Serotonin Effects on Adult Stem Cell Behavior

Alec DiVitoCentral Catholic High School

Question

• Does serotonin affect adult stem cell differentiation and/or proliferation?

Tissue Engineering• 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 the potential to replace or supplement the function of tissues destroyed or compromised in any variety of ways, including:

• Inherent design flaws• Hereditary/congenital defects or conditions• Disease• Trauma• Damage from an individual’s environment• Aging

• TE has great potential for supplementing muscle tissue.

Cells ECM

Hormones BloodSupply

Defect Regeneration

Phil Campbell, Carnegie Mellon

Principles of Tissue Engineering

Chemical Signaling

• Cells responsive to signaling molecules and growth factors such as BMP-5 and IGF.

• Signaling molecules control migration, proliferation, differentiation, attachment, and continuity. This is essential for stem cells.

• Do therapeutic drugs alter this signal driven cell regulation?

Neurotransmitters

• Endogenous chemicals transmit signals from neurons to target cells across a synapse.

• Release of neurotransmitters follows arrival of an action potential at the synapse.

• Synthesized from precursors molecules.

Serotonin• Neurotransmitter, is made via a unique

biochemical conversion process.• Can affect the functioning of other

organ systems.• Imbalance may influence mood in a

way that leads to depression.• Regeneration of brain cells is regulated

by serotonin throughout lifetime.• No way to measure its levels in the

living brain.• Don't know whether the dip in

serotonin causes the depression, or the depression causes serotonin levels to drop.

Adult Stem Cells• Undifferentiated cells

multiply to replenish dying cells.

• Somatic stem cells, can be found in juvenile and adult animals and humans.

• Self-renew indefinitely, and generate all cell types of the organ from which they originate.

• They have mainly been studied in humans and model organisms such as mice and rats.

C2C12• Subclone of the mus musculus

(mouse) myoblast cell line.• Mouse stem cell line is

frequently used as a model in tissue engineering experiments.

1. Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.

2. Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells.

3. Expresses the androgen receptor (AR).• AR- DNA binding

transcription factor which regulates gene expression.

3T3• Cell line established

from Swiss mouse embryo tissue.

• Has become the standard fibroblast cell line.

• Often used in the cultivation of keratinocytes, with the 3T3 cells secreting growth factors favorable to these kinds of cells.

Purpose

• To assess the effects of serotonin on adult stem cell behavior.

Null Hypothesis

• Serotonin will not affect adult stem cell behavior.

Alternative Hypothesis• Serotonin will decrease adult stem cell

proliferation and/or differentiation.

Materials• Serotonin Solution• 75mm2 tissue culture treated flasks• Twenty 25 mm2 tissue culture treated flasks• C2C12 Myoblastic Stem Cell Line• 3T3 Fibroblastic 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])

• 75 mL culture flask• Incubator• Nikon Inverted Microscope• Aspirating Vacuum Line• Laminar Flow Hood• Laminar Flow Hood UV Sterilizing Lamp• Labeling Tape• Hemocytometer• Sterile PBS• Ethanol (70% and 100%)• Distilled water

Procedure1. Five ml of adult stem cells and media were prepared in 12 T25 flasks. One ml of

C2C12 cells were also allowed to grow in nine well plates, six of which were 50% cells and 50% media and the other three were 30% cells and 70% media.

2. The serotonin was diluted with PBS into a low (10,000X) and high (1,000X). X = therapeutic dose.

3. One μl of each diluted serotonin was placed into the selected nine wells and one ml of serotonin into each flask.

4. Cells were imaged one and three days after (for C2C12) and one and two days after (for 3T3) exposure. Four images were taken per flask.

5. Cells were trypsinized prior to imaging and counting to release from bottom of flask. 0.5 ml of trypsin was inserted into each flask and allowed to incubate for four minutes and 1.5 ml of media was added to quench the reaction.

6. Twenty-five μl of trypsinized cells were loaded into hemacytometers. Eight cell counts were performed per flask.

7. The well plates of C2C12 cells were used to see how serotonin affected differentiation and the flasks represented proliferation of two different cell lines.

8. Six flasks for the 3T3 line and six for the C2C12 line, three flasks were counted and imaged as a representation for a day.

Proliferation of C2C12

Day 1 Day 3 Day 50

50000

100000

150000

200000

250000

300000

350000

400000

Low XHigh 10XControl 0X

Day

Cell

Coun

t

P-value = 0.008

Serotonin Concentration

Proliferation of 3T3

Day 1 Day 3 Day 50

50000

100000

150000

200000

250000

300000

350000

400000

Control 0XLow XHigh 10X

Day

Cell

Coun

t

P-value = 0.0282

Serotonin Concentration

Analysis of C2C12 Proliferation

T-value SignificanceLow 4.42 SignificantHigh 4.70 Significant

T-critical = 4.32

Analysis of 3T3 Proliferation

T-value Significance

Low 3.34 Significant

High 3.01 Significant

T-critical = 2.97

Differentiation of C2C12 – Day 1Control Low

High

Differentiation of C2C12 – Day 3Control Low

High

Differentiation of C2C12 – Day 5Control Low

High

C2C12 Differentiation AnalysisControl Low

Qualitative Analysis – Low concentration enhances myotube formation

Conclusion• From the ANOVA and Dunnett’s tests, the addition of

serotonin induced a statistically significant increase in proliferation in the C2C12 cells when added at one one-hundredth its suggested working concentration, X, and a statistically significant decrease in proliferation in the cells when added at one tenth its suggested working concentration, 10X.

• From the ANOVA and Dunnett’s tests, the addition of serotonin induced a statistically significant increase in proliferation when added at one tenth and one one-hundredth its suggested working concentration.

• From the qualitative analysis of the images gathered from the flasks, it appears that the addition of serotonin induced myotubule formation. This was especially apparent in the low concentration, while the high concentration showed less dramatic differentiation in comparison.

Limitations/Extensions• Evaluation of differentiation images is

qualitative and imprecise. A quantitative differentiation assay can be used, e.g. MyoD tagging.

• CyQUANT™ Cell Proliferation Assay can be used. More quantitative than counting cells on a Hemocytometer. Fluorescent dye binds to nucleic acid in the cell

• Test suggested working concentration and increased concentrations, to see if it would continue to be toxic to cells.

Sources and Acknowledgments• Dr. Phil Campbell• Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie

Mellon University• Mark Krotec, PTEI• Mr. Scott Windham, MSW, LCSW• C2C12 myoblastoma cell differentiation and proliferation is stimulated by

androgens and associated with a modulation of myostatin and Pax7 expression – German Sport University, Cologne, Germany

• Chen Y, Zajac JD Maclean HE 2005 Androgen regulation of satellite cell function. Journal of Endocrinology 186 21-31.

• Sinha-Hikim I, Taylor WE, Gonzalez-Cadavid NF, Zheng W and Bhasin S 2004 Androgen receptor in human skeletal muscle and cultured muscle satellite cells: up-regulation by androgen treatment. Journal of Clinical Endocrinology and Metabolism 89 5245-5255.

• "Bone Morphogenetic Protein-2 Converts the Differentiation Pathway of C2C12 Myoblasts into the Osteoblast Lineage." The Journal of Cell Biology. Web. 31 Jan. 2012. <http://jcb.rupress.org/content/127/6/1755.abstract>.