1. The Effect of FGF on Mammalian Stem Cells
Tim Ungarean
11th Grade
Central Catholic High School

2. FGF (Fibroblast Growth Factor)
Signal through FGF receptors (FGFR’s)
Regulates cell proliferation, differentiation, survival, and migration
Often used for studies dealing with in vitro functions of tissue regeneration
The properties of FGF are often modified to increase its half-life and its effectiveness

3. Stem Cells
Unspecialized cells capable of renewing themselves through cell division
Cells that can produce lineages more specialized than themselves and can renew itself

4. C2C12 Stem Cells
Sub clone of the Mus musculus (mouse) myoblast cell line.
Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.
Used as a model in many tissue engineering experiments.
Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells.

5. Purpose
To determine if FGF has an effect on C2C12 stem cell differentiation and proliferation.

6. Hypotheses
Alternate Hypothesis- FGF will have a significant effect on the proliferation and differentiation of C2C12 Stem Cells.
Null Hypothesis- FGF will not have a significant effect on the proliferation and differentiation of C2C12 Stem Cells.

7. Materials Two 75cm^2 culturing flasks Twelve 25cm^2 culturing flasks
Trypsin
Hemocytometer
10% DMEM media
1% DMEM media
PBS
P1000 pipette
P100 pipette
Sterile tips
Micro pipette
Macro pipettes
Incubator
EVOS computer imaging system
Laminar sterilized hood
Sterile gloves
70% ethanol
C2C12 Cells
Pen and notebook
FGF (Variable)
Six well culture plates

8. Variable Concentrations
Low
1 nanogram/mL
High
10 nanograms/mL

9. Procedure Cell Culturing
Cell culture was added to two 75cm2 culturing flasks
The cell suspensions were trypsinized and pulled into one 75cm2 flask
3. 4mL of cell suspension was aliquoted into eight 25cm2 flasks
30ul of FGF, low and high concentrations, were added to two flasks respectively
Cells were imaged using EVOS computer imaging system

10. Procedure (continued)
Cell Counting
Cells imaged before counting
The cells were trypsinized and suspended in 2ml of media, and 20ul samples were removed and placed on a hemocytometer

11. Procedure (continued)
Differentiation
1. 3 ml of cell suspension were seeded in two six well plates.
2. 30ul of the low and high variable concentrations were added to two well plates respectively.
3. On the next day, the media was replaced with 1% serum differentiation media, and variable was replaced
4. The wells were incubated for a week
5. Fresh media and variables were replaced every three days.

13. Dunnett's Test Concentration of Variable T-Crit T-Value Interpretation
Day 1 Low
2.921
0.64
Not Significant
Day 1 High
5.53
Significant
Day 2 Low
0.09
Day 2 High
3.491

14. Day One Images
Control
Low
High

15. Day Two Images
Control
Low
High

16. Differentiation Assay Images Day Three
Control
Low
High

17. Differentiation Assay Images Day Seven
Control
Low
High

18. Conclusion
The data suggests that the proliferation of the cells was greatly effected.
The images suggest that FGF does not have an effect on the differentiation of C2C12 cells.

19. Limitations
Hemocytometer counts were subject to miscounts from clumping.
No differentiation marker, differentiation was a qualitative analysis.
Limited variable concentration range.

20. Further Studies Test greater concentrations of FGF
Test on different cell lines, or other types of stem cells
Increase exposure time of FGF
Test a different growth factor in addition to FGF
Quantitative differentiation analysis through fluorescent antibodies.

21. Sources https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042641/

22. Day One Single-Factor Anova
Treatments 1 2 3 4 5
Total N 16 48 ∑X
847
797
418
2062
Mean
26.125
42.958
∑X2
50495
41845
11554
103894
Std.Dev.
6.5
Day One Single-Factor Anova
Result Details
Source SS df MS
Between-treatments 2
F =
Within-treatments 45
Total 47

23. Day Two Single-Factor Anova1 2 3
Total N 16 48 ∑X
1375
1365
1737
4477
Mean
93.271
∑X2
125317
119427
192831
437575
Std.Dev.
Day Two Single-Factor Anova
Result Details
Source SS df MS
Between-treatments 2
F =
Within-treatments 45
Total 47