Efficient and Controlled Generation of 2D and 3D Bile Duct Tissue from Human Pluripotent Stem Cell-Derived Spheroids

Abstract

While in vitro liver tissue engineering has been increasingly studied during the last several years, presently engineered liver tissues lack the bile duct system. The lack of bile drainage not only hinders essential digestive functions of the liver, but also leads to accumulation of bile that is toxic to hepatocytes and known to cause liver cirrhosis. Clearly, generation of bile duct tissue is essential for engineering functional and healthy liver. Differentiation of human induced pluripotent stem cells (iPSCs) to bile duct tissue requires long and/or complex culture conditions, and has been inefficient so far. Towards generating a fully functional liver containing biliary system, we have developed defined and controlled conditions for efficient 2D and 3D bile duct epithelial tissue generation. A marker for multipotent liver progenitor in both adult human liver and ductal plate in human fetal liver, EpCAM, is highly expressed in hepatic spheroids generated from human iPSCs. The EpCAM high hepatic spheroids can, not only efficiently generate a monolayer of biliary epithelial cells (cholangiocytes), in a 2D differentiation condition, but also form functional ductal structures in a 3D condition. Importantly, this EpCAM high spheroid based biliary tissue generation is significantly faster than other existing methods and does not require cell sorting. In addition, we show that a knock-in CK7 reporter human iPSC line generated by CRISPR/Cas9 genome editing technology greatly facilitates the analysis of biliary differentiation. This new ductal differentiation method will provide a more efficient method of obtaining bile duct cells and tissues, which may facilitate engineering of complete and functional liver tissue in the future.

Keywords: 3D tissue engineering; Ductal differentiation; Genome editing; Induced pluripotent stem cells; Liver progenitor; Spheroids.

Figure 1. Generation of EpCAM^high hepatic spheroids from human PSCs

(A) A schematic diagram of ductal differentiation procedure. (B) Human iPSC derived hepatic spheroids. The human iPSC were differentiated into definitive endoderm (DE) and hepatic progenitor (HP) stage cells, and were subsequently cultured in a low-attachment culture dish for 3 to 5 days with CHIR99021, SB431542 and nicotinamide to support hepatic spheroid formation. (C, D) These hepatic spheroids expressed significantly higher levels of EpCAM, a bipotent liver progenitor marker, compared to other differentiation stages, by both protein and gene analyses (see Fig 2). The hepatic spheroids also expressed AFP, a hepatoblast marker. (D) Flow cytometric analysis shows the hepatic spheroids are enriched with exclusively EpCAM high cells. Scale Bar, 100μm

Figure 2. Generation of ductal epithelial cells in a 2D culture condition

(A) Immunofluorescence analyses of bile duct epithelial cells in a 2D differentiation culture of human iPSC-derived hepatic spheroids. When the hepatic spheroids were further attached to a regular cell culture dish for 5 or more days in EGF containing media, they were induced into monolayers of biliary epithelial cells with high (over 90%) efficiency. These cells expressed multiple bile duct cell markers. (B) Real-time PCR analysis of ductal cell marker genes and genes associated with ductal cell commitment, for each stage of ductal differentiation from undifferentiated human iPSCs. These data suggest that the human iPSC-derived hepatic spheroids efficiently differentiate into bile ductal epithelial cells in a defined 2D differentiation condition. DE: definitive endoderm cells derived from human iPSC/ESC, HP: hepatic progenitor cells derived from human iPSC/ESC, Scale Bar, 100μm. *: p<0.05, **: p<0.01.

Figure 3. Ductal structure formation in a 3D culture condition

(A, B) Ductal structure formation in a Matrigel based 3D culture. To recapitulate the epithelial polarity of bile duct cells in vivo and ductal structure formation, we cultured human iPSC-derived hepatic spheroids in a 3D differentiation condition containing thick Matrigel and EGF/HGF. Within 5 to 10 days in 3D condition, ductal cells formed round cysts with a central luminal space enclosed by a monolayer of cells as well as bile duct-like tubular structures expressing ductal marker proteins. (C) Secretory function of 3D ductal cysts. To determine the secretory function of differentiated 3D ductal structures, we incubated the ductal cysts with rhodamine123, which can be secreted into the luminal space by MDR, an ATP-dependent transmembrane export pump. The fluorescence intensity inside of the ductal cysts was much higher than the surroundings, which demonstrated that rhodamine123 was successfully transported into luminal space by ductal cells. In addition, this effect can be inhibited by verapamil, an MDR inhibitor. These data demonstrate that the iPSC-derived ductal structures in a 3D condition resemble characteristics and functionality of bile duct tissue in vivo. Scale Bar, 100μm

Figure 4. Ductal differentiation using a CK7-mCherry reporter human iPSC line

(A, B) To efficiently determine the kinetics of ductal tissue generation, we have generated a reporter human iPSC line, which is designed to express a ductal marker, CK7, when properly differentiated. The genomic locus of KRT7 (CK7) gene and the reporter construct are shown (A). Using CRISPR/Cas9 system, the mCherry reporter cassette and the puromycin-resistance cassette were inserted at the end of CK7 coding sequence to replace the endogenous “TGA” stop codon in exon 9. A T2A self-cleaving peptide was inserted between CK7 and mCherry. The PGK-Puro selection cassette, flanked by a piggyBac inverted terminal repeat sequences, was removed by transient expression of piggyBac transposase in the iPSC line with targeted integration. This reporter line can express pluripotency markers, including Oct4 and Tra 1–60, before differentiation (B). (C) This CK7 reporter line formed hepatic spheroids based on our ductal differentiation method, (D) expressed cherry signal in the 2D differentiation condition, and (E, F) formed cherry positive ductal cysts and tubes in the 3D culture condition. The time frame for each stage differentiation of this reporter iPSC was consistent with control iPSCs (Fig 1A). These data not only confirm our 2D/3D ductal differentiation protocols but also provide a great resource for kinetic or imaging studies of human bile duct development. Scale Bar, 100μm