Effects of DLX3 on the osteogenic differentiation of induced pluripotent stem cell‑derived mesenchymal stem cells

Li,Junyuan; Lin,Qiang; Lin,Yixin; Lai,Renfa; Zhang,Wen

doi:10.3892/mmr.2021.11871

Effects of DLX3 on the osteogenic differentiation of induced pluripotent stem cell‑derived mesenchymal stem cells

Authors:
- Junyuan Li
- Qiang Lin
- Yixin Lin
- Renfa Lai
- Wen Zhang
View Affiliations

Affiliations: The Medical Center of Stomatology, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong 510630, P.R. China, Department of Endodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
Published online on: January 26, 2021 https://doi.org/10.3892/mmr.2021.11871
Article Number: 232
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Osteoporosis is a disease characterized by the degeneration of bone structure and decreased bone mass. Induced pluripotent stem cell‑derived mesenchymal stem cells (iPSC‑MSCs) have multiple advantages that make them ideal seed cells for bone regeneration, including high‑level proliferation, multi‑differentiation potential and favorable immune compatibility. Distal‑less homeobox (DLX)3, an important member of the DLX family, serves a crucial role in osteogenic differentiation and bone formation. The present study aimed to evaluate the effects of DLX3 on the proliferation and osteogenic differentiation of human iPSC‑MSCs. iPSC‑MSCs were induced from iPSCs, and identified via flow cytometry. Alkaline phosphatase (ALP), Von Kossa, Oil Red O and Alcian blue staining methods were used to evaluate the osteogenic, adipogenic and chondrogenic differentiation of iPSC‑MSCs. DLX3 overexpression plasmids were constructed and transfected into iPSC‑MSCs to generate iPSC‑MSC‑DLX3. iPSC‑MSC‑GFP was used as the control. Reverse transcription‑quantitative PCR (RT‑qPCR) and western blotting were performed to measure the expression of DLX3 2 days after transfection. Subsequently, cell proliferation was assessed using a Cell Counting Kit‑8 assay on days 1, 3, 5 and 7. RT‑qPCR and western blotting were used to analyze osteogenic‑related gene and protein expression levels on day 7. ALP activity and mineralized nodules were assessed via ALP staining on day 14. Statistical analysis was performed using an unpaired Student's t‑test. Flow cytometry results demonstrated that iPSC‑MSCs were positive for CD73, CD90 and CD105, but negative for CD34 and CD45. iPSC‑MSC‑DLX3 had significantly lower proliferation compared with iPSC‑MSC‑GFP on days 5 and 7 (P<0.05). mRNA expression levels of osteogenic markers, such as ALP, osteopenia (OPN), osteocalcin (OCN) and Collagen Type I (COL‑1), were significantly increased in iPSC‑MSC‑DLX3 compared with iPSC‑MSC‑GFP on day 7 (P<0.05). Similarly, the protein expression levels of ALP, OCN, OPN and COL‑1 were significantly increased in iPSC‑MSC‑DLX3 compared with iPSC‑MSC‑GFP on day 7 (P<0.05). The number of mineralized nodules in iPSC‑MSC‑DLX3 was increased compared with that in iPSC‑MSC‑GFP on day 14 (P<0.05). Thus, the present study demonstrated that DLX3 serves a negative role in proliferation, but a positive role in the osteogenic differentiation of iPSC‑MSCs. This may provide novel insight for treating osteoporosis.

View Figures

View References

1	Feng X and McDonald JM: Disorders of bone remodeling. Annu Rev Pathol. 6:121–145. 2011. View Article : Google Scholar : PubMed/NCBI
2	Raisz LG: Pathogenesis of osteoporosis: Concepts, conflicts, and prospects. J Clin Invest. 115:3318–3325. 2005. View Article : Google Scholar : PubMed/NCBI
3	Cummings SR and Melton LJ: Epidemiology and outcomes of osteoporotic fractures. Lancet. 359:1761–1767. 2002. View Article : Google Scholar : PubMed/NCBI
4	Genant HK, Cooper C, Poor G, Reid I, Ehrlich G, Kanis J, Nordin BE, Barrett-Connor E, Black D, Bonjour JP, et al: Interim report and recommendations of the World Health Organization Task-Force for Osteoporosis. Osteoporos Int. 10:259–264. 1999. View Article : Google Scholar : PubMed/NCBI
5	Aspray TJ and Hill TR: Osteoporosis and the Ageing Skeleton. Subcell Biochem. 91:453–476. 2019. View Article : Google Scholar : PubMed/NCBI
6	Hassan MQ, Javed A, Morasso MI, Karlin J, Montecino M, van Wijnen AJ, Stein GS, Stein JL and Lian JB: Dlx3 transcriptional regulation of osteoblast differentiation: Temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene. Mol Cell Biol. 24:9248–9261. 2004. View Article : Google Scholar : PubMed/NCBI
7	Zhao N, Zeng L, Liu Y, Han D, Liu H, Xu J, Jiang Y, Li C, Cai T, Feng H, et al: DLX3 promotes bone marrow mesenchymal stem cell proliferation through H19/miR-675 axis. Clin Sci (Lond). 131:2721–2735. 2017. View Article : Google Scholar : PubMed/NCBI
8	Li Y, Han D, Zhang H, Liu H, Wong S, Zhao N, Qiu L and Feng H: Morphological analyses and a novel de novo DLX3 mutation associated with tricho-dento-osseous syndrome in a Chinese family. Eur J Oral Sci. 123:228–234. 2015. View Article : Google Scholar : PubMed/NCBI
9	Choi SJ, Song IS, Ryu OH, Choi SW, Hart PS, Wu WW, Shen RF and Hart TC: A 4 bp deletion mutation in DLX3 enhances osteoblastic differentiation and bone formation in vitro. Bone. 42:162–171. 2008. View Article : Google Scholar : PubMed/NCBI
10	Lin H, Sohn J, Shen H, Langhans MT and Tuan RS: Bone marrow mesenchymal stem cells: Aging and tissue engineering applications to enhance bone healing. Biomaterials. 203:96–110. 2019. View Article : Google Scholar : PubMed/NCBI
11	Sabapathy V and Kumar S: hiPSC-derived iMSCs: NextGen MSCs as an advanced therapeutically active cell resource for regenerative medicine. J Cell Mol Med. 20:1571–1588. 2016. View Article : Google Scholar : PubMed/NCBI
12	Kang R, Zhou Y, Tan S, Zhou G, Aagaard L, Xie L, Bünger C, Bolund L and Luo Y: Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity. Stem Cell Res Ther. 6:1442015. View Article : Google Scholar : PubMed/NCBI
13	Xie J, Peng C, Zhao Q, Wang X, Yuan H, Yang L, Li K, Lou X and Zhang Y: Osteogenic differentiation and bone regeneration of iPSC-MSCs supported by a biomimetic nanofibrous scaffold. Acta Biomater. 29:365–379. 2016. View Article : Google Scholar : PubMed/NCBI
14	Jungbluth P, Spitzhorn LS, Grassmann J, Tanner S, Latz D, Rahman MS, Bohndorf M, Wruck W, Sager M, Grotheer V, et al: Human iPSC-derived iMSCs improve bone regeneration in mini-pigs. Bone Res. 7:322019. View Article : Google Scholar : PubMed/NCBI
15	Villa-Diaz LG, Brown SE, Liu Y, Ross AM, Lahann J, Parent JM and Krebsbach PH: Derivation of mesenchymal stem cells from human induced pluripotent stem cells cultured on synthetic substrates. Stem Cells. 30:1174–1181. 2012. View Article : Google Scholar : PubMed/NCBI
16	Liu Y, Goldberg AJ, Dennis JE, Gronowicz GA and Kuhn LT: One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating. PLoS One. 7:e332252012. View Article : Google Scholar : PubMed/NCBI
17	TheinHan WW, Liu J, Tang M, Chen W, Cheng L and Xu HH: Induced pluripotent stem cell-derived mesenchymal stem cell seeding on biofunctionalized calcium phosphate cements. Bone Res. 1:371–384. 2013. View Article : Google Scholar
18	Chen YS, Pelekanos RA, Ellis RL, Horne R, Wolvetang EJ and Fisk NM: Small molecule mesengenic induction of human induced pluripotent stem cells to generate mesenchymal stem/stromal cells. Stem Cells Transl Med. 1:83–95. 2012. View Article : Google Scholar : PubMed/NCBI
19	Meng X, Su RJ, Baylink DJ, Neises A, Kiroyan JB, Lee WY, Payne KJ, Gridley DS, Wang J, Lau KH, et al: Rapid and efficient reprogramming of human fetal and adult blood CD34⁺ cells into mesenchymal stem cells with a single factor. Cell Res. 23:658–672. 2013. View Article : Google Scholar : PubMed/NCBI
20	Sun S, Yu M, Fan Z, Yeh IT, Feng H, Liu H and Han D: DLX3 regulates osteogenic differentiation of bone marrow mesenchymal stem cells via Wnt/β-catenin pathway mediated histone methylation of DKK4. Biochem Biophys Res Commun. 516:171–176. 2019. View Article : Google Scholar : PubMed/NCBI
21	Li X, Yang G and Fan M: Effects of homeobox gene distal-less 3 on proliferation and odontoblastic differentiation of human dental pulp cells. J Endod. 38:1504–1510. 2012. View Article : Google Scholar : PubMed/NCBI
22	Spandidos A, Wang X, Wang H and Seed B: PrimerBank: A resource of human and mouse PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res. 38 (Suppl 1):D792–D799. 2010. View Article : Google Scholar : PubMed/NCBI
23	Zhang W, Zhang X, Li J, Zheng J, Hu X, Xu M, Mao X and Ling J: Foxc2 and BMP2 induce osteogenic/odontogenic differentiation and mineralization of human stem cells from Apical Papilla. Stem Cells Int. 2018:23639172018. View Article : Google Scholar : PubMed/NCBI
24	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
25	Zou L, Luo Y, Chen M, Wang G, Ding M, Petersen CC, Kang R, Dagnaes-Hansen F, Zeng Y, Lv N, et al: A simple method for deriving functional MSCs and applied for osteogenesis in 3D scaffolds. Sci Rep. 3:22432013. View Article : Google Scholar : PubMed/NCBI
26	Xu M, Shaw G, Murphy M and Barry F: Induced pluripotent stem cell-derived mesenchymal stromal cells are functionally and genetically different from bone marrow-derived mesenchymal stromal cells. Stem Cells. 37:754–765. 2019. View Article : Google Scholar : PubMed/NCBI
27	Zhan Y, Li X, Gou X, Yuan G, Fan M and Yang G: Dlx3 inhibits the proliferation of human dental pulp cells through inactivation of canonical wnt/beta-catenin signaling pathway. Front Physiol. 9:16372018. View Article : Google Scholar : PubMed/NCBI
28	Beanan MJ and Sargent TD: Regulation and function of Dlx3 in vertebrate development. Dev Dyn. 218:545–553. 2000. View Article : Google Scholar : PubMed/NCBI
29	Ghoul-Mazgar S, Hotton D, Lézot F, Blin-Wakkach C, Asselin A, Sautier JM and Berdal A: Expression pattern of Dlx3 during cell differentiation in mineralized tissues. Bone. 37:799–809. 2005. View Article : Google Scholar : PubMed/NCBI
30	Lee SH, Oh KN, Han Y, Choi YH and Lee KY: Estrogen Receptor α Regulates Dlx3-Mediated Osteoblast Differentiation. Mol Cells. 39:156–162. 2016. View Article : Google Scholar : PubMed/NCBI
31	Li H, Marijanovic I, Kronenberg MS, Erceg I, Stover ML, Velonis D, Mina M, Heinrich JG, Harris SE, Upholt WB, et al: Expression and function of Dlx genes in the osteoblast lineage. Dev Biol. 316:458–470. 2008. View Article : Google Scholar : PubMed/NCBI
32	Duverger O, Isaac J, Zah A, Hwang J, Berdal A, Lian JB and Morasso MI: In vivo impact of Dlx3 conditional inactivation in neural crest-derived craniofacial bones. J Cell Physiol. 228:654–664. 2013. View Article : Google Scholar : PubMed/NCBI
33	Isaac J, Erthal J, Gordon J, Duverger O, Sun HW, Lichtler AC, Stein GS, Lian JB and Morasso MI: DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo. Cell Death Differ. 21:1365–1376. 2014. View Article : Google Scholar : PubMed/NCBI
34	Sözen T, Özışık L and Başaran NC: An overview and management of osteoporosis. Eur J Rheumatol. 4:46–56. 2017. View Article : Google Scholar : PubMed/NCBI
35	Zou Z, Liu W, Cao L, Liu Y, He T, Peng S and Shuai C: Advances in the occurrence and biotherapy of osteoporosis. Biochem Soc Trans. 48:1623–1636. 2020. View Article : Google Scholar : PubMed/NCBI
36	Yin X, Zhou C, Li J, Liu R, Shi B, Yuan Q and Zou S: Autophagy in bone homeostasis and the onset of osteoporosis. Bone Res. 7:282019. View Article : Google Scholar : PubMed/NCBI
37	Li H, Jeong HM, Choi YH, Kim JH, Choi JK, Yeo CY, Jeong HG, Jeong TC, Chun C and Lee KY: Protein kinase a phosphorylates Dlx3 and regulates the function of Dlx3 during osteoblast differentiation. J Cell Biochem. 115:2004–2011. 2014.PubMed/NCBI