Mouse Mesenchymal Stem Cell Functional Identification Kit

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Kit Summary

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.
 

 

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 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.

 

Data Examples
Verification of Multipotency using the Mouse Mesenchymal Stem Cell Functional Identification Kit
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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).

 

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.

Product Datasheets

Certificate of Analysis Lookup

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Preparation and Storage
  • Stability & 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.
Background: Mesenchymal Stem Cells
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.

  • Alternate Names:
    AEG1; LYRIC; LYRIC/3D3; Mesenchymal Stem Cells; metadherin
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 Provided

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.

 

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
  • 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
 

 

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.

Culture cells to 100% confluency.

Replace the medium with Adipogenic Differentiation Medium to induce adipogenesis.

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.

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.

Plate 4.2 x 103 MSCs/cm 2 in StemXVivo Osteogenic/Adipogenic Base Media.

Replace the medium with Osteogenic Differentiation Media to induce osteogenesis.

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 Osteopontin.

ICC detection of Osteocalcin.

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.

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

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

After 17-21 days, the chondrogenic pellet can be fixed.

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

Cryosection the chondrogenic pellet.

 

ICC detection Collagen II.

ICC detection of Aggrecan.
Citations:

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

20 Citations: Showing 1 - 10
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Applications
Sample Type
  1. Synergistic antibacterial effect of co-administering adipose-derived mesenchymal stromal cells and Ophiophagus hannah L-amino acid oxidase in a mouse model of methicillin-resistant Staphylococcus aureus-infected wounds
    Authors: YY Mot, I Othman, SH Sharifah
    Stem Cell Res Ther, 2017;8(1):5.  2017
  2. Osteogenic Factor Runx2 Marks a Subset of Leptin Receptor-Positive Cells that Sit Atop the Bone Marrow Stromal Cell Hierarchy
    Authors: M Yang, A Arai, N Udagawa, T Hiraga, Z Lijuan, S Ito, T Komori, T Moriishi, K Matsuo, K Shimoda, AH Zahalka, Y Kobayashi, N Takahashi, T Mizoguchi
    Sci Rep, 2017;7(1):4928.  2017
  3. Aldehyde dehydrogenase activity identifies a subpopulation of canine adipose-derived stem cells with higher differentiation potential
    Authors: H Itoh, S Nishikawa, T Haraguchi, Y Arikawa, S Etoh, T Iseri, K Tani, M Nakaichi, Y Taura, K Itamoto
    J. Vet. Med. Sci., 2017;0(0):.  2017
  4. Lnk is an important modulator of insulin-like growth factor-1/Akt/peroxisome proliferator-activated receptor-gamma axis during adipogenesis of mesenchymal stem cells
    Korean J Physiol Pharmacol, 2016;20(5):459-66.  2016
  5. Age-dependent modulation of vascular niches for haematopoietic stem cells
    Authors: AP Kusumbe, SK Ramasamy, T Itkin, MA Mäe, UH Langen, C Betsholtz, T Lapidot, RH Adams
    Nature, 2016;0(0):.  2016
  6. MFG-E8 drives melanoma growth by stimulating mesenchymal stromal cell-induced angiogenesis and M2 polarization of tumor-associated macrophages
    Cancer Res, 2016;0(0):.  2016
  7. Age-dependent modulation of vascular niches for haematopoietic stem cells
    Authors: AP Kusumbe, SK Ramasamy, T Itkin, MA Mäe, UH Langen, C Betsholtz, T Lapidot, RH Adams
    Nature, 2016;0(0):.  2016
  8. Fanconi Anemia Mesenchymal Stromal Cells-Derived Glycerophospholipids Skew Hematopoietic Stem Cell Differentiation Through Toll-Like Receptor Signaling.
    Authors: Amarachintha S, Sertorio M, Wilson A, Li X, Pang Q
    Stem Cells, 2015;33(11):3382-96.  2015
  9. Engineered mesenchymal stem cells with enhanced tropism and paracrine secretion of cytokines and growth factors to treat traumatic brain injury.
    Authors: Wang Z, Wang Y, Wang Z, Gutkind J, Wang Z, Wang F, Lu J, Niu G, Teng G, Chen X
    Stem Cells, 2015;33(2):456-67.  2015
  10. Skin-derived mesenchymal stem cells help restore function to ovaries in a premature ovarian failure mouse model.
    Authors: Lai D, Wang F, Dong Z, Zhang Q
    PLoS ONE, 2014;9(5):e98749.  2014
  11. Mesenchymal stromal (stem) cells suppress pro-inflammatory cytokine production but fail to improve survival in experimental staphylococcal toxic shock syndrome.
    Authors: Kim H, Darwish I, Monroy M, Prockop D, Liles W, Kain K
    BMC Immunol, 2014;15(0):1.  2014
  12. MiR-1 and miR-200 inhibit EMT via Slug-dependent and tumorigenesis via Slug-independent mechanisms.
    Authors: Liu Y, Yin J, Abou-Kheir W, Hynes P, Casey O, Fang L, Yi M, Stephens R, Seng V, Sheppard-Tillman H, Martin P, Kelly K
    Oncogene, 2013;32(3):296-306.  2013
  13. miR-126 and miR-126* repress recruitment of mesenchymal stem cells and inflammatory monocytes to inhibit breast cancer metastasis.
    Authors: Zhang Y, Yang P, Sun T, Li D, Xu X, Rui Y, Li C, Chong M, Ibrahim T, Mercatali L, Amadori D, Lu X, Xie D, Li Q, Wang X
    Nat Cell Biol, 2013;15(3):284-94.  2013
  14. Adipose stromal cells contain phenotypically distinct adipogenic progenitors derived from neural crest.
    Authors: Sowa, Yoshihir, Imura, Tetsuya, Numajiri, Toshiaki, Takeda, Kosuke, Mabuchi, Yo, Matsuzaki, Yumi, Nishino, Kenichi
    PLoS ONE, 2013;8(12):e84206.  2013
  15. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.
    Authors: Rosu-Myles M, She YM, Fair J
    PLoS ONE, 2012;7(6):e38954.  2012
  16. Contribution of stem cells to neointimal formation of decellularized vessel grafts in a novel mouse model.
    Authors: Tsai TN, Kirton JP, Campagnolo P, Zhang L, Xiao Q, Zhang Z, Wang W, Hu Y, Xu Q
    Am. J. Pathol., 2012;181(1):362-73.  2012
  17. Adipose-derived stem cells produce factors enhancing peripheral nerve regeneration: influence of age and anatomic site of origin.
    Stem Cells Dev., 2012;21(11):1852-62.  2012
  18. Quantification of protein isoforms in mesenchymal stem cells by reductive dimethylation of lysines in intact proteins.
    Authors: She Y, Rosu-Myles M, Walrond L, Cyr T
    Proteomics, 2012;12(3):369-79.  2012
  19. Age-related changes in rat bone-marrow mesenchymal stem cell plasticity.
    Authors: Asumda FZ, Chase PB
    BMC Cell Biol., 2011;12(0):44.  2011
  20. Toll-like receptor 2 mediates mesenchymal stem cell-associated myocardial recovery and VEGF production following acute ischemia-reperfusion injury.
    Authors: Abarbanell AM, Wang Y, Herrmann JL
    Am. J. Physiol. Heart Circ. Physiol., 2010;298(5):H1529-36.  2010
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