Differentiation and Characterization
of Human MSCs
Roxanne L. Reger, Alan H. Tucker, and Margaret R. Wolfe
Abstract One of the hallmark characteristics of human MSCs (hMSCs) is their
ability to differentiate into adipocytes, chondrocytes and osteocytes in culture. The
default fate for hMSCs appears to be bone: if late-passage cultures are left in basic
culture medium, the hMSCs will become confluent and produce mineral, an indica-
tion of bone formation. However, when grown under certain culture conditions or in
media containing specific components, the cells can be driven to become a number
of other specific cell types including neural cells, myocytes, and cardiomyocytes.
The protocols given here are the basic differentiation procedures for inducing
osteogenesis, adipogenesis, and chondrogenesis in cultures of hMSCs. Although
there is still no clear consensus on the antigen expression pattern that will define
hMSCs, a protocol is also presented for the flow cytometric analysis using a series
of antibody panels. The analysis of these surface epitope patterns can aide in the
isolation and characterization of hMSCs.
Keywords: MSCs; differentiation; surface epitopes; culture; stem cells; multipo-
The plastic adherent adult stem/progenitor cells from bone marrow originally
referred to as fibroblastoid colony forming units, then in the hematological litera-
ture as marrow stromal, subsequently as mesenchymal stem cells, and most recently
as multipotent mesenchymal stromal cells (MSCs) have generated a great deal of
interest in the last few years. Human MSCs (hMSCs) not only are easily obtained
from a simple bone marrow aspiration and easily isolated by plastic adherence, but
they also have the ability to differentiate into several different cell and tissue types,
including bone (1–3), fat (1,4,5) and cartilage (1,6). This makes hMSCs an attrac-
tive candidate for possible cellular therapies. However, not all MSCs preparations
are optimal and not all will differentiate down multiple pathways. To assess various
hMSC preparations for potential for multidifferentiation, the cells should be set up
From: Methods in Molecular Biology, vol. 449, Mesenchymal Stem Cells: Methods and Protocols
Edited by D.J. Prockop, D.G. Phinney, and B.A. Bunnell © Humana Press, Totowa, NJ
94 R.L. Reger et al.
in specific culture conditions to “drive” the MSCs to become specific cell types.
Although there are many differentiation procedures depending on the desired type
of cell, provided here are the full protocols for simple and basic differentiation into
osteocytes, adipocytes, and chondrocytes.
The characterization of hMSCs can be augmented with a battery of monoclonal
antibodies and flow cytometry analyses to determine the presence or absence of
particular cell surface proteins (Table 7.1). Unlike hematopoietic cells, however,
there is little consensus on the antigen expression pattern that can precisely define
hMSCs. The use of flow cytometric analysis of hMSCs for surface proteins lies in
determining the type of cells obtained and establishing continuity of results among
cell preparations and over time in culture. Owing to the large number of antibodies
needed to evaluate hMSCs, a very large number of cells are needed to complete the
procedure. This problem can be alleviated in part using a panel of antibodies in each
analysis thus reducing the number of cells needed from 1 million to 500,000/panel.
The number of antibodies that can be mixed is determined by the type of flow
cytometer, the number of channels that instrument has available and the cross-
reactivity of the antibodies with each other. The list of surface antigens that can be
examined on hMSCs is extensive. The protocol presented here includes the antibody
panels that we commonly use for basic characterization of hMSC preparations.
1. α minimum essential medium (αMEM) with l-glutamine, without ribonucleosides
or deoxyribonucleosides (Invitrogen/GIBCO; catalog # 12561-056).
2. Fetal bovine serum (FBS), premium select, hybridoma qualified, not heat inac-
tivated (Atlanta Biologicals; catalog # S11550) – selected from a screen of 4–5
lots as providing the most rapid growth of hMSCs.
3. l-Glutamine (200 mM) in solution of 0.85% NaCl (Invitrogen/GIBCO; catalog
4. Penicillin G (10,000 units/mL) and streptomycin sulfate (10,000 µg/mL) in
solution of 0.85% NaCl (Invitrogen/GIBCO; catalog # 15140-122) (Optional).
5. Phosphate buffered saline (PBS) without Ca2+ or Mg2+, 1× (Invitrogen/GIBCO;
catalog # 10010-031).
6. 0.25% trypsin and 1 mM ethylene diamine tetraacetic acid (EDTA) in Hanks’
balanced salt solution (Invitrogen/GIBCO; catalog # 25200-056).
7. 0.4% Trypan blue in solution of 0.85% NaCl (Invitrogen/GIBCO; catalog #
8. Ethanol (70%, 95%, 100%).
9. Isopropanol (100%).
10. Methanol (100%).
7 Differentiation and Characterization of Human MSCs 95
Table 7.1 Descriptions of CD markers used for MSC updated flow cytometry panel
CD3 OKT3, integral membrane glycoproteins that associates with T cell antigen
receptor (TCR), and is required for TCR cell surface expression and signal
CD11b Aka integrin alpha M, Mac-1; Mediates adhesion to substrates by opsoni-
zation with iC3b and subsequent phagocytosis, neutrophil aggregation,
CD14 Aka lipopolysaccharide (LPS) receptor, monocyte differentiation antigen
CD19 Co-receptor with CD21; Earliest B cell antigen in fetal tissue
CD29 fibronectin receptor
CD34 Cell-cell adhesion molecule and cell surface glycoprotein
CD36 Aka platelet GPIV or GPIIIb; thrombospondin receptor. Cell adhesion
molecule in platelet adhesion and aggregation, platelet-monocyte and
platelet-tumor cell interaction
CD44 Family of cell surface glycoproteins with isoforms generated by alternate
splicing of mRNA. Important in epithelial cell adhesion to hyaluronate in
basement membranes and maintaining polar orientation of cells; also binds
laminin, collagen and fibronectin
CD45 leukocyte common antigen (LCA)
CD49b very late antigen (VLA) alpha 2 chain - on T cells, Aka GPIa/IIa when
expressed on platelets
CD49c very late antigen (VLA) alpha 3 chain. Receptor for laminin, collagen,
CD49f Aka very late antigen (VLA) alpha 6 chain; Laminin receptor
CD59 Aka protectin, regulates complement mediated cell lysis by inhibiting for-
mation of membrane attack complex (MAC)
CD73a Aka ecto-5′-nucleotidase
CD79a Aka MB-1, B-cell antigen receptor complex associated protein alpha-chain
CD105 Aka endoglin, regulatory component of TGF-beta receptor complex; medi-
ates cellular response to TGF-beta 1
CD106 Aka VCAM-1; alpha 4 beta 1 ligand
CD117 Aka c-kit, stem cell factor receptor
CD147 Aka neurothelin, extracellular matrix metalloproteinase inducer
CD166 Aka Activated Leukocyte Cell Adhesion Molecule (ALCAM)
CD184 Aka CXCR4, Stromal cell Derived Factor 1 (SDF1). Receptor for the CXC
CD271 Aka Nerve Growth Factor Receptor (NGFR)
HLA-1, ABC The antigen corresponds to a monomorphic determinant of human HLA
class I molecules. HLA-ABC is associated with beta-2 microglobulin.
HLA-2, DR, DQ All major histocompatibility Class II HLA-DR, DP and most DQ antigens
expressed on B cells, antigen presenting cells and activated T cells
Isotype Controls Mouse IgG1/Mouse IgG2a
11. Deionized (DI) water.
12. Distilled water (dH2O).
13. Neutral buffered formalin, 10% (NBF) (Sigma; catalog # HT50-1-128).
14. Clear-rite 3 (Richard–Allan Scientific; catalog # 6901).
15. Permount mounting media (Fisher; catalog # SP15-500).
96 R.L. Reger et al.
16. CD36 FITC IgG1 (Beckman-Coulter; catalog # IM0766).
17. CD34 PE IgG1 (Beckman-Coulter; catalog # IM1871).
18. CD19 ECD IgG1 (Beckman-Coulter; catalog # IM2708).
19. CD11b PeCy5 IgG1 (Beckman-Coulter; catalog # IM3611).
20. CD45 PeCy7 IgG1 (Beckman-Coulter; catalog # IM3548).
21. CD44 FITC IgG1 (Beckman-Coulter; catalog # IM1219).
22. CD166 PE IgG1 (Beckman-Coulter; catalog # A22361).
23. CD90 PeCy5 IgG1 (Beckman-Coulter; catalog # IM3703).
24. CD49b FITC IgG1 (Beckman-Coulter; catalog # IM1425).
25. CD105 PE IgG1 (Beckman-Coulter; catalog # A07414).
26. CD117 PeCy5 IgG1 (Beckman-Coulter; catalog # IM2657).
27. CD147 FITC IgG1 (BD Biosciences; catalog # 555962).
28. CD49c PE IgG1 (BD Biosciences; catalog # 556025).
29. CD14 ECD IgG2a (Beckman-Coulter; catalog # IM2707).
30. CD29 PeCy5 IgG1 (BD Biosciences; catalog # 559882).
31. CD59 FITC IgG1 (Beckman-Coulter; catalog # IM3457).
32. CD184 PE IgG1 (Beckman-Coulter; catalog # A07409).
33. CD79a PeCy5 IgG1 (Beckman-Coulter; catalog # IM3456).
34. HLA-Class I: ABC FITC IgG1 (BD Biosciences; catalog # 555552).
35. CD271 PE IgG1 (BD Biosciences; catalog # 557196).
36. CD49f PeCy5 IgG1 (BD Biosciences; catalog # 551129).
37. HLA-Class II: DR, DP, DQ FITC IgG1 (BD Biosciences; catalog # 555558).
38. CD73a PE IgG1 (BD Biosciences; catalog # 550257).
39. CD106 PeCy5 IgG1 (BD Biosciences; catalog # 551148).
40. Isotype control IgG1/IgG2a (Beckman-Coulter; catalog # A17599).
1. Electric or manual pipet filler/dispenser for mouth-free pipeting of solutions
(0.1 to 25 mL).
2. Laminar flow hood (Biosafety Cabinet, Class II).
3. Water bath (37 °C).
4. Tissue culture incubator (37 °C) with controlled and humidified gas with 5%
5. Vacuum aspiration source.
6. Hemocytometer with coverslips.
7. General laboratory centrifuge with swinging bucket rotor with buckets and car-
riers to accommodate various tube sizes (2 mL up to 250 mL).
8. Inverted (phase) microscope for checking culture confluence and counting
9. General purpose laboratory balance (0.01 g sensitivity; 600 g capacity).
10. Multi-channel Flow Cytometer capable of 5 color analysis (Beckman-Coulter
FC500 5 Channel Flow Cytometer with HeNe Laser).
7 Differentiation and Characterization of Human MSCs 97
11. General laboratory mini-vortex.
12. General laboratory microcentrifuge for 1.5-mL microcentrifuge tubes.
1. Sterile plastic disposable serological pipets: 5 mL, 10 mL, 25 mL, and 50 mL.
2. Sterile plastic disposable pipets for vacuum aspiration.
3. Single channel pipetors, air displacement, capable of accurately measuring
from 10 µL–1000 µL, i.e., Eppendorf Research Series 2100 or similar.
4. Sterile aerosol barrier pipet tips, 10, 20, 200, and 1,000 µL.
5. Sterile plastic disposable conical centrifuge tubes: 15 and 50 mL.
6. Plastic disposable snap-cap centrifuge tubes: 1.5 mL.
7. Costar 6-well TC treated microplates (Corning; catalog #3516).
8. Sterile 250-mL filter units 0.22-µm pore size (Millipore, Stericup; catalog #
9. Sterile 500-mL filter units, 0.22-µm pores (Millipore, Stericup, catalog #
10. Sterile 1,000-mL filter units, 0.22-µm pore (Millipore, Stericup, catalog #
11. Sterile tissue culture dishes/flasks: 15 cm diameter (145 cm2) dishes (Nunc;
catalog # 168381), or T175 (175 cm2) flasks (Nunc; catalog # 159910).
12. Whatman #1 filter paper.
13. Disposable transfer pipets, 3 mL.
14. 5 mL polystyrene culture test tubes, 12 × 75 mm (Fisher, catalog # 14-961-10)
or tubes recommended by the flow cytometer manufacturer.
1. Complete culture medium (CCM) with 16.5% FBS: 500 mL αMEM with
L-glutamine, 100 mL FBS, 6 mL l-glutamine, 6 mL penicillin/streptomycin
(Optional; see Note 1).
Filter medium through sterile filter unit. Divide into aliquots you are likely
to use for an experiment and store at 4 °C for up to 2 wk. Before an experiment,
warm the aliquot to 37 °C.
2. Osteogenesis differentiation media (ODM): 192 mL CCM; 10 nM dexametha-
sone (Sigma; catalog # D2915, water soluble—see Note 2; 200 µL of 1:100 dilu-
tion of 1 mM stock solution in DI water); 20 mM β-glycerol phosphate (Sigma;
catalog #G9891; 8 mL of 0.5 M stock in CCM); 50 µM l-ascorbic acid 2-phos-
phate (Sigma; catalog # A8960; 200 µL of 50 mM stock solution in DI water).
Filter medium through sterile filter unit and store at 4 °C for the duration of
the osteogeneic culture period.
98 R.L. Reger et al.
3. Adipogenesis differentiation media (ADM): 200 mL CCM; 0.5 µM dexamethasone
(Sigma; catalog # D2915, 100 mg, water soluble – see Note 1; 100 µL of 1 mM
stock in DI water); 0.5 µM isobutylmethylxanthine (Sigma; catalog # I5879;
20 µL of 5 mM stock in methanol); 50 µM Indomethacin (Sigma; catalog # I7378;
333 µL of 30 mM stock in methanol).
Filter medium through sterile filter unit and store at 4 °C for the duration of
the adipogenic culture period.
4. Chondrogenic media without cytokines (CMwoC)—STOCK: 500 mL bottle of
high-glucose DMEM (GIBCO; catalog # 10569-010) supplemented with: 50 µg/mL
l-ascorbic-2-phosphate (Sigma; catalog # A8960; 500 µL of 50 mg/mL stock in DI
water); 40 µg/mL l-proline (Sigma; catalog # P0380; 500 µL of 40 mg/mL stock in
DI water); 100 µg/mL sodium pyruvate (Sigma; catalog # P5280; 500 µL of 100 mg/
mL stock in DI water); 5 mL ITS+ Culture Supplement that consists of 6.25 µg/mL
insulin, 6.25 µg/mL transferrin, 6.25 ng/mL selenous acid, 1.25 mg/mL bovine serum
albumin, 5.35 mg/mL linoleic acid (B&D Biosciences; catalog # 35-4352).
CMwoC can be stored for the duration of the chondrogenic culture at
5. Chondrogenic media with cytokines (CMwC) – STOCK: Needed volume of
CMwoC supplemented with: 10 ng/mL rhTGF-β3 (R&D Systems; catalog # 243-B3;
from 10 µg/mL stock in 4 mM HCl); 10−7M dexamethasone (Sigma; catalog # D2915,
100 mg, water soluble; from 1 mM stock in DI water); 500 ng/mL BMP-2 (rhBMP-2,
CHO-derived, R&D Systems, catalog # 355-BM OR rhBMP-6, CHO-derived R&D
Systems, catalog # 507-BP; from 10 µg/mL stock in PBS, see Note 3).
CMwC should be prepared fresh with every use. Stocks of cytokines should
be aliquoted and frozen at -20 °C to avoid several freeze-thaw cycles, which can
6. Alizarin red S stain WORKING (Osteogenesis): 1 g alizarin red S (Sigma;
catalog # A5533); 100 mL DI water.
Adjust pH of solution between 4.1 and 4.3 using 0.1% ammonium hydroxide.
Filter stain through sterile filter unit and store tightly capped at room tempera-
ture (RT) protected from light for up to 3 mo.
7. 0.5 Oil red O STOCK: 2.5 g oil red O (Sigma; catalog # 198196); 500 mL
Dissolve completely. Store in a tightly capped bottle at RT protected from
light for up to 3 mo.
8. Oil red-O stain WORKING (Make fresh for each use): 3 parts 0.5% oil-red-O
Stock; 2 parts PBS
Mix and wait 10 min. Filter stain through sterile filter unit. Wait 10 min before
use. Discard any unused stain.
9. Toluidine blue/1% sodium borate stain - WORKING (Chondrogenesis): 1 g
of toluidine blue (Richard-Allan Scientific; catalog # 90047); 1 g of sodium (Na)
borate (Sigma; catalog # S-9640); 100 mL dH2O.
First, make 1% Na borate solution (1 g/100 mL dH2O). Dissolve completely
until water is clear. Once clear, add 1 g toluidine blue, dissolve completely.
Prefilter using Whatman #1 filter paper and then filter using sterile filter unit.
Store tightly capped in an amber bottle for up to 1 mo at RT.
7 Differentiation and Characterization of Human MSCs 99
3.1 Plating and Maintaining 6-Well Differentiation Plates
for Adipogenesis and Osteogenesis
1. Following the pattern suggested in Fig. 7.1, label 6-well plate with:
a. Sample number and date
b. Passage number and cell density per well
c. 2 wells for bone differentiation medium, 2 wells for fat differentiation
medium and 2 wells for control (CCM) medium
d. Any other pertinent information
2. Add 2 mL of CCM to each well.
3. Add 100,000 cells in a volume less than 2 mL to each well. (see Note 4)
4. Incubate cells in humidified incubator at 37 °C with 5% CO2.
5. Every 3–4 d before the cells reach 70% confluency, aspirate media from each
well, rinse with 2 mL of PBS, and add 2 mL of fresh CCM. Return to incubator.
Fig. 7.1 Layout of 6 well plate for differentiation of MSCs. First column of wells (1 and 4) gets
MSCs + ODM (bone medium), second column of wells (2 and 5) get MSCs plus ADM (fat
medium) and the third column of wells (3 and 6) gets MSCs plus CCM. The first row of wells
(1, 2 and 3) gets rinsed with DI water, and then stained with alizarin red S. The second row of
wells (4, 5 and 6) gets rinsed with PBS and then stained with oil red O. Alizarin red staining
for osteogenesis should be strong in Well 1 and absent to light in wells 2 & 3. Oil red O staining for
adipogenesis should be strong in Well 5 and absent to light in the wells 4 & 6. This arrangement
allows for controls of media and staining specificity
100 R.L. Reger et al.
6. After the cells have reached 70% confluency in 2–8 d, aspirate media from
each well and rinse with 2 mL of PBS.
7. Add 2 mL of the appropriate media to the wells:
CCM (Subsection 2.4, 1) to the 2 control wells for no differentiation
ODM (Subsection 2.4, 2) to the 2 wells for bone/mineral differentiation
ADM (Subsection 2.4, 3) to the 2 wells for adipogenic differentiation
8. Continue to incubate cells in humidified incubator at 37 °C with 5% CO2.
9. Every 3–4 d for 21 days wash with 2 mL PBS and replace the appropriate dif-
ferentiation or control media.
10. Monitor progress of differentiation using the inverted phase microscope. Refer
to Figs. 7.2 and 7.3 for appearance of unstained cultures producing fat and
3.2 Staining Osteogenic and Adipogenic Differentiation
1. At the end of 21 d, aspirate media and rinse each well with 2 mL PBS.
2. Add 2 mL of NBF to each well and incubate for 1 h at room temperature.
3. Aspirate NBF from each well and discard.
4. Rinse the wells to be stained with alizarin red S with 2 mL of DI water and
Fig. 7.2 Mineral deposition in MSCs in culture in osteogenic medium as seen under phase con-
trast. Dark areas indicate mineral that has been manufactured by the cells. Unstained. Mag: 10X
7 Differentiation and Characterization of Human MSCs 101
5. Rinse the wells to be stained with oil red O with 2 mL of PBS and aspirate.
6. Add 2 mL of alizarin red S to each the 3 wells across one row: 1 nondifferenti-
ated well (negative control), 1 fat well (specificity control) and 1 bone differ-
entiated well for actual sample.
7. Add 2 mL of oil red O to each of the 3 wells across one row: 1 nondifferentiated
well (negative control), 1 bone differentiated well (specificity control) and 1 fat
well for actual sample.
8. Incubate for 20 min at room temperature and then aspirate.
9. Rinse the wells stained with alizarin red S with 2 mL of DI water and
10. Repeat 2 times or until background is clear.
11. Rinse the wells stained with oil red O with 2 mL of PBS and aspirate. Repeat
two times or until background is clear.
12. Add a final 2 mL of DI water to the alizarin red S stained wells (Wells 1-3).
13. Add a final 2 mL of PBS to the oil red O stained wells (Wells 4-6).
14. Examine plate under inverted microscope for evidence of fat and/or bone dif-
ferentiation. Negative control wells should not stain at all. Also, fat differenti-
ated cells should not stain with alizarin red S and bone differentiated cells
should not stain with oil red O. (see Notes 5 and 6)
See Figs. 7.2 to 7.5 for illustration of osteogenic and adipogenic cultures, unstained
Fig. 7.3 Evidence of fat formation in MSCs cultured in adipogenic medium as seen under phase
contrast. Bright round circles are fat globules within the cell. Unstained. Mag: 10X
102 R.L. Reger et al.
3.3 Chondrogenesis Differentiation
Harvest cells when 70–80% confluent for this assay. Cells lifted during early to
mid-log of growth or those that have reached 100% confluence will not differentiate
as well, if at all.
Fig. 7.4 Mineral deposition by MSCs cultured in osteogenic medium indicating early stages of
bone formation. Stained with alizarin red S. Mag: 20X (See Color Plates)
Fig. 7.5 Fat globules seen in MSC culture grown in adipogenic medium indicating differentiating
into adipocytes. Stained with oil red O. Mag: 20X (See Color Plates)
7 Differentiation and Characterization of Human MSCs 103
1. Wash harvested MSCs in PBS and resuspend in 1.0 mL CMwC.
2. Do cell count and viability. Adjust to a concentration to 400 viable cells/µL
with Chondrogenic Media with Cytokines (CMwC).
For example, if cell count of 1 mL of cell suspension gives 1,000,000 cells/mL,
add 1.5 mL CMwC to give 2.5 mL of 400 cells/µL. Thus, 500 µL should contain
3. Transfer approx 200,000 MSCs in 500 µL CMwC into a 15-mL conical polypropylene
4. Screw caps on tightly while in hood (sterility is of the utmost importance as no
antibiotics are added to the media).
5. Centrifuge the 15-mL conical tube at 450 g for 10 min. DO NOT resuspend the
pellet and DO NOT aspirate the medium.
6. Place the conicals into the cell culture incubator, which is humidified at 37 °C
with 5% CO2. Loosen the caps on the conicals so that they are simply placed
on the conicals without screwing on, allowing for full air exchange. Be sure to
screw the caps on tightly before removing the conicals from the incubator. (see
7. Pellets should be visible within 24 h.
8. Change media every 3–4 d by using a P-1000 pipet to remove the old media and
add fresh CMwC, paying close attention to detach pellet from plastic with each
media change. Take care not to aspirate the pellet when removing old medium.
9. At 21 d, chondrocyte pellet should be 2-4 mm in diameter with BMP-2 or
1–2 mm with BMP-6.
10. At 21 d, chondrocyte pellet may be fixed with NBF, embedded in paraffin, cut
into 5-µm sections onto slides, and stained with 1% toluidine blue/1% sodium
borate. (see Note 8).
3.4 Preparing and Staining Paraffin Sections
of Chondrogenesis Pellets
1. Deparaffinize in Clear-rite 4 times for 5 min each at RT.
2. Hydrate in descending grades of alcohol from 100%, 95%, dH2O, 2 × 1 min
each at RT.
3. Incubate slides in 1% toluidine blue/1% Na borate solution for 5 min at RT.
4. Rinse in several changes of tap water, until water becomes clear.
5. Rinse slides in dH2O for 1 min at RT.
6. Dehydrate sections in ascending grades of alcohol from 95% and100%, 2 times
for 1 min each at RT.
7. Clear in 4 changes of Clear-rite, 1 min each, at RT.
8. Cover slip in Permount mounting media.
9. Examine by microscopy. See Figures 6 and 7 for illustrations of unstained and
stained chondrogenesis pellets.
End Result: purple = cartilage, blue = negative
104 R.L. Reger et al.
3.5 Staining Procedure for Surface Epitope Characterization
1. Flow cytometer startup and QC. The instrument procedure for startup and quality
control should be performed as described by the manufacturer. This should include
the analysis of fluorescent beads to validate the function of the lasers, flow sys-
tems, and detection systems. Any problems encountered during this phase should
be corrected before proceeding with the analysis of prepared samples.
2. Following the antibody manufacturer’s recommendations, the appropriate volume
of reagents should be dispensed into a series of eight (8) 1.5-mL microfuge
tubes (a panel) as follows:
Tube 1: CD36 FITC, CD34 PE, CD19 ECD, CD11b PeCy5, CD45 PeCy7
Tube 2: CD44 FITC, CD166 PE, CD90 PeCy5
Tube 3: CD49b FITC, CD105 PE, CD117 PeCy5
Tube 4: CD147 FITC, CD49c PE, CD14 ECD, CD29 PeCy5
Fig. 7.6 MSC micromass pellets, grown in chondrogenic medium. Scale: mm
7 Differentiation and Characterization of Human MSCs 105
Tube 5: CD59 FITC, CD184 PE, CD79a PeCy5
Tube 6: HLA-Class I: ABC FITC, CD271 PE, CD49f PeCy5
Tube 7: HLA-Class II: DR, DP, DQ FITC, CD73a PE, CD106 PeCy5
Tube 8: Isotype control, IgG1/IgG2a
The tubes containing the antibody cocktails can be made ahead and stored at 4°C
in the dark until needed.
3. Harvest cells and count using trypan blue or other method to determine viability.
Resuspend cells in PBS at a final concentration of 1 × 106 viable cells/mL.
Approx 4 × 106 cells will be required to complete this protocol.
4. Aliquot between 25 × 104 and 5 × 105 cells per tube set-up in Step 3.5, 2. Additionally,
set up a ninth tube containing only cell suspension as a control for autofluorescence.
Gently vortex to mix and incubate in the dark for 20 min at RT.
5. Wash the cells by adding PBS to the 1.5 mL mark on each tube. Pellet the cells
at 100 g for 1 min at RT. Remove the supernatant, resuspend the pellet in 1 mL
PBS and centrifuge again. Repeat one more time for a total of 3 washes.
Fig. 7.7 MSC micromass pellet, grown in chondrogenic medium and stained with Toluidine Blue
Na Borate. Mag: 10X (See Color Plates)
106 R.L. Reger et al.
6. Resuspend the final pellet in 500 µL PBS and gently vortex. Be sure no aggre-
gates are present.
7. Using a transfer pipet, place the cell suspensions into the 12 × 75 mm culture test
tubes (or recommended device) and analyze on the flow cytometer. Analyze the
unlabeled cells (Tube 9) first, followed by the isotype control (Tube 8). Use the
results of these 2 control tubes to set the gates and analysis regions. Then read
each of the antibody cocktail- labeled cells.
See Table 7.2 for expected expression levels of the panels of antibodies on MSCs
and an example of flow cytometry results performed on hMSCs at passage 2 (P2).
1. It is preferable to grow cells without penicillin/streptomycin because any contamination of the
culture will not be masked by the presence of antibiotics.
Table 7.2 Surface epitope expression on hMSCs at passage 2 for a single donor as determined
by flow cytometrya
Panel Expected result
No Antibody on MSCs
1 CD11b - PeCy5 Negative
CD19 - ECD Negative
CD34 - PE Negative
CD36 - FITC Negative
CD45 – PeCy7 Negative
2 CD44 - FITC Important Pos
CD90 - PeCy5 Important Pos
CD166 - PE Important Pos
3 CD49b - FITC Positive
CD105 - PE Important Pos
CD117 - PeCy5 Negative
4 CD14 - ECD Negative
CD29 - PeCy5 Positive
CD49c - PE Positive
CD147 - FITC Positive
5 CD59 - FITC Positive
CD79a – PeCy5 Negative
CD184 - PE Positive
6 CD49f - PeCy5 Positive
CD 271 - PE Negative
HLA-1:ABC - FITC Positive
7 CD73a – PE Positive
CD106 – PeCy5 Positive
HLA-class II – FITC Negative
Range of % gated for 5
hMSC preps for
aThe range of % gated of the 5 MSC preparations that are distributed as NIH resource through the
NCRR. The expected results are also listed. The unlabeled cells and the isotype controls should
be negative. ND = Not Done.
7 Differentiation and Characterization of Human MSCs 107
2. Water-soluble Dexamethasone (Sigma D2915) is supplied encapsulated in 2-hydroxypropyl-
b-cyclodextrin. Be aware that the actual amount of dexamethasone in the material can vary.
Check the label for the actual amount of dexamethasone in your lot and prepare stock
accordingly. For example, if the label states that there is 70 mg of dexamethasone per gram
of material, to get 3.92 mg of dexamethasone, you would need to weigh out 56 mg of material
3. 500 ng/mL BMP-2 may be replaced with 500 ng/mL BMP-6 as cost necessitates. However,
BMP-6 does not promote chondrogenesis differentiation as robustly as BMP-2 (data
4. If there is an insufficient amount of cells, a lower density can be substituted as long as each
well receives the same number of cells.
5. Cultures can be kept in this condition for extended periods to allow for microscopic analysis.
Add more DI water or PBS as evaporation decreases the levels.
6. Confluent culture in bone differentiation plates can tend to lift in sheets or form a “ball”. The
ball can be sliced and made into slides for microscopic analysis for mineral deposition.
7. Before harvesting the cells, place a clean, autoclaved test tube rack that is sufficiently large to
hold all of the 15 mL conical tubes and to allow an empty space on all 4 sides of each tube into
the incubator. Never remove the rack from the incubator during the course of the assay. Use
another clean rack to transport the tubes between the incubator and hood.
8. When staining, it is best to also stain a positive control, known to contain proteoglycans.
Chondrogenic pellets successfully differentiated and stained should appear as a positively stained
dark purple color, with a negative blue background.
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