Effects of Citrus Aurantium on Stem Cell Differentiation and Proliferation

By Kelly HylandOakland Catholic High School

Grade 11PJAS 2011

Stem Cell Overview

Un-specialized cells that have the potential to differentiate into mature body tissues

Can be either pluripotent (adult stem cells) or totipotent (embryonic stem cells)

Have potential to cure diseases such as heart disease, diabetes, and others

Subclone of the mus musculous (mouse myoblast) stem cell line

Differentiate rapidly to form myotubes, the cells that make up the myofibers of muscle tissue.

A useful model to study the differentiation from stem cells to mature skeletal muscle tissue

C2C12 Stem Cell Line

Strong herbal stimulant that modulates adrenaline pathways, increasing heart rate May be implicated in heart rhythm disorders

An additive in many weight loss drugs Extracted from orange oils Interferes with enzymatic activity

in the cholesterol production pathway, an essential part of the cell membrane (HMG-CoA reductase) May crystallize when reacting with certain sugars

Variable Citrus Aurantium

To test the effect(s) of Citrus Aurantium on C2C12 proliferation and differentiation.

Purpose

Null Hypothesis: Citrus Aurantium will not have a significant effect on C2C12 proliferation and differentiation.

Alternative Hypothesis: Citrus Aurantium will have a significant effect on stem cell proliferation and differentiation.

Hypothesis

Materials

Cryotank 75mm2 tissue culture treated flasks Six 25 mm2 tissue culture treated

flasks Fetal bovine serum (FBS) 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 Serum - 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])

Liquid Citrus Aurantium Extract

75 mL culture flask Incubator Nikon Inverted

Microscope Aspirating Vacuum Line Laminar Flow Hood Laminar Flow Hood UV

Sterilizing Lamp Hemocytometer Sterile PBS Ethanol (70% and 100%)

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 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

Made up stock solution with [10-2] of variable Seeded 6 flasks with C2C12 cells from the

original 3 flasks in 5mL of 10% DMEM media each

Allowed cells to incubate in CO2 incubator overnight and adhere to bottom of flask

Added 50μL of variable and removed 50μL of media from 2 flasks creating [10-4]

Added 5μL of variable and removed 5μL of media from 2 flasks creating [10-5] Recommended doses over 10 L of body fluid

2 flasks for control Allowed flasks to proliferate in incubator

overnight

Procedure: Proliferation Assay

Next day, aspirated media off of flasks Rinsed with 1mL of trypsin, aspirated off Added 1mL trypsin, incubate for 5 minutes Slap flask and confirm with microscope that cells are

no longer adhered to bottom of flask Quenched reaction with 5mL media. Re-add variable. Re-suspended cells before taking counts using pipette Loaded hemocytometer with approx 75uL from flask Took 4 counts per flask Counted cells in field of view of hemocytometer

under Nikon inverted microscope and multiplied count by 104 for total cells/mL

Repeated on Day 3

Procedure: Proliferation Continued

Proliferation: Results

jkl;jkk;ljk;jkkkkkkkkkkkCells/mL in 10,000

P Value: 5.89 * 10-9

0 0.0001 0.000010

100000

200000

300000

400000

500000

600000

700000

800000

Effect of Citrus Aurantium on C2C12 Proliferation

Day1 Day 3mL of variable/mL of solution

Aver

age

cells

/mL

T Values: 12.34, 17.33

Seeded 6 well plates with 3mL of C2C12 cells in 3mL 10% DMEM media

Allowed cells to proliferate until reached 80% confluence

Changed media to 1% DMEM to induce differentiation

Added 30μL of stock to and removed 30μL media from 2 plates, creating [10-4]

Added 3μL of stock to and removed 3μL media from 2 plates creating [10-5]

Kept 2 control plates Took images of plates Day 1, Day 3, and Day

4 Compared number of myotubes formed on each

plate

Procedure: Differentiation Assay

Differentiation Results:Day 1

Control Low Concentration

High Concentration

Differentiation Results: Day 3

Control Low Concentration

High Concentration

Differentiation Results:Day 4

Control Low Concentration

High Concentration

Interpretation:Differentiation

The variable significantly effected differentiation of the C2C12 cells

Crystallization observed in images: citrus aurantium may have crystallized with sugars in media and starved cells of nutrients

May have interfered with cholesterol synthesis and thus the cell membrane structure

Stat analysis showed that variation was significant

Must reject null hypothesis Data supports alternative hypothesis Results obtained may be due to either the

crystallization of citrus aurantium with media sugars or interference with cholesterol synthesis

Conclusion

http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl?10684

http://www.cmaj.ca/cgi/pmidlookup?view=long&pmid=15497209

http://www.nature.com/nature/journal/v270/n5639/abs/270725a0.html

"Dangerous Supplements: Twelve Supplements You Should Avoid" Consumer Reports Magazine, September 2010

Works Cited

Appendix

Tissue Engineering

What is TE? The development and

manipulation of artificial implants, laboratory-grown tissues, and genetically engineered cells and/or molecules to replace or support the function of defective or injured body parts

Has potential to replace damaged functional tissues in the body

Shows great promise in replacing functional muscle tissue

Repeat experiment to verify results To test leading hypothesis of crystallization,

measure amount of sugars remaining in fluid and compare with pure media

Further Research