Promoting effect of 1,25(OH)2 vitamin D3 in osteogenic differentiation from induced pluripotent stem cells to osteocyte-like cells

Abstract

We recently reported a new method to purify the induced pluripotent stem (iPS)-derived osteoprogenitors (iPSop). In this paper, we optimized the procedure and characterized cells at each process step. We observed that 10 days of treatment with FGF-2, IGF-1 and TGF-β (FIT) resulted in early-phase osteoblasts and 14 days of treatment resulted in late-phase osteoblasts. We found that treatment with 1,25(OH)2 vitamin D3 increased expression of osteocalcin and decreased expression of tissue-non-specific alkaline phosphatase and runt-related transcription factor 2 (RUNX2) in iPSop-day14 cells (cells treated with FIT for 14 days). Therefore, iPSop-day14 cells were promoted to mature osteoblasts by 1,25(OH)2 vitamin D3 treatment. In addition, we found that 1,25(OH)2 vitamin D3 treatment for 14 days enhanced not only mineralization but also expression of osteocyte markers, including dentin matrix protein-1 and fibroblast growth factor-23, in iPSop cells. Therefore, 1,25(OH)2 vitamin D3 is a potent promoter of osteoblast-osteocyte transition. The results of this study suggest that it is possible to evaluate both early- and late-phase osteoblasts and to apply cells to drug screening for anabolic drugs that stimulate bone formation.

Keywords: drug assessment; induced pluripotent stem cells; osteoblasts; osteocytes; vitamin D.

Figure 1.

The expression of TNAP during hiPSC-to-osteogenic cell differentiation and the expression of osteoblast markers. (a) The frequency of TNAP positive cells in hiPSCs, EBs and single cells derived from EBs cultured in OBM with FGF-2/IGF-1/TGF-β (FIT). (b) Flow cytometrical analysis was performed on hiPSCs, EBs and single cells cultured in OBM with FIT for 0, 4, 10 and 14 days. (c) qRT-PCR analysis of COL1A1, TNAP, OSX and RUNX2 was performed on iPSop-day0, 4, 10 and 14 cells. mRNA levels were normalized to those of 18S rRNA. Experiments were performed in triplicate. Values represent the mean ± s.d., n = 3. Bonferroni correction for multiple comparisons was applied. *p < 0.05, **p < 0.01.

Figure 2.

Expression of osteoblast markers after 1,25(OH)₂ vitamin D₃ treatment. (a) After isolation by flow cytometry, iPSop-day14 cells were treated with vehicle (control), 10 nM or 50 nM 1,25(OH)₂ vitamin D₃ for 6 days or (b) 12 days. Expression of COL1A1, TNAP, OCN and RUNX2 was analysed with qRT-PCR, and mRNA levels were normalized to those of 18S rRNA. Experiments were performed in triplicate. Values represent the mean ± s.d., n = 4. Bonferroni correction for multiple comparisons was applied. *p < 0.05, **p < 0.01.

Figure 3.

Expression of osteoblast markers in iPSop and MSCop cells at different differentiation stages after 1,25(OH)₂ vitamin D₃ treatment. iPSop and MSCop cells express various osteoblast markers. Comparison of expression of osteoblast marker genes between cells treated with vehicle and 1,25(OH)₂ vitamin D₃. (a) In iPSop- and MSCop-day4 cells. (b) In iPSop- and MSCop-day14 cells. These cells were treated with vehicle (white bar) or 50 nM 1,25(OH)₂ vitamin D₃ (black bar) for 6 or 12 days. Expressions of COL1A1, TNAP, OCN, RUNX2 and OSX were analysed with qRT-PCR, and mRNA levels were normalized to those of 18S rRNA. Experiments were performed in triplicate. Values represent the mean ± s.d., n = 3. Bonferroni correction for multiple comparisons was applied. *p < 0.05, **p < 0.01.

Figure 3.

Expression of osteoblast markers in iPSop and MSCop cells at different differentiation stages after 1,25(OH)₂ vitamin D₃ treatment. iPSop and MSCop cells express various osteoblast markers. Comparison of expression of osteoblast marker genes between cells treated with vehicle and 1,25(OH)₂ vitamin D₃. (a) In iPSop- and MSCop-day4 cells. (b) In iPSop- and MSCop-day14 cells. These cells were treated with vehicle (white bar) or 50 nM 1,25(OH)₂ vitamin D₃ (black bar) for 6 or 12 days. Expressions of COL1A1, TNAP, OCN, RUNX2 and OSX were analysed with qRT-PCR, and mRNA levels were normalized to those of 18S rRNA. Experiments were performed in triplicate. Values represent the mean ± s.d., n = 3. Bonferroni correction for multiple comparisons was applied. *p < 0.05, **p < 0.01.

Figure 4.

Expression of osteocyte-specific marker genes and the mineralization by alizarin red staining. (a) Expression of osteocyte-specific markers in iPSop-day14 cells treated with vehicle (vitamin D−) and 50 nM 1,25(OH)₂ vitamin D₃ (vitamin D+) for 14 days. The expression of dentin matrix protein-1 (DMP-1), fibroblast growth factor-23 (FGF-23), matrix extracellular phosphoglycoprotein (MEPE) and podoplanin (PDPN) were analysed by RT-PCR. β-Actin was used as an internal control. RT-PCR was performed using ExTaq DNA polymerase. Amplified PCR products were electrophoresed on 2% agarose gels. PCR primers are presented in table 2. (b) Mineralization in iPSop-day14 cells treated with vehicle (vitamin D−) and 50 nM 1,25(OH)₂ vitamin D₃ (vitamin D+) for 14 days with alizarin red staining.