The most clinically important, and imagination stimulating application of human embryonic stem cells (hESCs), is of course the generation of normal differentiated cells for replacement of those lost in disease or injury. However, there are several additional important ways in which hESCs can be used productively. Use of these cells has the potential to reveal important details about early human embryonic development, because early human embryos, for

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

both practical and ethical reasons, are not accessible to researchers. Arguments in the scientific and general community now seem to preclude the production of early chimeras using hESCs, whether the host blastocyst is human or mammalian (mouse) (1). By contrast, transplantation of hESCs into adult animals is routine today and does not appear to raise any ethical problems. However, the adult tissue environment likely lacks many of the factors required to direct differentiation of the hESCs, and the complex and compact three-dimensional structure can impede migration and integration of hESCs. This is clearly shown by the development of teratomas from hESCs when injected in adult mice (2,3).

The chick embryo has been studied intensively at the anatomical, cell biological, and molecular levels, and the precise timing and placement of many of the inductions and morphogenetic events underlying its development are known. In addition, experimental manipulations of the embryo are quite uncomplicated because of the easy accessibility of the embryo by simply opening a "window" in the shell. A number of studies have shown that mammalian cells survive and differentiate in response to environmental signals of avian embryos. For example, when chick neural tube is replaced with that of mouse embryos, mouse neural structures develop, including a spinal cord, a neural crest, and their derivatives the peripheral ganglia (4). Mouse embryonic stem cells and their derivatives have also been transplanted successfully to the chick embryo. In one elegant study, motoneu-ron precursors derived from mouse embryonic stem cells were transplanted to the neural tube of the chick, and mouse motoneurons not only differentiated, but sent their axons out chick nerves, and innervated peripheral muscles (5).

We therefore have transplanted hESC colonies into the developing tissues of early chick embryos in an attempt to provide hESC with an environment of developing tissues, while avoiding the ethical problem of generating embryos with extensive chimerism (6). We found that placement of the hESC in direct contact with the host neural tube consistently resulted in the generation of neural tube-like structures ("neural rosettes") that grew with the same orientation as the host central nervous system, with their axis parallel to that of the chick. In contrast, interspersing the hESC within somites led to integration of individual human cells into chick structures such as peripheral ganglia and vertebral antagen. The present chapter details the techniques that were involved in these experiments, with the primary focus on the surgical techniques themselves (see Note 1).

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