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Introduction: Stem cell therapy within the heart has proven so far to be a daunting task. Stem cells delivered to the heart exhibit poor survival, migration, and differentiation. [1] This may be in part due to the toxic environment in the myocardium following infarction. [2] Our prior studies show that self assembling peptides are an efficient delivery scaffold for cell transplantation following infarction and may improve cellular retention.[3] However. following infarction levels of reactive oxygen species, hydrogen peroxide in particular, increase in a time-dependent manner.
Methods: To determine the effects of the hydrogen peroxide on stem cell differentiation, we isolated rat primary mesenchymal stem cells and cultured them in 2D and 3D environments prior to hydrogen peroxide stimulation. To accomplish this, cells were immediately placed either on tissue culture plastic (2D) or 3D peptide nanofibers for 24 hours and then treated with increasing doses of hydrogen peroxide. The cells were harvested after 1 day and 5 days and protein levels were examined for markers of cardiac differentiation. Additionally, we looked at the effects of jagged protein (a pro-differentiation factor for embryonic stem cells).
Results: On day 1, we found that cell differentiation was not affected by the hydrogen peroxide; however, our day 5 samples suggest that hydrogen peroxide does inhibit stem cell differentiation in a dose-dependent manner as measured by expression of GATA-4 and Nkx2.5. Our study suggests that jagged protein does help to promote cell differentiation.
Conclusion: After heart attack, damaged cells release many harmful factors into the cardiac microenvironment. Our data suggest that great care needs to be taken toward the effects of oxidative stress on potential stem cell treatments.
Myocardial infarction (MI), commonly known as a heart attack, occurs when
flow of the blood to the heart is interrupted. This restriction of blood flow results in significant cell death of the cardiac myocytes and possibly endogenous stem cells. If enough heart tissue is damaged, it can eventually lead to heart failure, for which there is currently no cure. In vitro studies have shown that different stem cells can help regenerate myocytes which can lead to better healing of the heart. Unfortunately these studies have not translated to an improvement in vivo, due to poor survival and differentiation of the implanted stem cells. One solution is to create a microenvironment to direct the survival and differentiation of stem cells in order to regenerate damaged myocardial tissue. Self assembling peptides are an excellent delivery vehicle for cell therapy and the microenvironment can be easily altered. These peptides self assemble when exposed to physiological pH and osmolarity and would allow the stem cells to be retained in the local delivery
Our project goal is to determine the effect of oxidative stress on the differentiation of mesenchymal stem cells in both 2-D and 3-D culture. We will also study the effect of jagged protein on oxidative stress.
We first isolated primary stem cells from adult rats. After culturing the cells, we conducted experiments within the self assembling peptide scaffolds. We tested the differentiation of the cells exposed to various doses of hydrogen peroxide in the 3D and the 2D environment using Western blotting, measuring both Gata-4 and NKX-2.5 protein markers.. We then cultured the cells with jagged protein, a growth factor, in an effort to improve differentiation following oxidant exposure.
CELL DIFFERENTIATION
Acute hydrogen peroxide has no effect on cardiac differentiation
Image caption: Proteins were measured by western blot, and our data show that hydrogen peroxide has no effect on 2d or 3d differentiation in day 1 samples. The measurement of Gata-4, an early cardiac protein marker, was equal among all samples which displays that hydrogen peroxide has no effect on cardiac differentiation.
Hydrogen peroxide has negative effect on differentiation in 2d
Image caption: The data show that the Gata-4 samples in 2d were inhibited by increasing levels of hydrogen peroxide. Hydrogen peroxide in the Gata-4 samples in 3d culture had no real effect, but the results produced had variability . The hydrogen peroxide media was administered to cells after 24 hours of incubation,. Hydrogen peroxide media was changed every 2 days.
Chronic hydrogen peroxide (5 days) has opposing effect on differentiation in 2d and 3d culture
Image Caption: On 2D culture plates, Nkx-2.5 samples displayed increased cell differentiation while the 3D cultures displayed decreased amounts of differentiation at certain doses. Data are expressed as mean + SEM of 3 separate experiments. These data demonstrate that while 2d culture may show positive effects, great care must be taken as cell therapy will be a 3-dimensional approach to cardiac regeneration.
Jagged Protein increases cell differentiation and is modulated by hydrogen peroxide differentially
Image caption: Mesenchymal stem cell media was incubated with jagged protein, which is a pro-differentiation factor for other stem cell types. The data display that jagged protein does increase cell differentiation in both 2d and 3d samples while measuring the Nkx-2.5 marker. However, while Jagged protein-induced differentiation is positively affected by hydrogen peroxide in 2d culture, opposing effects are seen in 3d culture.
-On day 1, hydrogen peroxide did not affect cell differentiation in 2D or 3D culture.
-On day 5, cell differentiation was affected by hydrogen peroxide dosage levels.
-Jagged Protein did increase differentiation in both 3D and 2D samples.
-We speculate that 2D displayed more positive results due to spatial orientation within the cells. Understanding possible 3D and 2D mechanisms would be imperative to future studies.
-Further research also would include using different growth factors to achieve better differentiation of cells.
-Understanding the mechanisms of oxidative stress caused by hydrogen peroxide could lead to more successful stem cell therapy treatments within the heart after suffering a heart attack
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No.52005873 and by Division of Biological and Biomedical Sciences, Emory University.
1. Heish PCH, Davis ME, Gannon J, MacGillivray C, Lee RT. Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. The Journal of Clinical Investigation. 2006;116:237-247.
2. Davis ME, Motion JPM, Narmoneva DA, Takahashi T, Hakuno D, Kamm RD, Zhang S, Lee RT. Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells. Circulation. 2005; 111: 442-450.
3. Gelain F, Horii A, Zhang S. Designer self-assembling peptides scaffolds for 3-D tissue cell cultures and regenerative medicine. Macromol Biosci. 2007 May 10; 7 (5): 544-51.
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