Transplantation In vitro differentiation

Figure 1 Stepwise differentiation of mES cells Into multipotent neural precursors, with subsequent conversion into glial precursors (schematic). Mouse ES cells are proliferated to large numbers, on feeder layers and in the presence of LIF, in preparation for controlled neural differentiation (Protocol 1), as follows. Inducing aggregation by suspension culture in the absence of LIF initiates mES cell differentiation via EBs. The EBs are plated in defined medium promoting the survival of neural precursors. These cells are proliferated in the presence of FGF2; and sequential culture in defined media with FGF2 and EGF, followed by FGF2 and PDGF-AA, gradually converts the cells into glial precursors. Terminal differentiation is induced by growth factor withdrawal. By combining controlled differentiation with lineage selection, highly purified neurons and glia can be isolated from populations at individual stages (for details see Protocols 3 and 4). Letters A-K refer to corresponding phase-contrast micrographs shown in Figure 2.

Figure 2 (see Plate 4) Representative photomicrographs of differentiating mES cells at various stages of neural development. (A) Undifferentiated mES cells grown on a feeder monolayer of mitotically inactivated MEFs prior to differentiation according to Protocol 1. (B) EBs generated by aggregation of undifferentiated mES cells. (C) Selection of neural precursors after plating EBs in ITSFn medium. (D) Proliferation of multipotent neural precursors (i.e. ESNPs) in FGF2-supplemented medium. After withdrawal of FGF2, cells differentiate into post-mitotic neurons and glia: (E) Phase-contrast micrograph of cells 4d following FGF2 withdrawal; the corresponding immunofluorescence micrograph (F) depicts p-III-tubulin in mES cell-derived neurons (red). Further proliferation of ESNPs with FGF2 and EGF (G, H), and subsequently with FGF2 and PDGF-AA (I, J), enables the generation of highly purified glial precursors (ESGPs). (K, L) After growth factor withdrawal, ESGPs differentiate into GFAP-positive astrocytes (green) and 04-positive oligodendrocytes (red). (G-L) Phase-contrast micrographs (G, I, K) and corresponding immunfluorescence analyses with antibodies to nestin (H), A2B5 (J), GFAP (L, green), and 04 (L, red). Nuclei are counterstained with Hoechst (blue). Scale bars = 80mm in (A-C) and (G-H), 40mm in (D) and (I), 20mm in (E-F) and (J-L). Adapted from Wernig et al. (65), with kind permission of Springer Science + Business Media.

outgrowths are dissociated into a single-cell suspension, replated, and further proliferated in the presence of fibroblast growth factor-2 (FGF-2). At this stage the cells represent multipotent, mES cell-derived neural precursors (ESNPs (6), and see Figure 2D) which can be induced to differentiate into neurons and glia by growth factor withdrawal (Figure 2E, F). Alternatively, proliferating ESNPs can be shifted towards a predominantly glial precursor stage by further propagation with FGF-2 and epidermal growth factor (EGF). These mES cell-derived glial precursors (ESGPs; Figure 2G, H) are stable for several passages, and also can be differentiated further by growth factor withdrawal. An additional culture step in the presence of FGF-2 and platelet-derived growth factor-AA (PDGF-AA) can be used to obtain oligoastrocytic precursors, which efficiently give rise to myelinating oligodendrocytes ((7) and see Figure 2I-L).

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