Embryonic stem (ES) cells are in vitro counterparts of the inner cell mass of a preimplantation embryo at the blastocyst stage and can be expanded in an artificial culture environment for a prolonged period with a stable genetic background (1,2). They have the ability to generate almost all cell types of the body including neural cells and, thus, offer an in vitro model for tracing early cell lineages in mammals. ES cells established from human embryos (3) are also a source for potential future cell therapy. A first step toward the use of ES cells is a directed differentiation to a specific cell lineage such as neuroectodermal lineage.

From: Methods in Molecular Biology, vol. 331: Human Embryonic Stem Cell Protocols Edited by: K. Turksen © Humana Press Inc., Totowa, NJ

Developmental principles learned from animal studies form the basis of designing protocols for directed neural differentiation. Studies in low vertebrate animals such as amphibian, zebrafish, and chick indicate that activation of fibroblast growth factor (FGF) signaling or inhibition of bone morphogenetic protein signaling is necessary for specification of the neuroectoderm from the embryonic ectoderm (4-6) (for review, see ref. 7). The neuroectoderm folds to form the neural tube, during which it is patterned into regionally specialized domains along rostrocaudal and ventral-dorsal axes. Progenitor cells in each region differentiate to postmitotic neurons with unique transmitter and positional identities. In humans, the neuroectoderm is specified in the third week of gestation and the neural tube is formed by the end of the third week of gestation (8).

The most commonly used approach for neural differentiation from mouse ES cells is the spontaneous aggregation of ES cells into the so-called embryoid bodies and treatment of these ES cell aggregates with retinoic acid to promote neural differentiation (9) or with other morphogens such as FGF2 to preferentially promote neuroepithelial proliferation (10). Recently, protocols are developed to guide mouse ES cells toward neuroectoderm (11) and then to specialized neurons such as dopaminergic and motor neurons based on developmental principles (12,13) (for review, see ref. 14).

We have designed a chemically defined colony culture system to induce human ES cells toward a neural fate based on the developmental principles. This neural differentiation protocol takes into consideration the similarities and differences between human and mouse ES cells. We also expect that this in vitro system will mimic in vivo neural development in humans and that it will allow us to dissect mechanisms of early human neural development. Because human ES cells survive better as clusters as opposed to disaggregated individual cells, the neural differentiation process is initiated by detachment of ES cell colonies from the fibroblast feeder layer by enzymes such as dispase or collagenase and grown as aggregates in suspension (15,16). Most differentiation protocols involve treatment of these ES cell aggregates with morphogens or growth factors in suspension for neural differentiation. However, such suspension culture system has significant drawbacks. ES cell aggregates grown in suspension culture for an extended period often form cysts, resulting in stochastic rather than directed differentiation. An unusually high concentration of morphogens or growth factors is required for the factors to reach cells inside the aggregates (15-18). Even so, cells on the surface and those inside the aggregates will have a varied degree of exposure to morphogens, thus, creating a wide range of cell lineages or cells at various developmental stages. The cluster nature also makes it difficult to visualize the continual change in cell morphology in response to treatments. For these reasons, we plate the ES aggregates to a plastic culture surface in a serum-free neural medium at a low density so that the aggregates form individual colonies (19). The colony culture allows semiquantitation analysis and the monolayer nature also permits continual assessment of changes in cell morphology. Cells in the colony center transform into small columnar cells, whereas those in the periphery gradually become flattened. The small columnar cells organize into neural tube-like rosette formations by 7-10 d after plating the aggregates. The rosette-forming columnar cells express transcription factors such as Pax6 and Soxl, confirming the neural identity. Thus the neuro-ectodermal differentiation process resembles normal human neural development in terms of timing and morphology.

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