUANG, Chongbo Chen, LINGPING CEN, Jianhuan Chen, Mingzhi Zhang; Differentiation potential of adipose-derived stem cells into retinal lineage cells.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3595.
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© ARVO (1962-2015); The Authors (2016-present)
Human embryonic stem cells had been directed to retinal progenitor identity with Inhibition of bone morphogenic protein (BMP) and Wnt signaling. The objectives of this study are to investigate the differentiation potential of adipose-derived stem cells (ASCs) into retinal lineage cells by chemically inhibiting BMP and Wnt signaling , a protocol to promote anterior neural plate development and retinogenesis, as well as to study the temporal expression patterns of genes known to participate in differentiation regulation.
ASCs at passage 5 were recruited for a two-step induction procedure using media containing Noggin (antagonist of BMP signaling ) and Dkk1 (antagonist of Wnt/β-catenin signaling). Step 1 was to induce ASCs into neurospheres on ultra-low attachment culture plates for 3 days using induction medium 1 (IM1), which containing DMEM/F12, 2% B27, 1 ng/mL Noggin and 1 ng/ml Dkk-1. Step 2 was to plate neurospheres on matrigel-coated culture plates and to induce them to differentiate for up to 21 days using Induction medium 2 (IM2), which containing DMEM/F12, 2% B27, 1% N2, 10 ng/ml Noggin and 10ng/ml Dkk-1. Gene expression of neurospheres was revealed by Immunocytochemistry and RT-PCR. Cells at various time points of induction were collected and expression of markers labeling retinal progenitors and photoreceptors was revealed by RT-PCR and real-time PCR.
ASCs derived neurospheres formation could be observed as early as 24 h after IM1 induction and expressed nestin and p75/NGFR, markers for neural progenitors, as well as Nrl, markers for photoreceptor precursor. Neurospheres started to attach down the matrigel-coated culture plates 2h after transferring and exhibited rosette-like outgrowth. The treated cells showed up-regulated expression of Rhodopsin, OPN1SW and RCVRN, markers for photoreceptor, 3 days after induction by real-time PCR. None of the markers for retinal progenitor cells, such as Pax6, Chx10, Lhx2 and Rx were observed in the process of induction.
Our results suggest ASCs may have the potential to differentiate into photoreceptor-like cells under Noggin and Dkk1 induction conditions. Further examination needs to confirm the function of these ASCs derived differentiation cells and whether this induction process may bypasses the differentiation of retinal progenitors.
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