Browsing by Subject "Mesenchymal stem cells"

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Open Access
Chitosan scaffolds with BMP-6 loaded alginate microspheres for periodontal tissue engineering
(2012) Soran, Z.; Aydin, R.S.T.; Gumusderelioglu, M.
The aim of this study is to develop an effective growth factor releasing scaffold-microsphere system for promoting periodontal tissue engineering. Bone morphogenetic protein-6 (BMP-6)-loaded alginate microspheres in narrow size distribution were produced by optimising electrospraying conditions. The addition of these microspheres to chitosan gels produced a novel scaffold in which not only the pore sizes and interconnectivity were preserved, but also a controlled release vehicle was generated. Loading capacity was adjusted as 50ng or 100ng BMP-6 for each scaffold and the controlled release behaviour of BMP-6 from chitosan scaffolds was observed during seven days. Cell culture studies were carried out with rat mesenchymal stem cells derived from bone marrow in three groups; chitosan scaffolds, chitosan scaffolds containing BMP-6-loaded alginate microspheres and chitosan scaffolds with free BMP-6 in culture medium. Results showed that controlled delivery of BMP-6 from alginate microspheres has a significant effect on osteogenic differentiation. © 2012 Informa UK Ltd All rights reserved.
Open Access
Chondrogenic differentiation of mesenchymal stem cells on glycosaminoglycan-mimetic peptide nanofibers
(American Chemical Society, 2016) Yaylaci, S .U.; Sen, M.; Bulut, O.; Arslan, E.; Güler, Mustafa O.; Tekinay, A. B.
Glycosaminoglycans (GAGs) are important extracellular matrix components of cartilage tissue and provide biological signals to stem cells and chondrocytes for development and functional regeneration of cartilage. Among their many functions, particularly sulfated glycosaminoglycans bind to growth factors and enhance their functionality through enabling growth factor-receptor interactions. Growth factor binding ability of the native sulfated glycosaminoglycans can be incorporated into the synthetic scaffold matrix through functionalization with specific chemical moieties. In this study, we used peptide amphiphile nanofibers functionalized with the chemical groups of native glycosaminoglycan molecules such as sulfonate, carboxylate and hydroxyl to induce the chondrogenic differentiation of rat mesenchymal stem cells (MSCs). The MSCs cultured on GAG-mimetic peptide nanofibers formed cartilage-like nodules and deposited cartilage-specific matrix components by day 7, suggesting that the GAG-mimetic peptide nanofibers effectively facilitated their commitment into the chondrogenic lineage. Interestingly, the chondrogenic differentiation degree was manipulated with the sulfonation degree of the nanofiber system. The GAG-mimetic peptide nanofibers network presented here serve as a tailorable bioactive and bioinductive platform for stem-cell-based cartilage regeneration studies.
Open Access
Contribution of toll-like receptors to mesenchymal stem cell differentiation and immunomodulation
(2010) Taş, İbrahim Fırat
characteristics along with several stem cell features such as lineage dependent differentiation and self-renewal capacity. MSCs are known to induce immunomodulatory activity and homing capacity to damaged tissue sites. Such diverse capabilities of MSCs make them distinct from adult stem cells and can be harnessed in several therapeutic applications. Toll-like receptors (TLR) can recognize conserved microbial byproducts and are mainly expressed by innate immune system cells as well as epithelial or endothelial cells. Recent findings suggest that in vitro generated MSCs express some of these pathogen recognition receptors. In our view, to broaden the breath of the therapeutic potential, TLR mediated activation of MSCs and demonstrate its impact on differentiation and immunomodulatory activity is critical. First, bone marrow-derived MSCs were generated and characterized via their surface marker expression by FACS (CD90, CD106 and CD45) at protein level and their message transcripts by RT-PCR (CD11b, CD29, CD34, CD45, CD71, CD73, CD90 and CD166). The most abundant marker was found to be CD90 over several passages. Following determination of TLR expression profile by RT-PCR, contribution of TLR ligands addition (TLR2, TLR3, TLR7 and TLR9) to MSCs during adipogenic or osteogenic differentiation was studied. TLR3 was found to be the most abundant type over several passages. The adipogenic differentiation of rMSCs was found to be facilitated in the presence of TLR2 TLR3 and TLR7 ligands. Additionally, changes in the adipogenic and osteogenic markers (LPL, PPAR-g for adipogenesis, and ALP, OC-1, RUNX for osteogenesis) were analyzed by RT-PCR. While adipogenic markers upregulated osteogenic markers were downregulated in response to TLR ligand treatment. The final part of this study was performed with mouse mesenchymal stem cells. In order to define the immunostimulatory/immunosuppressive potential of mouse MSCs, immunomodulatory character of MSCs were examined in the presence or absence of mouse spleen cells. Our data suggested that when mMSCs are primed with TLRL, a pro-inflammatory cascade as evidenced by increased IL-6 and IFN-γ secretion is initiated either alone or in co-culture with splenocytes. In conclusion, TLR priming of MSCs augments their differentiation primarily into adipogenesis, and mainly these cells are immunostimulatory.
Open Access
Development and characterization of peptide nanofibers for cartilage regeneration
(2015-09) Yaylacı, Seher
Articular cartilage is a tissue that is continuously exposed to cyclical compressive stresses, but exhibits no capacity for self-healing following trauma. Cartilage has a dense extracellular matrix that is sparsely populated with cells, and the whole tissue lacks blood and lymphatic vessels, which complicates the cell infiltration response that ordinarily occurs during inflammation. In addition, the only cell type capable of synthesizing new cartilage matrix lies deeper in the tissue, near the bone boundary, and due to the dense extracellular matrix, chondrocytes cannot migrate to the defect site following injury. Consequently, cartilage tissue cannot effectively respond to treatment options. Treatment options exist for the short-term reduction of pain in smaller defects, but larger injuries necessitate tissue donation, and there is a severe shortage of articular cartilage that can be donated for autografting. Microfracture and autologous chondrocyte implantation are the current treatment options that use cellular therapy for the repair of cartilage. However, the cartilage tissue that forms in the course of these treatments is not the functional hyaline cartilage, but rather fibrous cartilage, which is mechanically weaker and degenerates over time. Tissue engineering studies using biodegradable scaffolds and autologous cells are gaining importance as effective long-term treatment options for the postinjury production of hyaline cartilage. Such scaffold systems are designed to be biodegradable and bioactive, which allows them to induce new tissue formation in shorter periods of time. In this dissertation, peptide nanofibers mimicking glycosaminoglycan molecules, which are important constituents of cartilage extracellular matrix, are designed and the effectiveness of these materials in terms of chondrocyte differentation are tested under in vitro conditions. As a follow-up study to in vitro experiments, the capacity of bioactive peptide nanofibers to support cartilage regeneration is evaluated in the rabbit osteochondral defect model. Structural and mechanical properties of newly deposited cartilage are highly dependent on the quality and quantity of its extracellular matrix, which also has a major impact on the integration of replacement cartilage into the surrounding healthy tissue. Signals provided by bioactive peptide nanofibers to cells at the defect site can strongly alter the quality of the newly synthesized extracellular matrix. Consequently, we designed glycosaminoglycanmimetic peptide nanofibers that closely imitate the structure of the native cartilage extracellular matrix and demonstrated that these nanofiber networks are able to induce the synthesis of collagen II and aggrecan molecules, which are the main constituents of cartilage tissue, during chondrogenic differentiation.
Open Access
Effect of double growth factor release on cartilage tissue engineering
(2013) Ertan, A.B.; Yilgor P.; Bayyurt, B.; Çalikoǧlu, A.C.; Kaspar Ç.; Kök F.N.; Kose G.T.; Hasirci V.
The effects of double release of insulin-like growth factor I (IGF-I) and growth factor β1 (TGF-β1) from nanoparticles on the growth of bone marrow mesenchymal stem cells and their differentiation into cartilage cells were studied on PLGA scaffolds. The release was achieved by using nanoparticles of poly(lactic acid-co-glycolic acid) (PLGA) and poly(N-isopropylacrylamide) (PNIPAM) carrying IGF-I and TGF-β1, respectively. On tissue culture polystyrene (TCPS), TGF-β1 released from PNIPAM nanoparticles was found to have a significant effect on proliferation, while IGF-I encouraged differentiation, as shown by collagen type II deposition. The study was then conducted on macroporous (pore size 200-400μm) PLGA scaffolds. It was observed that the combination of IGF-I and TGF-β1 yielded better results in terms of collagen type II and aggrecan expression than GF-free and single GF-containing applications. It thus appears that gradual release of a combination of growth factors from nanoparticles could make a significant contribution to the quality of the engineered cartilage tissue. © 2011 John Wiley & Sons, Ltd.
Open Access
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
(Springer Science+Business Media, 2012) Ayaloglu-Butun, F.; Terzioglu-Kara, E.; Tokcaer-Keskin, Z.; Akcali, K. C.
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.
Open Access
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
(Springer Science+Business Media, 2010) Tokcaer-Keskin, Z.; Dikmen, Z. G.; Ayaloglu-Butun, F.; Gultekin, S.; Gryaznov, S. M.; Akcali, K. C.
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.
Open Access
Immunomodulatory potential of human umbilical cord tissue-derived mesenchymal stromal cell (UCX®) exosomes in combination with immunosuppressive “A151” oligodeoxynucleotide
(2019-07) Bulut, Özlem
Mesenchymal stromal or stem cells (MSCs) modulate immune responses apart from their regenerative capacities. Accumulating evidence suggests that MSCs exert their paracrine effects through extracellular vesicles known as exosomes. In this study, we utilized a particular human umbilical cord tissue-derived MSC type termed as UCX®. UCX® is superior to the gold-standard bone marrow-derived MSCs in terms of immunosuppressive properties. We aimed to characterize and employ UCX® exosomes as cell-free immunosuppressive therapeutic agents. Another aim was to compare the functionalities of exosomes either isolated from 2-dimensional (2D) cultures or isolated from 3-dimensional (3D) spheroid cultures. 3D culture provides better cell-to-cell and cell-to-matrix interactions thereby mimics the in vivo environment better. A synthetic oligodeoxynucleotide called A151 ODN, which consists of 4 repeats of the mammalian telomeric TTAGGG motif, has broad immunosuppressive effects. Delivery of A151 ODN within liposomes or exosomes protects it from degradation by nucleases and improve the desired outcome. We also aimed to combine the immunomodulatory potentials of UCX® exosomes and A151 ODN through direct loading of A151 ODN into exosomes with ~95% efficiency via a dehydration-rehydration-based lyophilization method. First, we determined the binding and internalization kinetics of exosomes with different immune cells. 3D-exosomes interacted with the target cells much faster and more efficiently. Next, we investigated how UCX® exosomes influence Toll-like receptor (TLR) signaling in mouse splenocytes and bone marrow-derived macrophages (BMDMs). 3D-exosomes compared to 2D-exosomes were more potent to suppress IFNγ, IL6, IL12, and to a lesser extent TNFα production mediated by TLR1/2, TLR4, TLR7/8 and TLR9 but not by TLR3 triggering. A151 ODN-loading to either 2D- or 3D-exosomes improved the inhibition of all the above mentioned pro-inflammatory cytokines. Especially 3D-exosomes downregulated co-stimulatory molecules CD80 and CD86 along with MHC-II on BMDMs following TLR stimulation. Macrophage polarization experiments revealed that UCX® exosomes reprogram BMDMs to produce high amounts of nitric oxide and arginase-1 which are the key immunomodulatory factors induced by myeloid-derived suppressor cells (MDSCs) to inhibit T- and NK-cell activity. Besides shifting macrophages to an MDSC-like suppressive phenotype, exosomes also supported expansion of MDSC populations in vivo upon intraperitoneal injection. Next, we tested the therapeutic efficiency of UCX® exosomes with or without A151 ODN in zymosan-induced peritonitis and dextran sodium sulfate (DSS)-induced colitis models in mice. Exosomes could not alleviate zymosan-induced peritonitis which is an acute and severe inflammation. However, 3D-exosomes and A151 ODN-loaded versions of both 2D- and 3D-exosomes remarkably prevented DSS-induced colitis progression. A151 ODN itself was also therapeutic, albeit to a lesser degree. Standalone 3D-exosomes and A151 ODN-loaded exosomes prevented weight loss and colon shortening. All treatments except for 2D-exosomes could restore DSS-induced loss of T-cell numbers and cytokine-producing capacities in mesenteric lymph nodes and spleen. All treatments except for A151 ODN prevented DSS-induced macrophage accumulation in the lymph nodes. 3D-exosomes and A151 ODN-loaded versions of both exosomes normalized serum IL6 levels while only A151 ODN-loaded 3D-exosomes could impact the cytokine production capacities of macrophages. Finally, we tested the effects of UCX® exosomes with or without A151 ODN on wound healing. In vitro, 2D- and 3D-exosomes differentially upregulated the productions of wound healing-related cytokines and growth factors such as IL1α, TGFβ and VEGFα from fibroblast and keratinocyte cell lines. In vivo, in an excisional wound healing model, free or A151 ODN-loaded exosomes did not accelerate wound closure. However, they caused systemic immunosuppression at the late stages of wound healing. Systemic outcomes include reduced inflammatory capacity of macrophages and higher granulocytic MDSC numbers in spleen. A151 ODN-loaded 3D-exosomes also reduced T-cell numbers in spleen and pro-inflammatory cytokine levels in circulation. Taken together, this study revealed that 2D- but more importantly 3D-culturing of umbilical cord MSCs result in functionally different exosomes, 3D culture-derived exosomes display higher immunosuppressive potential, A151 ODN-loading into these exosomes improves immunosuppressive capacity and A151 ODN-loaded UCX® exosomes could be a valuable therapeutic agent for inflammatory and autoimmune disorders.
Open Access
Liver regeneration : potential roles of mesenchymal stem cells and toll like receptors
(2008) Koçak, Hande
Akçalı
Open Access
Mesenchymal stem cell derived extracellular vesicles: promising immunomodulators against autoimmune, autoinflammatory disorders and SARS-CoV-2 infection
(Scientific and Technical Research Council of Turkey, 2020) Bulut, Özlem; Gürsel, İhsan
Discovery of novel and broad-acting immunomodulators is of critical importance for the prevention and treatment of disorders occurring due to overexuberant immune responseincluding SARS-CoV-2 triggered cytokine storm leading to lung pathology and mortality during the ongoing viral pandemic. Mesenchymal stem/stromal cells (MSCs), highly regarded for their regenerative capacities, also possessesremarkable immunoregulatory functions affecting all types of innate and adaptive immune cells. Owing to that, MSCs have been heavily investigated in clinic for the treatment of autoimmune and inflammatory diseases along with transplant rejection. Extensive research in the last decaderevealed that MSCs carry out most of their functions through paracrine factors which are soluble mediators and extracellular vesicles (EVs). EVs, including exosomes and microvesicles, are an efficient way of intercellular communication due to their unique ability to carry biological messages such as transcription factors, growth factors, cytokines, mRNAs and miRNAs over long distances. EVs originate through direct budding of the cell membrane or the endosomal secretion pathway and they consist of the cytosolic and membrane components of their parent cell. Therefore, they are able to mimic the characteristics of the parent cell, affecting the target cells upon binding or internalization. EVs secreted by MSCs are emerging as a cell-free alternative to MSC-based therapies. MSC EVs are being tested in preclinical and clinical settings where they exhibit exceptional immunosuppressivecapacity. They regulate the migration, proliferation, activation and polarization of various immune cells, promoting a tolerogenic immune response while inhibiting inflammatory response. Being as effective immunomodulators as their parent cells, MSC EVs are also preferable over MSC-based therapies due to their lower risk of immunogenicity, tumorigenicity and overall superior safety. In this review, we present the outcomes of preclinical and clinical studies utilizing MSC EVs as therapeutic agents for the treatment of a wide variety of immunological disorders.
Open Access
Mesenchymal stem cell mechanics on osteoinductive peptide nanofibers
(2017-07) Topal, Ahmet Emin
In this thesis, changes in Young's modulus of mesenchymal stem cells (MSCs) were investigated during their osteogenic differentiation on bioactive peptide nanofibers that bear triple glutamic acid sequence (EEE), a non-collagenous protein sequence of some extracellular matrix (ECM) proteins (e.g. bone sialoprotein) found in bone tissue. MSCs formed spherical cell aggregates on the osteoinductive peptide nanofibers, here also called osteospheroids, of which their cells made intensive cell-cell contacts and showed osteoblast-like cell morphology. Mechanical characterization of the osteospheroids on the peptide nanofiber hydrogel was performed using atomic force microscope (AFM) where AFM probes modified with a thin film coating of octa uorocyclobutane (C4F8) were used to measure force maps of the cells at days 3, 7 and 14 of osteogenic differentiation. Hertz Cone model, same as Sneddon, was applied to approach curves of 12 force curves per cell to calculate the Young s modulus values. As a result, an increasing pattern was observed in the average Young's modulus of rMSCs on the peptide nanofibers throughout the osteogenic differentiation. Mineral deposition by the cells on the peptide nanofibers was checked by Alizarin red staining and a large amount of mineral deposition by the osteospheroids was observed, proving that an efficient osteogenic differentiation of the rMSCs happens on the osteoinductive peptide nanofibers. In the literature, a gradual decrease is shown in AFM measured average Young's modulus values of adhered MSCs throughout their osteogenic differentiation. When gelatin-coated glass was used as substrate instead of the peptide nanofibers, rMSCs showed a decreasing pattern in their Young's moduli, similarly to the literature, due to osteogenic differentiation. However, there was no significant calcium mineral deposition on the gelatin group, even until day 14 of osteogenic differentiation, indicating that a limited or a slower progression of osteogenic differentiation of rMSCs is present on the gelatin compared to rMSCs on the osteoinductive peptide nanofibers. A correlation between the observed increase in average Young's moduli of osteospheroid rMSCs and their mineral production on the peptide nanofibers may suggest a predominant role of biomineralization on the cell mechanics.
Open Access
Nanomechanical characterization of osteogenic differentiation of mesenchymal stem cells on bioactive peptide nanofiber hydrogels
(Wiley-VCH Verlag, 2017-08) Topal, A. E.; Tansik, G.; Ozkan A.D.; Güler, Mustafa O.; Dana, A.; Tekinay, A. B.
Stem cell differentiation is known to be influenced by the mechanical properties of the surrounding extracellular matrix (ECM); however, little is known about the mechanical phenotypes of differentiating stem cells within the ECM. Here, this study uses osteoinductive, ECM-mimetic peptide nanofibers to investigate the changes in the mechanical properties of rat mesenchymal stem cells (rMSCs) during osteogenic differentiation. In addition, octafluorocyclobutane (C4F8)-coated atomic force microscopy (AFM) cantilevers are developed to minimize tip–sample adhesion during the nanomechanical characterization of rMSCs, and osteogenic differentiation is monitored through molecular analysis in conjunction with AFM measurements. rMSCs cultured on osteoinductive peptide nanofibers differentiate at substantially higher rates, form osteogenic cell clusters, deposit calcium to the surrounding matrix, and strikingly increase their Young's moduli throughout the osteogenic differentiation process compared to controls. These results show that the elasticity profiles of differentiating rMSCs may change significantly depending on environmental factors and especially the degree of biomineralization, and that the natural elasticity responses of cells cultured on scaffolds may be considerably different from those observed on non-bioactive surfaces. This is important for the identification of cell elasticity as a biophysical marker of osteogenic differentiation of MSCs, and indicates that biomineralization might have a predominant role on cell mechanics.
Open Access
Preparation and characterization of poly(hydroxyethyl methacrylate-co-poly(ethyleneglycol-methacrylate)/hydroxypropyl-chitosan) hydrogel films: Adhesion of rat mesenchymal stem cells
(Hangug Gobunja Haghoi, 2010-12-02) Bayramoglu, G.; Akcalı, K. C.; Gultekin, S.; Bengu, E.; Arica, M. Y.
This study examined the effects of the surface properties of the materials, such as the hydroxyl, methyl and amino groups, on rat bone marrow derived Mesenchymal Stem Cell (MSC) seeding. A series of hydrogels were prepared in film form using 2-hydroxyethyl methacrylate (pHEMA), poly(ethyleneglycol) methacrylate (PEG-MA), and/or hydroxypropyl-chitosan (HPC). The physicochemical properties of these hydrogel films, such as water content, functional groups, contact angle, surface energy and thermal properties were affected by the composition of the materials. The ability of the MSCs to form colonies, as well as their viability on these materials were also analyzed. The water content of the hydrogel films increased with increasing PEG-MA and HPC ratio in the hydrogel. Contact angle measurements of the surface of the hydrogel films demonstrated that all the materials gave rise to a significantly hydrophilic surface compared to pure pHEMA. The blood protein interactions and platelet adhesion were reduced significantly on the surface of the materials upon the incorporation of PEG-MA compared to the control pure pHEMA and vice versa for HPC. The ability of the MSCs to adhere and form colonies on these materials was also analyzed. The results showed that these materials are suitable candidates to isolate and expand MSCs.
Open Access
Timing of induction of cardiomyocyte differentiation for in vitro cultured mesenchymal stem cells: a perspective for emergencies
(NRC Research Press, 2009) Tokçaer-Keskin, Zeynep; Akar, A. R.; Ayaloğlu-Bütün, Fatma; Terzioğlu-Kara, Ece; Durdu, S.; Özyurda, U.; Uğur, M.; Akçalı, Kamil C.
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.