Derivation of embryonic stem-cell lines from human blastocysts.

Embryonic stem cells have the unique ability to form all adult cell types. Harnessing this potential may provide a source of cells to replace those that are lost or impaired as a result of disease. Moreover, the derivation of human embryonic stem cells opens a unique window into the study of early human development. At present, approximately 15 human embryonic stem-cell lines are publicly available, and they vary considerably in their usefulness for research and the extent of their characterization (see http://stemcells.nih.gov/registry/index.asp). To promote further research with human embryonic stem cells, we sought to derive and characterize more fully cell lines that meet strict criteria for ease of manipulation, including enzymatic passage with trypsin, streamlined freezing and thawing procedures, well-defined culture mediums, and straightforward methods for in vitro differentiation. We report the derivation and characterization of 17 additional human embryonic stem cell lines. We obtained frozen cleavageand blastocyststage human embryos, produced by in vitro fertilization for clinical purposes, after obtaining written informed consent and approval by a Harvard institutional review board. A total of 286 frozen and thawed cleaved embryos (6 to 12 cells each) were cultured to the blastocyst stage, and 58 frozen and thawed blastocysts were allowed to re-expand in culture, whereupon they were treated with Tyrode’s solution to remove the zona pellucida, followed by immunosurgery to isolate inner cell masses. 1 Many of these embryos were of such poor quality that they did not develop or divide after thawing. Nevertheless, 97 inner cell masses were isolated, and 17 individual human embryonic stem-cell lines (HUES1 through HUES17) were derived according to published protocols that we modified in terms of medium composition, enzymatic dissociation, and procedures for freezing and thawing (Fig. 1A, 1B, and 1C, next page). 2-4 Too few embryos were available for us to determine systematically whether the procedural changes we used contributed substantially to the success rates we achieved. A detailed manual of our methods for culturing blastocysts and isolating embryonic stem cells is available elsewhere. 5