Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment

Abstract

BACKGROUNDTo 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.AIMTo characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cellsMETHODSAECs, 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.RESULTSAECs 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.CONCLUSIONHuman amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness. Under a 3D co-culture system, functional hepatic organoids were generated in...

Effects of Isorhamnetin in Human Amniotic Epithelial Stem Cells in vitro and Its Cardioprotective Effects in vivo

“…One of the major obstacles of translating hAESCs into the clinic is the heterogeneity in primary amnion epithelial cell population and discrepancies in their cell surface profiling (Centurione et al, 2018) that can be primarily attributed to isolation protocols, gestational age, passage number, and epithelial to mesenchymal transition (Ghamari et al, 2020). However, hAESCs isolated from the adherent subpopulations of passaged primary cells, and cultured in 3D environment may successfully overcome these limitations and may express a higher level of stemness properties (Ferdousi et al, 2019;Furuya et al, 2019). Additionally, protocol for immortalized human amniotic epithelial cells with high expressions of stem cell markers has been reported (Zhou et al, 2013).…”

Global Gene Expression Profiling Reveals Isorhamnetin Induces Hepatic-Lineage Specific Differentiation in Human Amniotic Epithelial Cells

“…Furthermore, studies have reported hepatic differentiation potentials of hAECs using multistep proliferation and differentiation protocols involving several combinations of growth factors and cytokines (Marongiu et al, 2011;Lin et al, 2015;Liu et al, 2018;Maymó et al, 2018). In our previous study, we have reported that the hAECs isolated from the adherent subpopulations of passaged primary cells have widely expressed stemness markers (Furuya et al, 2019). We have also shown that hAECs cultured in a 3D microenvironment as spheroids have highly expressed the stemness-related genes compared to their 2D counterpart (Ferdousi et al, 2019).…”

“…In our previous studies, a detailed methodology of hAEC extraction, culture, and 3D spheroid formation have been documented (Ferdousi et al, 2019;Furuya et al, 2019). Briefly, hAECs were extracted from the term placenta.…”

“…At this time, ICG uptake was detected by light microscopy. ICG clearance was detected after 6 h following previous protocols (Lin et al, 2015;Furuya et al, 2019).…”

“…We have also shown that hAECs cultured in a 3D microenvironment as spheroids have highly expressed the stemness-related genes compared to their 2D counterpart (Ferdousi et al, 2019). In 3D culture of hAECs, we could generate functional hepatic organoids using several growth factors (Furuya et al, 2019).…”