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By Mikkel L. Sorensen

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1. Free-Floating Culture in Astrocyte-Conditioned Medium In EB methods, one to several ES cells are cultured to form aggregates in culture medium with or without serum. In our method, however, colonies of undifferentiated ES cells grown on a mouse fibroblast feeder layer are picked up whole and transferred into non-adhesive bacteriological dishes. In addition, direct differentiation from ES cells into neural stem cells is induced by astrocyte-conditioned medium (ACM) in free-floating culture [6].

Magid, T. Ponomaryov, T. Byk, A. Nagler, H. Ben-Hur, A. Many, L. Shultz, O. Lider, R. Alon, D. Zipori, and T. Lapidot, Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4, Science 283, 845–848. (1999). D. Abbott, Y. Huang, D. Liu, R. S. J. Giordano, Stromal cellderived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation 110, 3300-3305 (2004).

Blood, 98, 1166–1173. E. (1994). A cell initiating human acute myeloid leukemia after transplantation into SCID mice. Nature, 367, 645– 648. E. (1997). Human acute leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. , 3, 730–737. E. (2007). Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc. Natl. Acad. Sci. U S A. 104, 973-978. , and Mori, M. (2006). Characterization of a side population of cancer cells from human gastrointestinal system.

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