Mouse Mesenchymal Stem Cell Functional Identification Kit
R&D Systems, part of Bio-Techne | Catalog # SC010
Key Product Details
Summary for Mouse Mesenchymal Stem Cell Functional Identification Kit
To verify multipotency of mouse mesenchymal stem cells by in vitro functional differentiation.
Key Benefits
- Confirms that the starting MSC population is multipotent
- Reduces experimental variation
- Reliably induces MSC trilineage differentiation using defined supplements
- Includes premium quality antibodies to confirm differentiation
Why Functionally Verify Mouse 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.
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
This kit contains the following reagents to drive mouse 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: Sheep Anti-Mouse Collagen II Antigen-affinity Purified Polyclonal Antibody
- Osteocyte marker: Goat Anti-Mouse Osteopontin Antigen-affinity Purified Polyclonal Antibody
This kit requires media (not included), such as Human/Mouse/Rat StemXVivo® Osteogenic/Adipogenic Base Media (Catalog #CCM007) or equivalent.
This is enough media for the differentiation of 16 wells of a 24-well plate for osteogenic and adipogenic lineages and 10 chondrocyte pellets.
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.
The term 'mesenchymal stem cells' (MSCs) is most commonly used to describe multipotent self-renewing cells that can be differentiated in vitro to generate adipocytes, chondrocytes, and osteoblasts. However, because these biological properties and hierarchical relationships remain to be clearly demonstrated in vivo, the term 'multipotent mesenchymal stromal cells' is often used to distinguish cultured cells from their in vivo precursors. Originally discovered in mouse bone marrow, multipotent mesenchymal stromal cells cultured from a variety of species and tissue types, have been shown to differentiate into progeny of additional lineages including, cardiomyocytes, endothelial cells, hepatocytes, and neural cells. Again, the physiological relevance of these findings remains to be determined.
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Verification of Multipotency using the Mouse Mesenchymal Stem Cell Functional Identification Kit Mouse 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 Mouse Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC010). The kit also contains a Goat Anti-Mouse FABP-4 Antigen Affinity-purified Polyclonal Antibody (adipocytes), a Sheep Anti-Mouse Collagen II Antigen Affinity-purified Polyclonal Antibody (chondrocytes), and a Goat Anti-Mouse Osteopontin Antigen Affinity-purified Polyclonal Antibody (osteocytes) for the confirmation of differentiation status. The cells were stained using the NorthernLights™557-conjugated Donkey Anti-Goat (Catalog # NL001; red) or Anti-Sheep (Catalog # NL010; red) IgG Secondary Antibodies, and the nuclei were counterstained with DAPI (blue). |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, mouse 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
Reagents supplied in the Mouse Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC010):
- Adipogenic Supplement
- Chondrogenic Supplement
- Osteogenic Supplement
- ITS Supplement
- Adipocyte marker: Goat Anti-Mouse FABP4 Antigen-affinity Purified Polyclonal Antibody
- Chondrocyte marker: Sheep Anti-Mouse Collagen II Antigen-affinity Purified Polyclonal Antibody
- Osteocyte marker: Goat Anti-Mouse Osteopontin Antigen-affinity Purified Polyclonal 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.
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
- Zinc Formalin
- 1% BSA in PBS
- Mounting medium (Catalog # CTS011 or equivalent)
- NorthernLights™ 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001 and NL010 or equivalent)
- 0.05% Tween® 20 in PBS
- 0.3% Triton® X-100, 1% BSA, 10% normal donkey serum in PBS
- 1% BSA, 10% normal donkey serum in PBS
- Fibronectin (optional; Human Fibronectin, Catalog # 1918-FN, Bovine Fibronectin, Catalog # 1030-FN, or equivalent)
- Universal Antigen Retrieval Reagent (Catalog # CTS015)
- Deionized or distilled water
Materials
- Mouse 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
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 3-4 days, replace with fresh Adipogenic Differentiation Medium.
After 10-14 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 Media to induce osteogenesis.
Every 3-4 days, replace with fresh Osteogenic Differentiation Medium.
After 14-21 days, osteocytes can be fixed.
ICC detection of Osteopontin.
Chondrogenic Differentiation
Transfer 2.5 x 105 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 17-21 days, the chondrogenic pellet can be fixed.
Cryosection the chondrogenic pellet.
ICC detection Collagen II.
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