2024-03-29T12:25:21Z
https://tsukuba.repo.nii.ac.jp/oai
oai:tsukuba.repo.nii.ac.jp:00054041
2022-04-27T09:28:10Z
160:523
2780:1493
2780:2612
2780:536
2780:5416
2780:5608
3:62:5612:7778
Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment
鄭, 允文
テイ, インブン
ZHENG, Yunwen
八木, 洋也
ヤギ, ヒロヤ
YAGI, Hiroya
濱田, 洋実
ハマダ, ヒロミ
HAMADA, Hiromi
礒田, 博子
イソダ, ヒロコ
ISODA, Hiroko
小田, 竜也
オダ, タツヤ
ODA, Tatsuya
大河内, 信弘
オオコウチ, ノブヒロ
OHKOHCHI, Nobuhiro
Furuya, Kinji
Sako, Daisuke
Iwasaki, Kenichi
Zheng, Dong-Xu
Ge, Jian-Yun
Liu, Li-Ping
Furuta, Tomoaki
Akimoto, Kazunori
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.
journal article
Baishideng Publishing Group
2019-09
application/pdf
World Journal of Stem Cells
9
11
705
721
1948-0210
https://tsukuba.repo.nii.ac.jp/record/54041/files/WJSC_11-9-705.pdf
eng
31616545
10.4252/wjsc.v11.i9.705
©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
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/