Name: Rat Mesenchymal Stem Cell Functional Identification Kit
Brand: R&D Systems
SKU: SC020

Kit Summary

To verify multipotency of rat 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 Rat 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 rat 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 (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.

Product Specifications for Rat Mesenchymal Stem Cell Functional Identification Kit

Species	Rat
Source	N/A

Scientific Data Examples for Rat Mesenchymal Stem Cell Functional Identification Kit

	Verification of Multipotency using the Rat Mesenchymal Stem Cell Functional Identification Kit. Rat 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 Rat Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC020). 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 NorthernLights^™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).

Preparation & Storage

Shipping Conditions	The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage	Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, rat 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 Rat Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC020):

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

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
Mounting medium (Catalog # CTS011 or equivalent)
NorthernLights^™ 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001 and NL007 or equivalent)
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)
Deionized or distilled water

Materials

Rat 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.