Cultivation And Identification Of Human Amniotic Fluid-derived Multipotential Stem Cells And Their Differentiation Potentiality

With the rapid growth of regenerative medicine, stem cells have played more and more important role in the basic research and clinical application of modern medicine. Because of ethical problem,the research of embryonic stem cells and clinical application has greatly restricted; adule stem cells have limited ability to differentiate into some cells and tissues. When the human amniotic fluid-derived stem cells have been discovered , they pioneer a new field for study the stem cells. Human amniotic fluid-derived stem cells (hAFS) may be a potential source of cells for tissue engineering and cellular therapy. Although several hAFS cells derivation techniques have now been developed, the existing techniques are unsuitable for hAFS cells production for medical purposes because these methods often result in contamination with other cell types or contamination with antibodies raised from animals. Additionally, these techniques require a long period of time for stem cell production. Hence, a better method which allows utilization of these cells for cell-based therapy needs to be developed. In the current study, we present an improved method as an efficient technique that is suitable to derive hAFS cells for therapeutic purposes.Objective1. To establish an improved device of culture amniotic fluid-derived multipotential stem cells in vitro and study their biological characteristics.2. The phenotypes of hAFS cells were tested by flow cytometry.3. Inducing hAFS cells into neurogenic, adipogenic and osteogenic by definite culture medium to detection their differentiation potentiality.Methods1. The technique starts by selecting adherent stem cells from second-trimester human amniotic fluid primary culture. A selective individual hAFS cell is called a "starter cell". The starter cell is used as a beginner cell for generating a clonal hAFS cell line.2. hAFS cell markers were characterized using flow cytometry analysis and RT-PCR technique, the tested antibody marker of some embryonic stem cells and mesenchymal stem cells such as: Oct-4,SSEA-4,Nanog,CD29,CD44,CD105 and CD117.3. Inducing amniotic fluid-derived stem cells into neurogenic, adipogenic and osteogenic by definite culture medium, The osteogenesis was assessed by determinating cell mineralization,using alizarin red S staining. The Oil Red O staining was used for detection of intracellular lipid droplet formation for evaluating adipogenesis. For evaluation of neural differentiation, the neuron specific marker nestin was used.Results1. The hAFS cells derived by our technique proliferated rapidly and have a high purity, with a population doubling time of 24h,it is significantly accelerated compared with the previous research.2. The hAFS cells showed high positive signals for embryonic stem cells and mesenchymal stem cells markers :Oct-4,SSEA-4,CD29,CD44,CD105and CD117. These cells show a negative signal for hematopoietic stem cell markers CD45 and CD133 by flow cytometry. RT-PCR show that the mRNA of Oct-4 and Nanog were expressed.3. Osteogenic differentiation for 3 weeks , calcium mineralization were appeared in cytoplasm of hAFS-derived osteogenic cells, they verified by a photochemical reaction using alizarin red S .For adipogenic differentiation, the appearance of endogenous lipid droplets was observed by staining with Oil Red O after 3 weeks of culture, the lipid droplets appeared in cytoplasm and bright red. For neurogenic differentiation, the cells showed positive staining for neuron-specific marker nestin. Above all,hAFS cells can differentiation into neurogenic,adipogenic and osteogenic in vitro.Conclusions1. The starter cell method as a simple approach to provide good quality hAFS cells that fit the requirements for use in therapeutic purposes. This technique should allow for future cell-based therapy.2. hAFS cells have a great potential for proliferation, which express both embryonic and mesenchymal stem cells markers. Their characteristics are in accord with multipotential stem cells.3. AFS cells have the ability to differentiate into neurogenic, adipogenic and osteogenic in suitable condition in vitro.