Isolation of amniotic stem cell lines with potential for therapy

Stem cells capable of differentiating to multiple lineages may be valuable for therapy. We report the isolation of human and rodent amniotic fluid‐derived stem (AFS) cells that express embryonic and adult stem cell markers. Undifferentiated AFS cells expand extensively without feeders, double in 36 h and are not tumorigenic. Lines maintained for over 250 population doublings retained long telomeres and a normal karyotype. AFS cells are broadly multipotent. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. Examples of differentiated cells derived from human AFS cells and displaying specialized functions include neuronal lineage cells secreting the neurotransmitter L-glutamate or expressing G-protein-gated inwardly rectifying potassium channels, hepatic lineage cells producing urea, and osteogenic lineage cells forming tissue-engineered bone. Amniotic fluid is known to contain multiple cell types derived from the developing fetus 1,2 . Cells within this heterogeneous population can give rise to diverse differentiated cells including those of adipose, muscle, bone and neuronal lineages 3‐6 . We now describe lines of broadly multipotent AFS cells, and use retroviral marking to verify that clonal human AFS cells can give rise to adipogenic, osteogenic, myogenic, endothelial, neurogenic and hepatic lineages, inclusive of all embryonic germ layers. In this respect, they meet a commonly accepted criterion for pluripotent stem cells, without implying that they can generate every adult tissue. Neurons, hepatocytes and osteoblasts are among the cell types for which improved stem cell sources may open up novel therapeutic applications. For each of these examples we show that AFS cells can yield differentiated cells that express lineage-specific markers and acquire characteristic functions in vitro. In addition, we present initial studies indicating that AFS cells induced toward particular lineages can generate specialized cells after implantation in vivo .W e show that AFS cells directed to neural lineage differentiation by exposure to nerve growth factor (NGF) are able to widely engraft the developing mouse brain in a manner similar to that observed previously for neural stem cells 7 . In addition, we document the formation of tissueengineered bone from printed constructs of osteogenically differentiated human AFS cells in immune-deficient mice. RESULTS Clonal stem cell lines from amniotic fluid Approximately 1% of the cells in cultures of human amniocentesis specimens obtained for prenatal genetic diagnosis express the surface