Isolation and gene modification of amniotic fluid derived progenitor cells / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 492-503, 2014.
Artigo
em Chinês
| WPRIM
| ID: wpr-279500
ABSTRACT
We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIXAg reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Células-Tronco
/
Coagulação Sanguínea
/
Fator IX
/
Transfecção
/
Engenharia Genética
/
Separação Celular
/
DNA Complementar
/
Técnicas de Cultura de Células
/
Biologia Celular
/
Vetores Genéticos
Limite:
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2014
Tipo de documento:
Artigo
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