The effect of telomerase template antagonist GRN163L on Bone-Marrow-Derived rat mMesenchymal stem cells is reversible and associated with altered expression of cyclin d1, cdk4 and cdk6

Telomerase activity is essential for the continued growth and survival of malignant cells, therefore inhibition of this activity presents an attractive target for anti-cancer therapy. The telomerase inhibitor GRN163L, was shown to inhibit the growth of cancer cells both in vitro and in vivo. Mesenchymal stem cells (MSCs) also show telomerase activity in maintaining their self-renewal; therefore the effects of telomerase inhibitors on MSCs may be an issue of concern. MSCs are multipotent cells and are important for the homeostasis of the organism. In this study, we sought to demonstrate in vitro effects of GRN163L on rat MSCs. When MSCs were treated with 1 μM GRN163L, their phenotype changed from spindle-shaped cells to rounded ones and detached from the plate surface, similar to cancer cells. Quantitative-RT-PCR and immunoblotting results revealed that GRN163L holds MSCs at the G1 state of the cell cycle, with a drastic decrease in mRNA and protein levels of cyclin D1 and its cdk counterparts, cdk4 and cdk6. This effect was not observed when MSCs were treated with a mismatch control oligonucleotide. One week after GRN163L was removed, mRNA and protein expressions of the genes, as well as the phenotype of MSCs returned to those of untreated cells. Therefore, we concluded that GRN163L does not interfere with the self-renewal and differentiation of MSCs under short term in vitro culture conditions. Our study provides additional support for treating cancers by administrating GRN163L without depleting the body's stem cell pools. © 2010 Springer Science+Business Media, LLC

Mesenchymal stem cells: Possibilities of new treatment options

Stem cell research evolved as a new hope and has gained tremendous interest during the last two decades in developing potential strategies for many debilitating diseases. Mesenchymal stem cells (MSCs) are bone marrow-derived multipotent stem cells capable of self-renewal and of differentiating into multiple lineages, such as osteocytes, adipocytes, chondrocytes, myoblasts, cardiomyocytes, and hepatocytes. MSCs are an important source for cellular therapies. They can easily be obtained and expanded in vitro in large numbers without significantly altering their properties. MSCs not only migrate to the injured site in vivo but also have immunomodulatory effects that make their use attractive for allogeneic grafting. MSCs can also be frozen for preservation; and when thawed, they retain their normal physiological function, allowing future ''off-the-shelf'' therapy approaches. Because of these features, MSCs have high therapeutic value in tissue engineering and regenerative medicine. In this chapter, the contribution of the MSCs to cardiovascular repair and liver regeneration are summarized. © Springer Science+Business Media, LLC 2012. All rights reserved

The Effect of Estrogen on Bone Marrow-Derived Rat Mesenchymal Stem Cell Maintenance: Inhibiting Apoptosis Through the Expression of Bcl-x L and Bcl-2

Mesenchymal Stem Cells (MSCs) have high therapeutic value for regenerative medicine and tissue engineering due to their differentiation potential and non-immunogenic characteristics. They are also considered as an effective in vivo delivery agent because of their ability to migrate to the site of injury. A major roadblock in their use for cell-based therapies is their rareness in vivo. Therefore, it is important to obtain increased number of functional MSCs in vitro in order to have adequate numbers for therapeutic regiments. We aimed to investigate the role of estrogen and its mechanism in obtaining more MSCs. MSCs were isolated from female and ovariectomized rats and cultured in the presence and absence of 10 -7 M estrogen. In the presence of estrogen, not only their CFU-F activity increased but also apoptotic rate decreased as shown by TUNEL staining leading to obtain more MSCs. Also the number of the cells in the colonies increased upon estrogen treatment. To reveal the mechanism of this effect, we focused on Bcl-2 family of proteins. Our immunoblotting experiments combined with knockdown studies suggested a critical role for anti-apoptotic Bcl-x L and Bcl-2. Estrogen treatment up regulated the expression Bcl-x L and Bcl-2. When we knocked down the expression of bcl-x L and bcl-2, MSCs lacking these genes showed an increase in the apoptotic rate in contrast to normal MSCs following estrogen treatment. Therefore, estrogen treatment will be of great advantage for cell-based therapies in order to get more functional MSCs and may provide opportunities to develop new strategies for debilitating diseases. © 2011 Springer Science+Business Media, LLC

Timing of induction of cardiomyocyte differentiation for in vitro cultured mesenchymal stem cells: A perspective for emergencies

Mesenchymal stem cells (MSCs) have the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, and cardiomyocytes. Several established methods are presently available for in vitro isolation of MSCs from bone marrow. However, the duration necessary to culture them can be a major handicap to cell-based therapies needed for such urgent cardiovascular conditions as acute myocardial infarction and acute hindlimb ischemia. The best timing of car- diomyocyte differentiation induction after MCS isolation and expansion is still an unresolved issue. Our goal was to investigate the possibility of obtaining functional cardiomyocytes from rat MSC within a shorter time period. We examined MSCs' colony-forming capacity, CD90 and CD34 immunoreactivity during the 14 days of culturing. Cardiomyocyte differentiation was induced by 5-azacytidine. Immunohistochemic staining, together with intracellular Ca2+ measurement experiments, revealed that MSCs do not differentiate into any specific cell lineage but show the characteristics of MSCs on both the 9th and 14th days of the culture. To check the potential for differentiation into cardiomyocytes, experiments with caffeine application and depolarization with KCl were performed. The cells possessed some of the specific biochemical features of contracting cells, with slightly higher capacities on the 14th day. Cells from 9th and 14th days of the culture that were treated with 5-azacytidine had a higher expression of cardiac-specific markers such as troponin I, α-sarcomeric actin, and MEF2D compared with the control groups. This study illustrates that it is possible to get functional cardiomyocytes from in vitro MSC culture in a shorter time period than previously achieved. This reduction in time may provide emergency cases with access to cell-based therapies that may have previously been unavailable

Mesenchymal stem cells or cardiac progenitors for cardiac repair? A comparative study

Gene regulation and cell differentiatio