Human embryonic stem cells cultured on hydrogels grafted with extracellular matrix protein-derived peptides with polyethylene glycol joint nanosegments

Abstract

Human pluripotent stem cells (hPSCs) can be proliferated on completely synthetic materials under xeno-free cultivation conditions using biomaterials grafted with extracellular matrix protein (ECM)-derived peptides. However, cell culture biomaterials grafted with ECM-derived peptides must be prepared using a high concentration of peptide reaction solution (e.g. 1000 μg/ml), whereas the ECM concentration of the ECM-coated surface for hPSC culture is typically 5 μg/ml. We designed a polyethylene glycol (PEG) joint nanosegment (linker) to be used between base cell culture biomaterials and bioactive ECM-derived peptides to enhance the probability of contact between ECM-derived peptides and cell binding receptors of hPSCs. Vitronectin-derived peptides with glycine joint nanosegments (GCGG) were conjugated onto poly (vinyl alcohol-co-itaconic acid) hydrogels via PEG joint nanosegments, and human embryonic stem cells (hESCs) were cultivated on these hydrogels. hESCs could successfully be cultivated on hydrogels while maintaining their pluripotency and differentiation potential to differentiate into cells that are induced from three germ layers in vitro and in vivo, where only a 50 μg/ml ECM-derived peptide concentration was used when the PEG joint nanosegments were introduced into peptides that were grafted onto hydrogel surfaces. The joint nanosegments between bioactive peptides and base cell culture biomaterials were found to contribute to efficient hESC attachment and proliferation.

FIGURE 1

Outline of the preparation method of the poly (vinyl alcohol‐co‐itaconic acid) (PVI) hydrogels grafted with VN4C (GCGGKGGPQVTRGDVFTMP) via polyethylene glycol (PEG) joint nanosegments

FIGURE 2

Surface analysis of poly (vinyl alcohol‐co‐itaconic acid) (PVI) hydrogels. (a) High‐resolution X‐ray photoelectron spectroscopy (XPS) spectra of the C 1s peaks on the surfaces of the (a) tissue cultivation polystyrene (TCP) dishes, (b) PVI hydrogels, (c) PVI‐PEG hydrogels, (d) PVI‐PEG‐VN4C(50) hydrogels, (e) PVI‐PEG‐VN4C(200) hydrogels and (f) PVI‐PEG‐VN4C(500) hydrogels. (b) High‐resolution XPS spectra of the N 1s peaks on the surfaces of the (a) TCP dishes, (b) PVI hydrogels, (c) PVI‐PEG hydrogels, (d) PVI‐PEG‐VN4C(50) hydrogels, (e) PVI‐PEG‐VN4C(200) hydrogels and (f) PVI‐PEG‐VN4C(500) hydrogels. (c) The nitrogen to carbon (N/C) atomic ratios on the surfaces of the PVI, PVI‐PEG, PVI‐VN4C and PVI‐PEG‐VN4C hydrogels. *p < 0.05

FIGURE 3

Human ESCs (WA09) cultured on poly (vinyl alcohol‐co‐itaconic acid) (PVI) hydrogels grafted with VN4C peptide. Morphologies of human embryonic stem cells (hESCs) on (a) PVI‐VN4C(50) hydrogels, (b) PVI‐VN4C(200) hydrogels, (c) PVI‐VN4C(500) hydrogels, (d) Matrigel‐coated tissue cultivation polystyrene (TCP) dishes, (e) PVI‐PEG‐VN4C(50) hydrogels, (f) PVI‐PEG‐VN4C(200) hydrogels and (g) PVI‐PEG‐VN4C(500) hydrogels. The scale bar indicates 100 μm

FIGURE 4

Human ESCs (WA09) cultured on poly (vinyl alcohol‐co‐itaconic acid) (PVI) hydrogels grafted with VN4C peptide. (a) Expansion folds of human embryonic stem cells (hESCs) on Matrigel‐coated tissue cultivation polystyrene (TCP) dishes, PVI‐VN4C hydrogels and PVI‐PEG‐VN4C hydrogels. *p < 0.05. (b) Differentiation rates of hESCs on Matrigel‐coated TCP plates, PVI‐VN4C hydrogels and PVI‐PEG‐VN4C hydrogels. **p > 0.05

FIGURE 5

Effect of human embryonic stem cell (hESC) expansion fold on the surface density (N/C atomic ratios) of bioactive peptide VN4C, where hESCs were cultured on poly (vinyl alcohol‐co‐itaconic acid) (PVI)‐VN4C hydrogels and PVI‐PEG‐VN4C hydrogels for one passage

FIGURE 6

Pluripotency evaluation of human embryonic stem cells (hESCs) after cultivation on PVI‐PEG‐VN4C(50) hydrogels for one passage. (A) Pluripotent protein expressions of Oct3/4 (a, green), Nanog (b, red), Sox2 (e, green), and SSEA‐4 (f, red) in hESCs evaluated by immunohistochemical staining with nuclear staining by Hoechst 33,342 (blue, (c), (g). Images (d) and (h) were made by merging (a)–(c) and (e)–(g), respectively. The scale bar indicates 100 μm. (B) (a) Morphologies of embryoid body (EB) cells differentiated from hESCs. (b)–(h) Expressions of a mesodermal protein marker (b, green, α‐SMA), an ectodermal protein marker (e, red, GFAP) and an endodermal protein marker (f, green, AFP) from EB cells analysed by immunohistochemical staining with nuclear staining from Hoechst 33,342 (c, g, blue). Photographs (d) and (h) were made by merging (b)–(c) and (e)–(g), respectively. The scale bar indicates 500 μm for (a) and 100 μm for (b)–(h). (C) (a) A teratoma generated by transplantation of hESCs (H9) into NOD/SCID mice. (b–d) Tissues including glandular ducts composed of cylindrical epithelium (b, endoderm), bone‐like tissue (c, mesoderm) and melanin‐producing cells (d, ectoderm) can be observed. The scale bar indicates 100 μm (b) and 200 μm (c,d)