Hepatitis B virus infection modeling using multi-cellular organoids derived from human induced pluripotent stem cells

Table 1 Current modeling strategies and applications of human multi-cellular liver organoids

Model/application	Multiple cells (ratio)	Culture system	Advances/significance	Limitations	Ref.
ALF	iPSC-HEs, HUVECs, BM-MSCs (10:8:2)	3D, Matrigel	Multi-cellular LOs with vascularization	Low reproducibility; Time-consuming	2013[65]
ALF	HEs, MCs, ECs (all from iPSCs) (10:8:2)	3D, ULA	All-iPSC-based strategy	Time-consuming; High cost	2017[16]
ALF	iPSC endoderm cells, HUVECs, UC-MSCs (10:7:1)	3D, ULA	LOs generated from single donor-derived cells	Low reproducibility; Time-consuming	2018[75]
ALF	iPSCs, HAMECs (3:1)	EB, Agarose	HAMECs improved hepatic functions	Unable to reflect the nature cellular composition of liver	2019[102]
Liver fibrosis	HepaRG, THP-1, hTERT-HSC	3D, Hanging drop	LOs derived from cell lines	Functional deficiency	2017[103]
Liver fibrosis and steatohepatitis	PHHs, KCs, HSCs, SECs (16:2:1:1)	3D, ULA	LOs derived from primary cell sources	Low reproducibility; High cost	2018[104]
Steatohepatitis	Hepatocytes, HSCs, BCs, KCs (all from iPSC)	3D, ULA	Co-differentiation of multiple cell lineages for iPSC- LOs	Functions undetermined; Potential inter/intra-batch variability	2019[51]
HBV infection ex vivo	iPSC endoderm cells, HUVECs, BM- MSCs (10:7:1)	3D, ULA	Validation of advantages of iPSC-LOs in HBV modeling	Low reproducibility; Time-consuming	2018[15]
Hepatic differentiation	iPSC-HEs, MSCs, HUVECs (10:2:7)	3D, Matrigel	Platform to identify developmental paracrine signals involved in hepatocyte differentiation	Low reproducibility; Time-consuming	2017[105]
Hepatic differentiation	iHEPs, ECs, HSCs, cholangiocytes (10:7:2:1)	3D, ULA	Cholangiocytes impaired the hepatic functions in LOs and were associated with the liver disease relevant phenotype	Cholangiocyte activation in LOs was unclear	2019[106]
Liver development and angiogenesis	Hepatocytes, BCs, ECs, HSCs (all from iPSC)	3D, Matrigel	Engineered iPSC-LOs by programming of the gene regulatory network	Not completed for liver functions	2021[66]

ALF: Acute liver failure; BCs: Biliary cells; BM: Bone marrow; EB: Embryoid body; ECs: Endothelial cells; HAMECs: Human adipose microvascular endothelial cells; HBV: Hepatitis B virus; HEs: Hepatic endoderm cells; HSCs: Hepatic stellate cells; HUVECs: Human umbilical vein endothelial cells; iHEPs: Induced hepatocytes; KCs: Kupffer cells; Los: Liver organoids; MCs: Mesenchymal cells; MSCs: Mesenchymal stem cells; NPCs: Non-parenchymal cells; PHHs: Primary human hepatocytes; SECs: Sinusoidal endothelial cells; UC: Umbilical cord; ULA: Ultralow adhesion microwell plate.

Table 2 Up-to-date methods for obtaining expandable human liver organoids

Initial cells	Expansion systems (substrate for embedding)	Medium	Expansion capability (Split ratio/passage days/expansion duration)	Ref.
Fetal and adult hepatocytes	3D, Matrigel	AdDMEM/F12, B27, N-Acetylcysteine, gastrin, RSPO1, Noggin, Wnt, EGF, FGF7, FGF10, HGF, TGFa, Nicotinamide, A83-01, CHIR99021 and Y27632	1:3/7-8 d/> 16 passages	2018[70]
EpCAM+ bile duct cells	3D, Matrigel or BME gel	AdDMEM/F12, N2, B27, N-Acetylcysteine, gastrin, RSPO1, Noggin (d0-3), Wnt (d0-3), EGF, FGF10, FGF19, HGF, Nicotinamide, A83-01, FSK, and Y27632 (d0-3)	1:4-1:8/7-10 d/6 mo	2015[69]
EpCAM+ bile duct cells	3D, BME gel	AdDMEM/F12, B27, N-Acetylcysteine, gastrin, RSPO1, Noggin (d0-3), Wnt (d0-3), EGF, FGF10, FGF19, HGF, Nicotinamide, A83-01, FSK, and Y27632 (d0-3)	1:5/7-10 d/> 6 mo	2019[107]
iPSC derived EpCAM+ hepatic progenitors	3D, Matrigel	AdDMEM/F12, B27, N-Acetylcysteine, gastrin, RSPO1, Noggin (d0-3), Wnt (d0-3), EGF, FGF10, HGF, Nicotinamide, A83-01, FSK and Y27632 (d0-3)	1:4-1:8/7-10 d/9-12 mo	2019[71]
PSC-derived hepatocytes	3D, Matrigel	AdDMEM/F12, N2, B27, N-Acetylcysteine, gastrin, RSPO1, EGF, FGF10, HGF, Nicotinamide, A83-01, FSK	1:3-1:10/7 d/3 mo	2019[72]
PSCs transduced with PROX1, ATF5 and CYP3A4	3D, Matrigel	APEL medium	NA/10 d/17 d	2021[66]

APEL: A commercial medium from Stem Cell Technologies; BME: Basement membrane extract; bFGF: Basic fibroblast growth factor; BME: Basement membrane extract; EGF: Epidermal growth factor; FGF: Fibroblast growth factor; FSK: Forskolin; HGF: Hepatocyte growth factor; iPSCs: Induced pluripotent stem cells; PSCs: Including embryonic stem cells and induced pluripotent stem cells; RSPO1: R-spodin1.