Human Mesenchymal Stem Cell Functional Identification Kit Summary
To verify multipotency of human mesenchymal stem cells by in vitro functional differentiation.
- Confirms that the starting MSC population is multipotent
- Can be used with the Human MSC Verification Flow Kit to define MSCs according to the ISCT recommendations
- Reliably induces MSC trilineage differentiation using defined supplements
- Includes premium quality antibodies to confirm 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.
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 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.
- 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 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. As no testing can offer complete assurance of freedom from infectious agents, this reagent should be handled as if capable of transmitting infection.
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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).
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.
- Dominici, M. et al. (2006) Cytotherapy 8:315.
- Keating, A. (2012) Cell Stem Cell 10:709.
Preparation and Storage
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.
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
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.
- 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
- 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
- 37 °C and 5% CO2 incubator
- Inverted microscope
- 2 °C to 8 °C refrigerator
- 37 °C water bath
- Fluorescence microscope
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.
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.
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.
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.
Citations for Human Mesenchymal Stem Cell Functional Identification Kit
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.
Citations: Showing 1 - 10
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Multilineage-differentiating stress-enduring (Muse)-like cells exist in synovial tissue
Authors: E Toyoda, M Sato, T Takahashi, M Maehara, Y Nakamura, G Mitani, T Takagaki, K Hamahashi, M Watanabe
Regen Ther, 2019;10(0):17-26. 2019
Comparative characterization of SHED and DPSCs during extended cultivation inï¿½vitro
Authors: H Wang, Q Zhong, T Yang, Y Qi, M Fu, X Yang, L Qiao, Q Ling, S Liu, Y Zhao
Mol Med Rep, 2018;0(0):. 2018
Characterization of the interaction between human decidua parietalis mesenchymal stem/stromal cells and natural killer cells
Authors: MH Abumaree, E Bahattab, A Alsadoun, A Al Dosaima, FM Abomaray, T Khatlani, B Kalionis, MF El-Muzaini, AO Alawad, AS AlAskar
Stem Cell Res Ther, 2018;9(1):102. 2018
Evaluation of the effects of ascorbic acid on metabolism of human mesenchymal stem cells
Authors: K Fujisawa, K Hara, T Takami, S Okada, T Matsumoto, N Yamamoto, I Sakaida
Stem Cell Res Ther, 2018;9(1):93. 2018
Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter
Authors: L Mezzanotte, JD Iljas, I Que, A Chan, E Kaijzel, R Hoeben, C Löwik
Cell Transplant, 2017;26(12):1878-1889. 2017
Are Adipose-Derived Stem Cells From Liver Falciform Ligaments Another Possible Source of Mesenchymal Stem Cells?
Authors: SW Lee, JU Chong, SO Min, SY Bak, KS Kim
Cell Transplant, 2017;26(5):855-866. 2017
Identification of multipotent stem cells in human brain tissue following stroke
Authors: K Tatebayash, Y Tanaka, A Nakano-Doi, R Sakuma, S Kamachi, M Shirakawa, K Uchida, H Kageyama, T Takagi, S Yoshimura, T Matsuyama, T Nakagomi
Stem Cells Dev, 2017;0(0):. 2017
Rapid Rapamycin-Only Induced Osteogenic Differentiation of Blood-Derived Stem Cells and Their Adhesion to Natural and Artificial Scaffolds
Authors: C Arianna, C Eliana, A Flavio, R Marco, D Giacomo, S Manuel, B Elena, G Alessandra
Stem Cells Int, 2017;2017(0):2976541. 2017
Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Transition in Colon Cancer Cells through Direct Cell-to-Cell Contact
Authors: H Takigawa, Y Kitadai, K Shinagawa, R Yuge, Y Higashi, S Tanaka, W Yasui, K Chayama
Neoplasia, 2017;19(5):429-438. 2017
Expression pattern of neurotrophins and their receptors during neuronal differentiation of adipose-derived stem cells in simulated microgravity condition
Authors: V Zarrinpour, Z Hajebrahim, M Jafarinia
Iran J Basic Med Sci, 2017;20(2):178-186. 2017
Transcriptome sequencing wide functional analysis of human mesenchymal stem cells in response to TLR4 ligand
Sci Rep, 2016;6(0):30311. 2016
Exendin-4 enhances the differentiation of Wharton's jelly mesenchymal stem cells into insulin-producing cells through activation of various ?-cell markers
Stem Cell Res Ther, 2016;7(1):108. 2016
Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells
Stem Cells Int, 2016;2016(0):5457132. 2016
Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice
PLoS ONE, 2016;11(7):e0158745. 2016
Equine metabolic syndrome impairs adipose stem cells osteogenic differentiation by predominance of autophagy over selective mitophagy
J Cell Mol Med, 2016;0(0):. 2016
In vitro characterization of human dental pulp stem cells isolated by three different methods
Authors: Euiseong Kim
Restor Dent Endod, 2016;41(4):283-295. 2016
Human endometrial mesenchymal stem cells exhibit intrinsic anti-tumor properties on human epithelial ovarian cancer cells
Sci Rep, 2016;6(0):37019. 2016
TLR3 preconditioning enhances the therapeutic efficacy of umbilical cord mesenchymal stem cells in TNBS-induced colitis via the TLR3-Jagged-1-Notch-1 pathway
Mucosal Immunol, 2016;0(0):. 2016
Generation of CCR5-defective CD34 cells from ZFN-driven stop codon-integrated mesenchymal stem cell clones.
Authors: Manotham, Krissana, Chattong, Supreech, Setpakdee, Anant
J Biomed Sci, 2015;22(0):25. 2015
Gene expression profile analysis of human mesenchymal stem cells from herniated and degenerated intervertebral discs reveals different expression of osteopontin.
Authors: Marfia G, Navone S, Di Vito C, Tabano S, Giammattei L, Di Cristofori A, Gualtierotti R, Tremolada C, Zavanone M, Caroli M, Torchia F, Miozzo M, Rampini P, Riboni L, Campanella R
Stem Cells Dev, 2015;24(3):320-8. 2015
Prospectively Isolated Human Bone Marrow Cell-Derived MSCs Support Primitive Human CD34-Negative Hematopoietic Stem Cells.
Authors: Matsuoka Y, Nakatsuka R, Sumide K, Kawamura H, Takahashi M, Fujioka T, Uemura Y, Asano H, Sasaki Y, Inoue M, Ogawa H, Takahashi T, Hino M, Sonoda Y
Stem Cells, 2015;33(5):1554-65. 2015
Umbilical cord mesenchymal stromal cells affected by gestational diabetes mellitus display premature aging and mitochondrial dysfunction.
Authors: Kim J, Piao Y, Pak Y, Chung D, Han Y, Hong J, Jun E, Shim J, Choi J, Kim C
Stem Cells Dev, 2015;24(5):575-86. 2015
Amide-type local anesthetics and human mesenchymal stem cells: clinical implications for stem cell therapy.
Authors: Dregalla, Ryan C, Lyons, Nicolett, Reischling, Patrick, Centeno, Christop
Stem Cells Transl Med, 2014;3(3):365-74. 2014
Coculture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes.
Authors: Lehmann T, Filipiak K, Juzwa W, Sujka-Kordowska P, Jagodzinski P, Zabel M, Glowacki J, Misterska E, Walczak M, Glowacki M
Mol Med Rep, 2014;9(2):574-82. 2014
Magnetic Nanoparticle Based Nonviral MicroRNA Delivery into Freshly Isolated CD105(+) hMSCs.
Authors: Schade A, Muller P, Delyagina E, Voronina N, Skorska A, Lux C, Steinhoff G, David R
Stem Cells Int, 2014;2014(0):197154. 2014
Human adipose-derived mesenchymal stem cells as a new model of spinal and bulbar muscular atrophy.
Authors: Dossena M, Bedini G, Rusmini P, Giorgetti E, Canazza A, Tosetti V, Salsano E, Sagnelli A, Mariotti C, Gellera C, Navone S, Marfia G, Alessandri G, Corsi F, Parati E, Pareyson D, Poletti A
PLoS ONE, 2014;9(11):e112746. 2014
Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.
Authors: Raynaud C, Halabi N, Elliott D, Pasquier J, Elefanty A, Stanley E, Rafii A
PLoS ONE, 2013;8(1):e54524. 2013
Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling.
Authors: Reinhardt P, Glatza M, Hemmer K, Tsytsyura Y, Thiel C, Hoing S, Moritz S, Parga J, Wagner L, Bruder J, Wu G, Schmid B, Ropke A, Klingauf J, Schwamborn J, Gasser T, Scholer H, Sterneckert J
PLoS ONE, 2013;8(3):e59252. 2013
WNT3A promotes hematopoietic or mesenchymal differentiation from hESCs depending on the time of exposure.
Authors: Gertow K, Hirst C, Yu Q, Ng E, Pereira L, Davis R, Stanley E, Elefanty A
Stem Cell Reports, 2013;1(1):53-65. 2013
Mesenchymal stem cells and endothelial progenitor cells decrease renal injury in experimental swine renal artery stenosis through different mechanisms.
Authors: Zhu X, Urbieta-Caceres V, Krier J, Textor S, Lerman A, Lerman L
Stem Cells, 2013;31(1):117-25. 2013
Efficient differentiation of human pluripotent stem cells into mesenchymal stem cells by modulating intracellular signaling pathways in a feeder/serum-free system.
Authors: Tran NT, Trinh QM, Lee GM
Stem Cells Dev., 2012;21(7):1165-75. 2012
Identification of spectral modifications occurring during reprogramming of somatic cells.
Authors: Sandt C, Feraud O, Oudrhiri N, Bonnet ML, Meunier MC, Valogne Y, Bertrand A, Raphael M, Griscelli F, Turhan AG, Dumas P, Bennaceur-Griscelli A
PLoS ONE, 2012;7(4):e30743. 2012
Malignant germ cell-like tumors, expressing Ki-1 antigen (CD30), are revealed during in vivo differentiation of partially reprogrammed human-induced pluripotent stem cells.
Authors: Griscelli F, Feraud O, Oudrhiri N, Gobbo E, Casal I, Chomel JC, Bieche I, Duvillard P, Opolon P, Turhan AG, Bennaceur-Griscelli A
Am. J. Pathol., 2012;180(5):2084-96. 2012
Isolation and characterization of synovial mesenchymal stem cells.
Authors: Harvanova D, Tothova T, Sarissky M, Amrichova J, Rosocha J
Folia Biol. (Praha), 2011;57(3):119-24. 2011
Isolation of alveolar epithelial type II progenitor cells from adult human lungs.
Authors: Fujino N, Kubo H, Suzuki T
Lab. Invest., 2011;91(0):363-78. 2011
Differentiation potential of human postnatal mesenchymal stem cells, mesoangioblasts, and multipotent adult progenitor cells reflected in their transcriptome and partially influenced by the culture conditions.
Authors: Roobrouck VD, Clavel C, Jacobs SA, Ulloa-Montoya F, Crippa S, Sohni A, Roberts SJ, Luyten FP, Van Gool SW, Sampaolesi M, Delforge M, Luttun A, Verfaillie CM
Stem Cells, 2011;29(5):871-82. 2011
Collection and culture of alveolar bone marrow multipotent mesenchymal stromal cells from older individuals.
Authors: Han J, Okada H, Takai H, Nakayama Y, Maeda T, Ogata Y
J. Cell. Biochem., 2009;107(6):1198-204. 2009
Generation of a human Ocular Albinism type 1 iPSC line, SEIi001-A, with a mutation in GPR143.
Authors: Baulier E, Garcia Diaz A, Corneo B, Farber D
Stem Cell Res, 0;33(0):274-277. 0
Can the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006) be used with non-human primate mesenchymal stem cells?
It is likely that the antibodies included in the kit are cross-reactive to other primates. The supplements included in the kit are not intended to be species-specific. However, the kit has not been tested with primate mesenchymal stem cells
For the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006), how can induction of differentiation be monitored?
For adipogenic differentiation, the appearance of vacuoles in cells after 5-7 days is a sign of differentiation and can be monitored by microscopic examination of the cells. For osteogenic differentiation, the beginning of cell detachment after about 14 days is a sign of differentiation. Cell detachment should be monitored in this case. For chondrogenic differentiation, there isn't an exact marker to look for other than fixing and staining the frozen pellet between differentiation days 14 - 21. The exact choice of time may take some empirical testing.
In the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006), are Part #'s 90415, 390416, and 390417 the same as the StemXVivo® Human Adipogenic Supplement (Catalog # CCM011), StemXVivo® Human Osteogenic Supplement (Catalog # CCM008), and StemXVivo® Human Chondrogenic Supplement (Catalog # CCM006), respectively?
Yes, the StemXVivo® Human Adipogenic Supplement (Catalog # CCM011), StemXVivo® Human Osteogenic Supplement (Catalog # CCM008), and StemXVivo® Human Chondrogenic Supplement (Catalog # CCM006) are the same as Part #'s 390415, 390416, and 390417, respectively, in the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006).
Are there any experimental tips/hints for successful chondrogenic differentiation of mesenchymal stem cells?
The following tips/hints are useful for chondrogenic differentiation:
a) The mesenchymal stem cells (MSCs) should not be from a late passage (passage 8 or less), b) if using the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006) or the StemXVivo® Chondrogenic Supplement (Catalog # CCM006), use the starting MSC cell number that is indicated in the protocol, c) Early during chondrogenic differentiation a pellet should form. As differentiation progresses, the pellet will grow and take up a ball-like appearance. d) The pellet should not attach to the tube, therefore care should be taken to not dislodge it while changing media.
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