Material surface conjugated with fibroblast growth factor-2 for pluripotent stem cell culture and differentiation

Abstract

Fibroblast growth factor-2 (FGF-2) is a critical molecule for sustaining the pluripotency of human pluripotent stem (PS) cells. However, FGF-2 is extremely unstable and cannot be stored long periods at room temperature. Therefore, the following FGF-2-conjugated cell culture materials were developed to stabilize FGF-2: FGF-2-conjugated polyvinyl alcohol (PVAI-C-FGF) hydrogels and FGF-2-conjugated carboxymethyl cellulose-coated (CMC-C-FGF) dishes. Human induced pluripotent stem (iPS) cells were proliferated on recombinant vitronectin (rVN)-coated PVAI-C-FGF hydrogels and CMC-C-FGF dishes in medium without FGF-2. Human iPS cells could not be cultivated on rVN-coated PVAI-C-FGF hydrogels for more than two passages but could proliferate on rVN-coated CMC-C-FGF dishes. These results indicated that the amount of immobilized FGF-2 and the base cell materials are important, including the amount of immobilized rVN and the conformation of FGF-2 on the surfaces. When human iPS cells were proliferated on rVN-coated CMC-C-FGF surfaces in medium containing no FGF-2 for 10 passages, their pluripotency and potential to differentiate into cells originating from three germ layers were maintained in vivo and in vitro. Furthermore, the cells could extensively differentiate into cardiomyocytes, which can be used for cardiac infarction treatment in future and retinal pigment epithelium for retinal pigmentosa treatment in future. The FGF-2-immobilized surface could enable human PS cell culture in medium that does not need to contain unstable FGF-2. The amount of FGF-2 immobilization on the rVN-coated CMC-C-5FGF and CMC-C-20FGF dishes was reduced to 93.6 and 52.2 times, respectively, which is less than the conventional amount of FGF-2 used in culture medium for one passage (6 days) of human iPS cell culture. This reduction resulted from the stabilization of unstable FGF-2 by the immobilization of FGF-2, which was achieved by utilizing optimal base materials (CMC), coating materials (rVN) and long-joint segment (PEG4-SPDP) design.

Keywords: carboxymethyl cellulose; cell differentiation; fibroblast growth factor-2; human pluripotent stem cells; material design.

Graphical Abstract

Figure 1.

Design of FGF-2-conjugated cell culture biomaterials. PVAI hydrogels and CMC-coated dishes were prepared. Subsequently, the carboxylic acid of the biomaterials was activated using the NHS/EDC reaction, and cysteine was conjugated on the surface of the biomaterials. FGF-2 was reacted with PEG4-SPDP, and FGF-2-conjugated PEG4-SPDP was reacted with cysteine-conjugated biomaterials using the Michael addition reaction. Finally, FGF-2-conjugated biomaterials, PVAI-C-FGF hydrogels and CMC-C-FGF dishes were generated.

Figure 2.

Physical characteristics of the PVAI hydrogel surface and CMC-coated dishes with and without FGF-2 conjugation. (A) Nitrogen to carbon (N/C) atomic ratios of the PVAI hydrogel surface (left bar) and CMC-coated dishes (right bar) with and without conjugation of FGF-2, which were analysed by XPS. * P < 0.05. ** P > 0.05. (B) Sulfur to carbon (S/C) atomic ratios of the surfaces of PVAI hydrogels (left bar) and CMC-coated dishes (right bar) with and without conjugation of FGF-2, which were analysed by XPS. * P < 0.05. ** P > 0.05. (C) Surface density of FGF-2 conjugated on the surfaces of PVAI hydrogels (left bar) and CMC-coated dishes (right bar) with and without FGF-2 conjugation, which were analysed by ELISA. * P < 0.05. ** P > 0.05. (D) Time dependence of FGF-2 density conjugated on the surfaces of PVAI hydrogels (closed square) and CMC-coated dishes (closed circle) with FGF-2 conjugation, which were immersed in PBS buffer solution for 7 days.

Figure 3.

Human iPS cell (HPS0077) culture on PVAI hydrogels conjugated with and without FGF-2 in the E8 and E7 medium, which contains and not contains FGF-2, respectively under xeno-free culture methods. (A) Morphologies of human iPS cells on rVN-coated TCPS (TCPS) dishes (a, f), rVN-coated PVAI-C hydrogels (b, g), rVN-coated PVAI-C-5FGF hydrogels (c, h), rVN-coated PVAI-C-20FGF hydrogels (d, i) and rVN-coated PVAI-C-80FGF hydrogels (e, j) in E8 medium (a–e) and E7 medium (f–j) at passage 1. The scale bar describes 100 μm. (B) Expansion fold of human iPS cells on rVN-coated TCPS (TCPS) dishes, rVN-coated PVAI-C hydrogels, rVN-coated PVAI-C-5FGF hydrogels, rVN-coated PVAI-C-20FGF hydrogels and rVN-coated PVAI-C-80FGF hydrogels at passage 1 (left bar) and passage 2 (right bar) in E8 medium, which contains FGF-2. (C) Expansion fold of human iPS cells on rVN-coated TCPS (TCPS) dishes, rVN-coated PVAI-C hydrogels, rVN-coated PVAI-C-5FGF hydrogels, rVN-coated PVAI-C-20FGF hydrogels and rVN-coated PVAI-C-80FGF hydrogels at passage 1 (left bar) and passage 2 (right bar) in E7 medium, which does not contain FGF-2. (D) Differentiation rate of human iPS cells on rVN-coated TCPS (TCPS) dishes, rVN-coated PVAI-C hydrogels, rVN-coated PVAI-C-5FGF hydrogels, rVN-coated PVAI-C-20FGF hydrogels and rVN-coated PVAI-C-80FGF hydrogels at passage 1 (left bar) and passage 2 (right bar) in E8 medium, which contains FGF-2. (E) Differentiation rate of human iPS cells on rVN-coated TCPS (TCPS) dishes, rVN-coated PVAI-C hydrogels, rVN-coated PVAI-C-5FGF hydrogels, rVN-coated PVAI-C-20FGF hydrogels and rVN-coated PVAI-C-80FGF hydrogels at passage 1 (left bar) and passage 2 (right bar) in E7 medium, which does not contain FGF-2.

Figure 4.

Long-term proliferation of human iPS cells on rVN-coated CMC dishes conjugated with FGF-2 in the E8 medium, which contains FGF-2, and E7 medium, which does not, under xeno-free culture methods. (A) Morphologies of human iPS cells on rVN-coated TCPS (TCPS) dishes (a, e), rVN-coated CMC-C dishes (b, f), rVN-coated CMC-C-5FGF dishes (c and g) and rVN-coated CMC-C-20FGF dishes (d and h) in E8 medium (a–d) and E7 medium (e–h) at 3 passages (e, f) or 10 passages (a–d, g, h). The white bar indicates differentiated cells. The scale bar depicts 100 μm. (B) Passage dependent of expansion fold of human iPS cells (HPS0077) on rVN-coated TCPS dishes (closed circle), rVN-coated CMC-C dishes (open square), rVN-coated CMC-C-5FGF dishes (closed square) and CMC-C-20FGF dishes (closed triangle) in E7 medium, which does not contain FGF. The data of human iPS cells cultured on rVN-coated TCPS dishes in E8 medium, which contains FGF-2, as a positive control are also shown (open circle). (C) Passage dependent of differentiation rate of human iPS cells (HPS0077) on rVN-coated TCPS dishes (closed circle), rVN-coated CMC-C dishes (open square), rVN-coated CMC-C-5FGF dishes (closed square) and CMC-C-20FGF dishes (closed triangle) in E7 medium, which does not contain FGF. The data of human iPS cells cultured on rVN-coated TCPS dishes in E8 medium, which contains FGF-2, as a positive control are also shown (open circle).

Figure 5.

Pluripotency and differentiation potential of human iPS cells (HPS0077) in vitro and in vivo after long-term (10 passages) proliferation on rVN-coated CMC-C-20FGF dishes in E7 medium, which does not contain FGF-2, using xeno-free proliferation methods. (A) Expression of pluripotency protein of (i) Oct3/4, (ii) Nanog, (v) Sox2 and (vi) SSEA-4 in human iPS cells analysed utilizing immunostaining with nuclear staining by (iii, vii) Hoechst 33342 after human iPS cell culture on rVN-coated CMC-C-20FGF dishes for 10 passages in E7 medium. The images (iv) and (viii) were created by merging (i)–(iii) and (v)–(vii), respectively. The scale bar describes 100 μm. (B) SSEA-4 expression on hiPSCs (HPS0077), after proliferation on CMC-C-5FGF dishes (i) and rVN-coated CMC-C-20FGF dishes (ii) for 10 passages in E7 medium. (C) Karyotyping evaluation of human iPS cells after long-term (10 passages) proliferation on rVN-coated CMC-C-20FGF dishes for 10 passages in E7 medium.

Figure 6.

Differentiation potential of human iPS cells in vitro and in vivo after human iPS cell culture for 10 passages on rVN-coated CMC-C-20FGF dishes in E7 medium. (A) Differentiation potential of human iPS cells in vitro using an EB formation assay. (i) Morphologies of EB cells derived from differentiation of human iPS cells. (ii–viii) Expression of a mesodermal protein marker (ii, α-SMA), an ectodermal protein marker (v, GFAP) and an endodermal protein marker (vi, AFP) from EB cells analysed utilizing immunostaining with stained nuclears from Hoechst 33342 (iii, vii ) the pictures (iv) and (viii) were prepared by merging (ii)–(iii) and (v–vii), respectively. The scale bar is 500 μm (i) and 100 μm (ii–viii). (B) Differentiation potential of human iPS cells in vivo using a teratoma assay. (i) A teratoma generated by the transplantation of human iPS cells. (ii–iv) Tissues describing ducts made of columnar epithelium (ii, endoderm), cartilage (iii, mesoderm) and undifferentiated neuroepithelium (iv, ectoderm) were detected. The white arrows describe teratoma (i) and specific tissues (ii, iii and iv). The scale bar describes 100 μm (ii–iv).

Figure 7.

Cardiac differentiation of human iPS cells (HPS0077) after long-term (10 passages) proliferation on rVN-coated CMC-C-5FGF and CMC-C-20FGF dishes in E7 medium, which does not contain FGF-2, using xeno-free proliferation methods. (A) Timeline of the cardiac differentiation method of human iPS cells utilized in this work. (B) The sequential morphological observation during cardiac differentiation of human iPS cells on CMC-C-5FGF (a–e) and CMC-C-20FGF (f–j) dishes on day 0 (a and f), 2 (b and g), 8 (c and h), 14 (d and i) and 18 (e and j). Scale bar indicates 200 µm. (C) Immunohistochemical staining evaluation of human iPS cell-derived cardiac cells on CMC-C-5FGF (a–d, i–l) and CMC-C-20FGF (e–h, m–p) dishes. Expression of ML2CV (a and e), cTnT (b and f), α-actinin (i), cTnT (j) Nkx2.5 (m) and α-actinin (n) on human iPS cell-derived cardiac cells investigated by an immunohistochemical staining, which were differentiated on CMC-C-5FGF (a–d, i–l) and CMC-C-20FGF dishes (e–h, m–p) on day 18. DAPI (c, g, k and o, blue) was utilized for nuclei staining. The photos (d), (h), (l) and (p) were created by merging (a–c), (e–g), (i–k), and (m–o), respectively. The scale bar shows 20 µm (a–l) and 50 µm (m–p).

Figure 8.

Differentiation of human iPS cells (HPS0077) into RPE cells after long-term (10 passages) proliferation on rVN-coated CMC-C-20FGF dishes in E7 medium, which does not contain FGF-2, using xeno-free proliferation methods. (A) Timeline of the RPE cell differentiation method of human iPS cells used in this work. (B) The morphological observation (a) and cell pellets (b) of human iPS cell-differentiated RPE cells after induction of 84 days. Scale bar shows 100 µm. Expression of RPE65 on human iPS cell-differentiated RPE cells analysed using flow cytometry after induction of 84 days (c). (C) Immunohistochemical staining evaluation of human iPS cell-differentiated RPE cells, after induction of 28 days. Expression of MITF (a) and PAX6 (b). DAPI (c) was utilized for nuclei staining. The photo (d) was created by merging (a–c). the scale bar shows 200 µm. (D) Immunohistochemical staining evaluation of human iPS cell-differentiated RPE cells, after induction of 86 days. Expression of ZO-1 (a) and RPE65 (b). DAPI (c) was utilized for nuclei staining. The photo (d) was created by merging (a–c). The scale bar shows 200 µm.