Stem Cell Study

The Riordan Clinic Research Institute has made interesting contributions to the field of stem cell research. Stem cells research at the Riordan Clinic grew out of our interest in the relationship between cancer and the body’s wound healing response.  We published a manuscript detailing our discovery of a method for harvesting adult stem cells from menstrual fluid.  These cells, which we termed “Endometrial Regenerative Cells” (ERC) were capable of differentiating into nine lineages: cardiomyocytic, respiratory epithelial, neurocytic, myocytic, endothelial, pancreatic, hepatic, adipocytic, and osteogenic.

Additionally, ERC produced MMP3, MMP10, GM-CSF, angiopoietin-2 and PDGF-BB at 10–100,000 fold higher levels than two control cord blood derived mesenchymal stem cell lines. Given the ease of extraction and pluripotency of this cell population, we proposed ERC as a novel alternative to current stem cells sources.

• Endometrial regenerative cells: a novel stem cell population. Meng X, Ichim T, Zhong J, Rogers A, Yin Z, Jackson J, Wang H, Ge W, Bogin V, Chan KW, Thébaud B, Riordan NH.  Journal of Translational Medicine , 2007, 5:57

This manuscript earned considerable interest, winning BioMed Central’s “Research Article of the Year in Medicine” award.

Using a rat brain tumor (glioma) model, we examined the interaction between tumor cells and stem cells injected into the tumor region.  Stem cells inhibited tumor growth compared to controls; moreover, growth medium conditioned by stem cells has a growth inhibitory effect on rat tumor cells.  Work with the protein array test had provided information concerning possible mechanisms of action for this effect.

• Inhibition of Intracranial Glioma Growth by Endometrial Regenerative Cells. Han X, Meng X, Yin Z, Rogers A, Zhong J, Rillema P, Jackson J, Ichim T, Minev B, Carrier E, Patel A, Murphy M, Min W, Riordan N. Cell Cycle, 2009, 8(4):1-5.

In this study we determined whether ERC would alter the growth of glioma using the aggressive C6/ LacZ7 (C6) into Sprague Dawley rat model. ERC administration by intravenous (i.v.) or intratumoral (i.t.) showed significant inhibition of glioma: volume reduction of 49% after i.v. treatment (p < 0.05), and about 46% i.t. treatment (p < 0.05). Tumor reduction was associated with inhibition of angiogenesis and reduced numbers of CD133 positive cells in the incranial tumor.

We have also developed methods for quantifying and isolating endothelial progenitor cells from circulating blood. We have shown that circulating endothelial cell numbers correlate with risk factors of cardiovascular disease, may be important in slowing down the aging process. Depletion of circulating endothelial progenitor cells (EPCs) may contribute endothelial dysfunction, both as an early event in the atherogenic process and as a late event leading to clinical manifestations of atherosclerosis and cardiovascular disease progression.

• Circulating endothelial progenitor cells: a new approach to anti-aging medicine?

Mikirova NA, Jackson JA, Hunninghake R, Kenyon J, Chan KWH, Swindlehurst CA, Minev B, Patel AN, Murphy MP, Smith L, Alexandrescu DT, Ichim TE, Riordan NH.  Journal of Translational Medicine 2009, 7:106

• Circulating Endothelial Progenitor Cells and Erectile Dysfunction: Possibility of Nutritional Intervention? Ichim TE, Zhong Z, Mikirova NA, Jackson JA, Hunninghake R, Mansilla E, Marin G, Núñez L, Patel AN, Angle N, Murphy MP, Dasanu CA, Alexan-drescu DT, Bogin V, Riordan NH. Panminerva Medica 2010, 52:(Suppl. 1 to No. 1) (June 2010)

Increased Level of Circulating Endothelial Microparticles and Cardiovascular Risk Factors. Mikirova NA, Casciari JJ, Hunninghake RE, Riordan NH. Journal of Clinical & Experimental Cardiology 2011, 2:4 (1 April 2011)

In addition, we identified the possible interventions for maintaining levels of circulating endothelial cells and reversing age-associated degeneration. Of particular interest was the level of vitamin D. To test whether vitamin D had an effect on the level of EPCs in circulation, the number of cells positive for EPC markers was compared for subjects with a level of vitamin D higher and lower than sufficient levels. The mean percentage of EPCs for subjects with a level of vitamin D lower than 40 ng/mL was significantly lower than the mean percentage of EPCs for subjects with a level of vitamin D higher than 40 ng/mL (0.045 versus  0.068, p<0.01).

As EPCs participate in vascular repair and angiogenesis, we characterized the effect of vitamin D on the ability of cultured mononuclear cells to differentiate into angiogenic cells. The percentage of cells differentiated in angiogenic cells in medium with endothelial growth factors was compared with the measured levels of vitamin D in the blood for all participants. The number of angiogenic cells demonstrated a positive association with the level of vitamin D.

As the result of this study, we demonstrated:

• vitamin D status has an effect on the number of EPCs in circulation and on the ability of peripheral mononuclear cells to differentiate into angiogenic cells.
• There were more circulating EPCs in subjects with a sufficient level of vitamin D than in subjects with an insufficient or deficient level of vitamin D.
• The number of circulating angiogenic cells developed from the peripheral mononuclear cells was also higher in subjects with a higher level of vitamin D in the blood.

 The results of the study are published in journal:

• Vitamin D Concentrations, Endothelial Progenitor Cells, and Cardiovascular Risk Factors. Mikirova NA, Belcaro G, Jackson JA, Riordan NH;  Panminerva Medica 2010, 52:(Suppl. 1 to No. 2)

In addition, we investigated whether a commercially available nutraceutical, Stem-Kine (Aidan Products, Chandler AZ), was capable of increasing the number of circulating stem cells and progenitor cells.   This proprietary food supplement is produced by fermentation of a combination of green tea, astralagus, goji berry extracts, with food-derived Lactobacillus fermentum together with ellagic acid, beta 1, 3 glucan and vitamin D3.  We performed a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period.

Stem-Kine administration was associated with significant mobilization of cells expressing hematopoietic stem cell markers. Quantification of peripheral blood cells expressing the hematopoietic stem cell markers CD133 and CD34 was performed before and after Stem-Kine supplementation.  Augmentation of both CD133 and CD34 cells in circulation was observed, as well as endothelial progenitor cells (KDR-1+/CD34+) capable of forming endothelial colonies. The average circulating CD133 cell numbers from all treated subjects peaked at 90% of pretreatment values (p= 0.01) on day 7, whereas circulating CD34 counts reached a maximal level of 53% (p=0.04) increase on day 2.

We published an article describing how the nutritional supplement “Stem-Kine” can increase circulating endothelial cell numbers.

• Nutraceutical Augmentation of Circulating Endothelial Progenitor Cells and Hematopoietic Stem Cells in Human Subjects Mikirova NA, Jackson JA, Hunninghake R, Kenyon J, Chan KWH, Swindlehurst CA, Minev B, Patel AN, Murphy MP, Smith L, Alexandrescu DT, Ichim TE, Riordan NH.  Journal of Translational Medicine 2010, 8:34

In next study, a flow cytometry based method was developed for quantifying circulating endothelial progenitor cells (stem cells of endothelial origin) in blood.  The normal ranges in healthy adults was established and  compared with circulating endothelial progenitor (EPC) cell counts in subjects with various disease states.  It appears that cancer patients have elevated EPC counts while subjects with type 2 diabetes and arthritis have below normal EPC counts. There was the effect of donor age on surface antigen expression of EPCs.

In addition, we developed protocol to derive stem cells from adipose tissue.  Adipose tissue is specialized connective cell (stromal cell) tissue that provides fat storage.  We developed the ways to collect cells from adipose tissue and to characterize their surface antigen expression using flow cytometry.