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Clinical Trial
. 2014 Aug;16(4):298-305.
doi: 10.1089/cell.2013.0090.

Characterization of amniotic stem cells

Chika Koike  1 , Kaixuan Zhou, Yuji Takeda, Moustafa Fathy, Motonori Okabe, Toshiko Yoshida, Yukio Nakamura, Yukio Kato, Toshio Nikaido
Affiliations
Clinical Trial

Characterization of amniotic stem cells

Chika Koike et al. Cell Reprogram. 2014 Aug.

Abstract

The amnion membrane is developed from embryo-derived cells, and amniotic cells have been shown to exhibit multidifferentiation potential. These cells represent a desirable source for stem cells for a variety of reasons. However, to date very few molecular analyses of amnion-derived cells have been reported, and efficient markers for isolating the stem cells remain unclear. This paper assesses the characterization of amnion-derived cells as stem cells by examining stemness marker expressions for amnion-derived epithelial cells and mesenchymal cells by flow cytometry, immunocytochemistry, and quantitative PCR. Flow cytometry revealed that amnion epithelial cells expressed CD133, CD 271, and TRA-1-60, whereas mecenchymal cells expressed CD44, CD73, CD90, and CD105. Immunohistochemistry showed that both cells expressed the stemness markers Oct3/4, Sox2, Klf4, and SSEA4. Stemness genes' expression in amnion epithelial cells, mesenchymal cells, fibroblast, bone marrow-derived mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) was compared by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Amnion-derived epithelial cells and mesenchymal cells expressed Oct3/4, Nanog, and Klf4 more than bone marrow-derived MSCs. The sorted TRA1-60-positive cells expressed Oct3/4, Nanog, and Klf4 more than unsorted cells or TRA1-60-negative cells. TRA1-60 can be a marker for isolating amnion epithelial stem cells.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Morphology of HAE and HAM cells. HAE and HAM cells are stained with anti-pan-CK or vimentin antibodies, respectively (green). Nuclear staining was performed with Hoechst (blue).
<b>FIG. 2.</b>
FIG. 2.
Histograms of flow cytometric analysis against TRA-1-60, TRA-1-81, SSEA3, SSEA4, CD24, CD73, CD90, and CD105 using HAE and HAM cells.
<b>FIG. 3.</b>
FIG. 3.
HAEs and HAMs are stained with anti-Oct3/4, Sox2, Klf4, c-Myc, SSEA3, or SSEA4, antibodies, respectively (green). Nuclear staining was performed with Hoechst (blue).
<b>FIG. 4.</b>
FIG. 4.
Quantitative RT-PCR was performed using cDNAs from fibroblasts, MSCs, iPSCs, HAE cells, or HAM cells. Relative expression patterns compared with the expression level of fibroblasts are shown. Bars, mean±standard error (SE). Statistical significance: (*) p<0.05, (**) p<0.01 compared to fibroblasts, (#) p<0.05, (##) p<0.01 compared to iPSCs are shown. (A) MSC markers, MMP1, ApoD, LIF, or MGP. (B) Stemness markers Oct3/4, Sox2, Klf4, c-Myc, Nanog, or Lin28.
<b>FIG. 5.</b>
FIG. 5.
(A) HAEs were sorted by MACS beads twice using anti-TRA-1-60 antibody. (B) Quantitative RT-PCR was performed using cDNAs from HAEs, TRA-1-60–negative cells, and TRA-1-60–positive cells. Relative expression patterns compared with the expression level of HAEs are shown. (*) p<0.05, (**) p<0.01 compared to unsorted cells are shown.
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