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Growth Dynamics of Fetal Human Neural Stem Cells

  • Walter D. Niles
  • Dustin R. Wakeman
  • Evan Y. SnyderEmail author
Chapter
  • 3.3k Downloads

Abstract

Human neural stem cells (hNSC) are useful for understanding neurogenesis, migration, other events in neural development, drug screening of developmental and disease targets, and potential neurological disease therapeutics. These uses require expansion of isolated hNSC in culture with retention of multipotency and the ability to interact with the pathological environment of a host. Growth of fetal-derived non-immortalized, undifferentiated hNSC in chemically defined serum-free media on bare “tissue culture-grade” polystyrene and laminin-coated polystyrene was measured to understand critical growth parameters. On uncoated plastic, survival and proliferation required (1) seeding freshly dispersed hNSC within a specific range of surface area density and (2) allowing the seed 3–5 days of initial undisturbed growth. Cells grew with a stereotypical time course in a multilayer pattern characterized by extensive interactions both between cells and with the growth surface. Laminin relaxed the seeding requirement and promoted monolayer growth. Analysis of harvested cell yield per seeded cell as a function of growth time revealed that laminin did not increase proliferation rate. Number of cells seeded, seeding surface density, and growth time were greatest yield predictors. Biphasic relations between yield and seed size suggest both positive and negative interactions between fetal hNSC influencing growth.

Keywords

Seed Size Leukemia Inhibitory Factor Seeding Density Culture Vessel Growth Surface 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

bFGF

Basic fibroblast growth factor (fibroblast growth factor 2)

DMSO

Dimethylsulfoxide

PBS

Dulbecco’s phosphate-buffered saline

ECM

Extracellular matrix

EGF

Epidermal growth factor

FBS

Fetal bovine serum

hNSC

Human neural stem cells

LIF

Leukemia inhibitory factor

Notes

Acknowledgment

This work was supported by grants from the California Institute for Regenerative Medicine.

Supplementary material

Movie 5.1

Progression of fetal hNSC in culture seeded on an uncoated polystyrene surface. Cells were seeded at a density of 105/cm2 in a 35 mm Petri dish (see section “Methods and Materials”) that was transferred immediately to the stage of the microscope maintained in a 37 °C incubator. The sample was viewed under brightfield transmission illumination and the focus plane was positioned at the seeding surface. Successive images were acquired in time-lapse once every 10 min for a duration of 8,480 min (848 images) or about 5.9 days. Illumination was turned off between image acquisitions. The resulting movie (see section “Time-Lapse Microphotography” in “Materials and Methods” section) duration was 2 min and 21 s (designated 02:21). Cells interact with each other to form aggregates similar to early neurospheres, clearing the medium of single unassociated cells by 6.8 h, corresponding to 6 s (00:06) in the movie. By 46 h (00:46 in the movie) the spherical aggregates have attached to the surface and cells have begun expressing transitory lamellipodia. (The image was refocused at 50 h in the experiment, 00:50 in the movie.) By 70 h (01:10) lamellipodia projecting toward each other from adjacent cluster became longer-lived, suggesting chemotropism, with the first stable interconnecting branch between adjacent colonies forming by 70.8 h or 3 days (01:11 in the movie). Cell migration between colonies along the free surface and along branches and proliferation within the colonies resulting in the multilayer growth pattern become evident. Objective, ×20; total system magnification, ×150; horizontal field of view, 1.28 mm (MPG 19,819 kb)

Movie 5.2

Progression of fetal hNSC showing extension of lamellipodia and interaction of cells with surface during multilayer growth. One image was acquired in time-lapse every 5 min for a total of 1,005 images in 167.5 h or about 7 days with a ×40 objective. The total movie duration is 02:46. Cells within colonies interact extensively with each other and with cells in adjacent colonies by extension of lamellipodia, contact with adjacent cells, and migration of cells between colonies. The greater magnification and faster time-lapse sampling reveal details of the extension and retraction of lamellipodia, degree of contact between cells migrating between colonies, and mitosis. Total system magnification, ×300; horizontal field of view, 0.64 mm (MPG 23,427 kb)

Movie 5.3

Fetal hNSC growth dynamics on a laminin-coated glass surface. Cells were seeded at a density of 4 × 104/cm2 in a Fluorodish and viewed with the ×20 objective. Images were acquired in time-lapse at an interval of 10 min for a total of 570 images, 95 h, or 3.95 days. The duration of the subsequent movie is 01:34. Cells begin to interact with the laminin-coated surface, extend lamellipodia, and migrate within minutes of seeding. Migration continues until proliferation becomes so extensive as to encroach on the area of surface available for unobstructed interaction. Laminin induces much greater surface interaction at the expense of intercellular interactions, but cells still form small clusters that expand by proliferation. Total system magnification, ×150; horizontal field of view, 1.28 mm (MPG 13,328 kb)

Movie 5.4

Growth on laminin-coated glass observed with ×40 objective. Images were acquired in time-lapse once every 10 min for 606 images, 101 h, or 4.2 days. The movie duration is 01:40. (At 29.83 s in the movie, the illumination intensity was adjusted, and at 48.67 s, the field of view was moved to the right ~0.5 mm.) Greater magnification reveals more detail of lamellipodia dynamics and mitosis during the first 4 days of culture. Objective, ×40; total system magnification, ×300; horizontal field of view, 0.64 mm (MPG 14,122 kb)

Suggested Readings

  1. Nethercott H, Sheridan M, Schwartz PH, Nethercott H, Sheridan M, Schwartz PH. Neural stem cell culture. In: Loring JF, Wesselschmidt RL, Schwartz PH, Loring JF, Wesselschmidt RL, Schwartz PH, editors. Human stem cell manual: a laboratory guide. San Diego, CA: Academic; 2007.Google Scholar
  2. Ryder EF, Snyder EY, Cepko CL. Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer. J Neurobiol. 1990;21:356–75.PubMedCrossRefGoogle Scholar
  3. Snyder EY, Deitcher DL, Walsh C, Arnold-Aldea S, Hartwieg EA, Cepko CL. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell. 1992;68:33–51.PubMedCrossRefGoogle Scholar

References

  1. 1.
    Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst LL, Jendoubi M, Sidman RL, Wolfe JH, Kim SU, Snyder EY. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol. 1998;16:1033–9.PubMedCrossRefGoogle Scholar
  2. 2.
    Carpenter MK, Cui X, Hu Z-Y, Jackson J, Sherman S, Seiger A, Wahlberg LU. In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol. 1999;158:265–79.PubMedCrossRefGoogle Scholar
  3. 3.
    Gottlieb DI. Large-scale sources of neural stem cells. Annu Rev Neurosci. 2002;25:381–407.PubMedCrossRefGoogle Scholar
  4. 4.
    Walsh K, Megyesi J, Hammond R. Human central nervous system tissue culture: A historical review and examination of recent advances. Neurobiol Dis. 2005;18:2–18.PubMedCrossRefGoogle Scholar
  5. 5.
    Singec I, Quinones-Hinojosa A. Neurospheres. In: Gage FH, Kempermann G, Song H, editors. Adult neurogenesis. New York, NY: Cold Spring Harbor Press; 2008. p. 119–34.Google Scholar
  6. 6.
    Singec I, Jandial R, Crain A, Nikkah G, Snyder EY. The leading edge of stem cell therapeutics. Annu Rev Med. 2007;58:313–28.PubMedCrossRefGoogle Scholar
  7. 7.
    Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992;255:1707–10.PubMedCrossRefGoogle Scholar
  8. 8.
    Wachs F-P, Couillard-Despres S, Engelhardt M, Wilhelm D, Ploetz S, Vroeman M, Kaesbauer J, Uyanik G, Klochen J, Karl C, Tebbing J, Svendsen C, Weidner N, Kuhn HG, Winkler J, Aigner L. High efficacy of clonal growth and expansion of adult neural stem cells. Lab Invest. 2003;83:949–62.PubMedCrossRefGoogle Scholar
  9. 9.
    Rietze RL, Reynolds BA. Neural stem cell isolation and characterization. Methods Enzymol. 2006;419:3–23.PubMedCrossRefGoogle Scholar
  10. 10.
    Singec I, Knoth R, Meyer RP, Maciaczyk J, Volk B, Nikkhah G, Frotscher M, Snyder EY. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods. 2006;3:801–6.PubMedCrossRefGoogle Scholar
  11. 11.
    Ray J, Raymon HK, Gage FH. Generation and culturing of precursor cells and neuroblasts from embryonic and adult central nervous system. Methods Enzymol. 1995;254:20–37.PubMedCrossRefGoogle Scholar
  12. 12.
    Ray J. Monolayer cultures of neural stem/progenitor cells. In: Gage FH, Kempermann G, Song H, editors. Adult neurogenesis. New York, NY: Cold Spring Harbor Press; 2008. p. 135–57.Google Scholar
  13. 13.
    Wakeman DR, Hofmann MR, Redmond DE, Teng YD, Snyder EY. Long-term multilayer adherent network (MAN) expansion, maintenance, and characterization, chemical and genetic manipulation, and transplantation of human fetal forebrain neural stem cells. Curr Protoc Stem Cell Biol. 2009;Chapter 2:Unit2D.3.PubMedGoogle Scholar
  14. 14.
    Parker MA, Anderson JK, Corliss DA, Abraria VE, Sidman RL, Park KI, Teng YD, Cotanche DA, Snyder EY. Expression profile of an operationally-defined neural stem cell clone. Exp Neurol. 2005; 194:320–32.PubMedCrossRefGoogle Scholar
  15. 15.
    Reynolds BA, Rietze RL. Neural stem cells and neurospheres–re-evaluating the relationship. Nat Methods. 2005;2:333–6.PubMedCrossRefGoogle Scholar
  16. 16.
    Svendsen CN, Smith AG. New prospects for human stem-cell therapy in the nervous system. Trends Neurosci. 1999;22:357–64.PubMedCrossRefGoogle Scholar
  17. 17.
    Rajan P, Snyder EY. Neural stem cells and their manipulation. Methods Enzymol. 2006;419:23–52.PubMedCrossRefGoogle Scholar
  18. 18.
    Deloulme JC, Baudier J, Sensenbrenner M. Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor. J Neurosci Res. 1991;29:499–509.PubMedCrossRefGoogle Scholar
  19. 19.
    Ray J, Gage FH. Spinal cord neuroblasts proliferate in response to basic fibroblast growth factor. J Neurosci. 1994;14:3548–64.PubMedGoogle Scholar
  20. 20.
    Svendsen CN, Fawcett JW, Bentlage C, Dunnett SB. Increased survival of rat EGF-generated CNS precursor cells using B27 supplemented medium. Exp Brain Res. 1995;102:407–14.PubMedCrossRefGoogle Scholar
  21. 21.
    Wakeman DR, Hofmann MR, Teng YD, Snyder EY. Neural progenitors (Chap. 1). In: Masters JR, Palsson BØ, editors. Human adult stem cells, human cell culture, vol. 7. New York, NY: Springer Science+Business Media B.V.; 2009. p. 1–44.CrossRefGoogle Scholar
  22. 22.
    Selmeczi D, Mosler S, Hagedorn PH, Larsen NB, Flyvbjerg H. Cell motility as persistent random motions: Theories from experiments. Biophys J. 2005;89:912–31.PubMedCrossRefGoogle Scholar
  23. 23.
    Menezes JR, Luskin MB. Expression of neuron-specific tubulin defines a novel population in the proliferative layers of the developing telencephalon. J Neurosci. 1994;14:5399–416.PubMedGoogle Scholar
  24. 24.
    Liu L, Geisert EE, Frankfurter A, Spano AJ, Jiang CX, Yue J, Dragatsis I, Goldowitz D. A transgenic mouse class-III β tubulin reporter using yellow fluorescent protein. Genesis. 2007;45:560–9.PubMedCrossRefGoogle Scholar
  25. 25.
    Vescovi AL, Snyder EY. Establishment and properties of neural stem cell clones: Plasticity in vitro and in vivo. Brain Pathol. 1999;9:569–98.PubMedCrossRefGoogle Scholar
  26. 26.
    Pearle E, Collett B, Bart K, Bilderback D, Newman D, Samuels S. What Brown saw and you can too. Am J Phys. 2010;78:1278–89.CrossRefGoogle Scholar
  27. 27.
    Imitola J, Raddassi K, Park KI, Mueller FJ, Nieto M, Teng YD, Frenkel D, Li D, Sidman RL, Walsh CA, Snyder EY, Khoury SJ. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1α/CXC chemokines receptor 4 pathway. Proc Natl Acad Sci U S A. 2004;101:18117–22.PubMedCrossRefGoogle Scholar
  28. 28.
    Nakaji-Hirabayashi T, Kato K, Iwata H. Improvement of neural stem cell survival in collagen hydrogels by incorporating laminin-derived adhesive polypeptides. Bioconjug Chem. 2012;23:212–21.PubMedCrossRefGoogle Scholar
  29. 29.
    Fietz SA, Lachmann R, Brandl H, Kircher M, Samusik N, Schröder R, Lakshmanaperumal N, Henry I, Vogt J, Riehn A, Distler W, Nitsch R, Enard W, Pääbo S, Huttner WB. Transcriptomes of germinal zones of human and mouse fetal neocortex suggest a role of extracellular matrix in progenitor self-renewal. Proc Natl Acad Sci USA. 2012;109:11836–41.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media New York 2013

Authors and Affiliations

  • Walter D. Niles
    • 1
  • Dustin R. Wakeman
    • 2
  • Evan Y. Snyder
    • 3
    Email author
  1. 1.Sanford Consortium for Regenerative MedicineSanford Burnham Medical Research InstituteLa JollaUSA
  2. 2.Department of Neurological SciencesRush Medical CollegeChicagoUSA
  3. 3.Sanford Burnham Medical Research InstituteLa JollaUSA

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