TY - JOUR
T1 - Effect of Passage, Sorting, and Media on Differentiation Capacity and Marker Expression in Amniotic Fluid Stem Cells
AU - Connell, Jennifer P.
AU - Augustini, Emily
AU - Cheng, Shannon
AU - Benavides, Omar M.
AU - Ruano, Rodrigo
AU - Jacot, Jeffrey G.
N1 - Funding Information:
The authors would like to thank Luis Juarez for assistance in RNA purification and cell culture, Madeline Monroe for assistance in neurogenic differentiation image analysis, and Patrick Connell for assistance with statistical analysis. The FACS and flow cytometry analysis in this project was supported by the Texas Children’s Hospital Cancer Center Flow Cytometry Core Laboratory. This work was supported by Texas Children’s Hospital, a National Science Foundation Graduate Fellowship (grant no. 0940902 to JPC), and a National Science Foundation CAREER Award (JGJ).
Publisher Copyright:
© 2015, Biomedical Engineering Society.
PY - 2016/3/1
Y1 - 2016/3/1
N2 - Amniotic fluid-derived stem cells (AFSC) represent a promising source for tissue engineering applications; however, wide variation exists in the methods and medium used in AFSC culture. To address the maintenance of stem cell markers and differentiation capacity of AFSC, both c-kit sorted and unfractionated adherent AFSC from human patient samples were cultured in a variety of media and evaluated over six passages. Medium supplements included Chang medium, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and both bFGF and EGF. Protein and gene expression of pluripotency markers, including c-kit, SSEA4, Sox2, Oct4, and cMyc were measured in undifferentiated AFSC. Differentiation capacities of AFSC towards endothelial, osteogenic, and neurogenic lineages were analyzed at passages 5 and 6. Expression of c-kit was higher in c-kit sorted cells compared to unfractionated cells; however, SSEA4, Sox2, cMyc, Tra-1-60, and Tra-1-81 were higher in the unfractionated population. Pluripotency marker expression was significantly lower at passage 6 than earlier passages. Correspondingly, cells differentiated more efficiently at passage 5 than passage 6. Media had distinct effects on differentiation towards each lineage. These results indicate that AFSC should be utilized prior to passage 6 and that optimal isolation and culture conditions depend on final differentiation intent.
AB - Amniotic fluid-derived stem cells (AFSC) represent a promising source for tissue engineering applications; however, wide variation exists in the methods and medium used in AFSC culture. To address the maintenance of stem cell markers and differentiation capacity of AFSC, both c-kit sorted and unfractionated adherent AFSC from human patient samples were cultured in a variety of media and evaluated over six passages. Medium supplements included Chang medium, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and both bFGF and EGF. Protein and gene expression of pluripotency markers, including c-kit, SSEA4, Sox2, Oct4, and cMyc were measured in undifferentiated AFSC. Differentiation capacities of AFSC towards endothelial, osteogenic, and neurogenic lineages were analyzed at passages 5 and 6. Expression of c-kit was higher in c-kit sorted cells compared to unfractionated cells; however, SSEA4, Sox2, cMyc, Tra-1-60, and Tra-1-81 were higher in the unfractionated population. Pluripotency marker expression was significantly lower at passage 6 than earlier passages. Correspondingly, cells differentiated more efficiently at passage 5 than passage 6. Media had distinct effects on differentiation towards each lineage. These results indicate that AFSC should be utilized prior to passage 6 and that optimal isolation and culture conditions depend on final differentiation intent.
KW - Amnion
KW - C-kit
KW - Multipotential differentiation
KW - Perinatal stem cells
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U2 - 10.1007/s12195-015-0407-6
DO - 10.1007/s12195-015-0407-6
M3 - Article
AN - SCOPUS:84958115348
VL - 9
SP - 139
EP - 150
JO - Cellular and Molecular Bioengineering
JF - Cellular and Molecular Bioengineering
SN - 1865-5025
IS - 1
ER -