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. 2021 Nov 19;11:22593. doi: 10.1038/s41598-021-02060-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Differentiation of MPC cells abundantly upregulates osteocyte and osteoblast genes. RNA from both MPC1 (white bars) and MPC2 (gray bars) cells were analyzed for several key osteoblast and osteocyte genes over the course of osteogenic differentiation and normalized to β-actin. (A) Sost mRNA gradually increased in both cell types over time. MPC2 cells exhibited higher Sost mRNA induction at Day 7 compared to MPC1, which normalized at day 14 and 21. By day 28 MPC1 cells expressed significantly more Sost mRNA compared to MPC2. Runx2 mRNA levels in undifferentiated cells at Day 0 were modestly elevated in MPC2 cells compared to MPC1. (B) Dmp1 mRNA was similar at baseline and displayed a dramatic increase at Day 7 of differentiation in both cell types. MPC2 cells maintained elevated Dmp1 levels over MPC1 cells until Day 28. MPC2 cells expressed significantly greater levels of Dmp1 compared to MPC1 at 7, 14, and 21 days of culture. (C) Fgf23 mRNA levels between the cell lines were similar at Day 0. Fgf23 mRNA showed a gradual induction over time with differentiation with both cell types. MPC2 cells maintained elevated Fgf23 mRNA over MPC1 cells at all time points except for Day 14. (D) Mepe mRNA was elevated in MPC2 cells at Day 0 compared to MPC1 cells. With differentiation Mepe mRNA significantly rose in MPC2 cells at all time points. For MPC1, Mepe expression was only modestly elevated until Day 21. By Day 28 Mepe mRNA levels were significantly elevated over MPC2 cells. (E) Compared to MPC1, E11 mRNA was highly elevated in MPC2 cells at day 0. With differentiation, MPC1 cells showed a gradual increase in E11 mRNA which surpassed expression levels of MPC2 cells at Day 21 and 28. E11 mRNA was only significantly elevated vs Day 0 in MPC2 cells after Day 21 and 28 of differentiation. (F) At day 0 Bglap mRNA was modestly, but significantly, elevated in MPC2 cells compared to MPC1. With differentiation, Bglap mRNA was dramatically upregulated over time in both cell types. Significant differences between MPC1 and MPC2 cells were evident at each time point until Day 28. (G) With differentiation, Runx2 mRNA significantly increased at day 7 in both cell types compared to day 0. These levels were maintained both throughout differentiation and the elevations between MPC2 cells. (n = 3; mean + standard deviation). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 between MPC1 and MPC2 at the time point designated; ^ap < 0.05, ^bp < 0.01, ^cp < 0.001, ^dp < 0.0001, and ^ep < 0.00001 vs day 0 within the same cell line.