Fibroblast growth factor 2 fgf2

L-ascorbic acid (LAA), ethylenediaminetetraacetic acid (EDTA), heparin, laminin from Engelbreth-Holm-Swarm murine sarcoma, Lowry assay kit (Peterson’s modification), poly-D-lysine hydrobromide, protease inhibitor cocktail (Cat#P8340), sodium selenite, and Terg-A-Zyme detergent were purchased from Millipore Sigma (Oakville, ON, Canada). Transforming growth factor-β1 (TGF-β1; cat #100–21) and fibroblast growth factor 2 (FGF2; cat#100-18B) were purchased from Peprotech (Cranbury, NJ, United States) was purchased from Peprotech (Cranbury, NJ, United States). Recombinant human transferrin (cat#777TRF029) was purchased from InVitria (Fort Collins, CO, United States). Radioimmunoprecipitation assay (RIPA) 10x buffer and the ROCK inhibitor Y-27632 (cat# 13624S) were purchased from Cell Signaling Technologies (Whitby, ON, Canada). BrainPhys™ Neuronal Medium, EasySep™ Release Human CD45 Positive Selection Kit, and the STEMdiff™ Cerebral Organoid Kit (cat#08570) were obtained from STEMCELL Technologies (Vancouver, BC, Canada). A full list of antibodies and their suppliers is provided in Table 2. All other reagents were sourced from Fisher Scientific (Ottawa, ON, Canada).

AD mice [B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J] (Jankowsky et al., 2001 (link)) were purchased from the Jackson Laboratory (Bar Harbor, ME), and ST-II-knockout (KO) mice were originally provided by the courtesy of Dr. Richard Proia (NIDDK, NIH, Bethesda, MD). Wild-type (WT) littermates of these mice were used for control. Mouse NSCs were prepared from embryonic brains in the form of neurospheres, which were floating clonal aggregates formed by NSCs in vitro (Nakatani et al., 2010 (link)). In brief, single-cell suspensions were prepared from the striata of embryonic day (E)-14.5 mouse brains. NSCs were cultured in Neurobasal A medium (Life Technologies, Carlsbad, CA) supplemented with B27 (Life Technologies) and 20 ng/ml of fibroblast growth factor 2 (FGF2; Peprotech, Rocky Hill, NJ) and 20 ng/ml of EGF (Peprotech). Neurospheres formed after 1 week were collected for further passages and analyses. The use of animals for this study was approved by the Institutional Animal Care and Use Committees at Georgia Regents University and the VA Medical Center, Augusta, GA.

Human umbilical cord matrix-derived stem cells (UCM-MSCs) were provided by the Asan Stem Cell Center (Asan Institute for Life Sciences, Seoul, Korea). The cells were obtained from the previously described protocols [23 (link)]. Both stem cell types (UCM-MSCs and AM-MSCs) were cultured on 0.1% gelatin-coated culture dishes with a culture medium. The culture medium was based on DMEM/F12 supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA), 10 ng/ml fibroblast growth factor 2 (FGF2; Peprotech, Rocky Hill, NJ, USA), 1% NEAA (Gibco), 1% Penicillin/Streptomycin (GeneDirex). Cells were passaged 1 to 3–5 every 3 to 4 days using trypsin/EDTA (Gibco, NY, USA).

The differentiation of iPSCs to CNs was performed following a previously established protocol 34. Briefly, the iPSCs were grown on Matrigel as monolayers, and neural differentiation was induced by changing the medium to basic neural medium DMEM/F12: Neurobasal 1:1, 1× N2, 1× B27, insulin 5 µg/mL (Sigma), nonessential amino acids 100 µM (Life Technologies), 2‐mercaptoethanol 100 µM, antibiotic‐antimycotic (Life Technologies) and supplementing it with 500 ng/mL Noggin (PeproTech, Rocky Hill, NJ, http://www.peprotech.com), and 10 µM SB431542 (Enzo Life Sciences). Medium was changed every 24 hours until neural rosettes could be observed (12–16 days). The neuroepithelial layer was lifted using Dispase (Life Technologies), and the medium was supplemented with 20 ng/mL Fibroblast growth factor 2 (FGF2) (PeproTech) for 4–6 days. The neural precursors were split using Accutase (Life Technologies) at a low density. The medium was then supplemented with 10 ng/mL BDNF and 10 ng/mL GDNF, while FGF2 was withdrawn. The neurons were allowed to extend processes and to mature for 60–70 days before functional assays.

Human iPSC were differentiated towards retinal organoids following an optimized protocol based on the one published by Reichman et al.²³ . Briefly,hiPSC-2 cell line was expanded to 80% confluence in Essential 8™ medium were switched in Essential 6™ medium (Thermo Fisher Scientific). After 3 days, cells were moved to the Proneural medium (Supplementary Table 2). The medium was changed every 2–3 days. After 4 weeks of differentiation, neural retina-like structures grew out of the cultures and were mechanically isolated. Pigmented parts, giving rise to RPE were carefully removed. The extended 3D culture in Maturation medium (Table S1) allowed the formation of retinal organoids. Addition of 10 ng/ml Fibroblast growth factor 2 (FGF2, Preprotech) at this point favoured the growth of retinal organoids and the commitment towards retinal neurons instead of RPE lineage^{53 (link)}. In order to promote the commitment of retinal progenitors towards photoreceptors, we specifically blocked Notch signalling for a week starting at day 42 of differentiation using the gamma secretase inhibitor DAPT (10 µM, Selleckchem)^{54 (link)}. Floating organoids were cultured in 6 well-plates (10 organoids per well) and medium was changed every 2 days. Supplementary Table 2 summarizes the formulations for the different media used.