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Abstract
It is the hope of both patients and investigators that human progenitor cells and stem cells can be widely used to replace dysfunctional cells within a tissue. It is speculated that such cells may prove to have the potential to treat or cure a myriad of diseases, including Parkinson’s and Alzheimer’s diseases, heart disease, diabetes, stroke, spinal cord injuries, and burns. A major goal in this area of research is to identify potential new sources for the isolation of progenitor cells or stem cells, without raising the ethical issues involved in embryonic stem cell research.
The amniotic fluid could provide the least invasive access to embryonic and fetal stem cells. Ethical objections to the isolation of these cells could be completely avoided given that amniocentesis is a widely accepted form of prenatal diagnostic testing.
The main goal of the present study was to identify a novel source of pluripotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells, while the specific goals of the study are the investigation of amniotic fluid as a potential source of oct-4 expressing cells at different gestational ages after amniotic fluid cell culture as well as the investigation of the effect of culture vessel on the expression of Oct-4 cells after amniotic fluid cell culture in rat model.
The present study was carried out on 29 healthy adult pregnant female rats of the Wistar strain which were divided into 3 groups. Group A consisted of 10 animals, where amniocentesis was performed at midgestation, while Group B consisted of 10 animals, where amniocentesis was performed at late gestation. Groups A and B were cultured on non-tissue culture Petri dishes. Group C comprised 6 random amniotic fluid samples, that where cultured on tissue culture treated vessels. Lastly, 3 full term amniotic fluid samples were handled like Groups A and B.
The study was conducted in 3 steps: Surgical Amniotic Fluid Collection, total Amniotic Fluid (AF) cell population expansion as well as characterization of cultured Amniotic Fluid-derived cells using inverted microscope/live cell imaging and Oct-4 flow cytometry assay.
Amniotic fluid cells were successfully cultured from 19amples. Failure of culture in the rest of the samples was either due to blood contamination of amniotic fluid, low amniotic fluid volume with very small cell yield (full term samples) to allow for cell attachment and/or bacterial infection.
Amniotic fluid cells exhibited a heterogeneous morphology ranging between spindle fibroblastoid and polygonal shaped cells and achieved 70% confluence in 14 to 21 days.
Regarding Oct-4 expression, there was no significant difference between groups A and B (mid and late gestation groups), while a significant reduction in Oct-4 expression was observed in group C.
from the present study it can be concluded that amniotic fluid may represent a new exciting source of pluripotent stem cells. However further investigation in this regard is recommended. Moreover, gestational age has no effect on the expression of Oct-4, while the seeding vessel greatly affects the pluripotency marker.