Abstract
The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10 µg/l) and basic fibroblastic growth factor (bFGF, 10 µg/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10 µM). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20 ± 8.677 (positive ratio 0.2140 ± 0.0119) to 109.20 ± 8.430 (positive ratio 0.3193 ± 0.0161) per visual field in the presence of Y-27632, while GFAP-positive cell number increased from 96.30 ± 8.486 (positive ratio 0.18 ± 0.0152) to 107.50 ± 8.683 (positive ratio 0.27 ± 0.0115) (P < 0.05 for both). Both NSE and GFAP protein expression levels were enhanced significantly by Y-27632 treatment (NSE: 0.74 ± 0.05 vs. 1.03 ± 0.06; GFAP: 0.64 ± 0.08 vs. 0.97 ± 0.05, both P < 0.01) as indicated by Western blots. The Rho kinase inhibitor Y-27632 concomitant with EGF and bFGF stimulation promotes BMSC differentiation into neural cells. Control of Rho kinase activity may enhance the efficiency of stem cell-based treatments for neurodegenerative diseases.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- BMSCs:
-
Bone-marrow mesenchymal stem cells
- EGF:
-
Epidermal growth factor
- bFGF:
-
Basic fibroblastic growth factor
- NSE:
-
Neuron-specific enolase
- GFAP:
-
Glial fibrillary acidic protein
References
Asumda FZ, Chase PB (2011) Age-related changes in rat bone-marrow mesenchymal stem cell plasticity. BMC Cell Biology 12:44. doi:10.1186/1471-2121-12-44
David M, Petit D, Bertoglio J (2012) Cell cycle regulation of Rho signaling pathways. Cell Cycle 11:3003–3010. doi:10.4161/cc.21088
Delorme B et al (2008) Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells. Blood 111:2631–2635. doi:10.1182/blood-2007-07-099622
Dolgin E (2011) Flaw in induced-stem-cell model. Nature 470:13. doi:10.1038/470013a
Estes BT, Diekman BO, Gimble JM, Guilak F (2010) Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc 5:1294–1311. doi:10.1038/nprot.2010.81
Grassel S, Stockl S, Jenei-Lanzl Z (2012) Isolation, culture, and osteogenic/chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Methods Mol Biol 879:203–267. doi:10.1007/978-1-61779-815-3_14
Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9:690–701. doi:10.1038/nrm2476
Hussein SM et al (2011) Copy number variation and selection during reprogramming to pluripotency. Nature 471:58–62. doi:10.1038/nature09871
Kazemnejad S et al (2009) Biochemical and molecular characterization of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel three-dimensional biocompatible nanofibrous scaffold. J Gastroenterol Hepatol 24:278–287. doi:10.1111/j.1440-1746
Kim K et al (2010) Epigenetic memory in induced pluripotent stem cells. Nature 467:285–290. doi:10.1038/nature09342
Kocamaz E, Gok D, Cetinkaya A, Tufan AC (2012) Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells. J Mol Histol 43(5):497–508. doi:10.1007/s10735-012-9430-2
Koyanagi M et al (2008) Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors. J Neurosci Res 86:270–280. doi:10.1002/jnr.21502
Kumar BM et al (2012) Neurogenic and cardiomyogenic differentiation of mesenchymal stem cells isolated from minipig bone marrow. Res Vet Sci 93:749–757. doi:10.1016/j.rvsc.2011.09.012
Li J et al (2008) Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness. Cell Res 18:360–371. doi:10.1038/cr.2007.103
Li X, Zhang Y, Qi G (2013) Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells. Cytotechnology 65:323–334. doi:10.1007/s10616-012-9497-3
Linseman DA, Loucks FA (2008) Diverse roles of Rho family GTPases in neuronal development, survival, and death Frontiers. Bioscience 13:657–676
Lo B, Parham L (2009) Ethical issues in stem cell research. Endocr Rev 30:204–213. doi:10.1210/er.2008-0031
Loucks FA, Le SS, Zimmermann AK, Ryan KR, Barth H, Aktories K, Linseman DA (2006) Rho family GTPase inhibition reveals opposing effects of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase and Janus kinase/signal transducer and activator of transcription signaling cascades on neuronal survival. J Neurochem 97:957–967. doi:10.1111/j.1471-4159.2006.03802.x
Maggini J et al (2010) Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile. PLoS ONE 5:e9252. doi:10.1371/journal.pone.0009252
Mayshar Y et al (2010) Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7:521–531. doi:10.1016/j.stem.2010.07.017
Murakoshi H, Wang H, Yasuda R (2011) Local, persistent activation of Rho GTPases during plasticity of single dendritic spines. Nature 472:100–104. doi:10.1038/nature09823
Ozdemir D et al (2012) The effect of Rho kinase inhibitor Y-27632 on endotoxemia-induced intestinal apoptosis in infant rats. J Mol Histol 43(1):81–87. doi:10.1007/s10735-011-9379-6
Parekkadan B, Milwid JM (2010) Mesenchymal stem cells as therapeutics. Annu Rev Biomed Eng 12:87–117. doi:10.1146/annurev-bioeng-070909-105309
Ramasamy R, Tong CK, Seow HF, Vidyadaran S, Dazzi F (2008) The immunosuppressive effects of human bone marrow-derived mesenchymal stem cells target T cell proliferation but not its effector function. Cell Immunol 251:131–136. doi:10.1016/j.cellimm.2008.04.009
Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG (2000) Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med 6:278–282. doi:10.1038/73128
Rizzino A (2010) Stimulating progress in regenerative medicine: improving the cloning and recovery of cryopreserved human pluripotent stem cells with ROCK inhibitors. Regenerative Med 5:799–807. doi:10.2217/rme.10.45
Shi Y et al (2010) Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair. Cell Res 20:510–518. doi:10.1038/cr.2010.44
Sun J et al. (2014) Overexpression of the PLAP-1 gene inhibits the differentiation of BMSCs into osteoblast-like cells J Mol Histol. [Epub ahead of print]
Takakura A et al (2000) Involvement of a small GTP-binding protein (G protein) regulator, small G protein GDP dissociation stimulator, in antiapoptotic cell survival signaling. Mol Biol Cell 11:1875–1886
Uccelli A, Laroni A, Freedman MS (2011) Mesenchymal stem cells for the treatment of multiple sclerosis and other neurological diseases. Lancet Neurol 10:649–656. doi:10.1016/S1474-4422(11)70121-1
Wislet-Gendebien S et al (2012) Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences. Cell Mol Life Sci CMLS 69:2593–2608. doi:10.1007/s00018-012-0937-1
Woodbury D, Schwarz EJ, Prockop DJ, Black IB (2000) Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 61:364–370
Wu B et al (2013) Lentiviral delivery of biglycan promotes proliferation and increases osteogenic potential of bone marrow-derived mesenchymal stem cells in vitro. J Mol Histol 44(4):423–431. doi:10.1007/s10735-013-9497-4
Yamachika E et al (2012) Basic fibroblast growth factor supports expansion of mouse compact bone-derived mesenchymal stem cells (MSCs) and regeneration of bone from MSC in vivo. J Mol Histol 43(2):223–233. doi:10.1007/s10735-011-9385-8
Acknowledgments
The authors thank the authorities of the Central Laboratory of the Affiliated Hospital of Xuzhou Medical College for providing research facilities for this work. Furthermore, this study was supported by a grant of the National Natural Science Foundation of China (No. 81371243).
Conflicts of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
Xiao Liu, Zhengzheng Zhang and Xianliang Yan have contributed equally to this work.
Rights and permissions
About this article
Cite this article
Liu, X., Zhang, Z., Yan, X. et al. The Rho kinase inhibitor Y-27632 facilitates the differentiation of bone marrow mesenchymal stem cells. J Mol Hist 45, 707–714 (2014). https://doi.org/10.1007/s10735-014-9594-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10735-014-9594-z