Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Sep 26, 2019; 11(9): 705-721
Published online Sep 26, 2019. doi: 10.4252/wjsc.v11.i9.705
Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment
Kinji Furuya, Yun-Wen Zheng, Daisuke Sako, Kenichi Iwasaki, Dong-Xu Zheng, Jian-Yun Ge, Li-Ping Liu, Tomoaki Furuta, Kazunori Akimoto, Hiroya Yagi, Hiromi Hamada, Hiroko Isoda, Tatsuya Oda, Nobuhiro Ohkohchi
Kinji Furuya, Yun-Wen Zheng, Daisuke Sako, Kenichi Iwasaki, Dong-Xu Zheng, Jian-Yun Ge, Li-Ping Liu, Tomoaki Furuta, Tatsuya Oda, Nobuhiro Ohkohchi, Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
Yun-Wen Zheng, Li-Ping Liu, Institute of Regenerative Medicine and Affiliated Hospital, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
Yun-Wen Zheng, Department of Regenerative Medicine, School of Medicine, Yokohama City University, Yokohama 236-0004, Japan
Daisuke Sako, Kazunori Akimoto, Department of Medicinal and Life Sciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda 278-8510, Japan
Hiroya Yagi, Hiromi Hamada, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan
Hiroko Isoda, Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan
Author contributions: Furuya K, Sako D, Iwasaki K, Ge JY, Liu LP and Furuta T performed the experiments; Furuya K, Sako D and Iwasaki K analyzed the data; Zheng DX performed bioinformatics; Akimoto K, Yagi H, Hamada H, Isoda H supplied experimental materials and resources; Ohkohchi N and Zheng YW conceived the study; Furuya K drafted the manuscript; Zheng YW and Oda T contributed to discuss and review the final manuscript; all the authors approved the final manuscript; Furuya K, Zheng YW and Sako D contributed equally to this work.
Supported by National Natural Science Foundation of China, No. 81770621; Ministry of Education, Culture, Sports, Science, and Technology of Japan, KAKENHI, No. 16K15604, No. 18H02866; Japan Science and Technology Agency-Japan International Cooperation Agency's (JST-JICA) Science and Technology Research Partnership for Sustainable Development (SATREPS) Project.
Institutional review board statement: All specimens and cells from the patients were obtained after their informed consent and ethical permission was obtained for participation in the study.
Conflict-of-interest statement: The authors report no relevant conflicts of interest.
Data sharing statement: Transcriptome datasets of primary amniotic epithelial cells from the Sequence Read Archive (SRA) of the NCBI are available in the website. SRA number is listed in Table 2. Participants gave informed consent for publication.
Open-Access: 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/
Corresponding author: Yun-Wen Zheng, PhD, Associate Professor, Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, University of Tsukuba Faculty of Medicine, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan. ywzheng@md.tsukuba.ac.jp
Telephone: +81-29-8533221 Fax: +81-29-8533222
Received: February 26, 2019
Peer-review started: February 27, 2019
Revised: August 6, 2019
Accepted: August 27, 2019
Article in press: August 27, 2019
Published online: September 26, 2019
Processing time: 211 Days and 2.6 Hours
ARTICLE HIGHLIGHTS
Research background

Cell transplantation is a promising method to solve the problem of organ donor deficiency. Recently, amniotic epithelial cells (AECs) have been studied as a somatic stem cell source for regenerative medicine. It has been reported that AECs possess hepatic differentiation capability and they are more cost-effective and non-tumorigenic compared to other cell types. Therefore, AECs could provide a new cell source for cell transplantation in the future, particularly for liver diseases.

Research motivation

However, the general profile for AECs has not been comprehensively analyzed and hepatic differentiation protocol for AECs has also not been established. Therefore, by clarifying the comprehensive characteristics of AECs and refining the hepatic differentiation protocol, we could use it effectively as a transplantation cell source in the future.

Research objectives

This study aimed to elucidate the comprehensive characteristics of human AECs, and to establish a novel hepatic differentiation protocol. Additionally, 3D multicellular culture condition and purification of stem cells obtained from AECs were studied, with an intention to improve their differentiation capability.

Research methods

All examined AECs, mesenchymal stem cells (MSCs), and human umbilical vein endothelial cells (HUVECs) were isolated from the placentae and umbilical cords after cesarean section. Stemness characteristics and heterogeneity of amnion and primary AECs were analyzed by immunofluorescence and AP staining, and flow cytometry. In addition, AEC transcriptomes were analyzed and compared with those for other cell sources based on bioinformatics. An adherent AEC subpopulation was selected from primary isolated AECs and their amniotic stem cell (ASC) purification quality was evaluated based on a colony formation assay. Hepatic differentiation capacities of AECs which were cultured in varying 2D or 3D conditions were compared according to their relative gene expression. Finally, ASCs, MSCs and HUVECs were co-cultured in a 3D system to generate hepatic organoids, and the organoid structure and hepatic function were compared with those of 3D AEC spheres using immunofluorescence imaging, Periodic acid Schiff staining, and an indocyanine green (ICG) test.

Research results

AECs expressed stemness markers such as EPCAM, SSEA4, and E-cadherin, whereas only limited cells in the AEC subpopulation were AP-positive, or expressed TRA-1-60 and TRA-1-81 stemness marker in the flow cytometry. The colony formation assay revealed that primary AECs could form colonies and the frequency was enhanced ten-fold in the adherent subpopulation. According to bioinformatics analysis of RNA sequencing, the primary AEC gene expression was different from those of pluripotent stem cells and hepatocytes; however, some overlapped genes were detected. Compared with the 2D system, AECs could retain their viability for a longer time in 3D culture conditions and the hepatic gene expressions were comparatively elevated using a two-step differentiation protocol. Furthermore, organoids derived from 3D multicellular culture condition using ASCs, MSCs and HUVECs, showed a 3D hepatic structure with polarity, hepatic-like glycogen storage, and ICG absorption/elimination.

Research conclusions

Human AECs are heterogeneous and certain subpopulations exhibit high stemness. AEC transcriptome analysis suggests some advantages and factors related to hepatic differentiation. 3D multicellular culture conditions improve the differentiation of ASCs into functional hepatic organoids.

Research perspectives

The results of this comprehensive analysis indicate that AECs have high hepatic differentiation capability which should be optimized. By optimizing the selected high stemness subpopulations of AECs and using a 3D co-culture system, AEC-derived hepatic organoid can be used for performing transplantation experiments to evaluate its in vivo function.