Speaker
Description
Stem cells, especially human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells, are attractive sources of cells for regenerative medicine and have been used in clinical trials. One of the difficulties of applying human pluripotent stem cells (hPS cells, including hES and hiPS cells) in the clinic is that hPS cells cannot be cultured on conventional tissue culture polystyrene (TPS) dishes using the way that adult stem cells can be cultured; rather, hPS cells should be cultivated on mouse embryonic fibroblasts (MEFs), Matrigel-coated surfaces, extracellular matrix (ECM) protein-coated surfaces, and ECM protein-derived peptide-immobilized surfaces. In particular, ECM protein-derived peptide-grafted cell culture biomaterials (peptide-grafted biomaterials) are attractive because completely synthesized surfaces can be prepared for the peptide-grafted biomaterials. In this study, we developed poly(vinyl alcohol-co-itaconic acid) (PV) hydrogels grafted with laminin-derived peptides that had different joint segments and several specific designs, including dual chain motifs. PV hydrogels grafted with a peptide derived from laminin-β4 (PMQKMRGDVFSP) chain containing a joint segment, dual chain motif and cationic amino acid insertion could attach human pluripotent stem (hPS) cells where the PV hydrogels were adjusted to have an optimal elasticity for hPS cell cultivation. The PV hydrogels grafted with a laminin-β4 peptide having a joint segment, dual chain motif and cationic amino acid promoted high expansion folds in long-term culture (over 10 passages) with low differentiation rates, whereas hPS cells attached poorly on PV hydrogels grafted with laminin-α5 (PASYRGDSC) peptides that had joint segments with and without a cationic amino acid or on PV hydrogels grafted with laminin-β4 peptides containing the joint segment only. The inclusion of a cationic amino acid in the laminin-β4 peptide was critical for hPS cell attachment on PV hydrogels, which contributed to the zeta potential shifting to higher values (3–4 mV enhancement). The expansion fold of hPS cells and the zeta potential of the materials have good relationship on the PV hydrogels having the same bioactive site of the peptide. The novel peptide segment-grafted PV hydrogels developed in this study supported hPS cell proliferation, which induced better hPS cell expansion than recombinant vitronectin-coated dishes (gold standard of hPS cell culture dishes), vitronectin-derived peptide (KGGPQVTRGDVFTMP)-grafted dishes or Synthemax-coated dishes (commercially available coating material) in xeno-free culture conditions. After long-term (10 passages) culture on peptide-grafted hydrogels, hPS cells showed their pluripotency from evaluation of immunohistochemical staining of Oct4, Sox2, Nanog, and SSEA-4 expression and differentiated into the cells derived from three germ layers in vitro (embryoid body formation assay) and in vivo (teratoma formation assay). Furthermore, hPS cells could be induced to differentiate into specific lineages of cells, such as cardiomyocytes, with high efficiency (expression of cardiac marker, cTnT > 90%).
