Differentiation of Dopaminergic Neurons from Mesenchymal-Like Stem Cells Derived from Human Umbilical Cord Vein

  • Published : 2009.09.30

Abstract

One of the most extensively studied populations of multipotent adult stem cells are mesenchymal stem cells (MSCs). MSCs derived from the human umbilical cord vein (HUC-MSCs) are morphologically and immunophenotypically similar to MSCs isolated from bone marrow. HUC-MSCs are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. Since neural tissue has limited intrinsic capacity of repair after injury, the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesenchymal-like stem cells from the human umbilical cord vein, and studied transdifferentiation-promoting conditions in neural cells. Dopaminergic neuronal differentiation of HUC-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulfoxide (DMSO) and butylated hydroxyanisole (BHA) in N2 medium and N2 supplement. The immunoreactive cells for $\beta$-tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. HUC-MSCs treated with bFGF, SHH and FGF8 were differentiated into dopaminergic neurons that were immunopositive for tyrosine hydroxylase (TH) antibody. HUC-MSCs treated with DMSO and BHA rapidly showed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including NeuroD1, $\beta$-tubulin III, GFAP and nestin was markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after neural differentiation, we confirmed the differentiation of dopaminergic neurons by TH/$\beta$-tubulin III positive cells. In conclusion, HUC-MSCs can be differentiated into dopaminergic neurons and these findings suggest that HUC-MSCs are alternative cell source of therapeutic treatment for neurodegenerative diseases.

Keywords

References

  1. Bjorklund A, Lindvall O (2000) Cell replacement therapies for central nervous system disorders. Nat Neurosci 3:537-544. https://doi.org/10.1038/75705
  2. Deans RJ, Moseley AB (2000) Mesenchymal stem cells: biology and potential clinical use. Exp Hematol 28:875-884. https://doi.org/10.1016/S0301-472X(00)00482-3
  3. Deng W, Obrocka M, Fischer I, Prockop DJ (2001) 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 282:148-152. https://doi.org/10.1006/bbrc.2001.4570
  4. Fu YS, Cheng YC, Lin MY, Cheng H, Chu PM, Chou SC, Shih YH, Ko MH, Sung MS (2006) Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism. Stem Cells 24:115-124. https://doi.org/10.1634/stemcells.2005-0053
  5. Gajovic S, St-Onge L, Yokota Y, Gruss P (1997) Retinoic acid mediates Pax6 expression during in vitro differentiation of embryonic stem cells. Differentiation 62:187-192.
  6. Hirsch EC, Graybiel AM, Agid YA (1988) Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson's disease. Nature 334:345-348. https://doi.org/10.1038/334345a0
  7. Kageyama R, Ishibashi M, Takebayashi K, Takebayashi K, Tomita K (1997) bHLH transcription factors and mammalian neuronal differentiation. Int J Biochem Cell Biol 29:1389-1399. https://doi.org/10.1016/S1357-2725(97)89968-2
  8. Kan I, Ben-Zur T, Barhum Y, Levy YS, Burstein A, Charlow T, Bulvik S, Melamed E, Offen D (2007) Dopaminergic differentiation of human mesenchymal stem cells-utilization of bioassay for tyrosine hydroxylase expression. Neurosci Lett 419:28-33. https://doi.org/10.1016/j.neulet.2007.03.070
  9. Kestendjieva S, Kyurkchiev D, Tsvetkova G, Mehandjiev T, Dimitrov A, Nikolov A, Kyurkchiev S (2008) Characterization of mesenchymal stem cells isolated from the human umbilical cord. Cell Biol Int 32:724-32. https://doi.org/10.1016/j.cellbi.2008.02.002
  10. Kim BJ, Seo JH, Bubien JK, Oh YS (2002) Differentiation of adult bone marrow stem cells into neuroprogenitor cells in vitro. Neuroreport 13:1185-1188. https://doi.org/10.1097/00001756-200207020-00023
  11. Kopen GC, Prockop DJ, Phinney DG (1999) Marrow stromal cells migrate throughout forebrain and cerebellum and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 96:10711-10716. https://doi.org/10.1073/pnas.96.19.10711
  12. Lee JE, Hollenberg SM, Snider L, Turner DL, Lipnick N, Weintraub H (1995) Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268:836-844. https://doi.org/10.1126/science.7754368
  13. Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD (2000) Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 18:675-679. https://doi.org/10.1038/76536
  14. Lindvall O (1989) Transplantation into the human brain: present status and future possibilities. J Neurol Neurosurg Psychiatry suppl:39-54.
  15. Mahowald MB, Arren J, Hoffer BJ, Jonsen AR, King P, Silver J, Slader JR Jr, Walters L (1987) Transplantation of neural tissue from fetuses. Science 235:1307-1308.
  16. Maxwell SL, Li M (2005) Midbrain dopaminergic development in vivo and in vitro from embryonic stem cells. Anat 207:209-218. https://doi.org/10.1111/j.1469-7580.2005.00453.x
  17. McCormick MB, Tamimi RM, Snider L, Asakura A, Berqstrom D, Tapscott SJ (1996) NeuroD2 and Neuro D3: distinct expression patterns and transcriptional activation potentials within the neuroD gene family. Mol Cell Biol 16:5792-5800. https://doi.org/10.1128/MCB.16.10.5792
  18. Patapoutian A, Reichardt LF (2000) Roles of Wnt proteins in enural development and maintenance. Curr Opin Neurobiol 10:392-399. https://doi.org/10.1016/S0959-4388(00)00100-8
  19. Paul G, Li JY, Brundin P (2002) Stem cells: hype or hope? Drug Discov Today 7:295-302. https://doi.org/10.1016/S1359-6446(01)02155-9
  20. Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA 101:12543-12548. https://doi.org/10.1073/pnas.0404700101
  21. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143-147. https://doi.org/10.1126/science.284.5411.143
  22. Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi CX, Stedeford T, Willing A, Freeman TB, Saporta S, Zigova T, Sanberg PR, Synder EY (2000) Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 164:247-256. https://doi.org/10.1006/exnr.2000.7389
  23. Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T, Lenvik T, Johnson S, Hu WS, Verfaillie CM (2002) Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 109:1291-1302.
  24. Smidt MP, Asbreuk CH, Cox JJ, Chen H, Johnson RL, Burbach JP (2000) A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b. Nat Neurosci 3:337-341. https://doi.org/10.1038/73902
  25. Toma C, Pittenger MF, Cahill KS, Bvrne BJ, Kessler PD (2002) Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 105:93-98. https://doi.org/10.1161/hc0102.101442
  26. 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. https://doi.org/10.1002/1097-4547(20000815)61:4<364::AID-JNR2>3.0.CO;2-C
  27. Ye W, Shimamura K, Rubenstein JL, Hynes MA, Rosenthal A (1998) FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 93:755-766. https://doi.org/10.1016/S0092-8674(00)81437-3