Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture

Abstract

Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells.

BMP-4 induces a Smad-dependent apoptotic cell death of mouse embryonic stem cell-derived neural precursors

“…Belonging to the family of the high-mobility group of transcription factors, it is first expressed in the neural plate and subsequently maintained in neuroepithelial cells throughout the entire neuraxis, but is downregulated during neuronal and glial differentiation. 26 Using Sox-1-gfp ES cells, 8 we quantify by flow cytometry the % of Sox-1-positive cells during the differentiation process. As illustrated in Figure 6A, more than 50% of the differentiated ES cells are Sox-1 þ cells at day 4, with a percentage increasing up to 70% by day 6 (not shown).…”

“…The undifferentiated mouse ES cell lines CGR8 and CGR8 Sox1 GFP (ES cell line from transgenic mouse in which the enhanced GFP reporter is inserted into the sox1 gene via gene targeting 8 ) were routinely cultured in flasks coated with 0.1% gelatin (GIBCO-BRL, Invitrogen, France) diluted in PBS pH 7.2 (GIBCO-BRL). The culture medium was composed of BHK-21 (GIBCO-BRL) supplemented with 10% fetal bovine serum (Hyclone, Perbio Science, France), 1% nonessential amino acids (GIBCO-BRL), 1 mM sodium pyruvate (GIBCO-BRL), 0.1 mM b-mercaptoethanol (GIBCO-BRL) and 10 3 U/ml LIF (Leukemia Inhibitory Factor, our own production).…”

“…Recent data have shown that, in the absence of feeder-derived or serum-derived factors and at low cell densities, ES cells undergo a direct phenotypic change towards the neural fate, 7 a process that requires autocrine fibroblast growth factor (FGF). 8 Nevertheless, BMP-4 appears to be the central mediator in the switch from neuronal to epidermal commitment. The target genes regulated by the BMP signaling pathway and their roles during the specification of ventral cell types are poorly understood.…”

The role of Ca2+ signaling on the self-renewal and neural differentiation of embryonic stem cells (ESCs)

“…Multiple neuronal-like phenotypes, including neurite outgrowth, expression of class -tubulin, GFAP and the neurofilament M subunit, as well as neuron-like electrophysiological properties, are subsequently induced following EB initiation and RA treatment [36]. Whereas the role of RA in neural differentiation of ESCs is known to be pleiotropic and of indeterminate physiological relevance [37], it is also known to play a role in the regulation of the -catenin signaling, FGF/Erk1/2 signaling and C-JNK/CREB signaling pathways [16]. However, neural precursors and differentiating neurons can also be generated in the absence of RA, in serumfree medium containing insulin, transferrin and sodium selenite (referred to as serum-free ITS), and basic fibroblast growth factor (bFGF).…”

“…More recently, a more direct and more efficient approach, using an adherent monoculture system, was developed for the differentiation of mouse ESCs into neurons [37] [41]. In monoculture, the elimination of inductive signals from alternative fates and the addition of autocrine FGF is sufficient for mouse ESCs to develop into neural precursors.…”