Final research presentation of my work as a research intern at the University of South Carolina during the summer (2015) in tissue engineering.

1. The Effect of Signaling
Factors on the
Differentiation of
Progenitor Cells
By Amanda Steel
The South Carolina Governor’s School for Science and Mathematics
The University of South Carolina – Biomedical Engineering Department
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2. The Effect of Signaling Factors on the
Differentiation of Progenitor Cells
 An Overall Aspect of Tissue Engineering and
Cell Research
 Cells Preparation Methods
 Testing Methods: DNA, ALP, Ca, PCR testing
 2D Results and 3D Results
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3. Tissue Engineering
Figure 1. Principle of Tissue
Engineering
Figure 2. Tissue Engineering
Triangle
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4. The Cell Aspect
Human Mesenchymal Stem Cells (hMSCs)
 Multipotent
 Present in bone, muscle, cartilage, and
fat
 Research for bone repair
Endothelial Colony Forming Cells (ECFCs)
 Differentiate from endothelial cells
 Present in the vessels in the bone
 Important to oxygen and nutrient
delivery
http://www.bioind.com/mesenchymal-stem-cells http://www3.imperial.ac.uk/bhfcre/trainingandcareersx/phdmrestrainingprogramme/centrestudents
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5. A View of My Experiment
 Progenitor Cells
 Growth Factors
 Bone Scaffolds
http://pubs.rsc.org/en/content/articlelanding/2011/cs/c0cs00025f#!divAbstract
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6. Introduction to Experiment
Figure 1
Differentiation MediaBasic Media
2D environment
that cells were
cultured in
3D environment
that cells were
cultured in
Cell Type Media Type Differentiation Ingredients
hMSC Basic DMEM 1%PCN 10%FBS
Osteogenic DMEM 1%PCN 10%FBS Ascorbic acid BGP Dexamethasone
ECFC Basic EBM 1%PCN 20%FBS
Vasculogenic EBM 1%PCN 20%FBS 2.5X VEGF
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7. Preparation Methods
 Human Mesenchymal Stem cells (hMSC)
and Endothelial Colony Forming Cells
(ECFC) were cultured in the bottom of t-75
flask
 At 80% confluency, the cells were washed
and trypsinized
 Added to well plates and petri dishes
 Basic and differentiation media added
 Placed in the incubator and the media was
periodically replaced
 hMSCs was replaced daily and ECFCs it was
replaced every other day
 Frozen for hMSCs at 1, 3, 7, and 14 days and
for ECFCs at 7 and 14 days
http://www.genengnews.com/insight-and-intelligence/growth-factors-for-cell-culture/77899959/
https://www.emdmillipore.com/US/en/product/Millicell-24-well-Cell-Culture-Plate,MM_NF-C10077
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8. Testing Methods
2D Samples
• Measured alkaline phosphatase (ALP)
• Measured calcium content of hMSCs
3D Samples
• PCR testing using ALP and PECAM-1 as markers of differentiation
Thermal Cycler
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9. Results- 2D
• There was no significant difference
between the DNA content in basic and
differentiation media
• Higher amount of DNA was measured
in later time points which shows the
proliferation of cells
• There was more of an increase
between days one and three, than
between day three and the rest of
the time points
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1 3 7 14
Basic 533652.6103 791743.7417 839500.158 841296.4973
Differentiation 515813.874 619164.116 715776.4873 774052.0367
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
DNA
Number of Days
DNA in hMSCs
1 7
Basic 246906.35 543823.3424
Differentiation 282941.812 596198.4604
0
100000
200000
300000
400000
500000
600000
700000
DNAcount
Number of Days
DNA in ECFCs

10. Results- 2D
• There were no calcium content observed in any of the time points
• ALP expression results showed that at day 14 the cells have higher ALP
expression in the osteogenic medium
1 3 7 14
Basal 1.26092E-07 2.54965E-07 9.40085E-07 9.51794E-06
Osteogenic 6.52261E-07 1.30413E-06 4.56876E-06 9.11037E-05
0
0.00001
0.00002
0.00003
0.00004
0.00005
0.00006
0.00007
0.00008
0.00009
0.0001
ALP/DNA
Number of Days
Figure 2: ALP/DNA in hMSCs
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11. Results- 3D
• The expression of PECAM-1 gene was measured using PCR testing
• At Day 7 we saw an up regulation of the PECAM-1 gene, which marked differentiation
and was expected
• Day 14 of vasculogenic media should have been a little less than day 7
• This was most likely due to error in analysis
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Day1B Day7B Day14B Day1V Day7V Day14V
Series1 1.0000 0.5000 0.0019 0.0117 3.1383 0.0030
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
VEGFContent
Day and Media Type
VEGF in ECFCs

12. Conclusion
Results of 2D culture showed:
 hMSCs in osteogenic media increased the ALP activity
 No calcium content
Results of 3D culture showed:
 In hMSCs and ECFCs in a 3D hydrogel scaffold had an
effect on upregulation of PECAM-1 gene
 Results were not that clear, so more tests should be
conducted to minimize flawed results
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13. Future Use
http://pubs.rsc.org/en/content/articlelanding/2011/cs/c0cs00025f#!divAbstract
Bone Scaffolds
Data and research from growth factors and both cells will
contribute to bone scaffold research and tissue regeneration
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14. Acknowledgements
 I would like to thank:
 My mentor Dr. Esmaiel Jabbari
 Graduate student Nazli Gharraee
 My advisor Dr. Kristin Walker
 My humanities advisor Dr. Antonio de Ridder-Vignone
 The University of South Carolina, Biomedical Engineering Department
 The South Carolina Governor’s School for Science and Mathematics along
with the Summer Program for Research Interns
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15. References
1. Itskovitz-Eldor, Joseph, Maya Schuldiner, Dorit Darsenti, Amir Eden, Ofra Yanuka, Michal
Amit, Hermona Soreq, and Nissim Benvenisty. "Differentiation of Human Embryonic Stem
Cells into Embryoid Bodies Comprising the Three Embryonic Germ Layers." Molecular
Medicine 6.2 (2000): 88-95. Print.
2. Maes, Christa. "Role and Regulation of Vascularization Processes in Endochondral Bones."
Calcif Tissue Int Calcified Tissue International 92.4 (2013): 307-23. Web. 14 July 2015.
3. Schuldiner, M., O. Yanuka, J. Itskovitz-Eldor, D. A. Melton, and N. Benvenisty. "Effects of
Eight Growth Factors on the Differentiation of Cells Derived from Human Embryonic Stem
Cells." Proceedings of the National Academy of Sciences 97.21 (2000): 11307-1312. Web. 14
July 2015.
4. Tuan, Rocky S., Genevieve Boland, and Richard Tuli. "Adult Mesenchymal Stem Cells and
Cell-based Tissue Engineering." Arthritis Research and Therapy 5.1 (2002): 32-45. Print.
5. Vlierberghe, S. Van, P. Dubruel, and E. Schacht. "Biopolymer-Based Hydrogels As Scaffolds
for Tissue Engineering Applications: A Review." Biomacromolecules 12.5 (2011): 1387-408.
Web. 14 July 2015.
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