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