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| Establishment and identification of induced pluripotent stem cells sourced fetal amniotic fluid cells of a fetus with severe α-halassemia |
| ZHANG Juan LAI Chuntian WAND Haiyan ZOU Wenda PENG Juan ZHU Liyu LI Hui |
| Department of Reproductive Center, Zhuzhou Central Hospital, Hu′nan Province, Zhuzhou 412007, China |
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Abstract Objective To explore the establishment and identification of induced pluripotent stem cells sourced fetal amniotic fluid cells of a fetus with severe α-halassemia. Methods Ultrasound-guided amniocentesis was performed on pregnant women (16+ weeks) whose fetus was diagnosed with severe α-halassemia(--SEA/--SEA), 10 mL of amniotic fluid was extracted. The amniotic fluid cells of a fetus with severe α-halassemia which were reprogrammed into induced pluripotent stem cells (iPSCs) by non-integrated sendai virus mediated transcription factors of Sox2, Klf4, c-Myc, Oct 3/4. IPSCs were multifunctionally identified by alkaline phosphatase (ALP) test and immunofluorescence (IF) labeling. The ability of iPSCs to differentiate outward from three endodermal cells in vivo was identified by embryoid (EB) formation and spontaneous differentiation test. Cell karyotype analysis and genetic diagnosis of type α-thalassemia were performed. Results Sendai virus could successfully reprogram the amniotic fluid cells of a fetus with severe α-halassemia into non-integrated induced pluripotent stem cells (h-AF-SeV-iPSCs). H-AF-SeV-iPSCs were strongly positive of ALP staining, and embryonic stem cells specific proteins of SOX2, Oct4, SSEA-4 and Tra-1-81 were positive expressed of IF staining. After the suspension culture of h-AF-SeV-iPSCs, the cystic EB body could be formed, and after the adherent culture, it could also differentiate into the three germ layers. The karyotype analysis of chromosome in the 10th and 20th generation of h-AF-SeV-iPSCs were normal (46, XY). H-AF-SeV-iPSCs and its primary amniotic fluid cells were all detected as --SEA/--SEA. Conclusion The h-AF-SeV-iPSCs could be used as an ideal cell model for study of intrauterine treatment of a fetus with severe α-halassemia, and amniotic fluid cells could be used as an ideal cells source for iPSCs.
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