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Neural stem cells (NSCs) were initially identified in liquid suspension cultures by their ability to proliferate into neurospheres.1,2 The Neurosphere Assay has become the assay of choice for the detection and isolation of mammalian growth factor-responsive NSCs, and forms the core of many studies used to understand the cellular and molecular biology of stem cells in the central nervous system (CNS). The Neurosphere Assay has been used to screen exogenous signaling factors for their effects on stem cell function,3 and to help understand the biology of NSCs.4,5
the need for a new NSC assay
The understanding of NSCs and their therapeutic potential relies heavily on robust functional assays that can identify and measure NSC activity in vivo and in vitro. The Neurosphere Assay is widely used to isolate NSCs and even measure their frequency. However, recent publications have highlighted the limitations of the Neurosphere Assay as an accurate assay for measuring NSC frequency in relation to NSC regulation.6,7 One of the major limitations of the Neurosphere Assay is that not all neurospheres in the assay are derived from a NSC.7 Because the Neurosphere Assay does not disriminate between neurospheres derived from a NSC or a progenitor cell, enumerating neurosphere numbers and equating this read-out to NSC numbers would lead to the overestimation of NSC frequency. In addition, neurosphere fusions occur significantly even at limiting dilution plating8 and the lack of standardization in the neurosphere culture system makes it difficult to compare experimental results between laboratories.9,10 These findings imply that while the Neurosphere Assay provides a simple means to isolate and expand NSCs harvested from the embryonic and adult mammalian CNS, its application as a quantitative in vitro assay for measuring NSC frequency is limited.
In order to address the need for an assay that can reliably detect alterations in NSC frequency, STEMCELL Technologies has developed a new single-step assay, the Neural Colony-Forming Cell (NCFC) Assay, which allows disrimination between NSCs and progenitors by the size of colonies they produce (i.e. their proliferative potential). The NeuroCult® NCFC Assay Kit is a collagen-based culture system has been optimized to support the growth of neural stem and progenitor cells. The NeuroCult® NCFC Assay Kit allows the quantification of neural stem and progenitor cells. Primary or cultured neural cells are suspended in serum-free medium containing optimized levels of growth supplements and recombinant cytokines. Collagen is then mixed with the cell/medium suspension and dispensed into 35 mm culture dishes. At the end of the culture period, clonally-derived colonies of different sizes are scored.
proliferation potential and differentiation potential (optional)
If desired, colonies can be excised from the collagen matrix and incubated individually in collagenase. The excised colony can then be disrupted to produce a single cell suspension. The cells from a single colony can be used in assays such as the Neurosphere Assay, plated onto a glass chamber slide and differentiated, or used in any other desired protocol.
