Ann Hepatobiliary Pancreat Surg.  2023 Nov;27(4):342-349. 10.14701/ahbps.23-052.

Enhancing generation efficiency of liver organoids in a collagen scaffold using human chemically derived hepatic progenitors

• Kim M
• Kim Y
• Silva ESS
• Adisasmita M
• Kim KS
• Jung YK
• Lee KG
• Shin JH
• Choi D

• KMID: 2548500
• DOI: http://doi.org/10.14701/ahbps.23-052

Abstract

Backgrounds/Aims
Liver organoids have emerged as a powerful tool for studying liver biology and disease and for developing new therapies and regenerative medicine approaches. For organoid culture, Matrigel, a type of extracellular matrix, is the most commonly used material. However, Matrigel cannot be used for clinical applications due to the presence of unknown proteins that can cause immune rejection, batch-to-batch variability, and angiogenesis.
Methods
To obtain human primary hepatocytes (hPHs), we performed 2 steps collagenase liver perfusion protocol. We treated three small molecules cocktails (A83-01, CHIR99021, and HGF) for reprogramming the hPHs into human chemically derived hepatic progenitors (hCdHs) and used hCdHs to generate liver organoids.
Results
In this study, we report the generation of liver organoids in a collagen scaffold using hCdHs. In comparison with adult liver (or primary hepatocyte)-derived organoids with collagen scaffold (hALO_C), hCdH-derived organoids in a collagen scaffold (hCdHO_C) showed a 10-fold increase in organoid generation efficiency with higher expression of liver- or liver progenitor-specific markers. Moreover, we demonstrated that hCdHO_C could differentiate into hepatic organoids (hCdHO_C_DM), indicating the potential of these organoids as a platform for drug screening.
Conclusions
Overall, our study highlights the potential of hCdHO_C as a tool for liver research and presents a new approach for generating liver organoids using hCdHs with a collagen scaffold.

Keyword

Human primary hepatocytes (hPHs); Human chemically derived hepatic progenitors (hCdHs); Collagen organoid

Figure

• 

  Fig. 1 Generation of hCdHs. (A) Schematic of the hPHs culture procedure in vitro using human liver tissue. The viability of hPHs is dependent on the presence of HAC. (B) Bright-field image of hPHs during culture without HAC(-) or with HAC(+) medium on days 0 and 7 and after passage. Scale bar: 100 μm. (C) RT-qPCR of the hepatic marker (ALB) and hepatic progenitor markers (CD44, CD90, ITGA6, and FOXJ1) in hPHs and hCdHs. Data on the expression of each gene marker were normalized against GAPDH. Data were analyzed by a two-tailed t-test. They are presented as mean ± standard error of the mean (SEM) of three individual experiments performed in triplicate. (D) Immunocytochemistry of protein expression of hepatic progenitor markers (E-CAD, EPCAM, and Ki67) and a hepatocyte marker (ALB) in hPHs and hCdHs. hCdHs, human chemically derived hepatic progenitor cells; hPHs, human primary hepatocytes; HGF, hepatocyte growth factor. **p < 0.01, ***p < 0.001.

• 

  Fig. 2 Characterization of hALO_C and hCdHO_C. (A) Bright-field images showing the generation of organoids of different origin hPHs or hCdHs at the generation stage (P0) and after sub-culture (after passage). Scale bar: 500 μm (top) or 100 μm (bottom). (B) Number of cells in hCdHO_C and hALO_C. Both hPHs or hCdHs were seeded into a 24-well cell culture plate with collagen solution. The number of cells in each organoid (hCdHO_C and hALO_C) was counted using a Nikon Eclipse Ti-e microscope. Data were obtained from triplicate experiments for a donor, hALO_C (n = 12) and hCdHO_C (n = 12). (C) Doubling time of hCdHO_C and hALO_C. Organoids were dissociated into single cells and cells were counted to analyze the doubling time for 48 h. (D) RT-qPCR of the hepatic marker (ALB) and hepatic progenitor markers (SOX9, EPCAM, CD44, CK19, and CD90) in hPHs, hALO_C, and hCdHO_C. Data on the expression of each gene marker were normalized against GAPDH. Data were analyzed by a two-tailed t-test. They are presented as mean ± standard error of the mean of three individual experiments performed in triplicate. hPHs, human primary hepatocytes; hALO_C, hPH-derived organoids with collagen scaffold; hCdHs, human chemically derived hepatic progenitors; hCdHO_C, hCdH-derived organoids in a collagen scaffold; ns, not significant. ***p < 0.001.

• 

  Fig. 3 Hepatic differentiation of hCdHO_C (hCdHO_C_DM). (A) Schematic of hepatic differentiation with hALO_C and hCdHO_C. (B) Relative mRNA levels of hepatic markers (ALB, HNF4a, AAT, ASGR1, CK19, CYP1A2, and CYP3A4) in hALO_C, hCdHO_C, hALO_C_DM, and hCdHO_C_DM determined by RT-qPCR. Expression levels of each gene marker in all samples were normalized agasint GAPDH (triplicate experiments). (C) Immunocytochemistry of hepatic markers (CK18 and ASGR1) in hALO_C_DM and hCdHO_C_DM. Scale bar: 50 μm. hCdHs, human chemically derived hepatic progenitors; hCdHO_C, hCdH-derived organoids in a collagen scaffold; DM, differentiation medium; hALO_C, hPH-derived organoids with collagen scaffold; EM, extension medium; ns, not significant. ***p < 0.001.

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