Hepatic differentiation of human iPSCs in different 3D models: A comparative study

Area of Science:

• Stem Cell Biology
• Hepatology
• Biotechnology

Background:

• Human induced pluripotent stem cells (hiPSCs) are a valuable source for generating various somatic cell types.
• Current hepatic differentiation protocols predominantly rely on 2D cell culture methods.
• Limitations in 2D cultures necessitate exploration of advanced 3D systems for improved differentiation.

Purpose of the Study:

• To investigate hiPSC-derived hepatocyte-like cell generation using two distinct 3D culture systems.
• To compare the efficacy of 3D scaffold-free microspheroids and 3D hollow-fiber bioreactors against conventional 2D cultures.
• To evaluate differentiation outcomes based on molecular, protein, and metabolic markers.

Main Methods:

• Generation of hiPSC-derived hepatocyte-like cells in 3D scaffold-free microspheroids and 3D hollow-fiber perfusion bioreactors.
• Comparison with cells differentiated in conventional 2D cultures.
• Assessment using mRNA expression, protein secretion, antigen expression, and metabolic activity assays, with primary human hepatocytes as controls.

Main Results:

• 3D culture systems showed lower α-fetoprotein expression and higher cytochrome P450 1A2/3A4 activity compared to 2D cultures.
• Cells differentiated in the 3D bioreactor exhibited increased albumin and hepatocyte nuclear factor 4α expression, and α-1-antitrypsin secretion, indicating enhanced maturation.
• 3D scaffold-free microspheroids offer a robust method for generating defined spheroids for screening, while the bioreactor is suitable for complex, physiological investigations.

Conclusions:

• Two novel 3D culture systems provide superior hepatic differentiation of hiPSCs compared to 2D methods.
• The scaffold-free microspheroid system is advantageous for screening applications.
• The 3D bioreactor system is ideal for complex investigations under near-physiological conditions, advancing stem cell-based liver research.