Aims

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.

Procedure

Attempt 1

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.

Attempt 2

Attempt 3

25032019

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.

26032019

200µL TSM-C with 125nM LLY283 (or DMSO control) was added to each well for a 6 day pre-treatment.

01042019

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.

08042019

Colonies were called by eye from brightfield images. Colonies of only >3 cells were counted.

Attempt 1 analysis

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

Attempt 2

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