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

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

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

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.

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

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.

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

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

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

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

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.

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

Day One Images Control Low High

Day Two Images Control Low High

Differentiation Assay Images Day Three Control Low High

Differentiation Assay Images Day Seven Control Low High

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.

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

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.

Sources https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042641/ www.Hopkinsmedicine.org www.cancer.org https://stemcultures.com/fibroblast-growth-factor-what-is-it/

Day One Single-Factor Anova Treatments 1 2 3 4 5 Total N 16 48 ∑X 847 797 418 2062 Mean 52.9375 49.8125 26.125 42.958 ∑X2 50495 41845 11554 103894 Std.Dev. 19.4198 11.9567 6.5 18.0507 Day One Single-Factor Anova Result Details Source SS df MS   Between-treatments 6878.7917 2 3439.3958 F = 18.34861 Within-treatments 8435.125 45 187.4472 Total 15313.9167 47

Day Two Single-Factor Anova 1 2 3 Total N 16 48 ∑X 1375 1365 1737 4477 Mean 85.9375 85.3125 108.5625 93.271 ∑X2 125317 119427 192831 437575 Std.Dev. 21.8372 14.0841 16.8482 20.6292 Day Two Single-Factor Anova Result Details Source SS df MS   Between-treatments 5615.1667 2 2807.5833 F = 8.78205 Within-treatments 14386.3125 45 319.6958 Total 20001.4792 47