Effect of Lithium Chloride on Proliferation and Bone Differentiation of Rat Marrow-Derived Mesenchymal Stem Cells in Culture

Document Type : Original Article


1 Stem Cell Department, Cell Sciences Research Center, Royan Institute, ACECR, Tehran, Iran.

2 School of Biology, College of Science, University of Tehran, Tehran, Iran


It is believed that the mesenchymal stem cell (MSC) differentiation and proliferation are the results of activation of wnt signaling pathway. On the other hand, lithium chloride is reported to be able to activate this pathway. The objective of this study was to investigate the effect of lithium on in vitro proliferation and bone differentiation of marrow-derived MSC.
Materials and Methods
In this experimental study, rat marrow cells were plated in a medium supplemented either with or without       2-10 mM lithium and expanded through three successive subcultures. To explore the impact of lithium on cell growth, doubling time (DT) of marrow cell population was determined for all the cultures. To determine the lithium effects on osteogenesis, the proliferation medium of passged-3 cells from all cultures were replaced by osteogenic media, with or without 2-12 mM lithium. Osteogenesis was then quantified by measurement of the amount of matrix mineralization and the expression of bone-specific genes.
DT results indicated that the marrow cells in 4 mM lithium concentration were grown faster than the others (P<0.05). Intensive matrix mineralization and abundance of bone specific gene expression were observed in the cultures with 10-12 mM lithium concentration. All these differences were statistically significant. According to the results, all lithium -treated cultures possessed more differentiation than the control. Moreover, lithium low concentration was associated with more proliferation and its high concentration with more differentiating effects.
Lithium chloride at 4 mM concentration promotes MSC proliferation and at 10-12 mM enhances MSC osteogenic differentiation.


1.  Barry FP. Biology and clinical application of mesenchymal stem cells. Birth Defects Res 2003; 69:707-715.
2. Friedenstein AJ, Chailakhajan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 1970; 3:393-403.
3. Periera RF, Halford KW, OHara MD, Leeper DB, Sokolov BP, Pollard MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci USA 1995; 11:4857-4861.
4. Eslaminejad MB, Nikmahzar A, Taghiyar L, Nadri S, Massumi M. Murine mesenchymal stem cells isolated by low density primary culture system. Dev Growth Differ 2006; 48:361-370.
5. Meirelles Lda S, Nardi NB. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol 2003; 4:702-711.
6. Zuk PA, Zhu M, Mizuno H, Huang G, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: Implication for cell-based therapies. Tissue Eng 2001; 7:211-228.
7. Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver and bone marrow. Blood 2001; 98:2396-2402.
8. Pieternella S, Anker SA, Kleijburg-van der keur C, Noort WA, Class FH, Willemze R, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003; 102:1548-1549.
9. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 2000; 109:235-242.
10. Pittenger MF, Makay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Scince 1999; 248:143-147.
11. Yeon Lim J, Jeun SS, Lee KJ, Oh JH, Kim SM, Park SI, et al. Multiple stem cell traits of expanded rat bone marrow stromal cells. Exp Neurol 2006; 199:416-426.
12. Martin I, Muraglia A, Campanile G, Cancedda R, Quarto R. Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrin 1997; 138:4456- 4462.
13. Bianco P, Robey PG. Stem cells in tissue engineering. Nature 2001; 414:118-121.
14. Khojasteh A, Eslaminejad MB, Nazarian H. Mesenchymal stem cells enhance bone regeneration in rat calvarial critical size defect more than platelete rich plasma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 106:356-362.

15. Jafarian M, Eslaminejad MB, Khojasteh A,  Mashhadi Abbas F, Dehgan MM, Hassanizadeh R et al. Marrow-derived mesenchymal stem cells-directed bone regeneration in the dog mandible: A comparison between Biphasic calcium phosphate and Natural bone mineral. Oral Surg Oral Med Oral Pathol Oral Radiol Endod  2008; 105:e14-e24.

16. Stefan H, Claire LK. Wnt signalling: variety at the core. J Cell Sci 2007; 120:385-393.
17. Cadigan KM, Nusse R. Wnt signaling: a common theme in animal development. Genes Dev 1997; 15:3286-3305.
18. Smalley MJ, Dale TC. Wnt signalling in mammalian development and cancer. Cancer Metastasis Rev 1999; 18:215-230.
19. Willert K, Brown JD, Danenberg E, Duncan AW, Welssman IL, Reya Tet al. Wnt proteins are lipid-modified and can act as cell growth factors. Nature 2003; 423:448-452.
20. Williams RS, Cheng L, Mudge AW, Harwood AJ. A common mechanism of action for three mood-stabilizing drugs. Nature 2002; 417:292-295.
21. Stambolic V, Ruel L, Woodgett JR. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol 1996; 6:1664-1668.
22. Todd DG, Husseini KM. Glycogen synthase kinase-3: putative molecular target for lithium mimetic drugs. Neuropsychopharmacol 2005; 30:1223-1237.
23. Yoshino JE, DeVries GH. Effect of lithium on Schwann cell proliferation stimulated by axolemma- and myelin-enriched fractions. J Neurochem  1987; 48:1270-1277.
24. Smits VA, Essers MA, Loomans DS, Klompmaker R, Rijksen G, Medema RH. Inhibition of cell proliferation by lithium is associated with interference in cdc2 activation. FEBS Lett  1999; 457:23-27.
25. De Boer J, Wang HJ, Van Blitterswijk C. Effects of Wnt signaling on proliferation and differentiation of human mesenchymal stem cells. Tissue Eng 2004; 10:393-401.
26. Pioletti D, Montjovent MC, Zambelli PY, Applegate L. Bone tissue engineering using fetal cell therapy. Swiss Med Wkly 2002; 136:557-560.
27. Weissman IL. Translating stem and progenitor cell biology to the clinic: barrier and opportunities. Science 2000; 287:1442-1446.
28. Hessle L, Johnson KA, Anderson HC, Narisawa S, Sali A, Goding JW, et al. Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization. Proc Natl Acad Sci USA 2002; 99:9445-9.
29. Hauschka PV, Lian JB, Cole DE, Gundberg CM. Osteocalcin and matrix Gla protein: vitamin K- dependent proteins in bone. Physiol Rev 1989; 69:990-1047.
30. Sinha D, Wang Z, Ruchalski KL, Levine JS, Krishnan S, Lieberthal W, et al. Lithium activates the Wnt and phosphatidylinositol 3-kinase Akt signaling pathways to promote cell survival in the absence of soluble survival factors. Am J Physiol Renal Physiol  2005; 288: F703-F713.
31. Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004; 10:23-24.
32. Potten CS, Lajtha LG. Stem cells versus stem lines. Ann N Y Acad  Sci 1982;397: 49-61.
33. Garcia-Maya M, Anderson AA, Kendal CE, Kenny AV, Edwards-Ingram LC, Holladay A, et al. Ligand concentration is a driver of divergent signaling and pleiotropic cellular responses to FGF. 1. J Cell Physiol 2006; 206:386-393
34. Tropel P, Noel D, Platet N, Legrand P, Benabid AL, Berger F. Isolation and characterization of mesenchymal stem cells from adult mouse bone marrow. Exp Cell Res 2004; 295:395-406.
35. Peister A, Mellad JA, Larsen LL, Hall BM, Gibson LF, Prockop DJ. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 2004; 103:1662-1668.
36. Sun S, Guo Z, Xiao X, Liu B, Liu X, Tang P-H, et al. Isolation of mouse marrow mesenchymal progenitors by a novel and reliable methods. Stem cells 2003; 21:527-535.
37. Eslaminejad MB, Mirzadeh H, Mohamadi Y, Aghbibi N. Bone differentiation of the marrow-derived mesenchymal stem cells using β-tricalcium phosphate/alginate/gelatin hybrid scaffolds. J Tissue Eng Regen Med 2007; 6:417- 424.