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. 2013 Sep 4;23(2):342–354. doi: 10.1093/hmg/ddt425

Figure 1.

Figure 1.

Differentiation of iPSCs into NSCs and selection of the ALDHhiSSCloVLA4+ fraction. (A) Scheme of the differentiation protocol of NSCs from iPSCs. Initially, the iPSCs are positive for OCT4. After detaching from the plate, the iPSCs form aggregates that are cultured for 7 days in neural medium. The differentiated cells show features of neuroepithelial (NE) rosettes. The primitive NE cells were positive for PAX6. These cells were then selected by FACS for their ALDHhiSSClo and VLA4 positivity and expanded in vitro. The cells were cultured with RA and SHH and differentiated into OLIG2-expressing motor neurons. They could be further matured into their neuronal phenotype in the presence of neurotrophic factors and reduced concentrations of SHH and RA. (B) Representative flow cytometric analysis of ALDH activity and VLA4+ positivity on iPSC-derived NSCs. NSCs were selected according to forward and side scatter properties. Cells incubated with Aldefluor substrate and the specific inhibitor of ALDH (DEAB) were used to establish the baseline fluorescence and to define the ALDHhi region. After sorting, this cell population is enriched as shown in the right panel. The ALDHhiSSClo fraction was further selected by FACS for VLA4 expression (left panel) and seeded in culture. Scale bar: 70 µm. (C) iPSC cells positive for VLA4 (red; nuclei stained with DAPI, blue signal). (D) Sorted ALDHhiSSCloVLA4+ cells grown at clonal density in NEP medium form epithelial-like clones positive for NSC markers such as SOX1, SOX2 and nestin. Scale bar: 100 µm. After in vitro differentiation, ALDHhiSSClo cells could give rise to cells of the three neuroectodermal lineages including GFAP-positive astrocytes (red), O4-positive oligodendrocytes (red) and motor neurons (ChAT red, TuJ1, green). Nuclei are counterstained with DAPI (blue signal). Scale bar: GFAP, 100 µm; O4, 50 µm; and TuJ1 and ChAT, 100 µm. (e) Quantification of cells positive for NSC markers. (F) Quantification of cells showing positive staining for astrocyte and oligodendrocyte markers after appropriate differentiation. (G) Quantification of cells that are double-positive for motor neuron markers TuJ1 and ChAT.