Generation and Characterization of iPSCs

The maintenance and passaging of iPSCs were performed using a TECAN liquid handling platform as described^{41 (link)}. Briefly, iPSCs were generated by the nucleofection (Lonza) of episomal vectors expressing OCT-4, SOX2, KLF4, L-MYC, LIN28, and shRNA against p53^{122 (link)} in feeder- and serum-free conditions using TeSR™-E7™medium (Stem Cell Technologies) as described^{41 (link)}. Pluripotent cells were selected using anti-human TRA-1-60 Microbeads (Miltenyi)^{43 (link)} and subsequently maintained onto vitronectin XF™-coated plates (Stem Cell Technologies) in StemFlex™ (Thermo Fisher Scientific), with media changes every second day and weekly passaging using ReLeSR™ (Stem Cell Technologies). Pluripotency was assessed by expression of the markers OCT3/4 (sc-5279, dilution 1/40, Santa Cruz Biotechnology) and TRA-1-60 (MA1-023-PE, dilution 1/100, Thermo Fisher Scientific) by immunocytochemistry, and virtual karyotyping by CNV array on all lines, as described in ref. ^{41 (link)}. Only geographic atrophy lines were generated for this study, as all control lines were already generated, and characterized in ref. ^{44 (link)}.

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Senabouth A., Daniszewski M., Lidgerwood G.E., Liang H.H., Hernández D., Mirzaei M., Keenan S.N., Zhang R., Han X., Neavin D., Rooney L., Lopez Sanchez M.I., Gulluyan L., Paulo J.A., Clarke L., Kearns L.S., Gnanasambandapillai V., Chan C.L., Nguyen U., Steinmann A.M., McCloy R.A., Farbehi N., Gupta V.K., Mackey D.A., Bylsma G., Verma N., MacGregor S., Watt M.J., Guymer R.H., Powell J.E., Hewitt A.W, & Pébay A. (2022). Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration. Nature Communications, 13, 4233.

Publication 2022

nucleofection TeSR™-E7™medium vitronectin XF™-coated plates StemFlex™ ReLeSR™ sc-5279 MA1-023-PE

Cells Dilution Episomal Geographic atrophy Human Immunocytochemistry Ipscs Klf4 Microbeads Oct3 Serum Shrna Sox2 Stem cell Vectors Vitronectin

Corresponding Organization : Royal Melbourne Hospital

Other organizations : Macquarie University, QIMR Berghofer Medical Research Institute, Harvard University, University of Western Australia, Lions Eye Institute

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Variable analysis

independent variables

Nucleofection of episomal vectors expressing OCT-4, SOX2, KLF4, L-MYC, LIN28, and shRNA against p53

dependent variables

Pluripotency, assessed by expression of the markers OCT3/4 and TRA-1-60 by immunocytochemistry
Virtual karyotyping by CNV array

control variables

Feeder- and serum-free conditions using TeSR™-E7™medium
Pluripotent cells selected using anti-human TRA-1-60 Microbeads
Cells maintained onto vitronectin XF™-coated plates in StemFlex™ medium

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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