Isolation and differentiation of human mesenchymal stem cells obtained from second trimester amniotic fluid; experiments at Chang Gung Memorial Hospital.

BACKGROUND: The aims of this study were to evaluate the efficacy of current techniques for isolating mesenchymal stem cells (MSCs) from amniotic fluid obtained by second-trimester amniocentesis as well as to determine their differentiation potential.

METHODS: We collected 50 samples of amniotic fluid by second-trimester amniocentesis. To obtain MSCs from amniotic fluid, the fluid was cultured using the two-stage culture protocol described in previous literature. Reverse transcription-polymerase chain reaction (RT-PCR) of a stem cell-specific transcription factor, octamer-binding protein 4 (Oct-4), was used to identify the characteristics of the MSCs cultured from amniotic fluid. Osteogeneic differentiation of these MSCs was confirmed by the presence of osteocalcin (a mineral-binding protein uniquely synthesized by osteoblasts) using RT-PCR and Von Kossa staining. Adipogenic differentiation of these MSCs was displayed by RT-PCR of adipocyte lipid-binding protein (a lipid-binding protein specifically in adipocytes) and Oil Red O staining.

RESULTS: Amniotic fluid-derived MSCs were successfully isolated and cultured from six samples. These cells could express the pluripotent stem cell-specific transcription factor Oct-4 as confirmed by RT-PCR. Under specific culture conditions, amniotic fluid-derived MSCs could be successfully induced to differentiate into adipocytes and osteocytes, based on product analysis by RT-PCR and specific staining.

CONCLUSION: Based on our experiment, we estimate the efficacy of isolating mesenchymal stem cells from second-trimester amniotic fluid obtained by amniocentesis to be about 12%. Human MSCs from second-trimester amniotic fluid had the ability to differentiate in vitro into adipocytes and osteocytes under specific culture conditions. The multilineage differentiation potential of these amniotic fluid-derived MSCs may be applicable to cell transplantation and regenerative medicine.