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Influence of 2D and 3D Environments on Osteogeneic Differentiation in hMSCs
Jacqueline Mimnaugh, RET
Neuqua Valley High School
Dr. Richard Gemeinhart
Melanie Kollmer
UIC Department of Biopharmaceutical Sciences
Tracy Chuong, REU
University of California Berkley
Stem cells differentiate into other types of cells
Two major categories:
What are hMSCs?
Human Mesenchymal Stem Cells (hMSCs)
Isolated from marrow
Can differentiate into bone, cartilage, fat
Embryonic(Pluripotent)
Adult(Multipotent)
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?
Cells in the lab are typically cultured on plates
2 D
The Problem
Cells in vivo (in an organism)
3 D
It is possible that cells grown in a 3D scaffold would:1. Be more like cells in vivo
2. Would not reach confluence as quickly
3. Could be implanted directly
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
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
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
1. MTS – Cell Viability
2. BCA – Protein Levels
3. Calcium
4. Alkaline Phosphatase – Early Marker
5. ELISA: Osteopontin – Early and Late Marker
Osteocalcin – Late Marker
Comparing the cells
6. qPCR – Determine Gene Expression
- ALPL, RUNX2, OC, OP, BMP2
7. Von Kossa Staining - Mineralization
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
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