The Biological Characteristics Study Of Pluripotent Stem Cells Derived From The Amniotic Fluid Of Sheep

A class of stem cells was found in the amniotic fluid of humans and animals in recent years. The amniotic fluid-derived stem cells(AFSC) can grow without cytotrophoblast cells and express the marker gene of embryonic stem cell (ESC) and somatic stem cell(SSC), and they can form embryoid bodies in vitro suspension culture. Therefore, the AFSCs are considered that they have the developmental pluripotency and important physiological functions in body development.In this study, three types of pluripotent stem cells from sheep amniotic fluid and fetal kidney tissue which express Oct4and SSEA-1genes were isolated using two types of isolation and culture methods. Mouse AFSCs which only express MHC-Ⅰ but MHC-Ⅱwere got by generic method of separating stem cells and named AFSC-I. On this basis, the pluripotent stem cells were isolated and cultured from amniotic fluid and fetal kidney tissues in the the same conception mid-sheep by0.01%trypsin medium. The sheep AFSCs express MHC-Ⅱ, not expression of MHC-Ⅰ, called AFSC-Ⅱ. The pluripotent stem cells derived from sheep renal tissue (the renal tissue pluripotent stem cells, RTSC) expression of MHC-Ⅱ, did not express MHC-I, called RTSC-Ⅱ.The RT-PCR analysis showed that AFSC-Ⅰ expressed ESCs marker genes Oct-4, Nanog, primordial germ cells(EGCs) marker genes SSEA-1and mesenchymal stem cells(MSCs) marker genes CD117, SH2. They expressed MHC-Ⅰ and did not express MHC-Ⅱ. Real time PCR analysis showed that both AFSC-Ⅱ and RTSC-Ⅱ expressed genes of Oct-4, SSEA-1, MHC-Ⅱ, Bax, Bcl-2, and did not express genes of MHC-Ⅰ, Nanog, and Tert. Gene expression relative quantification analysis showed that, Oct-4, SSEA-1and Bax/Bcl-2genes expression in the AFSC-Ⅱ was1.8,1.12, and2fold of RTSC-II respectively. In AFSC-Ⅱ, Bax, Bcl-2gene relative expression levels is62,63.7folds of SSEA-1, Bax/Bcl-2is about0.97. In RTSC-Ⅱ, Bax, Bcl-2gene relative expression levels is133、105folds of Oct4, Bax/Bcl-2is about1.3. All AFSC-Ⅰ, AFSC-Ⅱ and RTSC-IIcan form embryoid bodies in vitro after suspension culturing7days. RT-PCR detection results showed that embryoid bodies express the three germ layers specific marker genes of fgf5(ectoderm), a-fetoprotein (within the embryo layer) and ξ-globin (in embryo layer). AFSC-Ⅰ, AFSC-Ⅱ, and RTSC-IIcan be cultured to40,10and10generations respectively. Karyotype testing showed that the normal rate is of94%,88%,90%in AFSC-Ⅰ, AFSC-Ⅱ and RTSC-Ⅱ. Abnormal karyotype is chromosome number doubling.pCMV-DsRed reporter plasmids were transfected to AFSC-Ⅱ and RTSC-Ⅱ by liposome-mediated transfection. Flow cytometry analysis results showed that the gene transient transfection efficiency were68.61%,69.17%. Transgenic AFSC-IIand RTSC-Ⅱ was isolated through the6mg/mL G418selection.Both transgenic AFSC-IIand RTSC-IIcan form embryoid bodies in vitro after suspension culturing7days. RT-PCR analysis showed that transgenic AFSC-Ⅱ and RTSC-Ⅱ express genes of Oct-4, SSEA-1, Bax, Bcl-2and MHC-Ⅱ, except MHC-Ⅰ, Nanog and Tert. In transgenic AFSC-Ⅱ, SSEA-1gene expression increased significantly, Oct-4and Bax expression decreased slightly, while Bcl-2was significantly decreased. In transgenic RTSC-Ⅱ, Oct-4gene expression is13folds of wild type, SSEA-1gene expression is normal, while Bax and Bcl-2gene expression decreased significantly. Relative quantitative analysis results showed that, Bax, Bcl-2relative expression levels is53.4、36.6folds of SSEA-1,Bax/Bcl-2is about1.5in AFSC-II after transfection, Bax, Bcl-2relative expression levels is5.5,3.1folds of Oct4, Bax/Bcl-2is about1.8in RTSC-Ⅱ after transfection.The results shown above, the biological characteristics of sheep amniotic fluid stem cells similar to embryonic stem cells, but they differ from embryonic stem cells. Meanwhile, the body tissues have similar amniotic fluid stem cells exist. This means that the amniotic fluid stem cells may play an important role in the body tissues of human and animals.Therefore, the biological characteristics study of amniotic fluid stem cells has important theoretical and practical significance.