Mesenchymal Stem Cell Osteogen

Mesenchymal Stem Cell Osteogenesis Kit

Chemicon (Millipore)

Bone undergoes a continual remodeling process that requires the coordinated activity of two types of cells. Osteoclasts break down the bone matrix while osteoblasts deposits collagen, calcium, and phosphorous and other minerals to form new bone. The balance between the activity of osteoclasts and osteoblasts determine the mass and density of the bone. Many diseases of bone including osteoporosis, a common age-related phenomenon in post-menopausal women in which the bone mass has been greatly reduced, and osteogenesis imperfecta, also known as brittle-bone disease, are likely caused by the misregulation of osteoblasts and osteoclasts. Understanding the molecular mechanisms that underlie osteogenesis, the process by which new bone is formed is thus of critical importance.



Stem cell technology, particularly embryonic stem cells and/or mesenchymal stem cells, offer attractive sources of osteoblasts for tissue culture studies and for the biochemical dissection of the earliest steps involved in osteoblast cell determination. Mesenchymal stem cells are multipotent progenitor cells that have the capacity to differentiate into several mesenchymal cell lineages, including bone, cartilage and fat.



CHEMICON®'s Mesenchymal Stem Cell Osteogenesis Kit contains all the reagents necessary to readily differentiate mesenchymal stem cells to an osteogenic lineage as assessed by Alizarin Red staining. Reagents in the kit include two ECM coating molecules (collagen Type I and vitronectin) that have been shown to promote osteogenic differentiation of mesenchymal stem cells (Salasznyk, 2004) along with inducing reagents, dexamethasone, ascorbic acid 2-phosphate and b-glycerophosphate. Also included is Alizarin Red Solution, a staining solution that is used to detect the presence of calcium in bone.



Using CHEMICON®'s Mesenchymal Stem Cell Osteogenesis Kit, we typically obtain >50% mature osteocytes from rat bone marrow derived mesenchymal stem cells. Efficiency of osteogenic differentiation may vary, depending upon the quality of the mesenchymal stem cells and if variations to the protocol are introduced.

For Research Use Only; not for use in diagnostic procedure.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Stem Cell Kits

Mesenchymal Stem Cells

Differentiation

• Dexamethasone Solution: (Catalog No. 90357) One vial containing 100 μL of 10 mM dexamethasone in ethanol. Store at -20°C.
• Ascorbic Acid 2-Phosphate Solution: (Catalog No. 2004011) One vial containing 500 μL of 100 μM ascorbic acid 2-phosphate in water. Store at -20°C.
• Glycerol 2- Phosphate Solution: (Catalog No. 2004010) Three vials containing 1 mL of 1 M glycerol 2-phosphate in water. Store at -20°C.
• Collagen, Type I: (Catalog No. 2004013) One vial containing 150 μg collagen Type I. Store at -20°C.
• Vitronectin: (Catalog No. 2004012) One vial containing 150 μg vitronectin. Store at -20°C.
• Alizarin Red Solution: (Catalog No. 2003999) One bottle containing 50 mL Alizarin Red Solution. Store at 2° to 8°C.

Note: Kit components require two different storage temperatures.

Dexamethasone Solution, Ascorbic Acid 2-Phosphate Solution, Glycerol 2- Phosphate Solution, Collagen Type I, and Vitronectin should be stored at -20ºC. Alizarin Red Solution should be stored at 2° to 8°C.

Precautions

· Dexamethasone is an irritant and potentially toxic. DMSO is readily absorbed through the skin. Wear a lab coat and gloves when handling these solutions.

Please refer to the Material Safety Data Sheet at www.chemicon.com for further precautions.

1. Human or rat mesenchymal stem cell and cells culture reagents

2. Mesenchymal Stem Cell Expansion Media (DMEM-low glucose, without glutamine), 10% heat-inactivated fetal bovine serum (Catalog No. ES-009-D), 2 mM L-Glutamine (Catalog No. TMS-002-C) and 1X solution of penicillin and streptomycin (Catalog No. TMS-AB2-C))

3. 24-well tissue culture plates™

4. Phosphate-Buffered Saline (1X PBS) (Catalog No. BSS-1005-B)

5. Accutase^TM (Catalog No. SCR005)

6. Fixative (e.g. 4% Paraformaldehyde in 1X PBS)

7. Hemacytometer

8. Microscope