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Differentiation of ES cells in culture
- Embryonic stem cell differentiation: emergence of a new era in biology and medicine


When removed from the factors that maintain them as stem cells, ES cells will differentiate and, under appropriate conditions, generate progeny consisting of derivatives of the three embryonic germ layers: mesoderm, endoderm, and ectoderm (Keller 1995Go; Smith 2001Go). Wild-type ES cells do not differentiate to trophectoderm in culture and, in this respect, reflect the potential of their founder embryonic population, the inner cell mass (Fig. 1). hES cells differ from mouse cells in this respect, as when induced with BMP4, they will give rise to cells that display characteristics of the trophoblast lineage (Xu et al. 2002Go). The reason for this difference is not clear, but may indicate that at least some of the hES cell lines represent earlier stages of development than the comparable mouse populations.

Three general approaches, outlined in Figure 2, are used to initiate ES cell differentiation. With the first method, ES cells are allowed to aggregate and form three-dimensional colonies known as embryoid bodies (EBs) (Doetschman et al. 1985Go; Keller 1995Go). In the second method, ES cells are cultured directly on stromal cells, and differentiation takes place in contact with these cells (Nakano et al. 1994Go). The most commonly used stromal cell line for such differentiation studies is OP9 (Nakano et al. 1994Go), originally isolated from CSF-1-deficient op/op mice (Yoshida et al. 1990Go). The third protocol involves differentiating ES cells in a monolayer on extracellular matrix proteins (Nishikawa et al. 1998Go). 

All three approaches to ES cell differentiation are effective and have specific advantages and disadvantages. EBs offer the advantage of providing a three-dimensional structure that enhances cell–cell interactions that may be important for certain developmental programs. The complexity of the EBs can also be a disadvantage as the generation of cytokines and inducing factors within these structures can complicate interpretations of experiments in which one is trying to understand the signaling pathways involved in lineage commitment. Coculture with stromal cells provides the beneficial growth promoting effects of the particular cell line used. However, undefined factors produced by these supportive cells may influence the differentiation of the ES cells to undesired cell types. An additional problem with this method is the difficulty that can be encountered when attempting to separate the ES-cell-derived cells from the stromal cells. Differentiation in monolayers on known substrates can minimize the influence of neighboring cells and supportive stromal cells and in this regard is one of the simplest protocols. With this protocol the matrix proteins are critical, and different proteins may dramatically influence the generation and survival of the developing cell types.

Three criteria should be considered when using the ES cell model for lineage-specific differentiation. First, protocols need to be established that promote the efficient and reproducible development of the cell type of interest. If possible, selection strategies should be combined with optimal differentiation schemes to enable the isolation of highly enriched cell populations. Second, lineage development from ES cells should recapitulate the developmental program that establishes the lineage in the early embryo. Third, the mature cell populations that develop in these cultures must display appropriate functional properties both in culture and when transplanted into appropriate animal models.

The three differentiation methods described above have been used to generate a broad spectrum of cell types from ES cells (Keller 1995Go; Smith 2001Go). For lineages that have been studied in detail, the first two criteria outlined above have been met as efficient protocols for their differentiation have been established and the sequence of events leading to their development in culture was found to faithfully recapitulate those in the early embryo. The third aim has yet to be fulfilled for most populations and represents one of the major challenges in the field today. Since their derivation, progress has been made with the differentiation of hES cells. Although not nearly as advanced as the studies with the mouse system, the findings to date indicate that it will be possible to generate a broad spectrum of cell types from them in culture (Schuldiner et al. 2000Go; Odorico et al. 2001Go; Pera and Trounson 2004Go).

Many differentiation protocols have been optimized using FCS as a growth supplement and/or as a source of inducing factors. Although these approaches have been successful for the development of certain lineages, the use of FCS has several serious drawbacks that include batch-to-batch variability and the lack of identity of the inducing factors contained in it. As discussed in a later section of this review, several recent studies have eliminated serum and have begun to identify factors required for lineage-specific differentiation. As more protocols incorporate these approaches, both mouse and human ES cell multilineage differentiation will become a routine technology in many labs.

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