Additional file 4: of A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors

Morphology of cell populations during the process of derivation of CoMo-NSCs from pluripotent hESCs. A—Representative image of hESC colony on mouse embryonic feeder layer. B—Manually dissociated hESCs into smaller clumps and induced to form embryoid bodies (EBs) in non-adherent cell culture conditions. C—Morphology of first neural rosettes observed at days 4–12 after plating of EBs. D—Manually separated neural rosettes, dissociated into smaller pieces and transferred to new poly-l-ornithine/laminin-coated cell culture dishes. Upon adhesion, dissected clumps of rosettes began to generate new groups of rosettes (termed “R1”). E, F—Newly enriched population of neural rosettes, both fully reformed (E) and partially reformed (F), with a very small number of contaminating cells termed as “R2”. G—Independent “clone-like populations” of NSCs visible outside of rosettes-like structures. H, I—Manually isolated single “clone-like population” of NSCs and re-plated into 24 wells plate. J, K, L—Established self-renewing population of clonal morphology NSCs, further referred to as CoMo-NSCs at low density (J), high density (K) and high magnification (L). (scale bars: A 250 μm; B, C 500 μm; D–G 250 μm; H, I 150 μm; J, K 250 μm; L 100 μm). (JPG 2540 kb)