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Human Mesenchymal Stem Cell Functional Identification Kit

Catalog # SC006 | R&D Systems, Inc. a Bio-Techne Brand

Key Product Details

Verification of Multipotency using the Human Mesenchymal Stem Cell Functional Identification Kit. 
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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
 

 

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.

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

After 14-21 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 Medium 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 Osteocalcin.

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.

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

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

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

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

Cryosection the chondrogenic pellet.

 

ICC detection of Aggrecan.

ICC detection of Aggrecan.

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  • Human Mesenchymal Stem Cell Functional Identification Kit
    Name: Anonymous
    Verified Customer | Posted 10/17/2016
    The MSC’s were generated from induced pluripotent stem cells and then tested using your kit for its potential to differentiate along the adipogenic and chondrogenic lineages.
    Human Mesenchymal Stem Cell Functional Identification Kit SC006

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FAQs for Human Mesenchymal Stem Cell Functional Identification Kit

  • 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.

Product Documents for Human Mesenchymal Stem Cell Functional Identification Kit

Certificate of Analysis

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