1. Mesenchymal stromal cells (MSCs) are a highly attractive
therapeutic as they possess various healing abilities ranging from
preventing apoptosis of surrounding cells to promoting
angiogenesis of new blood vessels. Further, these cells do not elicit
any immune response due to the lack of an MHC Class II antigen,
deeming them safe for all patients even in allogeneic use. However,
previous studies show that 90% of MSCs directly injected to the site
of injury die within 24 to 48 hours. By seeding the MSCs onto an
extracellular matrix derived from the small intestinal submucosa
(SIS ECM) of pigs, the cells would remain localized and viable in
high concentrations. Prior to additional research, the isolated cells
were first confirmed as hMSCs through positive display of known
hMSC surface markers CD73, CD90, and CD105 alongside
negative display of known hematopoietic cell surface marker CD45.
Continuing classification, the cells were differentiated into
osteocytes, adipocytes, and chondrocytes, proving their aptitude for
trilineage differentiation. An array then compared cell secretion
levels of various chemokines involved in angiogenesis, developing
more specific reasoning for why MSCs are so helpful to the human
body and the advantages of hMSCs on an ECM support. From the
array, hMSCs on ECM secreted a high amount of protein IL-8
where cells on plastic did not. Subsequently, an assay was used to
more precisely quantify the difference, finding that cells on ECM
released 40x the amount of IL-8. IL-8 is known to have angiogenic
effects and regulate other growth and migration proteins, perhaps
explaining the remedial properties of mesenchymal stromal cells.
Figure 2a. Controls
Above are the controls performed for each of the lines of differentiation. From left to
right are the osteocyte control, the adipocyte control, and the chondrocyte control. In
comparison to differentiated cells, growth and change is evident.
Differential expression of IL-8 from mesenchymal stromal cells grown on
plastic or an extracellular matrix support
Jacqueline Liu, Xin Lin Ph.D., Tye Petrie, Gerhard Bauer, and Claus Sondergaard Ph.D.
Institute for Regenerative Cures, Stem Cell Program, UC Davis, Sacramento, CA, United States, 95817
Abstract / Introduction
Materials & Methods
Conclusion
Results
Results Cont.
Acknowledgements
References
• Isolation of human mesenchymal stromal cells from bone marrow
• Expansion of hMSC in plastic T225 flask with D20 media (DMEM
high glucose and 20% fetal bovine serum)
• Differentiation of hMSCs into osteocytes, adipocytes, and
chondrocytes
• Staining of osteocytes (Alizarin Red) and adipocytes (Oil Red O)
for visualization after approximately two weeks of growth
• Cryosectioning of chondrocytes and staining with Alcian Blue for
visualization after approximately two weeks of growth
• Performance of flow cytometry to check for presence or lack of
known cell surface markers and confirm status of hMSC (CD73+,
CD90+, CD105+, CD45-)
• Using array to compare secretion levels from hMSCs on matrix
versus plastic of various angiogenic factors
• Immunoassay ELISA kit (R&D Systems) to compare secretion
levels of IL-8 from hMSCs grown on plastic versus matrix
• Usage of program ReaderFit to create graphical representation
of data from ELISA assay
Isolation and Expansion
Results Cont.
Figure 1. Human mesenchymal
stromal cells
On the left are hMSCs grown on a plastic
T225 flask. Cells display typical spindle
shaped morphology.
Trilineage Differentiation
Figure 2b. Osteocytes
As seen on the left, osteocytes are
stained with Alizarin Red. Red
staining of mineral deposits in the
cell cultures is evidence of
calcification.
Figure 2c. Adipocytes
As seen on the left, adipocytes are
stained with Oil Red O. The fat is
recognizable as the small red
spheres inside the cells.
Flow Cytometry
Human IL-8 Immunoassay
• Confirmation of hMSCs based on three defining characteristics was
successful
• ELISA assay of IL-8 shows almost 40x amount of protein from hMSCs
on matrix in comparison to plastic
• hMSCs seeded on matrix have higher secretion levels of a key
angiogenic factor which may make cells grown on matrix more
effective for healing than cells without support
A huge thanks to my mentor Dr. Claus Sondergaard for allowing me to
work in his lab alongside Dr. Xin Lin and Tye Petrie, and Dr. Gerhard
Bauer, Dr. Jan Nolta, CIRM, and the UC Davis Medical Center’s Institute
for Regenerative Cures for this incredible opportunity.
Id Name Response Independent Calculated
1 Std1 1.8425 2000 2003.635
2 Std2 1.127 1000 986.6755
3 Std3 0.6895 500 518.1953
4 Std4 0.3755 250 242.7631
5 Std5 0.217 125 123.5553
6 Std6 0.1265 62.5 62.7066
7 Std7 0.0715 31.2 29.2378
8 Std8 (0) 0.014 0 0
9 Matrix (diluted 1:100) 0.0665 26.376
10 Plastic 0.132 66.2211
Calculations
Actual concentration (cells on matrix)— 26.376*100=2637.6 pg/mL
Ratio of IL-8 from cells on matrix to plastic— 2637.6:66.2211 = 39.83:1
Osteocyte Control
Well 1 Well 2
Adipocyte Control
Well 1 Well 2
Standard
hMSC on
matrix
(diluted
1:100)
hMSC on
plastic
Figure 5b. DataFigure 5a. Assay
Figure 5c. Graph
b. CD90+
d. CD45-c. CD105+
a. CD73+
Isotype Control
Actual
Control for CD45 is not to scale.
• Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal
stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–317.
• Keating A. Mesenchymal stromal cells. Curr Opin Hematol. 2006;13:419–425.
• AE Koch, PJ Polverini, SL Kunkel, LA Harlow, LA DiPietro, VM Elner, SG Elner, and RM Strieter. Interleukin-8 as a macrophage-derived
mediator of angiogenesis. Science 11 December 1992: 258 (5089), 1798-1801.
Human Angiogenesis Array
Figure 4a. Array
As seen, IL-8 expression from
hMSCs on matrix is much greater
than that of cells on plastic.Cells on matrix Cells on plastic
IL-8 Expression
IL-8 Expression
200 µm 200 µm
200 µm 200 µm
200 µm 200 µm
200 µm
• Positive expression of
MSC markers CD73,
CD90, and CD105
• Negative expression
of hematopoietic cell
marker CD45
• Cells exhibit expected
aspects of hMSCs
Figure 2d. Chondrocytes
As seen on the left, sectioned
chondrocytes are stained with
Alcian Blue. Blue stained cells are
clearly identifiable from nuclear red
stain of others, as in the control.
Chondrocyte Control
200 µm
200 µm 200 µm
Slide 1 Slide 2
Figure 3