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We focused on amniotic epithelial cells (AECs) as a cell source because, unlike induced pluripotent stem cells, they are cost-effective and non-tumorigenic. The utilization of AECs in regenerative medicine, however, is in its infancy. A general profile for AECs has not been comprehensively analyzed. Moreover, no hepatic differentiation protocol for AECs has yet been established. To this end, we independently compiled human AEC libraries, purified amniotic stem cells (ASCs), and co-cultured them with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cell (HUVECs) in a 3D system which induces functional hepatic organoids.\n\nAIM\nTo characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells\n\nMETHODS\nAECs, MSCs, and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients. Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry, Alkaline phosphatase (AP) staining, and flow cytometry. An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay. AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics. The 2D and 3D culture were compared by relative gene expression using several differentiation protocols. ASCs, MSCs, and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging, periodic acid Schiff, and an indocyanine green (ICG) test.\n\nRESULTS\nAECs have certain stemness markers such as EPCAM, SSEA4, and E-cadherin. One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers. Moreover, it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage. Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells. TJP1, associated with epidermal growth factor receptor, and MET, associated with hepatocyte growth factor receptor, were upregulated and may be important for hepatic differentiation. In conventional flat culture, the cells turned unviable and did not readily differentiate into hepatocytes. In 3D culture, however, hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol. Furthermore, the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity, hepatic-like glycogen storage, and ICG absorption/elimination.\n\nCONCLUSION\nHuman amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness. 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Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment
http://hdl.handle.net/2241/00159458
http://hdl.handle.net/2241/001594584b3b6ad0-15f6-4d43-8209-7097854c3344
名前 / ファイル | ライセンス | アクション |
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WJSC_11-9-705 (8.1 MB)
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Item type | Journal Article(1) | |||||||||||
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公開日 | 2020-01-21 | |||||||||||
タイトル | ||||||||||||
言語 | en | |||||||||||
タイトル | Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment | |||||||||||
言語 | ||||||||||||
言語 | eng | |||||||||||
資源タイプ | ||||||||||||
資源 | http://purl.org/coar/resource_type/c_6501 | |||||||||||
タイプ | journal article | |||||||||||
著者 |
鄭, 允文
× 鄭, 允文× 八木, 洋也× 濱田, 洋実× 礒田, 博子× 小田, 竜也× 大河内, 信弘
WEKO
131
× Furuya, Kinji× Sako, Daisuke× Iwasaki, Kenichi× Zheng, Dong-Xu× Ge, Jian-Yun× Liu, Li-Ping× Furuta, Tomoaki× Akimoto, Kazunori |
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抄録 | ||||||||||||
内容記述タイプ | Abstract | |||||||||||
内容記述 | BACKGROUND To solve the problem of liver transplantation donor insufficiency, an alternative cell transplantation therapy was investigated. We focused on amniotic epithelial cells (AECs) as a cell source because, unlike induced pluripotent stem cells, they are cost-effective and non-tumorigenic. The utilization of AECs in regenerative medicine, however, is in its infancy. A general profile for AECs has not been comprehensively analyzed. Moreover, no hepatic differentiation protocol for AECs has yet been established. To this end, we independently compiled human AEC libraries, purified amniotic stem cells (ASCs), and co-cultured them with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cell (HUVECs) in a 3D system which induces functional hepatic organoids. AIM To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells METHODS AECs, MSCs, and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients. Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry, Alkaline phosphatase (AP) staining, and flow cytometry. An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay. AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics. The 2D and 3D culture were compared by relative gene expression using several differentiation protocols. ASCs, MSCs, and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging, periodic acid Schiff, and an indocyanine green (ICG) test. RESULTS AECs have certain stemness markers such as EPCAM, SSEA4, and E-cadherin. One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers. Moreover, it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage. Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells. TJP1, associated with epidermal growth factor receptor, and MET, associated with hepatocyte growth factor receptor, were upregulated and may be important for hepatic differentiation. In conventional flat culture, the cells turned unviable and did not readily differentiate into hepatocytes. In 3D culture, however, hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol. Furthermore, the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity, hepatic-like glycogen storage, and ICG absorption/elimination. CONCLUSION Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness. Under a 3D co-culture system, functional hepatic organoids were generated in a multicellular microenvironment. |
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書誌情報 |
en : World Journal of Stem Cells 巻 11, 号 9, p. 705-721, 発行日 2019-09 |
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収録物識別子タイプ | ISSN | |||||||||||
収録物識別子 | 1948-0210 | |||||||||||
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識別子タイプ | PMID | |||||||||||
関連識別子 | 31616545 | |||||||||||
DOI | ||||||||||||
識別子タイプ | DOI | |||||||||||
関連識別子 | 10.4252/wjsc.v11.i9.705 | |||||||||||
権利 | ||||||||||||
権利情報 | ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved. | |||||||||||
権利 | ||||||||||||
権利情報 | This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/ | |||||||||||
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値 | publisher | |||||||||||
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出版者 | Baishideng Publishing Group |