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Influence of 2D and 3D Environments on Osteogeneic Differentiation in hMSCs
Jacqueline Mimnaugh, RETNeuqua Valley High School
Dr. Richard GemeinhartMelanie KollmerUIC Department of Biopharmaceutical Sciences
Tracy Chuong, REUUniversity of California Berkley
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Stem cells differentiate into other types of cellsTwo major categories:
What are hMSCs?
Human Mesenchymal Stem Cells (hMSCs)
Isolated from marrow
Can differentiate into bone, cartilage, fat
Embryonic(Pluripotent)
Adult(Multipotent)
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Induce hMSCs to develop into bone cells Osteogenesis
Tissue Engineering• Bone diseases/defects, trauma, cancer,
mal-union/non-union fractures
Why are hMSCs important?
How could we make new bone tissue for therapeutic uses?
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Cells in the lab are typically cultured on plates
2 D
The Problem
Cells in vivo (in an organism)
3 DIt is possible that cells grown in a 3D scaffold would:
1. Be more like cells in vivo2. Would not reach confluence as quickly3. Could be implanted directly
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How will a 2D and 3D environment effect the osteogenic differentiation of hMSCs?
Compare cells grown in a 2D and a 3D environment:
• Viability?• Proteins?• Genes expressed?• Mineralization?
Research Project
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Superporous Hydrogels
• Poly (ethylene glycol) diacrylate or PEGDA
• Polymer network, hydrophilic
• Pores from 100 µm to 600 µm created by gel-foaming
Creating a 3d Scaffold
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1. Seed hMSCs onto 2D plates and 3D hydrogels
Project Overview
2. Add osteogenic differentiation medium
3. Compare cells at day 2, 7, 14, 21 and 28
After 24 hours
After 24 hours
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1. MTS – Cell Viability
2. BCA – Protein Levels
3. Calcium
4. Alkaline Phosphatase – Early Marker
5. ELISA: Osteopontin – Early and Late MarkerOsteocalcin – Late Marker
Comparing the cells
6. qPCR – Determine Gene Expression- ALPL, RUNX2, OC, OP, BMP2
7. Von Kossa Staining - Mineralization
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1. Seed hMSCs onto 2D plates and 3D hydrogels
Accomplished Tasks
2. Add osteogenic differentiation medium
3. Compare cells at day 2, 7, 14, 21 and 28
After 24 hours
After 24 hours
Prep: Make gels, order supplies, review protocols