To develop a limiting dilution assay to determine the fraction of cells in a DIPG cell line capable of forming a new colony from a single cell (an indication of stem cell-like behaviour). To test the fraction of cells in the HSJD-DIPG-007 cell line that are stem-like.
Used inappropriate method: Using a U-bottomed plate can push multiple cells together creating a sphere from more than one initial cell – the assay relies on colonies being formed from a single cell.
HSJD-DIPG-007 cells were split (P143+10). 5k cells were seeded in a 6 well plate in 1800µL TSM-C. Cells were allowed to grow as normal for 7 days.
200µL TSM-C with 125nM LLY283 (or DMSO control) was added to each well for a 6 day pre-treatment.
Cells were split, passed through a 0.2µm filter, counted and seeded in the following densities (per well): 8, 4, 2, 1, 0.5, 0.25.
Colonies were called by eye from brightfield images. Colonies of only >3 cells were counted.
Cell densities/well and scoring of each well:
## [,1] [,2] [,3] [,4] [,5] [,6]
## 8 TRUE TRUE TRUE TRUE TRUE TRUE
## 4 TRUE TRUE TRUE TRUE TRUE TRUE
## 2 TRUE TRUE TRUE TRUE TRUE TRUE
## 1 TRUE FALSE TRUE FALSE TRUE FALSE
## 0.5 FALSE TRUE FALSE FALSE FALSE TRUE
## 0.25 FALSE FALSE FALSE FALSE FALSE FALSE
Count of positive wells for each density:
## Count
## 8 6
## 4 6
## 2 6
## 1 3
## 0.5 2
## 0.25 0
Calculation of stem-like compartment:
The following analysis makes use of the “statmod” package from R. The elda() function “implements limiting dilution analysis using complemenary log-log binomial generalized linear model regression”. The package is outlined in (Hu and Smyth, 2009) and hosted at http://bioinf.wehi.edu.au/software/elda/
Citation: Hu, Y, and Smyth, GK (2009). ELDA: Extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. Journal of Immunological Methods 347, 70-78.
Stem fraction in HSJD-DIPG-007 cells (with 95% CI intervals):
## Lower Estimate Upper
## Group HSJD-DIPG-007 1.858599 1.076912 1
Significance for test of whether data fits the single hit model:
## Estimate Std. Error z value Pr(>|z|)
## NA NA 1.92350555 0.05441659
## Estimate Std. Error z value Pr(>|z|)
## 2.1578331 0.7356778 1.5738317 0.1155264
Same as previous, with a greater number of cell densities: 9, 6, 4, 2.67, 1.78, 1.185, 0.790, 0.527, 0.351, 0.234 (a 1.5x dilution each time). Cells were stained with Calcein AM as follows: Calcein AM was diluted in PBS, vortexed, and added to cells to a final concentration of 1:2000. An initial incubation at 37oC for 15mins was insufficient to produce a useful level of contrast in the images, so the final incubation time was 30mins.
Calculation of stem-like compartment:
## Lower Estimate Upper
## Group HSJD-DIPG-007 2.172753 1.436059 1
Significance for test of whether data fits the single hit model:
## Estimate Std. Error z value Pr(>|z|)
## NA NA 0.8485532 0.3961300
## Estimate Std. Error z value Pr(>|z|)
## 1.2498240 0.2978540 0.8387463 0.4016117