1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, 3STEMCELL Technologies, Inc.
Azari, H., Louis, S. A., Sharififar, S., Vedam-Mai, V., Reynolds, B. A. Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells. J. Vis. Exp. (49), e2639, doi:10.3791/2639 (2011).
The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies ≥2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies < 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are < 2mm and the ones that are ≥2mm in reference to the number of cells that were initially plated.
1. Items That Need to Be Prepared Before Proceeding to Cell Plating:
2. Cell Preparation:
Depending on your experiment, cells can be prepared from an adult or embryonic source (primary dissociated tissue or dissociated neurospheres). Dissect tissues from adult/embryonic mouse central nervous system (CNS) or dissociate adult/embryonic-derived neurospheres as described before 1,2 and then:
3. Plating Cells in Semi-solid N-CFCA Medium:
4. Preparing Replenishment Medium and Feeding the Culture:
As N-CFCA cultures are incubated for an extended period of time (21 days), cultures should be fed with the appropriate complete NeuroCult replenishment medium prepared fresh as follows:
5. Scoring N-CFC Assay Derived Colonies and Calculating the Frequency of bona Fide NSCs and Neural Progenitor Cells:
6. Representative Results:
As in neurosphere assay, cells plated in N-CFC assay start to proliferate and make small colonies of cells within 3 - 7 days after plating (Figure 1). In two weeks time, colonies of different sizes can be distinguished. While the majority of colonies tend to stop growing after 14 days, some colonies continue to increase in size. By day 21, colonies can be classified into one of the four categories: 1) less than 0.5 mm diameter, 2) 0.5 - 1 mm diameter, 3) 1 - 2 mm diameter and 4) ≥2mm in diameter. Practically, colonies smaller than 2 mm diameter are referred to as progenitor derived (Figure 2) and colonies ≥2mm in diameter are referred to as NSC derived (Figure 3). The number of colonies ≥2mm in diameter per total cells plated, represents the frequency of the actual bona fide neural stem cells with long-term self-renewal and multi-potential capabilities. The total neurosphere forming frequency in a particular cell population after 7-8 day in a parallel NSA experiment has been estimated to be similar to the total colony forming frequency of the same cell population after 21 days in a N-CFCA experiment. The N-CFCA however, provides a more permissive condition so as each cell can show its full proliferative potential.
|Component||2 replicates||3 replicates||4 replicates|
|NeuroCult NCFC Serum-Free Medium without Cytokines||1700||2550||3400|
|NeuroCult NSC Proliferation Supplements||330||495||660|
|EGF (10 μg/mL)||6.6||9.9||13.2|
|bFGF (10 μg/mL), only for adult cells||3.3||4.95||6.6|
|Heparin Solution (0.2%), only for adult cells||3.3||4.95||6.6|
2.2 x 105 cultured cells/mL OR
6.5 x 105 primary cells/mL
Table 1. Components of complete N-CFC assay culture.
Figure 1. Representative colonies in N-CFCA culture of passage one E14 mouse NSCs 7 days after plating. The colonies might display different morphology and size. Original magnification; 4x
Figure 2. Representative progenitor derived colonies of different sizes in N-CFCA culture of passage one E14 mouse NSCs 21 days after plating. As shown, the colonies have different morphology but all are less 2 mm in size. Original magnification; 4x
Figure 3. Representative bona fide stem cell derived colony in N-CFCA culture of passage one E14 mouse NSCs 21 days after plating. Stem cell derived colonies might display different morphology (see the video) but all are ≥2mm in diameter. Original magnification; 4x
Although neurosphere assay3,1,2 is the most common method to isolate and expand neural stem cells from a variety of sources like adult and embryonic CNS tissue, it cannot accurately measure the NSC frequency in a mixed population of neural precursor cells (stem and progenitors) as there is not a one to one relationship between the number of neurospheres and the number of bona fide stem cells4. To address this limitation, the original NSA has been adapted so as to allow the neural stem and progenitors to grow to their full proliferation capacity for three weeks 5-7. Unlike the liquid NSA, in the N-CFCA the colonies are actually clonally derived, as the semi-solid collagen matrix prevents migration of the single cells plated and aggregation of colonies. For consistent results with this assay we recommend:
One of the authors, Sharon A. Louis, is affiliated with StemCell Technologies Inc, a producer of some reagents used in this study.
This work was supported by funding from the Overstreet Foundation.
|NeuroCult NSC Basal Medium||Medium||Stem Cell Technologies||05700|
|NeuroCult NSC Proliferation Supplements||Medium supplement||Stem Cell Technologies||05701|
|NeuroCult NCFC medium||Medium||Stem Cell Technologies||05720|
|0.05% trypsin-EDTA||Reagent||GIBCO, by Life Technologies||25300-062|
|Soybean trypsin inhibitor||Reagent||Sigma-Aldrich||T6522|
|Cell strainer||Sieve||BD Biosciences||352340|
|Pen/Strep||Reagent||GIBCO, by Life Technologies||15140-122|
|T25 flask||Culture ware||Nalge Nunc international||136196|
|T80 flask||Culture ware||Nalge Nunc international||178905|
|Collagen||Reagent||Stem Cell Technologies||04902|
|EGF||Growth factor||R&D Systems||2028-EG|
|b-FGF||Growth factor||R&D Systems||3139-FB|
|Heparin||Growth factor||Sigma-Aldrich||H4784||Reconstituted in PBS|
|15 ml tubes||Culture ware||BD Biosciences||352096|
|50 ml tubes||Culture ware||BD Biosciences||352070|
|35 mm culture dishes||Culture ware||Stem Cell Technologies||27100|
|Gridded scoring dishes||Culture ware||Stem Cell Technologies||27500|