Collagen coated electrospun polyethersulfon nanofibers improved insulin producing cells differentiation potential of human induced pluripotent stem cells

Figures & data

Table 1. Primer sequences used for real time-PCR in this study.

Human genes	Primer sequence	Annealing temperature (°C)	Product size (bp)
β2m	Forward: 5′-ATGCCTGCCGTGTGAAC-3′	60	91
	Reverse: 5′-ATCTTCAAACCTCCATGATG-3′
Gcg	Forward: 5′-ACCAGAAGACAGCAGAAATG-3′	59	191
	Reverse: 5′-GAATGTGCCCTGTGAATG-3′
Glut2	Forward: 5′-TCACTGCTGTCTCTGTATTCC-3′	60	147
	Reverse: 5′-TGCTCACATAACTCATCCAAG-3′
Pdx1	Forward: 5′-ATGGATGAAGTCTACCAAAGC-3′	60	159
	Reverse: 5′-CGTGAGATGTACTTGTTGAATAG-3′
Ngn3	Forward: 5′-AGAGAGCGTGACAGAGGC-3′	60	182
	Reverse: 5′-GCGTCATCCTTTCTACCG-3′
Insulin	Forward: 5′-GCTTCTTCTACACACCCAAG-3′	60	171
	Reverse: 5′-GGTAGAGGGAGCAGATGC-3′

Figure 1. Morphological changes of cultured cells in TCPS group. (a) hiPSCs in DMEM (control group). (b) hiPSCs in IPCs differentiation media at day-21. Scale bars are 100 µm.

Figure 2. Morphological changes of cultured cells on the 3D nanofibrous scaffold. (a) Unseeded scaffold with interconnected pores. (b) hiPSCs-seeded on the nanofibrous scaffold (control group). The cells show spindle-shape morphology. (c) the hiPSCs-seeded scaffold in IPCs differentiation media at day-21. The cells are round in shape and form spherical dense clusters. Scale bars are a, and b: 30 µm, c: 50 µm.

Figure 3. MTT cell proliferation assay of hiPSCs on polyethersulfone-collagen nanofibrous scaffold (3D) and tissue culture polystyrene (2D) at days 1, 3, 5 and 7 of cell culture. *p < .05.

Figure 4. Relative expression of pancreatic-specific genes in end-stage derived-IPCs. Gene transcripts of the 3D group are compared to the 2D group. Relative gene expressions were normalized to the human β2M as a reference gene. The value is shown in each graph as mean ± SD. *p < .05, **p < .01, ***p < .001 and ****p < .0001.

Figure 5. Immunocytochemical analysis of differentiated cells in end-stage derived-IPCs. Nuclei localization of Pdx1 and cytoplasmic localization of insulin in differentiated IPCs at day-14 in the 3D group (Figure 5(a)) and 2D group (Figure 5(b)) were detected by immunofluorescence analysis. Counter-staining of the nucleus (blue) was performed by DAPI and images were obtained by a fluorescence microscope. Bar graph quantification of the immunocytochemistry assay. Each bar represents the relative value of insulin and Pdx1 markers, differences were observed between 3D and 2D groups. Scale bars are 100 µm.

Figure 6. In vitro release of insulin and C-peptide in IPCs derived at day-21 of differentiation. (a and b) insulin secretion and C-peptide release changes in two groups of IPCs and control group in response to various concentrations of glucose from 5.5 to 25 mM. Values are expressed as mean ± SEM. (n = 6). *p < .05 and **p < .01.