ISOLATION AND CULTURE OF INNER CELL MASS CELLS FROM HUMAN BLASTOCYSTS

Totipotent non-committed inner cell mass (ICM) cells from human blasto cysts, if demonstrated to be capable of proliferating in vitro without differentiation, will have several beneficial uses, not only in the t reatment of neurodegenerative and genetic disorders, but also as a mod el in studying the events involved in embryogenesis and genomic manipu lation. Nine patients admitted to an in-vitro fertilization programme donated 21 spare embryos for this study. All 21 embryos were grown fro m the 2-pronuclear until blastocyst stages on a human tubal epithelial monolayer in commercial Earle's medium (Medicult, Denmark) supplement ed with 10% human serum. The medium was changed after blastocyst forma tion to Chang's medium supplemented with 1000 units/ml of human leukae mia inhibitory factor (HLIF) and the embryos left undisturbed for 72 h to allow the hatched ICM and trophoblast to attach to the feeder mono layer. Nineteen of the 21 embryos from nine patients produced healthy ICM lumps which could be separated and grown in vitro. Two of the lump s differentiated into fibroblasts while the remaining 17 (eight patien ts) produced cells with typical stem cell-like morphology, were alkali ne phosphatase positive and could be maintained for two passages. It w as possible to retain the stem cell-like morphology, alkaline phosphat ase positiveness and normal karyotype through the two passages in all of them using repeated doses of HLIF every 48 to 72 h. This is the fir st report on the successful isolation of human ICM cells and their con tinued culture for at least two passages in vitro.