1. Human Mesenchymal Stem Cell Products Are Superior to VEGF Alone in In-vivo Angiogenesis
Amir H. Najafi, MD
MedStar Health Research Institute, Washington DC
Thank you Dr. X.
Ladies, Gentlemen, Good morning and thank you for your attendance.
MedStar Health
Research Institute

2. Amir H. Najafi, MD DISCLOSURES
I have no real or apparent conflicts of interest to report.

3. Background
Randomized controlled trials report conflicting data about beneficial effects of bone marrow cells for treating ischemic cardiovascular disease.
Randomized controlled trials report conflicting data on beneficial effects of direct use of stem cells for treating ischemic cardiovascular disease.

4. Background
Randomized controlled trials report conflicting data about beneficial effects of bone marrow cells for treating ischemic cardiovascular disease.
Evidence indicates that stem cells exert their pro-angiogenic functions through paracrine mechanisms.
Also there are some evidence showing that stem cells exert their pro-angiogenic functions through paracrine mechanism.
Epstein SE et. al. Circ Res. 2004

5. Aim
To evaluate the angiogenic efficacy of cell-free secretions of human mesenchymal stem cells (MSCs)
Therefore our aim was to evaluate the angiogenic efficacy of cell-free secretions of human mesenchymal stem cells or MCSs …

6. Aim
To evaluate the angiogenic efficacy of cell-free secretions of human mesenchymal stem cells (MSCs)
To compare it with that of VEGF as a known single potent angiogenic factor.
and to compare it with that of VEGF as a known single potent angiogenic factor.

7. Methods Preparation of CM Cells: Human MSCs (Lonza), Passage 7
Culture expanded human MCSs were washed to remove any trace of serum growth factors and …

8. Methods Preparation of CM Cells: Human MSCs (Lonza), Passage 7
Treatment: Desferroxamine (DFO), a chemical HIF-1a inducer
Duration: 72 hours
… bathed in serum-free medium containing 100 μM desferroxamine or DFO for 72 hours. DFO is a potent chemical inducer of hypoxia inducible factor-1, which is an upstream gene controlling the expression of multiple cytokines and growth factors in response to ischemia.
Semenza GL. Sci STKE. 2007

9. Methods Preparation of CM Cells: Human MSCs (Lonza), Passage 7
Treatment: Desferroxamine (DFO), a chemical HIF-1a inducer
Duration: 72 hours
Processing: Serial Ultrafilteration (Amicon, 3kD)
Then we aspirated conditioned medium and used serial rounds of ultrafilteration to concentrate it. Concentrated CM was characterized by ELISA for the presence of VEGF.
Semenza GL. Sci STKE. 2007

10. Methods VEGF (pg/mL) DFO + Hypoxia DFO
This diagram shows results of one of our optimizing studies. We compared VEGF secretion from MSCs grown under normoxia, hypoxia, DFO, or DFO plus hypoxia. Also different batches of cells were treated for different durations from 24 to 72 hours. As you can see, DFO after 72 hours produced the highest concentration of VEGF.
Hypoxia
Normoxia
Duration (hours)

11. Methods Animal studies: 8 week old male C57 mice (Jackson)
4 groups (n=6/group):
Concentrated CM 50 ng/mL of VEGF (CCM50)
50 ng/mL (VEGF50)
5 ng/mL of VEGF (CCM5)
Negative control (PBS)
We measured in vivo angiogenesis with a matrigel plug assay.
Matrigel is a Basement Membrane Matrix polymer providing a reliable scaffold for attachment and differentiation of endothelial cells.
Male C57 mice were subcutaneously injected with matrigel plugs in the abdominal wall region.
We measured VEGF concentration in CCM, normalized it and then mixed it with matrigel polymer so that at the end the concentration of VEGF in the matrigel plug was 50 ng/ml, or CCM50 if you will. In the second group we used recombinant human VEGF and mixed it with matrigel to yeild 50ng/ml of VEGF, and Let’s call this group VEGF50. Please note that VEGF50 and CCM50 had same concentration of VEGF, but CCM50 on top of VEGF had all other cytokines and growth factors that MSCs produce along with VEGF.
Similarly we used 10-fold diluted CCM resulting in a final VEGF concentration of 5ng/ml in the matrigel or CCM5.
And finally we used buffered saline mixed with matrigel in our negative control group. There were 6 mice in each group.

12. Methods Histologic Assessments H&E CD31 IHC *
After 14 days, we harvested the plugs (and here you see a sample picture of a harvested plug). Then we performed histologic assessment with H&E staining and anti-CD31 immunohistochemistry, which is an endothelial cell marker.
* Couffinhal T. et. al. Frontiers in Bioscience. 2009

13. Methods Histologic Assessments Endpoints H&E CD31 IHC
Cell density (cells/mm2)
Number of RBC-containing vessels/200x field
Number of CD31+ vessels/100x field
All measured by blind observers, 5 slides were counted for each animal. *
Our endpoints included Cell density, Number of red blood cell-containing vessels, and Number of CD31+ vessels. Five slides were quantified for each animal, and all measurements were performed in a blinded fashion using …
* Couffinhal T. et. al. Frontiers in Bioscience. 2009

14. Methods Histologic Assessments Endpoints
H&E
CD31 IHC
Endpoints
Cell density (cells/mm2)
Number of RBC-containing vessels/200x field
Number of CD31+ vessels/100x field
All measured by blind observers, 5 slides were counted for each animal.
Image Pro Plus software for image analyses *
… Image Pro Plus software for image analysis.
* Couffinhal T. et. al. Frontiers in Bioscience. 2009

15. Results Neg Ctrl (PBS) VEGF50
Well, here you see a few examples of histologic evaluation of matrigel plug stained with H&E at 100x magnification from Negative control, VEGF50, CCM5, and CCM50.
CCM5
CCM50

16. Results
CCM50
CCM50
At a higher magnification we could clearly see blood vessels in the matrigel plugs containing CCM50 …

17. Results
And here, an example of CD31 staining, showing blood vessels lined with endothelial cells.
CCM50

18. Cell Density
Compared to VEGF50, CCM50 resulted in a greater density of cells…

19. Cell Density
P = 0.003

20. RBC containing vessels
… higher number of red blood cell- containing vessels

21. RBC containing vessels
P = 0.04

22. CD31+ vessels
... and higher number of CD31 + vessels

23. CD31+ vessels
P < 0.001

24. Cell Density
P < 0.001
We also observed a dose response relationship: CCM5 that had 1/10th concentration of CCM50 resulted in lower density of cells …

25. RBC containing vessels
P = 0.002
… lower density of red blood cell-containing vessels …

26. CD31+ vessels
P = 0.02
and lower density of CD31 + vessels.

27. Conclusion
Paracrine factors released by human MSCs have superior angiogenic capacity in a matrigel plug assay than rhVEGF alone.
In conclusion, paracrine factors released by human MSCs have superior angiogenic capacity in a matrigel plug assay than rhVEGF alone.

28. Conclusion
Paracrine factors released by human MSCs have superior angiogenic capacity in a matrigel plug assay than rhVEGF alone.
Multiple pro-angiogenic agents secreted by MSCs exert synergistic angiogeneic effects when compared to that provided by a single potent angiogenic agent.
This suggests that the multiple pro-angiogenic agents secreted by MSCs have synergistic angiogeneic effects…

29. Conclusion
Paracrine factors released by human MSCs have superior angiogenic capacity in a matrigel plug assay than rhVEGF alone.
Multiple pro-angiogenic agents secreted by MSCs exert synergistic angiogeneic effects when compared to that provided by a single potent angiogenic agent.
Cell-free CM of MSCs may provide a promising therapeutic strategy for enhancing collateral function in ischemic vascular disease.
And finally we conclude that cell-free CM of MSCs may provide a promising therapeutic strategy for enhancing collateral function in ischemic vascular disease.

30. Acknowledgements Mentors: Colleagues: Stephen E. Epstein, MD
Mary Susan Burnett, PhD
Colleagues:
Nima Aghili, MD
Hajra Nashin
Xinzhi Peng, MD
Roberta M. Lassance Soares, PhD
I’d like to thank my mentors Dr Epstein and Dr. Burnett as well as my colleagues…
Thank you very much for your attention. I’d be glad to take any questions. [pause then click]
Thank you. That was an excellent question. Well, I should say that I don't have a definitive answer for that. All I can say is that we didn't see any gross or histologic evidence of excessive inflammatory reaction. Also we should recognize that many of the pathways and cytokines involved in angiogeneis and inflammatory repsonses are in common. Two clear examples of such cytokines are CXCL-5 and IL-8; these are well known chemotactic factors that have potent roles in angiogenesis as well.