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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

Abstract

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4α nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

Introduction

The liver is a highly metabolic organ that plays several roles, including deoxidation, storing glycogen, and secretion and synthesis of proteins1. Various pathogens, drugs and heredity can cause pathological changes in the liver and affect its functions2,3. Hepatocytes, as the main functional unit of the liver, play an important role in artificial liver support systems and drug toxicity elimination. However, the resource of primary human hepatocytes is limited in cell-based therapy, as well as in liver disease research. Therefore, developing new sources of functional human hepatocytes i....

Protocol

1. Stem cell maintenance

NOTE: The cell maintenance protocol described below applies to the hES03 cell line maintained in an adherent monolayer. For all the following protocols in this manuscript, cells should be handled under a biological safety cabinet.

  1. Prepare 1x mTesR stem cell culture medium by diluting 5x supplementary medium to mTesR basic medium.
  2. Prepare 30x hESC-qualified Matrigel medium by diluting 5 mL of hESC-qualified Matrigel with 5 mL of DMEM/F12 on the ice.......

Representative Results

The schematic diagram of HLC induction from hESCs and representative bright-field images of each differentiation stage are shown in Figure 1. In Stage I, Activin A and CHIR99021 were added for 3 days to induce stem cells to form endoderm cells. In Stage II, the endoderm cells differentiated into hepatic progenitor cells after being treated with differentiation medium for 5 days. In Stage III, early hepatocytes had matured and differentiated into HLCs after 10 days in HGF and OSM (

Discussion

Here, we present a stepwise method that induces HLCs from hESCs in three stages. In the first stage, Activin A and CHIR99021 were used to differentiate hESCs into DE. In the second stage, KO-DMEM and DMSO were used to differentiate DE into hepatic progenitor cells. In the third stage, HZM plus HGF, OSM and hydrocortisone 21-hemisuccinate sodium salt were used to continue to differentiate hepatic progenitor cells into HLCs.

The following critical steps need to be taken into consideration w.......

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81870432 and 81570567 to X.L.Z.), (No. 81571994 to P.N.S.); the Natural Science Foundation of Guangdong Province, China (No. 2020A1515010054 to P.N.S.), The Li Ka Shing Shantou University Foundation (No. L1111 2008 to P.N.S.). We would like to thank Prof. Stanley Lin from Shantou University Medical College for useful advice.

....

Materials

NameCompanyCatalog NumberComments
2-MercaptoethanolSigmaM7522For hepatic progenitor differentiation
488 labeled goat against mouse IgGZSGB-BIOZF-0512For IF,second antibody
488 labeled goat against Rabbit IgGZSGB-BIOZF-0516For IF,second antibody
AccutaseStem Cell Technologies7920For cell passage
Activin ApeproTech120-14EFor definitive endoderm formation
Anti - Albumin (ALB)Sigma-AldrichA6684For IF and WB, primary antibody
Anti - Human Oct4AbcamAb19587For IF, primary antibody
Anti - α-Fetoprotein (AFP)Sigma-AldrichA8452For IF and WB, primary antibody
Anti -SOX17Abcamab224637For IF, primary antibody
Anti-Hepatocyte Nuclear Factor 4 alpha (HNF4α)Sigma-AldrichSAB1412164For IF, primary antibody
B-27 SupplementGibco17504-044For definitive endoderm formation
BSABeyotimeST023-200gFor cell blocking
CHIR99021Sigma-AldrichSML1046For definitive endoderm formation
DAPIBeyotimeC1006For nuclear staining
DEPC-waterBeyotimeR0021For RNA dissolution
DM3189MCEHY-12071For definitive endoderm formation
DMEM/F12Gibco11320-033For cell culture
DMSOSigma-AldrichD5879For hepatic progenitor differentiation
DPBSGibco14190-144For cell culture
GlutaMAXGibco35050-061For hepatic progenitor differentiation
H&E staining kitBeyotimeC0105SFor H&E staining
Hepatocyte growth factor (HGF)peproTech100-39For hepatocyte differentiation
HepatoZYME-SFM (HZM)Gibco17705-021For hepatocyte differentiation
Hydrocortisone-21-hemisuccinateSigma-AldrichH4881For hepatocyte differentiation
IndocyanineSangon BiotechA606326For Indocyanine staining
Knock Out DMEMGibco10829-018For hepatic progenitor differentiation
Knock Out SR Multi-SpeciesGibcoA31815-02For hepatic progenitor differentiation
Matrigel hESC-qualifiedCorning354277For cell culture
MEM NeAAGibco11140-050For hepatic progenitor differentiation
mTesR 5X SupplementStem Cell Technologies85852For cell culture
mTesR Basal MediumStem Cell Technologies85851For cell culture
Oncostatin (OSM)peproTech300-10For hepatocyte differentiation
P450 - CYP3A4 (Luciferin - PFBE)PromegaV8901For CYP450 activity
PAS staining kitSolarbioG1281For PAS staining
Pen/StrepGibco15140-122For cell differentiation
Peroxidase-Conjugated Goat anti-Mouse IgGZSGB-BIOZB-2305For WB,second antibody
Primary Antibody Dilution Buffer for Western BlotBeyotimeP0256For primary antibody dilution
ReverTraAce qPCR RT KitTOYOBOFSQ-101For cDNA Synthesis
RNAiso PlusTaKaRa9109For RNA Isolation
RPMI 1640Gibco11875093For definitive endoderm formation
Skim milkSangon BiotechA600669For second antibody preparation
SYBR Green Master MixThermo Fisher ScientificA25742For RT-PCR Analysis
Torin2MCEHY-13002For definitive endoderm formation
TweenSigma-AldrichWXBB7485VFor washing buffer preparation

References

  1. Hou, Y., Hu, S., Li, X., He, W., Wu, G. Amino Acid Metabolism in the Liver: Nutritional and Physiological Significance. Advances in Experimental Medicine and Biology. 1265, 21-37 (2020).
  2. Huang, C., Li, Q., Xu, W., Chen, L.

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Human Embryonic Stem CellsHepatocyte like CellsDifferentiationDefinitive EndodermFOXA2SOX17GATA4CXCR4FOXA1MatrigelMTESRActivin ACHIR99021Cell CultureDisease ModelingDrug Screening

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