Whey Protein Influence on C2C12 Muscle Stem Cells

James ConstantinCentral Catholic High SchoolGrade 11

Stem Cell Overview

Basic Definition- cell that can produce lineages more specialized than themselves and can renew itself

Spectrum of Stem Cell Behavior: totipotent embryonic cells pluripotent cells multipotent cells unipotent cells.

C2C12 Cells Subclone of the mus musculus (mouse)

myoblast cell line. Differentiates rapidly, forming contractile

myotubes and produces characteristic muscle proteins.

Mouse stem cell line is 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.

Expresses muscle proteins and the androgen receptor (AR). AR- DNA binding transcription factor which

regulates gene expression.

Variable: Whey Protein

Whey protein is a mixture of globular proteins isolated from whey, the liquid material created as a by-product of cheese production. Some preclinical studies in rodents have suggested that whey protein may possess anti-inflammatory or anti-cancer properties; however, human data is lacking. The effects of whey protein on human health are of great interest and are currently being investigated as a way of reducing disease risk (such as muscle degeneration), as well as a possible supplementary treatment for several diseases. Whey protein is commonly marketed and ingested as a dietary supplement, and various health claims have been attributed to it in the alternative medicine community.

Why Whey

Whey is essential in the bodybuilding world today because of its ability to be digested very rapidly. This allows the protein to become available for muscle building very quickly. Most commonly it is used after workouts to help increase levels of amino acids in the blood, which are taken up by the muscles to ultimately increase mass. In addition, during exercise, whey helps open up blood flow by inhibiting an angiotensin-converting enzyme which originally constricts blood vessels; this allows better flow of nutrients to necessary areas to help repair and rebuild muscle tissues. The use of whey protein as a source of amino acids and its effect on reducing the risks of diseases such as heart disease and cancer is the focus of ongoing research.

Purpose

To examine the effect(s) of (GNC) Wheybolic Extreme on proliferation and differentiation of C2C12 murine adult stem cells

Hypotheses

Null: GNC Wheybolic Extreme will not have an effect on the proliferation, differentiation, and survivorship of C2C12 cells.

Alternate: GNC Wheybolic Extreme will have an effect on the proliferation, differentiation, and survivorship of C2C12 cells. Specifically the variable will promote growth and promote the cells to differentiate into myofibrils.

Materials

Cryotank Three 75mm2 tissue

culture treated flasks Twenty-four 25 mm2

tissue culture treated flasks

10% fetal bovine serum

C2C12 Myoblastic Stem Cell Line

Trypsin-EDTA Pen/strep Macropipette + sterile

macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL)

Micropipettes + sterile tips

DMEM media -1% and Complete Media (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete])

75 mL culture flask Incubator Aspirating Vacuum Line Nikon Inverted Compound

Optical Scope Laminar Flow Hood Laminar Flow Hood UV

Sterilizing Lamp Labeling Tape GNC Amplified Wheybolic

Extreme 60 Hemocytometer Sterile PBS Ethanol (70% and 100%) Distilled water Antibody staining system Fluorescent sensitive

microscope

Hemocytometer grid

Procedure (Stem Cell Line Preparation)

A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm2 culture flask yielding a cell density of approximately 106 to 2x106 cells.

The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4x106 to 5x106 cells/mL was reached.

The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

Procedure (Proliferation Experiment)

Day 1 After trypsinization, cells from all of the flasks were pulled into 1 common 75mm2 flask

(cell density of approximately 1 million cells/mL). 5 ml of the cell suspension was added to 6 25 mm2 ( 2 for each grouping) tissue

culture treated flasks, creating a cell density of approximately 10 5 cells per flask. The following concentrations of variable (next page) were added to the flasks. 4

flasks for each group (2 for proliferation, 2 for differentiation) Using one flask from each group, cell densities were determined as follows:

▪ The cells were trypsinized and collected into cell suspension.▪ 25 µl aliquots were transferred to a Hemacytometer for quantification (six

counts per flask).Day 2 and Day 5

Using the Nikon Inverted Microscope, images of several representative areas of each flask were taken.

Procedure Antibody Assay (Differentiation Experiment)

3mL of the cell suspension was added to three glass bottom well plates

The plates were incubated for one day The following day (Day 1) 30 µL of variable

was added to the two variable plates The plates were allowed to incubate for one

week until myotubules formed1. The three plates were fixed by washing with

PBS and fixing with ice-cold methyl alcohol (MeOH)

2. Stain plates with primary and secondary GFP, Desmin, and Dapi

3. Images were recorded utilizing a fluorescent inverted view microscope

Experimental Groups (Concentrations)

Mini Flasks (Proliferation) 2 flasks per group

Plates (Differentiation)3 plates per group

0.0% 0 ml’s of 1%stock (Wheybolic Extreme)5 ml’s Media

0 ml’s of 1% stock (Wheybolic Extreme) 3ml’s

0.01% 50 microliters of 1% stock (Wheybolic Extreme)4.95 ml’s Media

3 microliters 1% stock (Wheybolic Extreme) 2.95 ml’s Media

0.001% 5 microliters of 1% stock (Wheybolic Extreme)4.995 ml’s Media

3 microliters 1% stock (Wheybolic Extreme) 2.995 ml’s Media

Statistical Analyses

ANOVA: In statistics, analysis of variance is a collection of statistical models, and their associated procedures, in which the observed variance in a particular variable is partitioned into components attributable to different sources of variation. In its simplest form ANOVA provides a statistical test of whether or not the means of several groups are all equal, and therefore generalizes t-test to more than two groups

Differentiantiation a Myosin Quantification Template was used: Overlaps images from fluorescent scope and compares nuclei, myotube growth etc.By comparing the means you are comparing variation between the groups to the variation within the groups.

Dunnett's test compares each variable group individually to the control group to see if that particular variable caused them to vary significantly. A T-test will yield the same p-value as the ANOVA and therefore will show insignificant results.

Control 0.001 0.010

5

10

15

20

25

30

35

15.1666666666667 16.166666666666718.1666666666667

26.333333333333327.6666666666667

29

Whey Influence On C2C12 Proliferation

Day1Day3

Concentration

Cells

perF

lask

(1

0^

5)

Control

Day 3Day 1

0.001%

0.01%

Proliferation

Antibody Assay Results

Variable Concentration

% Differentiation Nuclei/Myotube

Control 22.645% 4.112

.001 25.897% 4.465

.01 27.844% 2.572

• An ANOVA was performed on the data, and the f-value was larger than the f-crit, so the results are SIGNIFICANT.

Differentiation

Normal

Dapi Stain

Desmin Stain

GFP Stain

Conclusions

Null hypothesis can be accepted for the parameters that GNC Wheybolic Extreme does not have an effect on C2C12 stem cell survivorship.

Null hypothesis can be accepted for the parameters that GNC Wheybolic Extreme does not have an effect on C2C12 stem cell proliferation on Day 1.

The null Hypothesis can be accepted for the parameters that GNC Wheybolic Extreme does not have an effect on C2C12 stem cell proliferation on Day 2.

Differentiation Experiment The Null was rejected for both variable concentrations.

Supporting previous studies that Whey protein promotes muscle growth

Between Days

Day 1 Proliferation

Day 2 Proliferation

P-value0.201521

Limitations and Extensions

Different cell lines 3T3 Possibility of Errant cell count

Over trypsinization Damages cells and has a negative effect on

colony health

Trypan Blue Dye Exclusion Assay▪ Tells if cells are alive so gives more quantifiable

information CyQUANT™ Cell Proliferation Assay

▪ More quantitative than counting cells on a Hemocytometer

▪ Fluorescent dye binds to nucleic acid in the cell

References

John Wilson, Biostatistician for the University of Pittsburgh

Conrad M. Zapanta, Ph.D BiomedicalEngineering Laboratory, Carnegie Mellon University

Mark Krotec, PTEI http://www.netdoctor.co.uk/focus/nutrition/

facts/oxidative_stress/oxidativestress.htm http://cropsoil.psu.edu/courses/AGRO518/

Oxygen.htm http://www.creatinejournal.com/ http://www.bodybuilding.com/store/creatine.ht

ml Dr. Duprimia CMU

ANOVAAnova: Single Factor

SUMMARYGroups Count Sum Average Variance

Column 1 6 158 26.33333 8.266667Column 2 6 166 27.66667 1.466667Column 3 6 174 29 14.8

ANOVASource of VariationSS df MS F P-value F crit

Between Groups21.33333 2 10.66667 1.304348 0.300421 3.68232Within Groups122.6667 15 8.177778

Total 144 17