Improving the neuronal differentiation efficiency of umbilical cord blood-derived mesenchymal stem cells cultivated under appropriate conditions

Document Type : Original Article


1 Department of Immunology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

2 Department of Medical Laboratory Sciences, School of Paramedicine, Hamadan University of Medical Sciences, Hamadan, Iran

3 Department of Stem Cell Biology, Stem Cell Technology Research Center, Tehran, Iran


Objective(s): Umbilical cord blood-derived mesenchymal stromal cells (UCB-MSCs) are ideally suited for use in various cell-based therapies. We investigated a novel induction protocol (NIP) to improve the neuronal differentiation of human UCB-MSCs under appropriate conditions.
Materials and Methods: This experimental study was performed in Iranian Blood Transfusion Organization (IBTO), Tehran, Iran. UCB-MSCs were cultured in DMEM medium supplemented with 10% FBS in a humidified incubator in equilibration with 5% CO2 at 37oC. For neuronal differentiation of UCB-MSCs, DMEM was removed and replaced with pre-induction medium containing RA, bFGF, EGF, and basal medium for two days. Then, NGF, IBMX, AsA, and Neurobasal medium were used for six days for this purpose. Real-time PCR was performed to analyze the neuronal differentiation of UCB-MSCs for the first time in Iran.
Results: We found that the maximum and minimum levels of gene expression were related to GFAP and nestin, respectively. In addition, our study showed that compared to other neuronal inducers, RA might play the main role in neuronal differentiation and fate of MSCs compared to other neuronal inducers.
Conclusion: Our data showed that the combination of chemical (RA, IBMX, AsA) and growth factors (NGF, EGF, bFGF) in NIP may improve the efficiency of neuronal differentiation of UCB-MSCs and may provide a new method for easy and quick application of UCB-MSCs in regenerative medicine in the future. However, the functionality of neuron-like cells must be carefully assessed in animal experiments prior to use in clinical applications.


1.  Aharonowiz  M, Einstein O, Fainstein N, Lassmann H, Reubinoff B, Ben-Hur T. Neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis. PLoS One 2008;  3: e3145.
2. Ben Hur T, Idelson M, Khaner H, Pera M, Reinhartz E, Itzik A, Reubinoff BE. Transplantation of human embryonic stem cell–derived neural progenitors improves behavioral deficit in parkinsonian rats. Stem Cells 2004; 22:1246-1255.
3. Einstein O, Grigoriadis N, Mizrachi-Kol R, Reinhartz E, Polyzoidou E, Lavon I, et al. Transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis. Exp Neurol  2006; 198:275-284.
4. Hellmann M, Panet H, Barhum Y, Melamed E, Offen D. Increased survival and migration of engrafted mesenchymal bone marrow stem cells in 6-hydroxydopamine-lesioned rodents. Neurosci Lett 2006; 395:124-128.
5. Safford KM, Hicok KC, Safford SD, Halvorsen Y-DC, Wilkison WO, Gimble JM, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 2002; 294:371-379.
6. Knoepfler PS. Deconstructing stem cell tumo-rigenicity: a roadmap to safe regenerative medicine.Stem Cells 2009; 27:1050-1056.
7. Chen FH, Tuan RS. Mesenchymal stem cells in arthritic diseases. Arthritis Res Ther 2008; 10:223.
8. Jung KH, Shin HP, Lee S, Lim YJ, Hwang SH, Han H, et al. Effect of human umbilical cord blood‚Äźderived mesenchymal stem cells in a cirrhotic rat model. Liver Int 2009; 29:898-909.
9. Park DH, Lee JH, Borlongan CV, Sanberg PR, Chung YG, Cho TH. Transplantation of umbilical cord blood stem cells for treating spinal cord injury. Stem Cell Rev 2011; 7:181-194.
10. Cao W, Yang Y, Wang Z, Liu A, Fang L, Wu F, et al. Leukemia inhibitory factor inhibits T helper 17 cell differentiation and confers treatment effects of neural progenitor cell therapy in autoimmune disease. Immunity 2011; 35:273-284.
11. Harris VK, Yan QJ, Vyshkina T, Sahabi S, Liu X, Sadiq SA. Clinical and pathological effects of intrathecal injection of mesenchymal stem cell-derived neural progenitors in an experimental model of multiple sclerosis. J Neurol Sci 2012; 313:167-177.
12. Liu S, Li C, Xing Y, Tao F. Effect of transplantation of human embryonic stem cell-derived neural progenitor cells on adult neurogenesis in aged hippocampus. Am J Stem Cell 2014; 3:21.
13. Yang J, Yan Y,  Ma CG, Kang T, Zhang N, Gran B, et al. Zangaladze, Accelerated and enhanced effect of CCR5-transduced bone marrow neural stem cells on autoimmune encephalomyelitis. Acta Neuropathol 2012; 124:491-503.
14. Jiang Y, Henderson D, Blackstad M, Chen A, Miller RF, Verfaillie CM. Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci 2003; 100:11854-11860.
15. Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A. Adult Bone Marrow Stromal Cells Differentiate into Neural Cells. in Vitro. Exp Neurol 2000; 164:247-256.
16. Rafieemehr S, Kheirandish M, Soleimani M. A New Two Step Induction Protocol for Neural  Differentiation of  Human  Umbilical  Cord  Blood Derived Mesenchymal Stem Cells. IJBC 2015; 7:111-116.
17. Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000; 61:364-370.
18. Greschat S, Schira J, Küry P, Rosenbaum C, de Souza Silva MA, Kögler G, et al. Unrestricted somatic stem cells from human umbilical cord blood can be differentiated into neurons with a dopaminergic phenotype. Stem Cells Dev 2008; 17:221-232.
19. Kögler G, Sensken S, Airey JA, Trapp T, Müschen M, Feldhahn N, et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med  2004; 200:123-135.
20. Lim JY, Park SI, Oh JH, Kim SM, Jeong CH, Jun J, et al. Brain derived neurotrophic factor stimulates the neural differentiation of human umbilical cord blood derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt dependent signaling pathways. J Neurosci Res 2008; 86:2168-2178.
21. Tio M, Tan KH, Lee W, Wang TT, Udolph G. Roles of db-cAMP, IBMX and RA in aspects of neural differentiation of cord blood derived mesenchymal-like stem cells. PLoS One 2010; 5:e9398.
22. Wang T, Tio M, Lee W, Beerheide W, Udolph G. Neural differentiation of mesenchymal-like stem cells from cord blood is mediated by PKA. Biochem Biophys Res Commun 2007; 357:1021-1027.
23. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell  2007; 131:861-872.
24. Venugopal P, Balasubramanian S, Majumdar AS, Ta M. Isolation, characterization, and gene expression analysis of Wharton’s jelly-derived mesenchymal stem cells under xeno-free culture conditions. Stem Cells 2011; 4:39.
25. Kuroda Y, Kitada M, Wakao S, Dezawa M. Mesenchymal stem cells and umbilical cord as sources for schwann cell differentiation: Their potential in peripheral nerve repair, Open Tissue Eng, Regen Med J 2011; 4:54-63.
26.  Zhang J, Chen G, Wang Y, Zhao Y, Duan H, Liao L, et al. Chen, Hydrogen peroxide preconditioning enhances the therapeutic efficacy of Wharton’s Jelly mesenchymal stem cells after myocardial infarction. Chin Med J 2012; 125:3472-3478.
27. Zhang YQ, He LM, Xing B, Zeng X, Zeng CG, Zhang W, et al. Neurotrophin-3 gene-modified Schwann cells promote TrkC gene-modified mesenchymal stem cells to differentiate into neuron-like cells in poly (lactic-acid-co-glycolic acid) multiple-channel conduit. Cells Tissues Organs 2011; 195:313-322.
28. Rebelatto C, Aguiar AM, Moretao MP, Senegaglia AC, Hansen P, Barchiki F, et al. Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue. Exp Biol Med 2008; 233:901-913.
29. Martins A, Paiva A, Morgado J, Gomes A, Pais M. Quantification and immunophenotypic characterization of bone marrow and umbilical cord blood mesenchymal stem cells by multicolor flow cytometry. Transplantation proceedings, Elsevier; 2009.p.943-946.
30. Vishnubalaji P, Al-Nbaheen M, Kadalmani A, Aldahmash A, Ramesh T. Comparative investigation of the differentiation capability of bone-marrow-and adipose-derived mesenchymal stem cells by qualitative and quantitative analysis. Cell Tissue Res 2012; 347:419-427.
31. Zhang X, Hirai M, Cantero S, Ciubotariu S, Dobrila L, Hirsh A, et al. Nishimura, Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue. J Cell Biochem 2011; 112:1206-1218.
32. Bieback K, Kern S, Klüter H, Eichler H. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 2004; 22:625-634.
33. Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006; 24:1294-1301.
34. Kondo T, Matsuoka AJ, Shimomura A, Koehler KR, Chan RJ, Miller JM, et al. Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3. Stem Cells 2011; 29: 836-846.
35. Taran R, Mamidi MK, Singh G, Dutta S, Parhar IS, John JP. In vitro and in vivo neurogenic potential of mesenchymal stem cells isolated from different sources. J Biosci 2014; 39:157-169.
36. Deng W, Obrocka M, Fischer I, Prockop DJ. In Vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun 2001; 282:148-152.