Isolation of human pluripotent mesenchymal stem cells from second-trimester amniotic fluid using two kinds of culture protocol and their differentiation into neuron-like cells

Abstract

Objective To isolate mesenchymal stem cells (MSCs) from second-trimester amniotic fluid using an improved two-stage culture protocol, and to induce these MSCs into neuron-like cells. Methods An improved two-stage culture protocol for MSCs from amniotic fluid was developed. MSCs from amniotic fluid were induced to differentiate with β-mercaptoethanol into neuron-like cells. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were employed for analysis of the phenotypic characteristics of the cultured MSCs from amniotic fluid. Results MSCs from amniotic fluid were successfully isolated, cultured and enriched without interfering with the routine process of fetal karyotyping. Flow cytometry analysis showed that they were positive for CD44, CD29, and CD105, but negative for CD45, CD34, or human leucocyte antigen-DR (HLA-DR). Importantly, a subpopulation of transcription factor Oct-4 positive cells could be detectable in the cultured MSCs from amniotic fluid. Moreover, MSCs from amniotic fluid obtained by two-stage culture protocol showed higher proliferation rate [(15.0±2.3)% vs. (10.0±1.8)%] and higher Oct-4 positive cell rate [(1.2±0.3)% vs. (0.9±0.2)%, both P<0.05]. After the induction, MSCs from amniotic fluid displayed processes that formed extensive networks. Positive cells for neurone specific enolase (NSE) constituted (54.76±3.65)%, and glial fibrillary acidic protein (GFAP) (36.28±4.27)%, respectively. Conclusion We demonstrate that human pluripotent MSCs are present in second-trimester amniotic fluid. The two-stage culture protocol could be a kind of simple one with high performance and it does not interfere with the routine fetal karyotyping. MSCs from amniotic fluid can be induced to differentiate into neuron-like cells in vitro by β-mercaptoethanol in optimal medium. Key words: Amniotic fluid cell ; Mesenchymal stem cell ; Transcription factor Oct-4 ; Neuron ;