Advertisement

Cytotechnology

, Volume 63, Issue 6, pp 621–631 | Cite as

Optimal time for passaging neurospheres based on primary neural stem cell cultures

  • Fangling Xiong
  • Huasong Gao
  • Yan Zhen
  • Xue Chen
  • Weiwei Lin
  • Jianhong Shen
  • Yaohua Yan
  • Xiaodong Wang
  • Mei Liu
  • Yilu Gao
Original Research

Abstract

Cultured neural stem cells (NSCs) provide a powerful means for investigating central nervous system disease, neuron development, differentiation, and regeneration. To obtain sufficient neurospheres, subculturing is essential following establishment of the primary NSC culture. Passaging the primary neurospheres is a key issue that is often ignored. We evaluated the influence of different passaging schedules on primary cultured NSCs. Passaging was performed on day 5, 7 or 9. We observed more neurospheres with diameters of 200–250 μm on day 7 than on day 5 or 9. Prolonging the time of primary culture reduced the cell metabolic activity by the MTT assay and cell proliferation by colony-forming assay and the differentiation to neurons from cells at P2 and later decreased. Additionally, more cells were in G0/G1 phase, and higher expression of p16 INK4a and lower expression of cyclin D1 was found when the time of primary culture was prolonged to 9 days compared to 7-days cultures. Thus, in this study, we established that the optimal time for subculturing aggregated NSCs was on day 7 based on the primary culture.

Keywords

Neurosphere Neural stem cells Primary culture Cell passage Optimal time 

Notes

Acknowledgments

This study was supported by a grant from the National Natural Science Foundation of China (No. 81070992), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Foundation of the Graduate Natural Science Innovation Project of Nantong University (No. YKC10050).

Conflict of interest

This study was supported by a grant from the National Natural Science Foundation of China (No. 81070992), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Foundation of the Graduate Natural Science Innovation Project of Nantong University (No. YKC10050). The authors have declared that no conflict of interest exists.

References

  1. Bartek J, Lukas J (2001) Pathways governing G1/S transition and their response to DNA damage. FEBS Lett 490:117–122. doi: 10.1016/S0014-5793(01)02114-7 CrossRefGoogle Scholar
  2. Bassiouny AE, Nosseir MM, Zoheiry MK, Ameen NA, Abdel-Hadi AM, Ibrahim IM, Zada S, El-Deen AH, El-Bassiouni NE (2010) Differential expression of cell cycle regulators in HCV-infection and related hepatocellular carcinoma. World J Hepatol 2:32–41. doi: 10.4254/wjh.v2.i1.32 Google Scholar
  3. Bez A, Corsini E, Curti D, Biggiogera M, Colombo A, Nicosia RF, Pagano SF, Parati EA (2003) Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization. Brain Res 993:18–29. doi: 10.1016/j.brainres.2003.08.061 CrossRefGoogle Scholar
  4. Bornfeldt KE (2003) The cyclin-dependent kinase pathway moves forward. Circ Res 92:345–347. doi: 10.1161/01.RES.0000061765.06145.10 CrossRefGoogle Scholar
  5. Brewer GJ, Torricelli JR (2007) Isolation and culture of adult neurons and neurospheres. Nat Protoc 2:1490–1498. doi: 10.1038/nprot.2007.207 CrossRefGoogle Scholar
  6. Chen Y, Balasubramaniyan V, Peng J, Hurlock EC, Tallquist M, Li J, Lu QR (2007) Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nat Protoc 2:1044–1051. doi: 10.1038/nprot.2007.149 CrossRefGoogle Scholar
  7. Dhara SK, Stice SL (2008) Neural differentiation of human embryonic stem cells. J Cell Biochem 105:633–640. doi: 10.1002/jcb.21891 CrossRefGoogle Scholar
  8. Favaro R, Valotta M, Ferri AL, Latorre E, Mariani J, Giachino C, Lancini C, Tosetti V, Ottolenghi S, Taylor V, Nicolis SK (2009) Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh. Nat Neurosci 12:1248–1256. doi: 10.1038/nn.2397 CrossRefGoogle Scholar
  9. Goldman S (2005) Stem and progenitor cell-based therapy of the human central nervous system. Nat Biotechnol 23:862–871. doi: 10.1038/nbt1119 CrossRefGoogle Scholar
  10. Kim JB, Zaehres H, Wu G, Gentile L, Ko K, Sebastiano V, Arauzo-Bravo MJ, Ruau D, Han DW, Zenke M, Scholer HR (2008) Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454:646–650. doi: 10.1038/nature07061 CrossRefGoogle Scholar
  11. Kim JB, Sebastiano V, Wu G, Arauzo-Bravo MJ, Sasse P, Gentile L, Ko K, Ruau D, Ehrich M, van den Boom D, Meyer J, Hubner K, Bernemann C, Ortmeier C, Zenke M, Fleischmann BK, Zaehres H, Scholer HR (2009) Oct4-induced pluripotency in adult neural stem cells. Cell 136:411–419. doi: 10.1016/j.cell.2009.01.023 CrossRefGoogle Scholar
  12. Krishan A (1975) Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188–193. doi: 10.1083/jcb.66.1.188 CrossRefGoogle Scholar
  13. Lange C, Huttner WB, Calegari F (2009) Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors. Cell Stem Cell 5:320–331. doi: 10.1016/j.stem.2009.05.026 CrossRefGoogle Scholar
  14. Li XG, Yang ZY, Zhang AF (2009) The effect of neurotrophin-3/chitosan carriers on the proliferation and differentiation of neural stem cells. Biomaterials 30:4978–4985. doi: 10.1016/j.biomaterials.2009.05.047 CrossRefGoogle Scholar
  15. Lobo MVT, Alonso FJM, Redondo C, Lopez-Toledano MA, Caso E, Herranz AS, Paino CL, Reimers D, Bazan E (2002) Cellular characterization of epidermal growth factor-expanded free-floating neurospheres. J Histochem Cytochem 51:89–103. doi: 10.1177/002215540305100111 CrossRefGoogle Scholar
  16. Lowe SW, Sherr CJ (2003) Tumor suppression by Ink4a-Arf: progress and puzzles. Curr Opin Genet Dev 13:77–83. doi: 10.1016/S0959-437X(02)00013-8 CrossRefGoogle Scholar
  17. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ (2003) Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425:962–967. doi: 10.1038/nature02060 CrossRefGoogle Scholar
  18. Molofsky AV, Slutsky SG, Joseph NM, He S, Pardal R, Krishnamurthy J, Sharpless NE, Morrison SJ (2006) Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing. Nature 443:448–452. doi: 10.1038/nature05091 CrossRefGoogle Scholar
  19. Ourednik V, Ourednik J, Xu YF, Zhang Y, Lynch WP, Snyder EY, Schachner M (2009) Cross-talk between stem cells and the dysfunctional brain is facilitated by manipulating the niche: evidence from an adhesion molecule. Stem Cells 27:2846–2856. doi: 10.1002/stem.227 CrossRefGoogle Scholar
  20. Prestoz L, Relvas JB, Hopkins K, Patel S, Sowinski P, Price J, ffrench-Constant C (2001) Association between integrin-dependent migration capacity of neural stem cells in vitro and anatomical repair following transplantation. Mol Cell Neurosci 18:473–484. doi: 10.1006/mcne.2001.1037 CrossRefGoogle Scholar
  21. Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1701–1710. doi: 10.1126/science.1553558 CrossRefGoogle Scholar
  22. Reynolds BA, Weiss S (1996) Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 175:1–13. doi: 10.1006/dbio.1996.0090 CrossRefGoogle Scholar
  23. Salomoni P, Calegari F (2010) Cell cycle control of mammalian neural stem cells: putting a speed limit on G1. Trends Cell Biol 205:233–243. doi: 10.1016/j.tcb.2010.01.006 CrossRefGoogle Scholar
  24. Sherr CJ (2001) The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol 2:731–737. doi: 10.1038/35096061 CrossRefGoogle Scholar
  25. Sicinski P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner H, Haslam SZ, Bronson RT, Elledge SJ, Weinberg RA (1995) Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82:621–630. doi: 10.1016/0092-8674(95)90034-9 CrossRefGoogle Scholar
  26. Sugimoto M, Nakamura T, Ohtani N, Hampson L, Hampson IN, Shimamoto A, Furuichi Y, Okumura K, Niwa S, Taya Y, Hara E (1999) Regulation of CDK4 activity by a novel CDK4-binding protein, p34(SEI-1). Genes Dev 13:3027–3033CrossRefGoogle Scholar
  27. Sun Y, Pollard S, Conti L, Toselli M, Biella G, Parkin G, Willatt L, Falk A, Cattaneo E, Smith A (2008) Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture. Mol Cell Neurosci 38:245–258. doi: 10.1016/j.mcn.2008.02.014 CrossRefGoogle Scholar
  28. Suslov ON, Kukekov VG, Ignatova TN, Steindler DA (2002) Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres. Proc Natl Acad Sci USA 99:14506–14511. doi: 10.1073/pnas.212525299 CrossRefGoogle Scholar
  29. Svendsen CN, Smith AG (1999) New prospects for human stem cell therapy in the nervous system. Trends Neurosci 22:357–364. doi: 10.1016/S0166-2236(99)01428-9 CrossRefGoogle Scholar
  30. Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, van der Kooy D (1999) Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 208:166–188. doi: 10.1006/dbio.1998.9192 CrossRefGoogle Scholar
  31. Vukicevic V, Jauch A, Dinger TC, Gebauer L, Hornich V, Bornstein SR, Ehrhart-Bornstein M, Müller AM (2010) Genetic instability and diminished differentiation capacity in long-term cultured mouse neurosphere cells. Mech Ageing Dev 131:124–132. doi: 10.1016/j.mad.2010.01.001 CrossRefGoogle Scholar
  32. Ye S, Su ZP, Zhang J, Qian X, Zhuge QC, Zeng YJ (2008) Differential centrifugation in culture and differentiation of rat neural stem cells. Cell Mol Neurobiol 28:511–517. doi: 10.1007/s10571-007-9194-5 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Fangling Xiong
    • 1
  • Huasong Gao
    • 4
  • Yan Zhen
    • 1
  • Xue Chen
    • 2
  • Weiwei Lin
    • 2
  • Jianhong Shen
    • 1
  • Yaohua Yan
    • 1
  • Xiaodong Wang
    • 2
  • Mei Liu
    • 3
  • Yilu Gao
    • 1
  1. 1.Department of NeurosurgeryAffiliated Hospital of Nantong UniversityNantongChina
  2. 2.Department of Histology and EmbryologyNantong UniversityNantongChina
  3. 3.Jiangsu Key Laboratory of NeuroregenerationNantong UniversityNantongChina
  4. 4.Department of NeurosurgeryHuashan Hospital affiliated to Fudan UniversityShanghaiChina

Personalised recommendations