Contribution of mesenchymal stem cells in cell based therapies

Ankara : The Department of Molecular Biology and Genetics and the Institute of Engineering and Science of Bilkent University, 2010.Thesis (Ph. D.) -- Bilkent University, 2010.Includes bibliographical references leaves 73-106.tem cell research evolved as a new hope and has gained tremendous interest in the last two decades to develop new strategies for many of debilitating diseases. Mesenchymal Stem Cells (MSCs) are multipotent cells capable of self-renewal and differentiating into multiple lineages such as osteocytes, adipocytes, chondrocytes, myoblasts, and hepatocytes. MSCs can migrate to the injured tissue and have immunomodulatory effects. Due to these features, MSCs have high therapeutic value in tissue engineering and regenerative medicine. In this thesis, our aim was to investigate the further contribution of the MSCs in different cellular therapies. We used two approaches to accomplish our aim. First, we investigated the possibility of obtaining functional cardiomyocytes from rat MSC within a shorter time period by determining the induction timing of cardiomyocyte differentiation of MSCs. Our data revealed 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. In the second part of this thesis, we examined in vivo and in vitro effects of a telomerase antagonist, imetelstat (GRN163L) on MSCs. 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. MSCs also show telomerase activity in maintaining their self-renewal; therefore the effects of telomerase inhibitors on MSCs may be an issue of concern. Our results showed that inhibiting the telomerase activity does not interfere with the self-renewal and differentiation of MSCs under short term in vitro culture conditions.Keskin, Zeynep TokcaerPh.D

Administration of bone marrow derived mesenchymal stem cells modulate tlr expression during liver regeneration

Liver cell transplantation is a powerful alternative to orthotopic cell transplantation in the treatment of liver failures. Recently, considerable effort is being channeled to understand the nature and kinetics of directing stem cells to effectively accumulate at the regenerating liver site. Mesenchymal stem cells are one of the promising cell sources modulating liver regeneration process. The present was designed to study how mesenchymal stem cells might modulate liver immune behaviors by changing Toll-like receptor (TLR) expression and increase regenerative potential during liver regeneration in rats. Normal and partially hepatectomized rats were treated with mesenchymal stem cells isolated and expanded from rat bone marrows. Accumulation of mesenchymal stem cells was confirmed by Real Time-Polymerase Chain Reaction (RT-PCR), Fluorescence-Activated Cell Sorting (FACS), and Immunofluorescence Staining (IFS). Student's t-test analysis was used to evaluate the significance of differences between sham and partially hepatectomized rat groups. Our results showed that mesenchymal stem cells expressed several TLRs, and their accumulation during regeneration was depended on the timing of injury. Mesenchymal stem cells isolated from bone marrow of normal rats were observed at the injured liver 3 days after the injection. There were no labeled mesenchymal stem cells in the liver sections of the uninjured animals. Mesenchymal stem cell administration significantly altered the expression of TLR2, 3 and 9 while retaining their migration potential to regenerating liver. Our findings implicated that mesenchymal stem cell administration during liver regeneration modulate the immune response through changing the expression of the TLRs in the remaining liver parts into which the cells are recruited or infused. This alteration may contribute to the regeneration process following partial hepatectomy.Karaciğer hücresi nakli, karaciğer yetmezliğinde ortotopik hücre nakline güçlü bir alternatiftir. Son yıllarda, kök hücrelerin doğalarının, kinetiklerinin ve yenilenen karaciğer bölgesinde etkili bir şekilde toplanmalarının sağlanmasının anlaşılması için hatırı sayılır gayretler sarf edilmektedir. Mesenkimal kök hücreler karaciğer yenilenme sürecini modüle eden ümit verici hücre kaynaklarından bir tanesidir. Bu çalışma, mezenkimal kök hücrelerin sıçanlarda toll benzeri reseptör (TLR) ifadesini değiştirmek suretiyle karaciğer immün yanıtını nasıl etkileyebildiklerini ve karaciğer yenilenmesi esnasında yenilenme potansiyelini arttırabildiklerini belirlemek için gerçekleştirilmiştir. Normal ve karaciğerleri kısmen çıkarılmış sıçanlar, sıçan kemik iliğinden elde edilip çoğaltılan mesenkimal kök hücreler ile muamele edilmişlerdir. Mesenkimal kök hücrelerin toplanması Eş Zamanlı Polimeraz Zincir Reaksiyonu (RT-PCR), Floresan Aktivite Hücre Ayırma (FACS), ve Immunfloresan Boyama (IFS) ile doğrulanmıştır. Sham ve karaciğeri kısmen alınmış sıçan grupları arasındaki farklılığın istatistiki analizinde Student's t-testi kullanılmıştır. Elde edilen sonuçlar mezenkimal kök hücrelerinde çeşitli TLR’lerin ifade edildiklerini ve bu hücrelerin yenilenme esnasında toplanmalarının meydana gelen hasarın zamanlamasına bağlı olduğunu göstermiştir. Normal sıçanların kemik iliğinden izole edilen mezenkimal kök hücreler hasarlı karaciğerde enjeksiyon sonrası 3. günde görülmüşlerdir. Hasarsız hayvanların karaciğer kesitlerinde işaretli bir mezenkimal kök hücre görülmemiştir. Mezenkimal kök hücre uygulaması TLR2, 3 ve 9'un ifadesinin anlamlı bir şekilde değiştirirken yenilenen karaciğere göç etme yeteneklerini devam ettirmişlerdir. Sonuçlar, karaciğer yenilenmesi esnasında mezenkimal kök hücre uygulamasının, hücrelerin uygulandığı hasarsız karaciğer parçalarında TLR’lerin ifadelerini değiştirme yoluyla immün yanıtı modüle ettiğini ortaya koymaktadır. TLR ifadesindeki bu değişim kısmı hepatoktemi sonrası yenilenme sürecine katkı sağlayabilir niteliktedir

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

Rapid differentiation of MSC into adipocytes and osteocytes is facilitated by Toll-Like receptor engagement

Mesenchymal stem cells are multipotent progenitor cells capable of differentiating into several lineages including adipocytes, and osteocytes. While they are regarded as non-immunogenic, accumulating evidence suggests that MSC expresses several TLRs. We checked the contribution of TLR ligands to MSC differentiation. Rat bone marrow derived MSC surface marker and TLR transcript levels were checked by FACS and RT-PCR respectively. MSCs were CD29hi, CD71dim, CD90hi, and negative for CD45 & CD34. They expressed TLR1,2,3,4,6, and low levels of 7 and 9 (no TLR5 was detected) at passage zero. Cells were incubated in adipogenic and osteogenic differentiation media either alone or supplemented with, TLR2L:PGN, TLR3L:pI:C, TLR7L:R848, and TLR9L:CpGODN as well as control ODN and followed for three weeks. Oil Red O staining (to assess adipogenesis) suggested that, induction in adipogenic media (by one week) gave no adipose positive cells whereas treatment with R848 or PGN induced significantly high adipose positive cells (1-2% vs 30-35%, respectively). The degree of adipogenic differentiation at wk1, was; TLR7≥TLR2>TLR3>TLR9>Control group≥medium. During osteogenesis (checked by alizarin red staining) by one week the highest differentiation was seen with CpG ODN treatment (TLR9≥TLR7>TLR2>TLR3>Control≥medium). These results strongly support the view that TLR ligands differentially contribute to transition of MSCs into either adipogenic or osteogenic differentiation

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