Objective: To investigate cardio-myogenic differentiation potential of human amniotic fluid colony derived stem cells (HAFCDSC) in the form of embryonic body (EB)-like structure in vitro.
Methods: HAFCDSC were isolated from second trimester amniotic fluid which was backup of amniocentesis specimens. The forth passage of HAFCDSC were cultured by hanging-drop preparation in complete medium for 5 days to form EB-like structures followed by inducing medium in regular tissue culture dishes for 2 weeks (experiment group). The EB-like structures cultured in complete medium were served as control group. Cardiomyocyte phenotypes were detected by RT-PCR and immunofluorescence staining.
Results: HAFCDSC could form EB-like structures 24 h after hanging drop culture, and the diameter of EB-like structures gradually increased with culture duration and the mean diameter of EB-like structures reached 237.3 ± 26.7 µm on 5(th) day. The formation rate of EB-like structures was 93.5%. RT-PCR analysis showed EB-like structures expressed stem cell related mRNA. Immunofluorescence staining demonstrated that some cardiomyocyte phenotypes such as Desmin, α-Actinin and Tn I were expressed by HAFCDSCs in EB-like structures after cardio-myogenic induction. Cardiomyocytes specific mRNA including Desmin, α-Actin, Tn I, Tn T, T-box and NK2.5 were also transcripted as detected by RT-PCR. No positive results was found in control group.
Conclusion: HAFCDSC can form uniform EB-like structures after hanging drop culture for 5 days. HAFCDSC differentiate into cardiomyocyte-like cells through the form of EB-like structures after induction by 5-Aza.