A method of harvesting mesenchymal stem cells from human amniotic fluid
uses a two-stage culture protocol comprising culturing human amniocytes
and then culturing mesenchymal stem cells. For culturing human
amniocytes, primary amniocyte cultures are set up using routine or
standard culture protocol in a cytogenetic laboratory. Non-adherent human
amniotic fluids cells in the supernatant medium are collected. For
culturing mesenchymal stem cells ("MSC"), the non-adherent cells are
centrifuged and then plated with an alpha-modified Minimum Essential
Medium supplemented with fetal bovine serum. Incubate with humidified
CO.sub.2 for MSC growth. Reverse transcription polymerase chain reaction
("RT-PCR") and immunocytochemical analyses reveal that Oct-4 mRNA and
OCT-4 protein expression is detectable in the cultured amniotic fluid
mesenchymal stem cells ("AFMSCs"). Under differentiation culture
conditions, the AFMSCs can be induced to develop into multi-lineage
cells, such as adipocytes, osteocytes, neuronal cells, etc.