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

What are hMSCs? Stem cells differentiate into other types of cells Two major categories: Embryonic (Pluripotent) Adult (Multipotent) Human Mesenchymal Stem Cells (hMSCs) Isolated from marrow Can differentiate into bone, cartilage, fat Before I begin explaining my research project, I want to talk a little about stem cells. Stem cells are a special type of cell that can change into, or differentiate, into other types of cells. There are two major types of stem cells, embryonic and adult. Embryonic stem cells are pluripotent, which means they can develop into any type of cells in the body (for example: muscle cells, bone cells, blood cells, neurons). Adult stem cells are different from embryonic stem cells because they have already partially differentiated. They are more limited in they number of types of cells they may possible become, and so we describe them as multipotent. In my research, I will be working with hMSCs, or human mesenchymal stem cells. These are adult stem cells which can be found in marrow and in other sources in the body. hMSCs can differentiate into osteoblasts which are bone cells, adipocytes which are fat cells, or chrondrocytes which are cartilage cells.

Induce hMSCs to develop into bone cells Osteogenesis Why are hMSCs important? Induce hMSCs to develop into bone cells Osteogenesis Tissue Engineering Bone diseases/defects, trauma, cancer, mal-union/non-union fractures How could we make new bone tissue for therapeutic uses?

The Problem 2 D 3 D Cells in the lab are typically cultured on plates Cells in vivo (in an organism) 3 D It is possible that cells grown in a 3D scaffold would: Be more like cells in vivo Would not reach confluence as quickly Could be implanted directly

Research Project 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?

Creating a 3d Scaffold Superporous Hydrogels Poly (ethylene glycol) diacrylate or PEGDA Polymer network, hydrophilic Pores from 100 µm to 600 µm created by gel-foaming

Project Overview 1. Seed hMSCs onto 2D plates and 3D hydrogels After 24 hours 2. Add osteogenic differentiation medium After 24 hours 3. Compare cells at day 2, 7, 14, 21 and 28

Comparing the cells 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 6. qPCR – Determine Gene Expression - ALPL, RUNX2, OC, OP, BMP2 7. Von Kossa Staining - Mineralization

Accomplished Tasks Prep: Make gels, order supplies, review protocols 1. Seed hMSCs onto 2D plates and 3D hydrogels After 24 hours 2. Add osteogenic differentiation medium After 24 hours 3. Compare cells at day 2, 7, 14, 21 and 28