Design of dual peptide-conjugated hydrogels for proliferation and differentiation of human pluripotent stem cells

Abstract

Completely synthetic cell cultivation materials for human pluripotent stem cells (hPSCs) are important for the future clinical use of hPSC-derived cells. Currently, cell culture materials conjugated with extracellular matrix (ECM)-derived peptides are being prepared using only one specific integrin-targeting peptide. We designed dual peptide-conjugated hydrogels, for which each peptide was selected from different ECM sites: the laminin β4 chain and fibronectin or vitronectin, which can target α6β1 and α2β1 or αVβ5. hPSCs cultured on dual peptide-conjugated hydrogels, especially on hydrogels conjugated with peptides obtained from the laminin β4 chain and vitronectin with a low peptide concentration of 200 μg/mL, showed high proliferation ability over the long term and differentiated into cells originating from 3 germ layers in vivo as well as a specific lineage of cardiac cells. The design of grafting peptides was also important, for which a joint segment and positive amino acids were added into the designed peptide. Because of the designed peptides on the hydrogels, only 200 μg/mL peptide solution was sufficient for grafting on the hydrogels, and the hydrogels supported hPSC cultures long-term; in contrast, in previous studies, greater than 1000 μg/mL peptide solution was needed for the grafting of peptides on cell culture materials.

Keywords: Cardiomyocyte; Human pluripotent stem cells; Hydrogel; Integrin; Peptide; Proliferation.

Graphical abstract

Fig. 1

Design of peptide-conjugated hydrogels. (A) Peptide sequences used for the grafting on PVA hydrogels. (B) Preparation method for peptide-conjugated PVA hydrogels. After PVA film was prepared in dishes, PVA was crosslinked with glutaraldehyde. The elasticity of PVA hydrogels was controlled by the reaction time. After the activation of carbonic acid on PVA hydrogels with EDC/NHS, peptide solution was added to the PVA hydrogels for the grafting of the peptides.

Fig. 2

XPS analysis of peptide-conjugated PVA hydrogels. (A) Nitrogen to carbon (N/C) atomic ratio for the surface of P-KKLB2CK hydrogels prepared with several concentrations of KKLB2CK peptide. (B) Nitrogen to carbon (N/C) atomic ratios for the surfaces of PVA (PV) hydrogels and peptide-conjugated PVA hydrogels. *p < 0.05. **p > 0.05. (C) Sulfur to carbon (S/C) atomic ratios for the surfaces of PVA (PV) hydrogels and peptide-conjugated PVA hydrogels. *p < 0.05. **p > 0.05.

Fig. 3

Physical characterization of peptide-conjugated PVA hydrogels. (A) Zeta potential of the peptide-conjugated PVA (P-KKLB2CK, PVN2CK, and P-VN2C + KKLB) hydrogels prepared with several peptide concentrations. (B) Zeta potentials of peptide-conjugated PVA hydrogel surfaces and ECM-coated dishes.

Fig. 4

hiPSC (HPS0077) culture on single or dual peptide-conjugated PVA hydrogels cultured under xeno-free proliferation conditions. (A) Morphologies of HPS0077 on P-VN2CK (i), P-KKLB2CK (ii), and P-VN2C + KKLB (iii) hydrogels prepared with peptide concentrations of 20 (a), 50 (b), 100 (c), 200 (d), 500 (e), and 1000 (f) μg/mL at passage 3. The scale bar represents 500 μm. (B) Dependence of fold expansion of HPS0077, cultured on single and dual peptide-conjugated PVA hydrogels, during passages 1–3 on the concentration of peptides used for the grafting of peptides to PVA hydrogels. Fold expansion of hiPSCs on T-rVN dishes is also plotted as open circle. (C) Dependence of fold expansion of HPS0077, cultured on single and dual peptide-conjugated PVA hydrogels, during passage 1–3 on the nitrogen to carbon atomic ratios for peptide-conjugated PVA hydrogels, as analyzed by XPS.

Fig. 5

Long-term proliferation of hiPSCs (HPS0077) on peptide-conjugated PVA hydrogels under xeno-free proliferation conditions. (A) Morphologies of hiPSCs on Matrigel-coated dishes (a), rVN-coated dishes (b), P-VN2CK hydrogels (c), P-KVN2CK hydrogels (d), P-LB2CKKK hydrogels (e), P-KKLB2CK (f), P-VN2C + KKLB hydrogels (g), and P-RGDK + KKLB hydrogels (h) at passage ten. The scale bar represents 500 μm. (B) Dependence of fold expansion of hiPSCs on passage on Matrigel-coated dishes (closed black triangle), rVN-coated dishes (closed reverse green triangle), P-VN2CK hydrogels (closed red circle), and P-KVN2CK hydrogels (closed blue square). (C) Dependence of fold expansion of hiPSCs on passage on rVN-coated dishes (closed reverse green triangle), P-LB2CKKK hydrogels (closed black triangle), P-KKLB2CK hydrogels (closed purple rhombus), P-RGDK + KKLB hydrogels (closed blue square), and P-VN2C + KKLB hydrogels (closed red circle). (D) Average fold expansion of hiPSCs on rVN-coated dishes and single or dual peptide-conjugated PVA hydrogels for 10 passages. *p < 0.05. **p > 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Analysis of the pluripotency of hiPSCs (HPS0077) after long-term (ten passages) proliferation on single or dual peptide-conjugated PVA hydrogels under xeno-free proliferation conditions. (A, B) Expression of the pluripotent proteins Oct3/4 (a, green), Nanog (b, red), Sox2 (e, green), and SSEA-4 (f, red) in hiPSCs, as determine with immunostaining and nuclear staining (Hoechst 33342) (blue, c, g) after the long-term (ten passages) proliferation of hiPSCs on P-VN2C + KKLB (A) and P-RGDK + KKLB (B) hydrogels. The images in (d) and (h) were generated by merging (a)–(c) and (e)–(g), respectively. The scale bar represents 100 μm. (C) Flow cytometry analysis of pluripotent marker (SSEA-4) expression in hiPSCs after long-term (ten passages) proliferation on Matrigel-coated dishes (a), rVN-coated dishes (b), P-VN2CK hydrogels (c), P-KVN2CK hydrogels (d), P-LB2CKKK hydrogels (e), P-KKLB2CK (f), P-VN2C + KKLB hydrogels (g), and P-RGDK + KKLB hydrogels (h). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Differentiation ability of hiPSCs (HPS0077) in vivo utilizing a teratoma analysis after long-term (ten passages) proliferation on single or dual peptide-conjugated PVA hydrogels under xeno-free proliferation conditions. (A–D) (i) A teratoma created by the transplantation of HPS0077. (ii-iv) Glandular ducts and squamous nests (ii, endoderm), bone-like tissue (iii, mesoderm), and immature neuroepithelium and choroid plexus-like tissue (iv, ectoderm) were detected. The white arrows indicate a teratoma (i) and specific tissues (ii, iii and iv) on P-VN2CK (A), P-KKLB2CK (B), P-RGDK + KKLB (C), and P-VN2C + KKLB (D) hydrogels. The scale bar represents 100 μm (B(ii), B(iii), C(i), C(iii), D(i), D(iii)) or 200 μm (A(ii)-A(iv), B(iv), C(iii), D(iii)).

Fig. 8

Cardiac differentiation of hiPSCs (HPS0077) after long-term (passage ten) culture on Matrigel-coated dishes and single or dual peptide-conjugated PVA hydrogels under xeno-free proliferation conditions. (A) Timeline of the cardiac induction protocol for hiPSCs used in this experiment. (B) Sequential morphological detection during cardiac induction of hiPSCs on Matrigel-coated surfaces and P-KVN2CK, P-LB2CKKK, P-KKLB2CK, P-VN2C + KKLB, and P-RGDK + KKLB hydrogels. Scale bar represents 500 μm. (C) Immunohistochemical staining of hiPSC-derived cardiomyocytes on P-VN2C + KKLB (a–c) and P-RGDK + KKLB (d–f) hydrogels after the long-term (ten passages) proliferation of hiPSCs on P-VN2C + KKLB hydrogels (a–c) and P-RGDK + KKLB hydrogels (d–f), respectively. Expression of cTnT (a, c, d, f, green) in hiPSC-derived cardiomyocytes, as determined with immunohistochemical staining, differentiated on P-VN2C + KKLB (a–c) and P-RGDK + KKLB (d–f) hydrogels on day 17. DAPI (b and e, blue) was used to stain nuclei. The photos in (c) and (f) were created by merging (a)–(b) and (d)–(e), respectively. The scale bar represents 100 μm. (D) Flow cytometry analysis of cardiac marker (cTnT) expression in hiPSC-derived cardiomyocytes differentiated after the long-term (ten passages) proliferation of hiPSCs on P-VN2C + KKLB hydrogels (a), and P-RGDK + KKLB hydrogels (b). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)