Define of Optimal Addition Period of Osteogenic Peptide to Accelerate the Osteogenic Differentiation of Human Pluripotent Stem Cells

Abstract

Background: The addition of growth factiors is commonly applied to improve the osteogenic differentiation of stem cells. However, for human pluripotent stem cells (hPSCs), their complex differentiation processes result in the unknown effect at different stages. In this study, we focused on the widely used bone forming peptide-1 (BFP-1) and investigated the effect and mechanisms of its addition on the osteogenic induction of hPSCs as a function of the supplementation period.

Methods: Monolayer-cultured hPSCs were cultured in osteogenic induction medium for 28 days, and the effect of BFP-1 peptide addition at varying weeks was examined. After differentiation for varying days (0, 7, 14, 21 and 28), the differentiation efficiency was determined by RT-PCR, flow cytometry, immunofluorescence, and alizarin red staining assays. Moreover, the expression of marker genes related to germ layers and epithelial-mesenchymal transition (EMT) was investigated at day 7.

Results: Peptide treatment during the first week promoted the generation of mesoderm cells and mesenchymal-like cells from hiPSCs. Then, the upregulated expression of osteogenesis marker genes/proteins was detected in both hESCs and hiPSCs during subsequent inductions with BFP-1 peptide treatment. Fortunately, further experimental design confirmed that treating the BFP-1 peptide during 7-21 days showed even better performance for hESCs but was ineffective for hiPSCs.

Conclusion: The differentiation efficiency of cells could be improved by determining the optimal treatment period. Our study has great value in maximizing the differentiation of hPSCs by adding osteogenesis peptides based on the revealed mechanisms and promoting the application of hPSCs in bone tissue regeneration.

Keywords: Bone forming peptide; Epithelial-mesenchymal transition; Human pluripotent stem cells; Osteogenic differentiation; Supplement period.

Fig. 1

Osteogenic induction of hPSCs with BFP-1 peptide treatment. An Illustrated image of the addition of BFP-1 peptide in osteogenic induction medium (OM) at various periods for monolayer cultured hPSCs. In addition, the whole induction process without or with BFP-1 treatment was conducted as pristine and positive controls, respectively. B The osteogenic differentiation of f-hiPSCs as a function of BFP-1 peptide concentration at day 14 was evaluated by alizarin red staining. C-D For BFP-1 peptide-treated hPSCs, the viabilities of H9 hESCs (C) and f-hiPSCs (D) during osteogenic induction were detected at the desired intervals (0, 7, 14, 21, and 28 days) using a CCK8 reagent. E-L After culturing for 7 days, the gene expression of RUNX2, ALP, COL1A1, and OPN in cell samples was measured by RT–PCR. The expression of these genes in undifferentiated hPSCs was standardized to 1. + indicates OM supplemented with peptide. Scale bar, 100 μm. n = 3. * represents p < 0.05, ** represents p < 0.01

Fig. 2

The expression of marker proteins in hPSCs during the 28 days of osteogenic differentiation. The protein expression of RUNX2 (red) in cells was measured every 7 days by immunofluorescence. Cell nuclei were stained blue with DAPI. Scale bars, 200 µm

Fig. 3

The expression of marker proteins in hPSCs during 28 days of osteogenic differentiation. The protein expression of COL1A1 (red) in cells was measured every 7 days by immunofluorescence. Cell nuclei were stained blue with DAPI. Scale bars, 200 µm

Fig. 4

The expression of marker proteins in hPSCs during 28 days of osteogenic differentiation. The protein expression of OPN (red) in cells was measured every 7 days by immunofluorescence. Cell nuclei were stained blue with DAPI. Scale bars, 200 µm

Fig. 5

Analyses of alizarin red staining for induced hPSCs. A-B Images of alizarin red-stained H9 hESCs (A) and f-hiPSCs (B) on different days (0, 7, 14, 21, and 28). C, D Cetylpyridinium bromide solution was applied to dissolve deposited alizarin red, and the absorbance at 490 nm was measured. Scale bars, 100 µm. n = 3. * represents p < 0.05, ** represents p < 0.01

Fig. 6

The osteogenic differentiation of H9 hESCs with peptide addition during 7–21 days of induction. A After 7 days of induction, H9 hESCs were treated with BFP-1 peptide for 14 days. After culturing for 21 days and 28 days, the viability of the cells was measured using a CCK8 reagent. B-E The gene expression of ALP (B), RUNX2 (C), COL1A1 (D) and OPN (E) in cell samples was measured by RT–PCR. The expression of these genes in undifferentiated H9 hESCs was standardized to 1. G–H The positive expression of RUNX2 protein in induced cell samples at days 21 (G) and 28 (H) was measured by flow cytometry. F-I Cells were stained with alizarin red. Images of cell samples (I) are shown. Then, the deposited calcium was quantitatively detected at a wavelength of 490 nm (F). J The protein expression of RUNX2, COL1A1 and OPN in cells was studied by immunofluorescence. Scale bars, 200 µm. n = 3. * represents p < 0.05, ** represents p < 0.01

Fig. 7

The osteogenic differentiation of f-hiPSCs treated with peptide addition at specific periods. A After 7 days of induction, f-hiPSCs were treated with BFP-1 peptide for 14 days. Then, the effect of peptide supplementation on the differentiation of cells was also studied during the period of 21–28 days. The viability of cells was measured using a CCK8 reagent. B-E The gene expression of ALP (b), RUNX2 (c), COL1A1 (d) and OPN (E) in cell samples was measured by RT–PCR. G-H The expression of RUNX2 protein in cells after induction for 21 (G) or 28 (H) days as measured by flow cytometry. F-I Cells were stained with alizarin red. Images of cell samples (I) are shown. Then, the deposited calcium was quantitatively detected at a wavelength of 490 nm (F). J The protein expression of RUNX2, COL1A1 and OPN in cells was studied by immunofluorescence. Scale bars, 200 µm. n = 3. * represents p < 0.05, ** represents p < 0.01

Fig. 8

The mechanism underlying the effect of BFP-1 peptide supplementation on the osteogenic differentiation of hPSCs. A-C The impact of BFP-1 peptide addition on the differentiation process of both H9 hESCs (A) and f-hiPSCs (C) was investigated after 7 days of induction. The relative expression of marker genes related to the germ layer (endoderm: AFP, GATA4; mesoderm: T, MEOX1 and MIXL1; ectoderm: PAX6, SOX1 and FOXD3) was measured using RT–PCR. B-D The expression of gene markers related to epithelial-mesenchymal transition (EMT), including Snail, Twist, Slug, E-cadherin, β-catenin, ZO-1, N-cadherin, Vimentin and α-SMA, was also investigated. E, F The gene expression of the mesenchymal marker CD73 was detected in hESCs (E) and hiPSCs (F) with peptide treatment at varying periods. The expression of these genes in hPSCs before differentiation was standardized to 1. G A dynamic map summarizing the impact of BFP-1 peptide addition on the osteogenic differentiation of hPSCs during various weeks of induction over 28 days. n = 3. * indicates p < 0.05, and ** indicates p < 0.01