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Neuronal Conditioning Medium and Nerve Growth Factor Induce Neuronal Differentiation of Collagen-Adherent Progenitors Derived from Human Umbilical Cord Blood

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Abstract

The aim of the study was to isolate and characterize a population of neuronal progenitors in the human umbilical cord blood (HUCB) mononuclear cell (MNC) fraction, for in vitro manipulation towards neuronal differentiation. Selection of the HUCB neuronal progenitors (HUCBNPs) was based on the neuronal prerequisite for adherence to collagen. Populations of collagen-adherent, nestin-positive (94.8 ± 2.9%) progenitors expressing α1/2 integrin receptors, as revealed by Western blot and adhesion assay using selective antagonists, were isolated and survived for more than 14 days. In vitro differentiation of the HUCBNPs was achieved by treatment with 10% human SH-SY5Y neuroblastoma cell-conditioning media (CM) supplemented with 10 ng/ml nerve growth factor (NGF). Some 83 ± 8.2% of the surviving progenitors acquired a neuronal-like morphology, expressed by cellular outgrowths of different lengths. About 35 ± 6% of the HUCBNPs had long outgrowths with a length/cell diameter ratio greater than 2, typical of developing neurons. The majority of these progenitors, analyzed by immunocytochemistry and/or RT-PCR, expressed common neuronal markers such as microtubule-associated protein 2 (MAP-2; 98.5 ± 2%), neurotrophin receptor (TrkA; 98.5 ± 0.06%), neurofillament-160 (NF-160; 94.2 ± 1%), beta-tubulin III (89.8 ± 4.2%) and neuron specific enolase (NSE). Combined CM and NGF treatment induced constitutive activation of the mitogen-activated protein kinases ERK2 (36-fold vs control), p38α (nine-fold vs control) and p38beta (23-fold vs control), most likely related to survival and/or differentiation. The results point to operationally defined conditions for activating neuronal differentiation of HUCBNPs ex vivo and emphasize the crucial role of neuronal CM and NGF in this process.

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References

  • Bracci-Laudiero, L., Celestino, D., Starace, G., Antonelli, A., Lambiase, A., Procoli, A., et al. (2003). CD34-positive cells in human umbilical cord blood express nerve growth factor and its specific receptor TrkA. Journal of Neuroimmunology, 136, 130–139.

    Article  PubMed  CAS  Google Scholar 

  • Bradshaw, A. D., McNagny, K. M., Gervin, D. B., Cann, G. M., Graf, T., & Clegg, D. O. (1995). Integrin alpha 2 beta 1 mediates interactions between developing embryonic retinal cells and collagen. Development, 121, 3593–3602.

    PubMed  CAS  Google Scholar 

  • Buzanska, L., Machaj, E. K., Zablocka, B., Pojda, Z., & Domanska-Janik, K. (2002). Human cord blood-derived cells attain neuronal and glial features in vitro. Journal of Cell Science, 115, 2131–2138.

    PubMed  CAS  Google Scholar 

  • Campos, L. S. (2005). Beta1 integrins and neural stem cells: Making sense of the extracellular environment. BioEssays, 27, 698–707.

    Article  PubMed  CAS  Google Scholar 

  • Carlson, K., & Ehrich, M. (2000). Human neuroblastoma cell viability and growth are affected by altered culture conditions. In Vitro & Molecular Toxicology, 13, 249–262.

    CAS  Google Scholar 

  • Chang, J. C., Brewer, G. J., & Wheeler, B. C. (2003). A modified microstamping technique enhances polylysine transfer and neuronal cell patterning. Biomaterials, 24, 2863–2870.

    Article  PubMed  CAS  Google Scholar 

  • Chao, M., Casaccia-Bonnefil, P., Carter, B., Chittka, A., Kong, H., & Yoon, S. O. (1998). Neurotrophin receptors: Mediators of life and death. Brain Research Brain Research Reviews, 26, 295–301.

    Article  PubMed  CAS  Google Scholar 

  • Chen, N., Hudson, J. E., Walczak, P., Misiuta, I., Garbuzova-Davis, S., Jiang, L., et al. (2005). Human umbilical cord blood progenitors: The potential of these hematopoietic cells to become neural. Stem Cells, 23, 1560–1570.

    Article  PubMed  CAS  Google Scholar 

  • Clegg, D. O. (2000). Novel roles for integrins in the nervous system. Molecular Cell Biology Research Communication, 3, 1–7.

    Article  CAS  Google Scholar 

  • Cohen, Y., & Nagler, A. (2004). Umbilical cord blood transplantation-how, when and for whom? Blood Review, 18, 167–179.

    Article  Google Scholar 

  • Czyz, J., & Wobus, A. (2001). Embryonic stem cell differentiation: The role of extracellular factors. Differentiation, 68, 167–174.

    Article  PubMed  CAS  Google Scholar 

  • Deschaseaux, F., & Charbord, P. (2000). Human marrow stromal precursors are alpha 1 integrin subunit-positive. Journal of Cellular Physiology, 184, 319–325.

    Article  PubMed  CAS  Google Scholar 

  • Eggert, A., Grotzer, M. A., Ikegaki, N., Liu, X. G., Evans, A. E., & Brodeur, G. M. (2002). Expression of the neurotrophin receptor TrkA down-regulates expression and function of angiogenic stimulators in SH-SY5Y neuroblastoma cells. Cancer Research, 62, 1802–1808.

    PubMed  CAS  Google Scholar 

  • Garbuzova-Davis, S., Willing, A. E., Zigova, T., Saporta, S., Justen, E. B., Lane, J. C., et al. (2003). Intravenous administration of human umbilical cord blood cells in a mouse model of amyotrophic lateral sclerosis: Distribution, migration, and differentiation. Journal of Hematotherapy & Stem Cell Research, 12, 255–270.

    Article  CAS  Google Scholar 

  • Goodwin, H. S., Bicknese, A. R., Chien, S. N., Bogucki, B. D., Quinn, C. O., & Wall, D. A. (2001). Multilineage differentiation activity by cells isolated from umbilical cord blood: Expression of bone, fat, and neural markers. Biology of Blood and Marrow Transplantation, 7, 581–588.

    Article  PubMed  CAS  Google Scholar 

  • Ha, Y., Choi, J. U., Yoon, D. H., Yeon, D. S., Lee, J. J., Kim, H. O., et al. (2001). Neural phenotype expression of cultured human cord blood cells in vitro. NeuroReport, 12, 3523–3527.

    Article  PubMed  CAS  Google Scholar 

  • Ha, Y., Lee, J. E., Kim, K. N., Cho, Y. E., & Yoon, D. H. (2003). Intermediate filament nestin expressions in human cord blood monocytes (HCMNCs). Acta Neurochirurgica (Wien), 145, 483–487.

    CAS  Google Scholar 

  • Habich, A., Jurga, M., Markiewicz, I., Lukomska, B., Bany-Laszewicz, U., & Domanska-Janik, K. (2006). Early appearance of stem/progenitor cells with neural-like characteristics in human cord blood mononuclear fraction cultured in vitro. Experimental Hematology, 34, 914–925.

    Article  PubMed  CAS  Google Scholar 

  • Hynes, R. O. (2002). Integrins: Bidirectional, allosteric signaling machines. Cell, 110, 673–687.

    Article  PubMed  CAS  Google Scholar 

  • Jang, Y. K., Park, J. J., Lee, M. C., Yoon, B. H., Yang, Y. S., Yang, S. E., et al. (2004). Retinoic acid-mediated induction of neurons and glial cells from human umbilical cord-derived hematopoietic stem cells. Journal of Neuroscience Research, 75, 573–584.

    Article  PubMed  CAS  Google Scholar 

  • Jeong, J. A., Gang, E. J., Hong, S. H., Hwang, S. H., Kim, S. W., Yang, I. H., et al. (2004). Rapid neural differentiation of human cord blood-derived mesenchymal stem cells. NeuroReport, 15, 1731–1734.

    Article  PubMed  CAS  Google Scholar 

  • Kale, V. P. (2005). MAP kinase: A switch in fate determination of stem cells. Stem Cells Development, 14, 248–251.

    Article  CAS  Google Scholar 

  • Kaplan, D. R., & Miller, F. D. (2000). Neurotrophin signal transduction in the nervous system. Current Opinion in Neurobiology, 10, 381–391.

    Article  PubMed  CAS  Google Scholar 

  • Katzir, I., Shani, J., Regev, K., Shabashov, D., & Lazarovici, P. (2002). A quantitative bioassay for nerve growth factor, using PC12 clones expressing different levels of trkA receptors. Journal of Molecular Neuroscience, 18, 251–264.

    Article  PubMed  CAS  Google Scholar 

  • Khalsa, P. S., Zhang, C., Sommerfeldt, D., & Hadjiargyrou, M. (2000). Expression of integrin alpha2beta1 in axons and receptive endings of neurons in rat, hairy skin. Neuroscience Letters, 293, 13–16.

    Article  PubMed  CAS  Google Scholar 

  • Kisiel, D. G., Calvete, J. J., Katzhendler, J., Fertala, A., Lazarovici, P., & Marcinkiewicz, C. (2004). Structural determinants of the selectivity of KTS-disintegrins for the alpha1beta1 integrin. FEBS Letters, 577, 478–482.

    Article  PubMed  CAS  Google Scholar 

  • Lee, O. K., Kuo, T. K., Chen, W. M., Lee, K. D., Hsieh, S. L., & Chen, T. H. (2004). Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood, 103, 1669–1675.

    Article  PubMed  CAS  Google Scholar 

  • Lendahl, U., Zimmerman, L. B., & McKay, R. D. (1990). CNS stem cells express a new class of intermediate filament protein. Cell, 60, 585–595.

    Article  PubMed  CAS  Google Scholar 

  • Leor, J., Guetta, E., Feinberg, M. S., Galski, H., Bar, I., Holbova, R., et al. (2006). Human umbilical cord blood-derived CD133+ cells enhance function and repair of the infarcted myocardium. Stem Cells, 24, 772–780.

    Article  PubMed  Google Scholar 

  • Lu, D., Sanberg, P. R., Mahmood, A., Li, Y., Wang, L., Sanchez-Ramos, J., et al. (2002). Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplantation, 11, 275–281.

    PubMed  Google Scholar 

  • Marcinkiewicz, C. (2005). Functional characteristic of snake venom disintegrins: Potential therapeutic implication. Current Pharmaceutical Design, 11, 815–827.

    Article  PubMed  CAS  Google Scholar 

  • O’Hare, M. M., & Schwartz, T. W. (1989). Expression and precursor processing of neuropeptide Y in human and murine neuroblastoma and pheochromocytoma cell lines. Cancer Research, 49, 7015–7019.

    PubMed  CAS  Google Scholar 

  • Oswald, J., Steudel, C., Salchert, K., Joergensen, B., Thiede, C., Ehninger, G., et al. (2006). Gene-expression profiling of CD34+ hematopoietic cells expanded in a collagen I matrix. Stem Cells, 24, 494–500.

    Article  PubMed  CAS  Google Scholar 

  • Pegram, C. N., Eng, L. F., Wikstrand, C. J., McComb, R. D., Lee, Y. L., & Bigner, D. D. (1985). Monoclonal antibodies reactive with epitopes restricted to glial fibrillary acidic proteins of several species. Neurochemical Pathology, 3, 119–138.

    PubMed  CAS  Google Scholar 

  • Raedt, R., & Boon, P. (2005). Cell therapy for neurological disorders: A comprehensive review. Acta Neurologica Belgica, 105, 158–170.

    PubMed  Google Scholar 

  • Rasouly, D., Rahamim, E., Lester, D., Matsuda, Y., & Lazarovici, P. (1992). Staurosporine-induced neurite outgrowth in PC12 cells is independent of protein kinase C inhibition. Molecular Pharmacology, 42, 35–43.

    PubMed  CAS  Google Scholar 

  • Romanova, E. V., Oxley, S. P., Rubakhin, S. S., Bohn, P. W., & Sweedler, J. V. (2006). Self-assembled monolayers of alkanethiols on gold modulate electrophysiological parameters and cellular morphology of cultured neurons. Biomaterials, 27, 1665–1669.

    Article  PubMed  CAS  Google Scholar 

  • Sanchez-Ramos, J. R. (2002). Neural cells derived from adult bone marrow and umbilical cord blood. Journal of Neuroscience Research, 69, 880–893.

    Article  PubMed  CAS  Google Scholar 

  • Sanchez-Ramos, J. R., Song, S., Kamath, S. G., Zigova, T., Willing, A., Cardozo-Pelaez, F., et al. (2001). Expression of neural markers in human umbilical cord blood. Experimental Neurology, 171, 109–115.

    Article  PubMed  CAS  Google Scholar 

  • Saporta, S., Kim, J. J., Willing, A. E., Fu, E. S., Davis, C. D., & Sanberg, P. R. (2003). Human umbilical cord blood stem cells infusion in spinal cord injury: Engraftment and beneficial influence on behavior. Journal of Hematotherapy & Stem Cell Research, 12, 271–278.

    Article  CAS  Google Scholar 

  • Sofroniew, M. V., Howe, C. L., & Mobley, W. C. (2001). Nerve growth factor signaling, neuroprotection, and neural repair. Annual Review of Neuroscience, 24, 1217–1281.

    Article  PubMed  CAS  Google Scholar 

  • Sun, W., Buzanska, L., Domanska-Janik, K., Salvi, R. J., & Stachowiak, M. K. (2005). Voltage-sensitive and ligand-gated channels in differentiating neural stem-like cells derived from the nonhematopoietic fraction of human umbilical cord blood. Stem Cells, 23, 931–945.

    Article  PubMed  CAS  Google Scholar 

  • Suzuki, A., Taniguchi, H., Zheng, Y. W., Takada, Y., Fukunaga, K., Seino, K., et al. (2000). Clonal colony formation of hepatic stem/progenitor cells enhanced by embryonic fibroblast conditioning medium. Transplantation Proceedings, 32, 2328–2330.

    Article  PubMed  CAS  Google Scholar 

  • Sweatt, J. D. (2001). The neuronal MAP kinase cascade: A biochemical signal integration system subserving synaptic plasticity and memory. Journal of Neurochemistry, 76, 1–10.

    Article  PubMed  CAS  Google Scholar 

  • Tabakman, R., Jiang, H., Schaefer, E., Levine, R. A., & Lazarovici, P. (2004a). Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38alpha and p38beta activation and confers neuroprotection in the pheochromocytoma PC12 Model. Journal of Molecular Neuroscience, 22, 237–250.

    Article  CAS  PubMed  Google Scholar 

  • Tabakman, R., Lecht, S., Sephanova, S., Arien-Zakay, H., & Lazarovici, P. (2004b). Interactions between the cells of the immune and nervous system: Neurotrophins as neuroprotection mediators in CNS injury. Progress in Brain Research, 146, 387–401.

    Article  PubMed  CAS  Google Scholar 

  • Tondreau, T., Meuleman, N., Delforge, A., Dejeneffe, M., Leroy, R., Massy, M., et al. (2005). Mesenchymal stem cells derived from CD133-positive cells in mobilized peripheral blood and cord blood: Proliferation, Oct4 expression, and plasticity. Stem Cells, 23, 1105–1112.

    Article  PubMed  CAS  Google Scholar 

  • White, D. J., Puranen, S., Johnson, M. S., & Heino, J. (2004). The collagen receptor subfamily of the integrins. International Journal of Biochemistry & Cell Biology, 36, 1405–1410.

    Article  CAS  Google Scholar 

  • Willing, A. E., Lixian, J., Milliken, M., Poulos, S., Zigova, T., Song, S., et al. (2003). Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. Journal of Neuroscience Research, 73, 296–307.

    Article  PubMed  CAS  Google Scholar 

  • Yan, Y., Lagenaur, C., & Narayanan, V. (1993). Molecular cloning of M6: Identification of a PLP/DM20 gene family. Neuron, 11, 423–431.

    Article  PubMed  CAS  Google Scholar 

  • Zhao, Z. M., Li, H. J., Liu, H. Y., Lu, S. H., Yang, R. C., Zhang, Q. J., et al. (2004). Intraspinal transplantation of CD34+ human umbilical cord blood cells after spinal cord hemisection injury improves functional recovery in adult rats. Cell Transplantation, 13, 113–122.

    PubMed  Google Scholar 

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Acknowledgments

PL is affiliated with and supported in part by the David R. Bloom Center for Pharmacy at The Hebrew University of Jerusalem. The authors are grateful to Mr. Shimon Lecht, Dr. Rinat Tabakman, Dr. Ezra Rahamim and Dr. Cezary Marcinkiewicz for their comments.

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Authors and Affiliations

  1. Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, POB 12065, Jerusalem, 91120, Israel

    Hadar Arien-Zakay & Philip Lazarovici

  2. Division of Hematology, Cord Blood Bank and Laboratory of Molecular Immunobiology, Chaim Sheba Medical Center, Tel-Hashomer, Israel

    Arnon Nagler & Hanan Galski

Authors
  1. Hadar Arien-Zakay
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  2. Arnon Nagler
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  3. Hanan Galski
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  4. Philip Lazarovici
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Correspondence to Philip Lazarovici.

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This study is part of a PhD thesis to be submitted to the Hebrew University of Jerusalem by H.A.Z.

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Arien-Zakay, H., Nagler, A., Galski, H. et al. Neuronal Conditioning Medium and Nerve Growth Factor Induce Neuronal Differentiation of Collagen-Adherent Progenitors Derived from Human Umbilical Cord Blood. J Mol Neurosci 32, 179–191 (2007). https://doi.org/10.1007/s12031-007-0027-2

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  • DOI: https://doi.org/10.1007/s12031-007-0027-2

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