Isolation of hES cells

2.1 Human embryos

Human ES cells have been isolated from in vitro fertilized embryos, in some cases following cryopreservation. Most jurisdictions that allow the use of human embryos for the production of hES cell lines now have in place a regulatory framework to ensure ethical conduct of the research. These regulations vary considerably internationally, and researchers must ensure that all proposed studies are in compliance with local and national standards and statutory requirements.

The comparative properties of human and mouse ES cells have been reviewed elsewhere (3, 4; and see Chapter 12). While there are certainly similarities in the biology of these pluripotent stem cells in the two species, differences in morphology, growth characteristics, and methods for propagation are considerable. Therefore, researchers new to the field should gain experience culturing extant lines of monkey or human pluripotent stem cells (i.e. ES or EC cell lines) prior to attempting to isolate new hES cell lines.

Protocol 1 outlines our procedure for isolating the inner cell mass (ICM) from human embryos for establishment of hES cell lines (Figure 1). All cell culture incubation is carried out in a humidified incubator set at 37 °C with 5% CO2.

Most, though not all, reports of hES cell derivation have used a culture system incorporating serum-supplemented medium, mouse embryonic fibroblast (MEF)

Figure 1 Isolation and primary culture of ICM from human blastocyst. (A) Expanded blastocyst. (B) Blastocyst following removal of zona pellucida. (C) ICM following isolation by immunosurgery. (D) Primary culture of ICM at 3days. Here the ICM has attached to the MEF feeder layer, and commenced forming an outgrowth. Magnification: (A) 40x; (B-D), 20x. Micrographs provided by Dr Carmel Obrien, Stem Cell Sceiences Pty., and Ms Nicole Merry, Melbourne IVF, Melbourne Australia.

Figure 1 Isolation and primary culture of ICM from human blastocyst. (A) Expanded blastocyst. (B) Blastocyst following removal of zona pellucida. (C) ICM following isolation by immunosurgery. (D) Primary culture of ICM at 3days. Here the ICM has attached to the MEF feeder layer, and commenced forming an outgrowth. Magnification: (A) 40x; (B-D), 20x. Micrographs provided by Dr Carmel Obrien, Stem Cell Sceiences Pty., and Ms Nicole Merry, Melbourne IVF, Melbourne Australia.

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