Growth Dynamics of Fetal Human Neural Stem Cells

  • Walter D. Niles
  • Dustin R. Wakeman
  • Evan Y. SnyderEmail author


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.


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.



Basic fibroblast growth factor (fibroblast growth factor 2)




Dulbecco’s phosphate-buffered saline


Extracellular matrix


Epidermal growth factor


Fetal bovine serum


Human neural stem cells


Leukemia inhibitory factor



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)

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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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