Human Mesenchymal Stem Cell Functional Identification Kit

To verify multipotency of human mesenchymal stem cells by in vitro functional differentiation.

Why Funtionally Verify Human MSC Multipotency In Vitro?

Mesenchymal stem/stromal cells (MSCs) can be characterized based on the expression of specific cell surface markers, the absence of hematopoietic markers, and adherence to plastic in vitro.

To more rigorously determine if a cell is truly an MSC, it is important to also verify its ability to differentiate into adipocytes, chondrocytes, and osteocytes.

Functional verification of MSC multipotency in vitro:

• Uses defined supplements to drive reproducible trilineage differentiation.
• Verifies a healthy, multipotent starting MSC population to increase consistency between studies and reduce unwanted experimental variability.
• Meets one of the three recommended minimal criteria for MSC identification.

Mesenchymal Stromal Cells or Mesenchymal Stem Cells?

The term ‘mesenchymal stromal cells’ is commonly used to describe a heterogeneous population of cultured cells that are adherent to plastic, have a distinct morphology, and express a specific set of marker proteins. Within this heterogeneous population are cells referred to as ‘mesenchymal stem cells.’

Mesenchymal stem cells are multipotent, self-renewing cells that have the ability to differentiate into adipocytes, chondrocytes, and osteoblasts when cultured in vitro. Read More about MSC Nomenclature

Kit Components

This kit contains the following reagents to drive MSC differentiation and a marker to analyze each of the three differentiated derivatives:

• Adipogenic Supplement
• Chondrogenic Supplement
• Osteogenic Supplement
• ITS Supplement
• Adipocyte marker: Goat Anti-Mouse FABP4 Antigen Affinity-purified Polyclonal Antibody
• Chondrocyte marker: Goat Anti-Human Aggrecan Antigen Affinity-purified Polyclonal Antibody
• Osteocyte marker: Mouse Anti-Human Osteocalcin Monoclonal Antibody

This kit requires media (not included), such as Human/Mouse/Rat StemXVivo Osteogenic/Adipogenic Base Media (CCM007) or equivalent.

The quantity of each media supplement in this kit is sufficient to make 50 mL of media for differentiation. This is enough media for the differentiation of 16 wells of a 24-well plate for osteogenic and adipogenic lineages and 10 chondrocyte pellets.

Precautions

• The Adipogenic Supplement contains 95% ethanol and is highly flammable. Keep the container tightly closed, and keep it away from sources of ignition.
• The acute and chronic effects of over-exposure to the reagents in this kit are unknown. Safe laboratory handling procedures should be followed and protective clothing should be worn when handling kit reagents.
• The ITS Supplement contains human transferrin. This transferrin was purified from donor plasma and tested at the donor level using an FDA licensed method and found to be non-reactive for anti-HIV-1/2 and Hepatitis B surface antigen. 

2006 Proposed Change to MSC Nomenclature

Although mesenchymal stromal cells were once referred to as ‘mesenchymal stem cells’, a change to ‘mesenchymal stromal cells’ was proposed by the International Society for Cellular Therapy in 2006.¹

The change in nomenclature originates from two important factors:

• Methods used to isolate mesenchymal stem cells yield a heterogeneous population of cells with only a fraction of these cells demonstrating multipotency.
• The absence of direct evidence that mesenchymal stem cells can self-renew and differentiate in vivo.

Use of Mesenchymal Stem and Stromal Cell Terminology

Data supporting MSC self-renewal and multipotency have been obtained using in vitro conditions, which does not adequately reflect the in vivo environment. The lack of in vivo data has led some researchers to question the validity of the term ‘mesenchymal stem cell’ providing further support for the use of ‘mesenchymal stromal cells’ to describe MSCs.² While ‘mesenchymal stromal cells’ may be the more scientifically accurate term for MSCs, the two terms are often used interchangeably in the literature. R&D Systems recognizes the use of both mesenchymal stem cells and mesenchymal stromal cells and uses ‘MSC’ to indicate mesenchymal stem/stromal cells to account for both designations.

Definitions of Mesenchymal Stromal Cells and Mesenchymal Stem Cells

• Mesenchymal Stromal Cells – A heterogeneous population of cultured cells with similar characteristics such as the ability to adhere to plastic and the expression of specific marker proteins.
• Mesenchymal Stem Cells – A subpopulation of mesenchymal stromal cells that have the capacity to self-renew and differentiate into mesodermal lineages when cultured in vitro. The capacity to self-renew and differentiate in vivo has yet to be clearly demonstrated for mesenchymal stem cells.

References

• Dominici, M. et al. (2006) Cytotherapy 8:315.
• Keating, A. (2012) Cell Stem Cell 10:709.

Scientific Data Examples for Human Mesenchymal Stem Cell Functional Identification Kit

Verification of Multipotency using the Human Mesenchymal Stem Cell Functional Identification Kit.  Human mesenchymal stem cells were cultured in StemXVivo®Mesenchymal Stem Cell Expansion Media (Catalog # CCM004) and differentiation was induced as indicated using the media supplements included in the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006). The kit also contains a Goat Anti-Mouse FABP-4 Antigen Affinity-purified Polyclonal Antibody (adipocytes), a Goat Anti-Human Aggrecan Antigen Affinity-purified Polyclonal Antibody (chondrocytes), and a Mouse Anti-Human Osteocalcin Monoclonal Antibody (osteocytes) for the confirmation of differentiation status. The cells were stained using the NorthernLightsTM 557-conjugated Donkey Anti-Goat (Catalog # NL001; red) or Anti-Mouse (Catalog # NL007; red) IgG Secondary Antibodies, and the nuclei were counterstained with DAPI (blue).

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, human MSC multipotency is verified using the following in vitro differentiation procedure:

• Culture multipotent cells of interest
• Induce adipocyte, chondrocyte, and osteocyte differentiation using media supplements
• Evaluate differentiation using mature phenotype marker antibodies and fluorescent ICC

View Graphic Procedure

 

 

Reagents Provided

Reagents supplied in the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006):

• Adipogenic Supplement
• Chondrogenic Supplement
• Osteogenic Supplement
• ITS Supplement
• Adipocyte marker: Goat Anti-Mouse FABP4 Antigen-affinity Purified Polyclonal Antibody
• Chondrocyte marker: Goat Anti-Human Aggrecan Antigen-affinity Purified Polyclonal Antibody
• Osteocyte marker: Mouse Anti-Human Osteocalcin Antigen-affinity Purified Monoclonal Antibody

Note: The quantity of each media supplement in this kit is sufficient to make 50 mL of media for differentiation. 50 mL can be used for 16 wells of a 24-well plate for osteogenic and adipogenic lineages and 10 chondrocyte pellets.

 

Other Supplies Required

Reagents

• StemXVivo^® Osteogenic/Adipogenic Base Media (Catalog # CCM007 or equivalent)
• D-MEM/F-12 (1X)
• Phosphate Buffered Saline (PBS)
• Penicillin-Streptomycin-Glutamate (100X)
• 4% Paraformaldehyde in PBS
• 1% BSA in PBS
• Triton^® X-100
• 10% normal donkey serum
• Fibronectin (optional; Human Fibronectin, Catalog # 1918-FN, Bovine Fibronectin, Catalog # 1030-FN, or equivalent)
• Mounting medium (Catalog # CTS011 or equivalent)
• NorthernLightsTM 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001 or equivalent)
• Deionized or distilled water

Materials

• Human MSCs
• 24-well culture plates
• 12 mm coverslips (Carolina Biologicals, Catalog # 633009 or equivalent)
• 15 mL centrifuge tubes
• Pipettes and pipette tips
• Serological pipettes
• Glass slides
• Fine pointed curved forceps
• Liquid barrier pen

Equipment

• 37 ^°C and 5% CO₂ incubator
• Centrifuge
• Hemocytometer
• Inverted microscope
• 2 ^°C to 8 ^°C refrigerator
• 37 ^°C water bath
• Fluorescence microscope
• Cryostat

 

Procedure Overview

This protocol has been tested using bone marrow- and/or adipose tissue-derived MSCs. If using a different tissue source or cell line, the protocol below may need to be optimized.

Adipogenic Differentiation

Plate 2.1 x 10⁴ MSCs/cm² in StemXVivo^® Osteogenic/Adipogenic Base Media.

Culture cells to 100% confluency.

Replace the medium with Adipogenic Differentiation Medium to induce adipogenesis.

Every 2-3 days, replace with fresh Adipogenic Differentiation Medium.

After 14-21 days, adipocytes can be fixed.

 

ICC detection of FABP4.

Osteogenic Differentiation

 

Plate 4.2 x 10³ MSCs/cm² in StemXVivo^® Osteogenic/Adipogenic Base Media.

Culture cells to 50-70% confluency.

Replace the medium with Osteogenic Differentiation Medium to induce osteogenesis.

Every 3-4 days, replace with fresh Osteogenic Differentiation Medium.

After 14-21 days, osteocytes can be fixed.

 

ICC detection of Osteocalcin.

Chondrogenic Differentiation

 

Transfer 2.5 x 10⁴ MSCs to a 15 mL conical tube.

Centrifuge and resuspend the cells in Chondrogenic Differentiation Media.

Centrifuge the cells but do not remove the medium.

Every 2-3 days, replace with fresh Chondrogenic Differentiation Media.

After 14-21 days, the chondrogenic pellet can be fixed.

Cryosection the chondrogenic pellet.

 

ICC detection of Aggrecan.