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.