Substrate-mediated commitment of human embryonic stem cells for hepatic differentiation.

Area of Science:

• Biomaterials Science
• Stem Cell Biology
• Regenerative Medicine

Background:

• Human embryonic stem cells (hESCs) are crucial for developing cellular therapies for diseases like liver failure.
• Soluble activin A (Act) is a common inducer of definitive endoderm (DE) in vitro.
• Effective differentiation protocols are needed to generate specific cell lineages from hESCs.

Purpose of the Study:

• To develop a nanofibrous poly(ɛ-caprolactone) substrate biofunctionalized with activin A for directed hESC differentiation into DE.
• To evaluate the bioactivity and stability of immobilized activin A (iAct) on the nanofibrous scaffold.
• To assess the potential of iAct-modified scaffolds for generating hepatocyte-like cells (HLCs).

Main Methods:

• Fabrication of nanofibrous poly(ɛ-caprolactone) meshes.
• Biofunctionalization of nanofibers with activin A via covalent conjugation.
• Confirmation of Act conjugation using ELISA and immunostaining.
• Cultivation of hESCs on functionalized nanofibers and assessment of endodermal marker expression (SOX17, FOXA2, CXCR4).
• Evaluation of DE-derived lineage potential by differentiating cells into HLCs.

Main Results:

• Covalently immobilized Act (iAct) on nanofibers significantly increased endodermal marker expression compared to physically adsorbed Act (aAct) or no Act.
• iAct retained its bioactivity after five days of storage without cell seeding.
• The iAct sample demonstrated enhanced hepatic marker expression in differentiated HLCs compared to aAct.
• Combining iAct with soluble Act (sAct) improved the conventional protocol for generating HLCs from hESCs.

Conclusions:

• Biofunctionalized nanofibrous scaffolds with covalently immobilized activin A provide an effective platform for directed hESC differentiation into definitive endoderm.
• This approach enhances the generation of endodermal cells and their subsequent differentiation into hepatocyte-like cells.
• The developed material offers a promising strategy for advancing cell-based therapies for liver diseases.