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Y. Arsenijevic, L. Michault, W. Gehring, B. Angénieux; Bmi1 and Pou5f1 Are Necessary for Adult Human Retinal Stem Cell Renewal . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5752.
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© ARVO (1962-2015); The Authors (2016-present)
We previously isolated and characterized adult human retinal stem cells (hRSCs, Coles et al, 2004), and presented a new protocol to expand them. By analyzing the transcriptom during expansion and differentiation, we aimed to identify new genes controlling hRSC renewal.
We isolated retinal stem cells able to proliferate extensively and analyzed their gene expression pattern by Microarray. Clonogenic expanded hRSCs gave rise to more than 100 billion cells in 120 days. To further characterize their stemness potential, we performed an expression analysis using the Affymetrix® U133 Plus 2.0 microarray. The function of candidate genes was investigated using siRNA on 3 different cultures.
Using a subtractive approach, microarray analysis revealed that, during expansion, hRSCs expressed 1386 specific transcripts and 2122 after differentiation. Proliferating hRSCs express transcripts common to other stem cells, such as nucleostemin, nestin, ABCG2, c–kit and its ligand, as well as cyclin D3 which is downstream from c–kit. Bmi1 is present during both proliferation and differentiation. Interestingly, Pou5f1 is 6 times more expressed during proliferation. We confirmed Pou5f1 expression by RT–PCR and Western blot. During proliferation, we transfected the hRSCs with Pou5f1 siRNA and observed cell growth arrest, whereas GFP siRNA–treated cells (controls) continued to expand. We investigated also the function of Bmi1 using a lentiviral vector coding for Bmi1 siRNA containing also a CMV–GFP transgene to follow the targeted cells. We infected proliferating hRSCs and observed, by immunocytochemistry, the inhibition of Bmi1 synthesis leading to the disappearance of the infected cell population through passages. Control cells, infected with a CMV–GFP vector, maintained all the time the same percentage of GFP–positive cells.
We showed that the hRSCs expand easily, share common characteristics with other stem cell populations, and that both Pou5f1 as well as Bmi1 are necessary for adult hRSC proliferation. This demonstrates that hRSCs and microarray analysis can serve to identify crucial genes regulating the stemness of hRSCs.
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