Human Reproduction · 1994 · 272 citations · 0 references
Human blastocyst inner cell mass cells, if shown to proliferate in vitro without differentiating, could aid neurodegenerative and genetic disorder therapies and serve as models for embryogenesis and genomic manipulation. Embryos from nine patients were cultured from the 2‑pronuclear stage to blastocyst on a human tubal epithelial monolayer in Earle’s medium with 10 % human serum, then switched to Chang’s medium with HLIF for 72 h to allow ICM and trophoblast attachment. Nineteen of 21 embryos yielded healthy ICM lumps that could be isolated and cultured; 17 displayed stem‑cell‑like morphology, alkaline phosphatase positivity, and normal karyotype for two passages with repeated HLIF, marking the first successful isolation and in‑vitro culture of human ICM cells.
Totipotent non-committed inner cell mass (ICM) cells from human blastocysts, if demonstrated to be capable of proliferating in vitro without differentiation, will have several beneficial uses, not only in the treatment of neurodegenerative and genetic disorders, but also as a model in studying the events involved in embryogenesis and genomic manipulation. Nine patients admitted to an in-vitro fertilization programme donated 21 spare embryos for this study. All 21 embryos were grown from the 2-pronuclear until blastocyst stages on a human tubal epithelial monolayer in commercial Earle's medium (Medicult, Denmark) supplemented with 10% human serum. The medium was changed after blastocyst formation to Chang's medium supplemented with 1000 units/ml of human leukaemia inhibitory factor (HLIF) and the embryos left undisturbed for 72 h to allow the hatched ICM and trophoblast to attach to the feeder monolayer. Nineteen of the 21 embryos from nine patients produced healthy ICM lumps which could be separated and grown in vitro. Two of the lumps differentiated into fibroblasts while the remaining 17 (eight patients) produced cells with typical stem cell-like morphology, were alkaline phosphatase positive and could be maintained for two passages. It was possible to retain the stem cell-like morphology, alkaline phosphatase 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 first report on the successful isolation of human ICM cells and their continued culture for at least two passages in vitro.