Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology

Ilyas Singec, Rolf Knoth, Ralf P Meyer, Jaroslaw Maciaczyk, Benedikt Volk, Guido Nikkhah, Michael Frotscher & Evan Y Snyder

Supplementary Fig. 1 (pdf 104K)
Beating cellular specializations of neurosphere cells.

Supplementary Note (pdf 132K)
Mathematical analysis of proliferative cells in secondary neurospheres.

Supplementary Video 1 (mov 2M)
Representative time-lapse video microscopy demonstrating frequent, rapid, and multiple "coalescence" of secondary neurospheres. Note that neurospheres are capable of active locomotion, apparently propelled by tiny beating cellular processes. The movie shows a 2.5 hr segment of the 10-hr time-lapse recording excerpted as well in Fig. 2. The video recorder captured images every 3.5 seconds. This neurosphere culture was generated from the dissociated forebrain of a newborn mouse, initially seeded at a density of 50 cells/l and 10⁴ cells/cm² (standard "medium density"), and cultured for 5 days in a humidified atmosphere at 37°C and 5% CO₂ at the time of this videomicrograph. For illustrative purposes, this movie presents an "overview" of a "medium" cell density culture in a 6-well plate. Numerous time-lapse video-microscopic recordings confirmed merging of spheres grown at even "clonal" cell density (Supplementary Video 2 online). The beating cellular processes are better appreciated at higher power in Supplementary Video 3 online.

Supplementary Video 2 (mov 680K)
Time-lapse video-microscopy demonstrating, at higher magnification, neurosphere locomotion and "coalescence" despite plating at "clonal" density. Cellular processes (see also Supplemental Fig. 1 and Supplemental Video 3) propel neurospheres over considerable distances increasing the probability of coalescence events in even low density cultures. The movie shows a time-lapse recording of 2 hr. This neurosphere culture (24-well plate) was generated from the dissociated forebrain of a newborn mouse, initially seeded at a density of only 5 cells/l and 10³ cells/cm² ("clonal" density), and cultured for 5 days in a humidified atmosphere at 37°C and 5% CO₂ at the time of this videomicrograph.

Supplementary Video 3 (mov 9M)
Time-lapse video microscopy of human fetal cortex-derived neurospheres at higher magnification. Note the tiny beating cellular processes covering the surface of two merging human neurospheres. These beating structures were abundant on human and rodent neurospheres irrespective of their origin (e.g. cortex, striatum, spinal cord) and seem to propel them in suspension culture.

Supplementary Video 4 (mov 8M)
Time-lapse video microscopy of human neurospheres grown in 96-well plates. Neurospheres are motile and merge in 96-well plates similar to the findings obtained in 6-well (Video 1) and 24-well (Video 2) plates. In this experiment with 96-well plates, the initial plating density was 10 cells/l or 2000 cells/well (personal communication, B. Reynolds), but nevertheless still generated polyclonal spheres. Note that movie speed here is 5 times faster than in the other Videos.

Supplementary Video 5 (mov 2M)
Time-lapse video microscopy of mouse cell culture showing neurosphere fusion and interaction of motile spheres with single cells attached to uncoated plastic dish. This example shows, besides the merging of two neurospheres, that single cells can attach to or detach from a motile neurosphere compromising their clonal boundaries even at "clonal" plating density (5 cells/l).

Supplementary Video 6 (mov 5M)
Time-lapse video microscopy of neurosphere-like cells that have become adherent. Note that adherent spheres, too, migrate and coalesce. Only if this concentrated culture dish had begun as a single isolated cell in a single isolated miniwell would an assumption of clonal boundaries and identity be justified and legitimate. In this experiment mouse forebrain stem cells (20 cells/l) were plated into 24-well plates with glass coverslips coated with poly-L-lysine and laminin. The videomicrograph was taken on day 7.

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