Abstract
The aim of this study was to isolate and characterize porcine amniotic fluid-derived multipotent stem cells (pAF-MSC). The porcine amniotic fluid (AF) from the amniotic cavity of pregnant gilts in the early stages of gestation (at E35) was collected and centrifuged for 5–10 min at 400 g to pellet cells. The primary culture of AF showed the multiple cell types, including the epithelial-like cells and fibroblast-like cells. By culturing in AMM medium for 6 to 8 days, the epithelial-like cells disappeared and the remaining cells presented the fibroblastoid morphology. The doubling time of pAF-MSCs was about 34.6 h, and the cells had been continually cultured over 60 passages in vitro. The flow cytometry results showed that pAF-MSCs were positive for CD44, CD117 and CD166, but negative for CD34, CD45 and CD54. Meanwhile, pAF-MSCs expressed ES cell markers, such as Oct4, Nanog, SSEA4, Tra-1-60 and Tra-1-81. The ratio of CD117+ CD44+ cells accounted for 98% of pAF-MSCs population. Three germ layer markers, including FGF5 (ectodermal marker), AFP (endodermal marker) and Bra (mesodermal marker), were detected in embryoid bodies derived from pAF-MSCs. Under the different induction conditions, the pAF-MSCs were capable of differentiating into neurocytes, adipocytes and beating cardiomyocytes. Furthermore, the pAF-MSCs didn't form teratoma when injected into immunodeficiency mice. These optimal features of pAF-MSCs provide an excellent alternative stem cell resource for potential cell therapy in regenerative medicine and transgenic animals.
Highlights
Amniotic fluid (AF) is composed of normal embryonic or fetal chipping cells derived from the three germ layers [1,2]
The porcine amniotic fluid-derived multipotent stem cells (pAF-MSC) isolated from porcine amniotic fluid The porcine AF was collected from amniotic cavity in the early stage gestation of porcine fetus
For 2 to 3 passages, most cells exhibited fibroblast-like phonotype (Fig. 1B), and occasionally, some ES cell-like colonies were observed and these colonies were positive for alkaline phosphatase (AP) staining (Fig. 1C)
Summary
Amniotic fluid (AF) is composed of normal embryonic or fetal chipping cells derived from the three germ layers (ectoderm, endoderm and mesoderm) [1,2]. It possesses the natural precursors of all differentiation lineages. AF has been used as a source of human mesenchymal stem cells (hMSCs) that express the specific markers, such as CD90, CD105, CD73 and CD166 [3,4,5,6]. Over 90% of hAFSCs express Oct (the specific marker of ES cells). The AFS may be an intermediate type of cells between ES cells and adult stem cells
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