Neurochemical Research

, Volume 35, Issue 9, pp 1455–1470 | Cite as

Characterization of Neural Stem/Progenitor Cells Expressing VEGF and its Receptors in the Subventricular Zone of Newborn Piglet Brain

  • Jahan Ara
  • Saskia Fekete
  • Anli Zhu
  • Melissa Frank


Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multipotent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.


Neural stem/progenitor cells Subventricular zone Proliferation Newborn brain Piglet VEGF 



We thank Dr. Judy Grinspan, Children’s Hospital of Philadelphia and University of Pennsylvania, for kindly providing the anti-O4 and Anti-GFAP antibodies. We also thank Dr. David Pleasure, UC Davis School of Medicine/Shriners Hospital, California, for critically reviewing the manuscript. This work was supported by American Heart Association #0835233 N; March of Dimes Foundation #6-FY09-321 and Saint Christopher’s Foundation for Children 270675 (to JA).


  1. 1.
    Lindvall O, Kokaia Z (2006) Stem cells for the treatment of neurological disorders. Nature 441:1094–1096CrossRefPubMedGoogle Scholar
  2. 2.
    Palmer TD, Ray J, Gage FH (1995) FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci 6:474–486CrossRefPubMedGoogle Scholar
  3. 3.
    Reynolds BA, Tetzlaff W, Weiss S (1992) A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 12:4565–4574PubMedGoogle Scholar
  4. 4.
    Sanai N, Tramontin AD, Quinones-Hinojosa A, Barbaro NM, Gupta N, Kunwar S, Lawton MT, McDermott MW, Parsa AT, Manuel-Garcia Verdugo J, Berger MS, Alvarez-Buylla A (2004) Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature 427:740–744CrossRefPubMedGoogle Scholar
  5. 5.
    Emsley JG, Mitchell BD, Kempermann G, Macklis JD (2005) Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells. Prog Neurobiol 75:321–341CrossRefPubMedGoogle Scholar
  6. 6.
    Scheffler B, Edenhofer F, Brustle O (2006) Merging fields: stem cells in neurogenesis, transplantation, and disease modeling. Brain Pathol 16:155–168CrossRefPubMedGoogle Scholar
  7. 7.
    Li Y, Chopp M (1999) Temporal profile of nestin expression after focal cerebral ischemia in adult rat. Brain Res 838:1–10CrossRefPubMedGoogle Scholar
  8. 8.
    Lindvall O, McKay R (2003) Brain repair by cell replacement and regeneration. Proc Natl Acad Sci USA 100:7430–7431CrossRefPubMedGoogle Scholar
  9. 9.
    Gu W, Brannstrom T, Wester P (2000) Cortical neurogenesis in adult rats after reversible photothrombotic stroke. J Cereb Blood Flow Metab 20:1166–1173CrossRefPubMedGoogle Scholar
  10. 10.
    Jin K, Minami M, Lan JQ, Mao XO, Batteur S, Simon RP, Greenberg DA (2001) Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc Natl Acad Sci USA 98:4710–4715CrossRefPubMedGoogle Scholar
  11. 11.
    Liu J, Solway K, Messing RO, Sharp FR (1998) Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. J Neurosci 18:7768–7778PubMedGoogle Scholar
  12. 12.
    Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH (1997) Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci 17:3727–3738PubMedGoogle Scholar
  13. 13.
    Scharfman HE, Goodman JH, Sollas AL (2000) Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis. J Neurosci 20:6144–6158PubMedGoogle Scholar
  14. 14.
    Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O (2002) Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 8:963–970CrossRefPubMedGoogle Scholar
  15. 15.
    Jin K, Sun Y, Xie L, Peel A, Mao XO, Batteur S, Greenberg DA (2003) Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum. Mol Cell Neurosci 24:171–189CrossRefPubMedGoogle Scholar
  16. 16.
    Parent JM, Valentin VV, Lowenstein DH (2002) Prolonged seizures increase proliferating neuroblasts in the adult rat subventricular zone-olfactory bulb pathway. J Neurosci 22:3174–3188PubMedGoogle Scholar
  17. 17.
    Nakatomi H, Kuriu T, Okabe S, Yamamoto S, Hatano O, Kawahara N, Tamura A, Kirino T, Nakafuku M (2002) Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 110:429–441CrossRefPubMedGoogle Scholar
  18. 18.
    Parent JM, Vexler ZS, Gong C, Derugin N, Ferriero DM (2002) Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann Neurol 52:802–813CrossRefPubMedGoogle Scholar
  19. 19.
    Doetsch F (2003) A niche for adult neural stem cells. Curr Opin Genet Dev 13:543–550CrossRefPubMedGoogle Scholar
  20. 20.
    Nadarajah B, Parnavelas JG (2002) Modes of neuronal migration in the developing cerebral cortex. Nat Rev Neurosci 3:423–432CrossRefPubMedGoogle Scholar
  21. 21.
    Kuhn HG, Winkler J, Kempermann G, Thal LJ, Gage FH (1997) Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J Neurosci 17:5820–5829PubMedGoogle Scholar
  22. 22.
    Ogunshola OO, Antic A, Donoghue MJ, Fan SY, Kim H, Stewart WB, Madri JA, Ment LR (2002) Paracrine and autocrine functions of neuronal vascular endothelial growth factor (VEGF) in the central nervous system. J Biol Chem 277:11410–11415CrossRefPubMedGoogle Scholar
  23. 23.
    Schanzer A, Wachs FP, Wilhelm D, Acker T, Cooper-Kuhn C, Beck H, Winkler J, Aigner L, Plate KH, Kuhn HG (2004) Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor. Brain Pathol 14:237–248CrossRefPubMedGoogle Scholar
  24. 24.
    Ferrara N, Davis-Smyth T (1997) The biology of vascular endothelial growth factor. Endocr Rev 18:4–25CrossRefPubMedGoogle Scholar
  25. 25.
    Lei J, Jiang A, Pei D (1998) Identification and characterization of a new splicing variant of vascular endothelial growth factor: VEGF183. Biochim Biophys Acta 1443:400–406PubMedGoogle Scholar
  26. 26.
    Poltorak Z, Cohen T, Sivan R, Kandelis Y, Spira G, Vlodavsky I, Keshet E, Neufeld G (1997) VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. J Biol Chem 272:7151–7158CrossRefPubMedGoogle Scholar
  27. 27.
    Ribeiro LA, Bacci ML, Seren E, Tamanini C, Forni M (2007) Characterization and differential expression of vascular endothelial growth factor isoforms and receptors in swine corpus luteum throughout estrous cycle. Mol Reprod Dev 74:163–171CrossRefPubMedGoogle Scholar
  28. 28.
    Robinson CJ, Stringer SE (2001) The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci 114:853–865PubMedGoogle Scholar
  29. 29.
    Whittle C, Gillespie K, Harrison R, Mathieson PW, Harper SJ (1999) Heterogeneous vascular endothelial growth factor (VEGF) isoform mRNA and receptor mRNA expression in human glomeruli, and the identification of VEGF148 mRNA, a novel truncated splice variant. Clin Sci (Lond) 97:303–312CrossRefGoogle Scholar
  30. 30.
    Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med 9:669–676CrossRefPubMedGoogle Scholar
  31. 31.
    Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z (1999) Vascular endothelial growth factor (VEGF) and its receptors. Faseb J 13:9–22PubMedGoogle Scholar
  32. 32.
    Ferrara N (2000) VEGF: an update on biological and therapeutic aspects. Curr Opin Biotechnol 11:617–624CrossRefPubMedGoogle Scholar
  33. 33.
    Flecknell PA, Wootton R, John M (1982) Accurate measurement of cerebral metabolism in the conscious, unrestrained neonatal piglet. II. Glucose and oxygen utilization. Biol Neonate 41:221–226CrossRefPubMedGoogle Scholar
  34. 34.
    Haaland K, Loberg EM, Steen PA, Thoresen M (1997) Posthypoxic hypothermia in newborn piglets. Pediatr Res 41:505–512CrossRefPubMedGoogle Scholar
  35. 35.
    Armstrong RJ, Tyers P, Jain M, Richards A, Dunnett SB, Rosser AE, Barker RA (2003) Transplantation of expanded neural precursor cells from the developing pig ventral mesencephalon in a rat model of Parkinson’s disease. Exp Brain Res 151:204–217CrossRefPubMedGoogle Scholar
  36. 36.
    Isacson O, Deacon TW (1996) Specific axon guidance factors persist in the adult brain as demonstrated by pig neuroblasts transplanted to the rat. Neuroscience 75:827–837CrossRefPubMedGoogle Scholar
  37. 37.
    Zigova T, Song S, Willing AE, Hudson JE, Newman MB, Saporta S, Sanchez-Ramos JR, Sanberg PR (2002) Human umbilical cells express neural antigens after transplantation into the developing rat brain. Cell Transplant 11:265–274PubMedGoogle Scholar
  38. 38.
    McGuckin C, Forraz N (2006) Mini-livers from umbilical cord blood stem cells.
  39. 39.
    Sanberg PR, Willing AE, Cahill DW (2002) Novel cellular approaches to repair of neurodegenerative disease: from Sertoli cells to umbilical cord blood stem cells. Neurotox Res 4(2):95–101CrossRefPubMedGoogle Scholar
  40. 40.
    Ara J, Bannerman P, Shaheen F, Pleasure DE (2005) Schwann cell-autonomous role of neuropilin-2. J Neurosci Res 79:468–475CrossRefPubMedGoogle Scholar
  41. 41.
    Ara J, Bannerman P, Hahn A, Ramirez S, Pleasure D (2004) Modulation of sciatic nerve expression of class 3 semaphorins by nerve injury. Neurochem Res 29:1153–1159CrossRefPubMedGoogle Scholar
  42. 42.
    Rosser AE, Tyers P, ter Borg M, Dunnett SB, Svendsen CN (1997) Co-expression of MAP-2 and GFAP in cells developing from rat EGF responsive precursor cells. Brain Res Dev Brain Res 98:291–295CrossRefPubMedGoogle Scholar
  43. 43.
    Roy NS, Wang S, Jiang L, Kang J, Benraiss A, Harrison-Restelli C, Fraser RA, Couldwell WT, Kawaguchi A, Okano H, Nedergaard M, Goldman SA (2000) In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus. Nat Med 6:271–277CrossRefPubMedGoogle Scholar
  44. 44.
    Carpenter MK, Cui X, Hu ZY, Jackson J, Sherman S, Seiger A, Wahlberg LU (1999) In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol 158:265–278CrossRefPubMedGoogle Scholar
  45. 45.
    Gage FH (2000) Mammalian neural stem cells. Science (New York, NY) 287:1433–1438Google Scholar
  46. 46.
    Svendsen CN, Clarke DJ, Rosser AE, Dunnett SB (1996) Survival and differentiation of rat and human epidermal growth factor-responsive precursor cells following grafting into the lesioned adult central nervous system. Exp Neurol 137:376–388CrossRefPubMedGoogle Scholar
  47. 47.
    Svendsen CN, ter Borg MG, Armstrong RJ, Rosser AE, Chandran S, Ostenfeld T, Caldwell MA (1998) A new method for the rapid and long term growth of human neural precursor cells. J Neurosci Methods 85:141–152CrossRefPubMedGoogle Scholar
  48. 48.
    Vescovi AL, Parati EA, Gritti A, Poulin P, Ferrario M, Wanke E, Frolichsthal-Schoeller P, Cova L, Arcellana-Panlilio M, Colombo A, Galli R (1999) Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 156:71–83CrossRefPubMedGoogle Scholar
  49. 49.
    Gage FH, Coates PW, Palmer TD, Kuhn HG, Fisher LJ, Suhonen JO, Peterson DA, Suhr ST, Ray J (1995) Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci USA 92:11879–11883CrossRefPubMedGoogle Scholar
  50. 50.
    Jin K, LaFevre-Bernt M, Sun Y, Chen S, Gafni J, Crippen D, Logvinova A, Ross CA, Greenberg DA, Ellerby LM (2005) FGF-2 promotes neurogenesis and neuroprotection and prolongs survival in a transgenic mouse model of Huntington’s disease. Proc Natl Acad Sci USA 102:18189–18194CrossRefPubMedGoogle Scholar
  51. 51.
    Jin K, Wang X, Xie L, Mao XO, Zhu W, Wang Y, Shen J, Mao Y, Banwait S, Greenberg DA (2006) Evidence for stroke-induced neurogenesis in the human brain. Proc Natl Acad Sci USA 103:13198–13202CrossRefPubMedGoogle Scholar
  52. 52.
    Zhang RL, LeTourneau Y, Gregg SR et al (2007) Neuroblast division during migration toward the ischemic striatum: a study of dynamic migratory and proliferative characteristics of neuroblasts from the subventricular zone. J Neurosci 27:3157–3162CrossRefPubMedGoogle Scholar
  53. 53.
    Pluchino S, Zanotti L, Rossi B, Brambilla E, Ottoboni L, Salani G, Martinello M, Cattalini A, Bergami A, Furlan R, Comi G, Constantin G, Martino G (2005) Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism. Nature 436:266–271CrossRefPubMedGoogle Scholar
  54. 54.
    Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703–716CrossRefPubMedGoogle Scholar
  55. 55.
    Frederiksen K, McKay RD (1988) Proliferation and differentiation of rat neuroepithelial precursor cells in vivo. J Neurosci 8:1144–1151PubMedGoogle Scholar
  56. 56.
    Hockfield S, McKay RD (1985) Identification of major cell classes in the developing mammalian nervous system. J Neurosci 5:3310–3328PubMedGoogle Scholar
  57. 57.
    Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585–595CrossRefPubMedGoogle Scholar
  58. 58.
    Lothian C, Lendahl U (1997) An evolutionarily conserved region in the second intron of the human nestin gene directs gene expression to CNS progenitor cells and to early neural crest cells. Eur J Neurosci 9:452–462CrossRefPubMedGoogle Scholar
  59. 59.
    Bonfanti L, Theodosis DT (1994) Expression of polysialylated neural cell adhesion molecule by proliferating cells in the subependymal layer of the adult rat, in its rostral extension and in the olfactory bulb. Neurosci 61:291–305CrossRefGoogle Scholar
  60. 60.
    Gates MA, Thomas LB, Howard EM, Laywell ED, Sajin B, Faissner A, Gotz B, Silver J, Steindler DA (1995) Cell and molecular analysis of the developing and adult mouse subventricular zone of the cerebral hemispheres. J Comp Neurol 361:249–266CrossRefPubMedGoogle Scholar
  61. 61.
    Lois C, Garcia-Verdugo JM, Alvarez-Buylla A (1996) Chain migration of neuronal precursors. Science 271:978–981CrossRefPubMedGoogle Scholar
  62. 62.
    Rousselot P, Alvarez-Buylla A, Lois C (1995) Embryonic (PSA) N-CAM reveals chains of migrating neuroblasts between the lateral ventricle and the olfactory bulb of adult mice. J Comp Neurol 351:51–61CrossRefPubMedGoogle Scholar
  63. 63.
    Zerlin M, Levison SW, Goldman JE (1995) Early patterns of migration, morphogenesis, and intermediate filament expression of subventricular zone cells in the postnatal rat forebrain. J Neurosci 15:7238–7249PubMedGoogle Scholar
  64. 64.
    Doetsch F (2003) The glial identity of neural stem cells. Nat Neurosci 6:1127–1134CrossRefPubMedGoogle Scholar
  65. 65.
    Quinones-Hinojosa A, Sanai N, Soriano-Navarro M, Gonzalez-Perez O, Mirzadeh Z, Gil-Perotin S, Romero-Rodriguez R, Berger MS, Garcia-Verdugo JM, Alvarez-Buylla A (2006) Cellular composition and cytoarchitecture of the adult human subventricular zone: a niche of neural stem cells. J Comp Neurol 494:415–434CrossRefPubMedGoogle Scholar
  66. 66.
    Heins N, Malatesta P, Cecconi F, Nakafuku M, Tucker KL, Hack MA, Chapouton P, Barde YA, Gotz M (2002) Glial cells generate neurons: the role of the transcription factor Pax6. Nat Neurosci 5:308–315CrossRefPubMedGoogle Scholar
  67. 67.
    Lee JA, Cole GJ (2000) Localization of transition mRNA, a nestin-like intermediate filament family member, in chicken radial glia processes. J Comp Neurol 418:473–483CrossRefPubMedGoogle Scholar
  68. 68.
    Parnavelas JG, Nadarajah B (2001) Radial glial cells are they really glia? Neuron 31:881–884CrossRefPubMedGoogle Scholar
  69. 69.
    Seri B, Garcia-Verdugo JM, McEwen BS, Alvarez-Buylla A (2001) Astrocytes give rise to new neurons in the adult mammalian hippocampus. J Neurosci 21:7153–7160PubMedGoogle Scholar
  70. 70.
    Aharoni R, Arnon R, Eilam R (2005) Neurogenesis and neuroprotection induced by peripheral immunomodulatory treatment of experimental autoimmune encephalomyelitis. J Neurosci 25:8217–8228CrossRefPubMedGoogle Scholar
  71. 71.
    Emery DL, Fulp CT, Saatman KE, Schutz C, Neugebauer E, McIntosh TK (2005) Newly born granule cells in the dentate gyrus rapidly extend axons into the hippocampal CA3 region following experimental brain injury. J Neurotrauma 22:978–988CrossRefPubMedGoogle Scholar
  72. 72.
    Song H, Stevens CF, Gage FH (2002) Astroglia induce neurogenesis from adult neural stem cells. Nature 417:39–44CrossRefPubMedGoogle Scholar
  73. 73.
    Bjorklund A, Lindvall O (2000) Self-repair in the brain. Nature 405:892–895CrossRefPubMedGoogle Scholar
  74. 74.
    Kuhn HG, Palmer TD, Fuchs E (2001) Adult neurogenesis: a compensatory mechanism for neuronal damage. Eur Arch Psychiatry Clin Neurosci 251:152–158CrossRefPubMedGoogle Scholar
  75. 75.
    Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA (2002) Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci USA 99:11946–11950CrossRefPubMedGoogle Scholar
  76. 76.
    Maurer MH, Tripps WK, Feldmann RE Jr, Kuschinsky W (2003) Expression of vascular endothelial growth factor and its receptors in rat neural stem cells. Neurosci Lett 344:165–168CrossRefPubMedGoogle Scholar
  77. 77.
    Silverman WF, Krum JM, Mani N, Rosenstein JM (1999) Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures. Neuroscience 90:1529–1541CrossRefPubMedGoogle Scholar
  78. 78.
    Tonchev AB, Yamashima T, Sawamoto K, Okano H (2005) Enhanced proliferation of progenitor cells in the subventricular zone and limited neuronal production in the striatum and neocortex of adult macaque monkeys after global cerebral ischemia. J Neurosci Res 81:776–788CrossRefPubMedGoogle Scholar
  79. 79.
    Tonchev AB, Yamashima T, Zhao L, Okano HJ, Okano H (2003) Proliferation of neural and neuronal progenitors after global brain ischemia in young adult macaque monkeys. Mol Cell Neurosci 23:292–301CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Jahan Ara
    • 1
  • Saskia Fekete
    • 2
  • Anli Zhu
    • 1
  • Melissa Frank
    • 1
  1. 1.Department of PediatricsDrexel University College of Medicine and Saint Christopher’s Hospital for ChildrenPhiladelphiaUSA
  2. 2.Faculdade de Medicina de BotucatuUniversidade Estadual PaulistaBotucatuBrazil

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