Elsevier

Methods in Enzymology

Volume 365, 2003, Pages 327-341
Methods in Enzymology

Defined Conditions for Neural Commitment and Differentiation

https://doi.org/10.1016/S0076-6879(03)65023-8Get rights and content

Publisher Summary

Pluripotent mouse embryonic stem (ES) cells can be expanded in culture indefinitely, retaining the capacity to produce seemingly every type of fetal and adult cell. Current methods are empirical with outcomes that are invariably heterogeneous and often poorly reproducible. Some progress has been made with controlling the intermediate stages of lineage progression, but mastering the full sequence of steps necessary for efficient generation of any particular terminally differentiated phenotype remains elusive. There are two major challenges: first to understand and manipulate lineage choices; second to develop culture conditions that support the viability and maturation of progenitor and terminal phenotypes in vitro. The most widely used method to trigger neural development from ES cells is cell aggregation in suspension culture followed by treatment with retinoic acid. This chapter describes defined conditions for conversion of ES cells to neural fates in monolayer culture.

Introduction

Pluripotent mouse embryonic stem (ES) cells can be expanded in culture indefinitely while retaining the capacity to produce seemingly every type of fetal and adult cell.1 ES cell differentiation in vitro is thought to recapitulate in vivo developmental programs2 and generation of various apparently fully specified and functional cell types has been described.3 However, we cannot yet claim an ability to “direct” ES cell differentiation. Current methods are empirical with outcomes that are invariably heterogeneous and often poorly reproducible. Some progress has been made with controlling intermediate stages of lineage progression,4, 5 but mastering the full sequence of steps necessary for efficient generation of any particular terminally differentiated phenotype remains elusive. There are two major challenges: first to understand and manipulate lineage choices; second to develop culture conditions that support the viability and maturation of progenitor and terminal phenotypes in vitro. Our laboratory has begun to investigate these issues in the context of neural differentiation.5a

The most widely used method to trigger neural development from ES cells is cell aggregation in suspension culture followed by treatment with retinoic acid. In suspension culture ES cells form multicellular multi-differentiated structures called embryoid bodies.6 Neural derivatives are present only at low frequency in embryoid bodies generated in serum-containing medium, but their proportion increases dramatically after addition of retinoic acid.7 Regardless of the concentration or duration or retinoic acid treatment, however, the final cultures are always a heterogeneous mixture of various cell types. Several strategies have been developed to purify or enrich neuroectodermal precursors or more mature neuronal or glial phenotypes from embryoid bodies. These include the introduction of a transgene marker conferring drug resistance and/or cell-sorting capacity specifically to neural lineage cells,8, 9 immunopanning for neural antigens to select neuronal or glial restricted progenitors,10 and a combination of growth factor stimulation and differential adhesion and proliferation in minimal media.11, 12 Although these techniques are effective, the primary process of neural determination remains unexplained and relatively inefficient.

It is difficult to dissect and manipulate differentiation within embryoid bodies because they are multicellular agglomerations of extraembryonic endoderm and definitive ectodermal, mesodermal and endodermal derivatives.2 Furthermore, retinoic acid has pleiotropic actions—it induces other lineages13 and affects positional specification5—and the route by which it influences neural commitment is obscure. Several groups have developed methods in which treatment with retinoic acid is avoided.14, 15, 16, 17 Neural cells appear in embryoid bodies in the absence of serum14, 15 or presence of conditioned medium extracts.17 They can also be obtained at high frequency upon coculture with a particular stromal cell line, PA6,16 an effect ascribed to an unidentified stromal cell-derived inducing activity (SDIA). In all cases, however, the mechanism of neural commitment remains elusive. Data from one study indicates that neural cells can be derived from individual ES cells when placed in suspension in the absence of serum.14 However, the interpretation that neural specification arises by default18 is challenged by the low frequency of this event (1 in 1000 cells).

We sought to develop a simple system that would allow direct observation, analysis, and manipulation of the process of neural specification without the confounding influences of cell aggregation, coculture, uncharacterized media constituents, or cell selection. We also wished to avoid retinoic acid because this is likely to restrict the regional identity of neural precursors.5 Here, we describe defined conditions for conversion of ES cells to neural fates in monolayer culture.

Section snippets

ES Cells

Parental ES cell lines are germline competent CGR819 and E14Tg2a20 derived from 129/Ola mice. Genetically manipulated ES cells are:

46C: generated by gene targeting in E14TG2a.21 The open reading frame of the Sox1 gene is replaced with the coding sequence for enhanced green fluorescent protein (GFP) and an internal ribosome entry site (IRES)-linked puromycin resistance gene. In embryos generated from 46C ES cells GFP is expressed in neuroepithelial cells throughout the neuraxis and in the lens

Trouble-Shooting Monolayer Differentiation

The monolayer protocol is a straightforward method for neural differentiation of mouse ES cells. However, due to variation between cell lines, reagents and laboratory practices, achieving consistent high efficiency neural differentiation may prove challenging. Below are some comments and tips on tackling problems that may arise during monolayer differentiation.

Acknowledgements

We are indebted to Marios Stavridis, Meng Li, Dean Griffiths and Katherine Rennie for their contributions to these studies. This work was supported by the International Human Frontiers Science Program Organisation, and by the Medical Research Council and the Biotechnology and Biological Sciences Research Council of the United Kingdom.

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