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Cardiovascular Research 2003 58(2):460-468; doi:10.1016/S0008-6363(03)00265-7
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Copyright © 2003, European Society of Cardiology

Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation?

Yu Liua, Jian Songa,b,*, Weixin Liua, Yu Wanc, Xichang Chena and Chengjun Hua

aFaculty of Anatomy and Embryology, Wuhan University School of Medicine, Wuhan, Hubei 430071, PR China
bCenter for Research in Structural Biology, Wuhan University School of Medicine, Wuhan, Hubei 430071, PR China
cDepartment of Physiology, Wuhan University School of Medicine, Wuhan, Hubei 430071, PR China

lyu_lyu{at}hotmail.com

s004184{at}sina.com

* Corresponding author. Department of Anatomy, Wuhan University School of Medicine, 135 Donghu Road, Wuhan, Hubei 430071, PR China. Tel.: +86-27-8733-1572; fax: +86-27-8730-7966.

Received 5 November 2002; accepted 29 January 2003


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The potential use of bone marrow stromal cells (MSCs) as a cellular therapy for chronic cardiac diseases relies on the ability of the cell to replicate extensively in vitro and to give rise to myogenic cells that can replace the damaged cardiomyocytes. For this reason the present study investigated the replication lifespan and chemical-induced cardiomyogenic differentiation of rat MSCs in vitro. Methods: The primary and the successively passaged Wistar rat MSCs were exposed to different concentrations (3, 5 and 10 µM) of 5-azacytidine using different methods (single- or repeat-treatment). The growth properties and the fate of the cells were compared to their untreated counterparts by cell counting, immunocytochemistry and Western analysis. Results: When seeded at a density of 2845 cells/cm2 and cultured under common conditions, rat MSCs could be expanded up to 21.94 cell doublings in 30 days of successive subcultures. This was accompanied by a gradual loss of their replication ability with passages. When treated with 5-azacytidine for 24 h at day 3 of primary culture and the first subculture, the growth properties of the MSCs were not obviously affected. Neither the spontaneously beating cells nor the formation of myotubes were found in the primary and first passaged MSCs after a single treatment with 5-azacytidine and in cultures which underwent repeated 5-azacytidine-treatments during continuous subculturing to passage 2. The expressions of cardiac troponin I, cardiac myosin heavy chain and connexin 43 by the 5-azacytidine-treated MSCs were also undetectable at both immunocytochemistry and Western blot levels. The specificity and reliability of the detection methods were technically confirmed with cultured rat cardiomyocytes. Conclusions: Rat MSCs cannot be extensively expanded in vitro or be induced to differentiate in an expected cardiomyogenic way by 5-azacytidine-treatment, if the cells are not immortalized.

KEYWORDS Experimental; Heart; Bone marrow stromal cells; Proliferation; Differentiation; 5-Azacytidine; Cell culture


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Since mammalian cardiomyocytes rarely regenerate after birth, a necrosis in myocardium is repaired by fibrous scar tissues. A progressive reduction of functional cardiomyocytes leads to heart failure—a final stage of the development of many chronic heart diseases [1]. Although heart transplantation has been a therapy for these cases for several decades, its usage is much limited by the shortage of donors and the host immuno-rejective reaction to the grafts.

As a newly developed strategy, cell transplantation, which aims to introduce healthy myogenic cells into the myocardium of the diseased heart, is showing a bright future in this field [2]. To date, a number of cell types, including skeletal myoblasts [3,4], fetal cardiomyocytes [5–7], smooth muscle cells [2], embryonic stem cells [8] and marrow stromal cells (MSCs) [9,10], have been employed for transplantation, and have shown benefits on heart functions in various animal models of myocardium injury. However, each of all these cell sources has its own inherent problems [2,11].

MSCs are nonhematopoietic multipotent stem-like cells that exist in bone marrow over the whole lifespan of mammals [12]. Considering their advantages, such as ease of obtaining by a simple routine bone marrow aspiration, ability to self-renew and recently reported potential to differentiate into cardiomyogenic cells [13], MSCs have been considered as one of the most promising candidates for this purpose.

The potential of MSCs to differentiate into myogenic cells was first reported by Wakitani et al. [14], and then by a number of other investigators [15,16]. More recently, a cardiomyogenic cell line was isolated from immortalized MSCs exposed to 5-azacytidine [13], followed by a report that primary cultured rat MSCs treated with 5-azacytidine were able to form myotubes and express myocardial specific proteins, such as cardiac troponin I and cardiac myosin heavy chain (MHC) [17]. These suggest that MSCs can be induced to differentiate into cardiomyocytes in vitro. In vivo studies also found that autologous MSCs transplanted into injured myocardium could differentiate into cardiac muscle-like cells [9,10,17] and establish intercalated discs with host cardiomyocytes [9]. Although these findings are really exciting, the underlying mechanisms are still unclear and the claim that adult stem cells can differentiate into multiple unexpected cell types is also questioned by some authors [18–21].

The use of MSCs in such therapeutic strategies relies on the ability of the cell to replicate extensively in vitro and to give rise to myogenic cells that can replace the damaged cardiomyocytes. For this reason the present study systematically examined the growth properties of rat MSCs in vitro and compared the fate of MSCs treated with 5-azacytidine to their untreated counterparts. Evidence shows that at least some of the reported in vitro characteristics of adult rat MSCs could not be confirmed.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The present investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Cell culture
MSCs were obtained from the femurae and tibiae of Wistar rats (140–160 g) with a modified method originally described by Dobson et al. [22]. To collect the cells more efficiently, the bones were mounted in microfuge tubes after their proximal ends were removed, and centrifuged at 2000 rpm for 2 min. The marrow pellet was washed in Hanks’ balanced salt solution (HBSS), centrifuged at 1000 rpm for 10 min and then resuspended in Dulbecco's modified Eagle's medium (DMEM) (Gibco, NY, USA). Nucleated cells were isolated with a density gradient (Ficoll/Paque) and cultured at 37°C in humid air with 5% CO2 in DMEM containing 20% fetal bovine serum (FBS) (Gibco), penicillin (100 U/ml), streptomycin (100 mg/ml) and amphotericin B (85 µg/ml). The medium was changed to remove the nonadherent cells at 24 h after seeding, and every 3 days thereafter. For subculture, cells were resuspended with 0.25% trypsin and passaged at a ratio of one to three plates. Since MSCs grows in colonies and cannot reach confluence over the whole dish, the criterion for passaging was that a half of colonies reached 70–80% confluence (usually 9–10 days after seeding for primary culture and 6–8 days for subcultures).

Cardiomyocytes, used as control cells in the present study, were prepared from Wistar rat neonates. Briefly, hearts were removed from 1- to 3-day-old rats and washed in HBSS. After being minced into fragments about 1 mm3 and rinsed several times in HBSS to remove blood and cellular debris, the tissue fragments were digested in successive changes of 0.1% collagenase for 10 min each at 37°C with gentle rocking. The digests after the second round of digestion were collected and resuspended in cold DMEM containing 5% FBS. The cell suspension was incubated in 90-mm dishes at 37°C for 2 h. Cardiomyocytes were recovered by collecting the nonadherent cells and plated at a density of 105 cells/ml. The medium was changed 12 h after seeding and every 2 days thereafter.

2.2 Quantification of MSC growth
To determine the initial density of MSCs in primary cultures, nonadherent cells were discarded by thoroughly washing three times with HBSS at 24 h after seeding. Cells were detached with 0.25% trypsin, and stained with 0.2% trypan blue. Triplicate samples from each well of 24-well plastic plates were resuspended in 2 ml HBSS and counted with a hemocytometer under a phase-contrast microscope. The average cell number calculated from four wells of each of three plates (0.2±0.06x104 cells/well; n = 12) was taken as the initial density of MSCs in primary culture and the seeding density for every following subcultures. This cell counting procedure was repeated every 24 h until the cells almost ceased proliferation (3 days after the first time point at which the cell number no longer significantly increased). The growth curves and the precise growth properties of MSCs in the primary culture (P0) and the successive four subcultures (P1–P4) were determined. The numbers of cell doublings (NCD) were calculated with the formula

Formula
where X represents cell numbers at the start and Y at the end of the logarithmic replication phase in every passage (see Results). Data were statistically analyzed with SIGMASTAT 1.02 using a t-test (Jandel Scientific, San Rafael, CA, USA).

2.3 Test of MSC differentiation induced by 5-azacytidine
To confirm if a chemical induction can trigger the differentiation of a MSC into a cardiomyocyte or a cardiac muscle-like cell, the primary and the first passaged MSCs were exposed to different concentrations (3, 5 and 10 µM) of 5-azacytidine (Sigma, St. Louis, MO, USA) for 24 h on day 3 of culture. In another group, a treatment with 5 µM 5-azacytidine was repeated in the same way in three continuous passages from the primary culture to passage 2. The dynamic changes in morphology and the growth properties were compared with the untreated cultures at indicated time points. The experiment was repeated in three 24-well plates with 12 wells for each treatment.

2.4 Immunocytochemistry
To determine if 5-azacytidine treatment can induce MSCs to express cardiac muscle specific or related proteins, an immunocytochemistry examination was performed with monoclonal antibodies against cardiac MHC {alpha}/β (Chemicon, Temecula, CA, USA), cardiac troponin I (Maxin Biochemistry, Fuzhou, China) and connexin 43 (Transduction Labs., Lexington, KY, USA). At day 14 and day 28 after 5 µM 5-azacytidine treatment in P0 and P1, and the last treatment in P2 (in repeat-treatment group), cells grown on glass coverslips in culture dishes were washed three times with PBS, permeabilized and fixed for 10 min with cold acetone (for connexin 43 detection) or 0.1% Triton X-100 in 4% paraformaldehyde (for cardiac MHC and troponin I detection). After being washed in PBS, cells were blocked with 1% BSA for 30 min, incubated with the primary antibodies at 4°C overnight, followed by a secondary antibody (FITC-conjugated anti-mouse IgG for cardiac MHC and troponin I detection, or horseradish peroxidase-conjugated anti-mouse IgG for connexin 43) for 1 h. Connexin 43 labelling was then visualized by DAB staining. Cultured cardiomyocytes and untreated MSCs were used as the positive and negative controls, respectively. The experiment was repeated in three different cultures (35-mm dish) with three coverslips for each antibody staining in each treatment.

2.5 Western blot analysis
MSCs after treatment with 5-azacytidine as described in Section 2.4, untreated MSCs cultured in 90-mm dishes and cultured cardiomyocytes as positive control were taken for Western blot analysis. The cells were washed with ice-cold PBS, scraped off the dish and transferred into centrifuge tubes. After centrifuging at 2000 rpm for 5 min at 4°C, the pellets were lysed in ice-cold lysing buffer (1 mM EDTA, 10 mM Hepes, 50 mM NaCl, 0.05% 2-mercaptoethanol, 0.5% Triton X-100, 5 µM leupeptin, and 10 µg/ml aprotinin). Following centrifugation at 8000 rpm for 10 min at 4°C, the supernatants were collected and stored at –65°C until use. Equal amounts of protein extracts (15 µg/lane) were subjected to SDS–PAGE on a 5% stacking gel and 8% separating gel. Duplicate gels were prepared with one for Coomassie staining and another for electrophoretical transferring of proteins onto PVDF membrane. After being blocked with 5% skim milk powder, 0.1% Tween 20 in TBS for 30 min, the membrane was incubated with a mixture of anti-cardiac MHC{alpha}/β and anti-cardiac troponin I monoclonal antibodies for 2 h at room temperature. After being washed, the membrane was incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG for 1 h followed by AEC staining.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Morphology and growth properties of MSC in culture
After discarding the nonadherent cells by the first medium change and by washing with HBSS three times at 24 h of primary culture, MSCs were seen to attach to culture dishes sparsely and the majority of the cells displayed a spindle-like shape (Fig. 1A). These cells began to proliferate at about day 4, and gradually grew to form small colonies (Fig. 1B). By day 7, the number of cellular colonies with different size had obviously increased (about 8–12 colonies/60-mm dish). In large colonies cells were more densely distributed and showed a spindle or triangle shape (Fig. 1C). As growth of cells continued, colonies gradually expanded in size with the adjacent ones interconnected with each other. However, the cultures failed to reach confluency by day 12, although the cells in the center of the colonies had formed several overcrowded layers (Fig. 1D) and nearly ceased proliferation at this time.


Figure 1
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  		Fig. 1 Phase-contrast micrographs of MSCs on day 1 (A), day 4 (B), day 7 (C) and day 12 (D) of primary culture, and on day 2 (E) and day 4 of passage 1 (F). (B) MSCs proliferated to form a small colony on day 4. (C) Large colony composed of densely distributed spindle (black arrow) and triangle (white arrow) shaped MSCs on day 7. (D) Center of a large colony with several overcrowded layers of MSCs on day 12. (E) Two types of MSCs seen on day 2 of passage 1, small spindle- or triangle-like cells (black arrow) and broad flattened cells (white arrow). (F) Flattened cells were surrounded by quickly proliferating spindle- or triangle-like cells in a MSC colony on day 4 of passage 1. Scale bar=50 µm.

 
Passaged MSCs behaved similarly to those in primary cultures. However, the cells were larger in size and more heterogeneous in morphology and in growth properties. Grossly, the MSCs in subcultures could be divided into two types: small spindle- or triangle-like, and broad flattened cells (Fig. 1E). The flattened cells seldom proliferated and were gradually surrounded by the small spindle- or triangle-like cells that replicated faster and were more inclined to form colonies (Fig. 1F). It seemed that the spindle- and triangle-like MSCs gradually transformed into broad flattened cells with passages. At the end of the first passage, about 15–18% of cells were flattened, but at the end of passage 4, at least more than 57% broad flattened cells were observed. This was accompanied by a progressive decrease in MSC proliferation and in the tendency of the cells to form colonies.

The growth curves of MSCs in primary culture and in the four successive passages are shown in Fig. 2. For primary cultures, the cells remained quiescent during the first 4 days of culture, and then quickly replicated until day 10 when the average cell number reached 15.37±0.10x104. The number of cells did not significantly increase during the following 4 days of culture (days 11–14), although the cells had not yet reached confluence over the culture dishes. The patterns of the growth curves of MSCs in passages 1 and 2 were similar to those of primary cells. However, the cells showed a shortened quiescent period (days 1–3) before proliferation and the cell number kept increasing significantly for a shorter period (days 4–7 in P1 and days 4–6 in P2). The increase of average cell numbers was considerably slowed in passage 3, and the cells almost ceased proliferation in passage 4.


Figure 2
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  		Fig. 2 Growth curves for MSCs in primary culture (P0) and 4 successive subcultures (P1, P2, P3, P4). The points on curves represent mean±S.D. *, P<0.05 compared with the previous adjacent time point in the same group.

 
To determine the changes in precise growth properties of MSCs with passages, the growth course of MSCs in the primary culture and in each successive subcultures was divided into three phases according their growth curves: the initial quiescent phase (from the seeding time to the last time point at which the cell number still did not significantly increase), the logarithmic replication phase (from the end of the initial quiescent phase to the first time point from which the cell number no longer significantly increased compared to the adjacent previous time point) and the phase after logarithmic replication. A comparison of the length of the initial and the logarithmic replication phases, the number of cell doublings (NCD) in different passages, and the accumulative NCD with passages are shown in Table 1.


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  		Table 1 Growth properties of MSCs in primary and passaged cultures

 
3.2 Effects of 5-azacytidine treatment on MSC growth and morphology
The primary and the first passaged MSCs treated with 3, 5 or 10 µM of 5-azacytidine were observed under the same culture conditions for control cells. Treatment of 5-azacytidine at any concentrations above did not obviously affect the growth properties of MSCs in both primary and passaged cultures (although the values of the average cell numbers after day 6 in P1 were slightly lower than those in other groups, these differences were not statistically important) (P>0.05; Fig. 3). Throughout the 30-day period of observation, there were no spontaneously beating cells, myotubes, and typical ball- or stick-like cells, which were suggested to be the myotube-forming cells [13], found in all 5-azacytidine-treated cultures.


Figure 3
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  		Fig. 3 Effect of different concentrations of 5-azacytidine on growth of cultured MSCs. The points on curves represent mean±S.D. There were no statistical importance in differences of average cell numbers between 5-azacytidine-treated and control cells.

 
3.3 Immunocytochemistry
Immunocytochemistry examination clearly detected the localizations of cardiac MHC and cardiac troponin I on the stress fibers with characteristic periodic striations (Fig. 4A and B), and connexin 43 at the interfaces between adjacent cells (Fig. 4C) in cardiomyocyte cultures. However, these proteins could not be detected in all 5-azacytidine-treated MSCs at day 14 and day 28 after treatment (Fig. 4D–I).


Figure 4
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  		Fig. 4 Immunocytochemistry detection for cardiac MHC, cardiac troponin I and connexin 43 in cultured MSCs. Representative photos of cardiomyocytes as positive controls (A–C), primary MSCs at day 28 after treatment with 5 µM 5-azacytidine (D–F) and repeatedly treated SMCs at day 14 after the last treatment with 5 µM 5-azacytidine in passage 2 (G–I) were shown. Cardiac MHC (A) and cardiac troponin I (B) visualized by FITC were located on the stress fibers with regular periodicity in cardiomyocytes; and connexin 43 stained by DAB (C) appeared at the interface between adjacent cardiac muscle cells. None of the three proteins were detectable in 5-azacytidine-treated MSCs (D–I). Scale bar=50 µm. Inserts are the phase-contrast micrographs of the same fields as (D), (E), (G) and (H), respectively.

 
3.4 Western blot analysis
SDS–PAGE showed that there were no visible differences in components of total proteins between 5-azacytidine treated and control MSCs (Fig. 5A, lanes 3–8), Immunoblot analysis could not detect expressions of cardiac MHC and cardiac troponin I in either 5-azacytine treated or untreated MSCs (Fig. 5B, lanes 3–8). The reliability of the analysis was confirmed with cultured cardiomyocytes under the same conditions (Fig. 5B, lane 2).


Figure 5
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  		Fig. 5 Western blot analysis for cardiac MHC and cardiac troponin I in 5 µM 5-azacytidine-treated MSCs in culture. (A) SDS–PAGE; (B) Immunoblots. Lanes: 1=molecular marker; 2=cultured cardiomyocytes; 3=primary MSCs at day 14 after treatment; 4=primary MSCs at day 28 after treatment; 5=untreated MSCs on day 17 of primary culture; 6=first passaged MSCs at day 14 after treatment; 7=repeatedly-treated SMCs at day 14 after the last treatment in passage 2; 8=untreated MSCs on day 17 of passage 2.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
MSCs were first described in 1968 by Friedenstein et al. [23], who discovered that MSCs adhered to tissue culture plates, resembled fibroblasts in morphology, and grew in the form of a colony [24]. These characteristics have been identified in MSCs from numerous species including human, rats, mice, rabbits and monkeys. However, the expandability of MSCs in vitro varied dramatically among different species and different methodologies for isolation and plating of the cells. It has been reported that murine MSCs were much more difficult to grow than other species [25,26] and this was probably due to the higher sensitivity of the murine MSC to the initial seeding density [27]; and if the cells were plated sparsely, for instance at a density of about 2 cells/cm2, they were able to be amplified up to 4000-folds in 12 days of culture [27]. Here, the present study showed that at a seeding density of about 2845 cells/cm2 (0.2x104 cells/well in 24-well plastic plates) and under the routine culture conditions, rat MSCs could be expanded to 11.85 cell doublings (about 3600-fold) after 17 days of culture (P0–P1) and up to a maximum of 21.94 cell doublings in 30 days. This resembles the reported in vitro lifespan of human MSCs (22–23 doublings beginning at primary culture [28] and 15, at passage 1 [29]). The main reason for the low yield of murine MSCs in early studies is probably due to the length of culture time for each passage, rather than the initial seeding density. MSCs grow in the form of colonies and the growth stages of cells in different colonies are widely different. It was frequently observed that large colonies with several overcrowded layers coexisted simultaneously with newly formed small ones. The cells in the former had already overgrown and become senescent while those in the latter were still in early stages of replication. In our pilot experiments (data not shown), it was found that if the cells were grown over 2 weeks before subculture, they quickly lost the ability to proliferate within 2–3 passages, and that if seeded at 2 cells/cm2, they grew very slowly with a considerably lengthened initial quiescent phase. It is known that proliferation of cultured cells is usually promoted when cells begin to release their own cytokines and inhibited by cell-to-cell contacts upon confluency. Thus, a much lower initial seeding density may delay the establishment of autocrine and paracrine of growth factors by MSCs in culture, and a long-time culture without passaging may result in the cells in the large colonies becoming senescent. In the present study the end of the logarithmic replication phase of the cells was chosen as the time point for cell passaging (usually 9–10 days after seeding for primary culture and 6–8 days for subcultures), at which time about half of the colonies had reached 70–80% confluence.

Although a more frequent subculture can delay the senescence of MSCs, they gradually lost the ability to proliferate with the increasing of cell doublings, and this was accompanied by a gradual morphological conversion of the small spindle- or triangle-like MSCs into the broad flattened cells. Together with the evidence from several other laboratories [28–30], this indicates that the in vitro expandability of MSCs as a whole population is quite limited. The short lifespan of MSCs in vitro would at least hinder the utility of the cell for gene-therapeutic strategies in which a much longer time for in vitro manipulation is needed to screen, select and expand the successfully transfected cells. On the other hand, it also raises the question as to how long the cells can maintain their ability to self-renew in the host heart after transplantation.

Another important question raised in the present study is if MSCs freshly isolated or in early passages are ready to be induced to differentiate into a cardiomyocyte or a cardiac muscle-like cell. Stem cells have been divided into two groups: embryonic stem cells that are pluri-potential, capable of generating all differentiated cell types in embryos, and organ- or tissue-specific stem cells that exist for a lifetime and are able to give rise to the cell types of the tissue in which they reside [18]. As a member of the second group, MSCs have been well documented to contain progenitors capable of multidifferentiating into lineages for skeletal tissues such as bone, cartilage, fat, tendon, ligament and marrow stroma [31–34]. However, a flurry of reports that adult MSCs can be induced in appropriate environments to differentiate into cells of other tissues in vitro or after transplantation has emerged recently [13,18,35,36]. These novel findings not only challenge the traditional stem cell biology, but also imply that MSCs have potential for wide clinical usages. The first report that MSCs could differentiate into cardiomyocytes came from Makino et al. [13]. In their studies, a cardiomyogenic cell line was established from immortalized MSCs by repeated treatments with 5-azacytidine and colony screenings for spontaneously beating cells. Following the report, Tomita et al. [17] claimed that after exposure to 5-azacytidine for 24 h, MSCs in primary culture could also differentiate into cardiomyocytes. However, this novel conversion of MSCs in vitro could not be confirmed in our study. We tested three different concentrations of 5-azacytidine (3, 5 and 10 µM), but neither the spontaneously beating cells nor the formation of myotubes were noted throughout 30 days of observation. The expressions of cardiac troponin I, cardiac MHC and connexin 43 by 5-azacytidine-treated MSCs were also undetectable at least at both immunocytochemistry and Western blot levels. The reason for the contradictory results is unknown. The specificity and reliability of the detection techniques in the present study were confirmed using cultured rat cardiomyocytes. The methods we employed for cell isolation and the components of culture medium are routinely used for MSC studies [9,14,17,22,29]. Although we slightly shortened the culture time for every passage, this should theoretically promote the induced myogenic differentiation of MSCs by delaying their senescence and their default osteogenic differentiations [37]. The dosage we tested covers the reported lowest to highest effective concentrations of this compound to induce the myogenic differentiation of MSCs without serious impact on their survival index [14]. We also mimicked different methods of treatment reported in the literature (single treatment for each passages from the primary culture to passage 1 and repeated treatments during continuous subculturing to passage 2). Thus, it seems difficult to interpret the discrepancies by technical differences alone.

The present study does not exclude the possibility that repeated treatments with 5-azacytidine may do this in an immortalized MSC cell line via reprogramming the cell's differentiation [13,38]. However, if this is so, the action of the drug may not be specific. As a cytosine analogue, 5-azacytidine has been reported to cause phenotype changes in a number of cell types by activating novel gene expressions both in vitro and in vivo [39]. Treatment with 5-azacytidine causes phenotypic conversions of fibroblasts to adipocytes and muscle cells [40] and induces erythroid differentiation in erytholeukemia cells [41]. The expression of {gamma}-globin gene in adult human and baboons is also found following administration of this drug [42,43]. Although the precise mechanisms for the action of 5-azacytidine are still unclear, it has been suggested that the cytosine analogue may activate some silent genes by inhibiting DNA methylation [39,41,44] or induce changes in some specific genes to trigger their response to exogenous regulators [39,45]. Thus, it is quite possible that the 5-azacytidine induced differentiation in immortalized MSC cell line is due to a random reprogramming of gene expression induced by the drug, rather than to the cell's potentiality of cardiomyogenic differentiation. This unpredictable differentiation of immortalized MSCs may imply unexpected behaviors of the cell if used in a therapeutic context.

In summary, the present study demonstrated that adult rat MSCs cannot be extensively expanded in vitro and be induced to differentiate to an expected cardiomyogenic way by 5-azacytidine treatment, if the cells are not immortalized. This implies that there is still a long way to go before the cells can be used for a cell/gene therapy for heart diseases.

Time for primary review 27 days.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Pfeffer M.A, Braunwald E. Ventricular remodeling after myocardial infarction: experimental observations and clinical implications. Circulation (1990) 81:1161–1172.[Abstract/Free Full Text]
  2. Reinlib L, Field L. Cell transplantation as future therapy for cardiovascular disease?: a workshop of the National Heart, Lung, and Blood Institute. Circulation (2000) 101:E182–E187.[Web of Science][Medline]
  3. Chiu R.C, Zibaitis A, Kao R.L. Cellular cardiomyoplasty: myocardial regeneration with satellite cell implantation. Ann Thorac Surg (1995) 60:12–18.[Abstract/Free Full Text]
  4. Taylor D.A, Atkins B.Z, Hungspreugs P, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med (1998) 4:929–933.[CrossRef][Web of Science][Medline]
  5. Delcarpio J.B, Claycomb W.C. Cardiomyocyte transfer into the mammalian heart: cell to cell interactions in vitro and in vivo. Ann NY Acad Sci (1995) 752:267–285.[Web of Science][Medline]
  6. Yoo K.J, Li R.K, Weisel R.D, et al. Heart cell transplantation improves heart function in dilated cardiomyopathic hamsters. Circulation (2000) 102(19 suppl 3):III204–III209.[Medline]
  7. Koh G.Y, Soonpaa M.H, Klug M.G, Field L.J. Long-term survival of AT-1 cardiomyocyte grafts in syngeneic myocardium. Am J Physiol (1993) 264(5 Pt 2):H1727–H1733.[Web of Science][Medline]
  8. Klug M.G, Soonpaa M.H, Koh G.Y, Field L.J. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest (1996) 98:216–224.[Web of Science][Medline]
  9. Wang J.S, Shum-Tim D, Galipeau J, et al. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J Thorac Cardiovasc Surg (2000) 120:999–1005.[Abstract/Free Full Text]
  10. Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation (2001) 104:1046–1052.[Abstract/Free Full Text]
  11. Paul G, Li J.Y, Brundin P. Stem cell: hype or hope? DDT (2002) 7:295–302.[Medline]
  12. Prockop D.J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science (1997) 276:71–74.[Abstract/Free Full Text]
  13. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from stromal cells in vitro. J Clin Invest (1999) 103:697–705.[Web of Science][Medline]
  14. Wakitani S, Saito T, Caplan A.I. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve (1995) 18:1417–1426.[CrossRef][Web of Science][Medline]
  15. Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science (1998) 279:1528–1530.[Abstract/Free Full Text]
  16. Gussoni E, Soneoka Y, Strickland C.D, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature (1999) 401:390–394.[CrossRef][Medline]
  17. Tomita S, Li R.K, Weisel R.D, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation (1999) 100(19 Suppl):II247–II256.[Medline]
  18. Anderson D.J, Gage F.H, Weissman I.L. Can stem cell cross lineage boundaries? Nat Med (2001) 7:393–395.[CrossRef][Web of Science][Medline]
  19. Morrison S.J. Stem cell potential: can anything make anything? Curr. Biol. (2001) 11:R7–R9.[CrossRef][Web of Science][Medline]
  20. Hawley R.G, Sobieski D.A. Somatic stem cell plasticity: to be or not to be. Stem Cells (2002) 20:195–197.[CrossRef][Web of Science][Medline]
  21. Wagers A.J, Sherwood R.I, Christensen J.L, Weissman I.L. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science (2002) 297:2256–2259.[Abstract/Free Full Text]
  22. Dobson K.R, Reading L, Haberey M, Marine X, Scutt A. Centrifugal isolation of bone marrow from bone: an improved method for the recovery and quantitation of bone marrow osteoprogenitor cells from rat tibiae and femurae. Calcif Tissue Int (1999) 65:411–413.[CrossRef][Web of Science][Medline]
  23. Friedenstein A.J, Petrakova K.V, Kurolesova A.I, Frolova G.P. Heterotopic transplants of bone marrow: analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation (1968) 6:230–247.[Web of Science][Medline]
  24. Friedenstein A.J, Deriglasova U.F, Kulagina N.N, et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol (1974) 2:83–92.[Web of Science][Medline]
  25. Clark B.R, Keating A. Biology of bone marrow stroma. Ann NY Acad Sci (1995) 770:70–78.[Web of Science][Medline]
  26. Phinney D.G, Kopen G, Isaacson R.L, Prockop D.J. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth and differentiation. J Cell Biochem (1999) 72:570–585.[CrossRef][Web of Science][Medline]
  27. Javazon E.H, Colter D.C, Schwarz E.J, Prockop D.J. Rat marrow stromal cell are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells. Stem Cells (2001) 19:219–225.[CrossRef][Web of Science][Medline]
  28. Banfi A, Muraglia A, Dozin B, et al. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol (2000) 28:707–715.[CrossRef][Web of Science][Medline]
  29. Digirolamo C.M, Stokes D, Colter D, et al. Propagation and senescence of human marrow stromal cells in culture:a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol (1999) 107:275–281.[CrossRef][Web of Science][Medline]
  30. Bruder S.P, Jaiswal N, Haynesworth S.E. Growth kinetics,self-renewal,and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem (1997) 64:278–294.[CrossRef][Web of Science][Medline]
  31. Friedenstein A.J, Chailakhyan R.K, Gerasimov U.V. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chamber. Cell Tissue Kinet (1987) 20:263–272.[Web of Science][Medline]
  32. Ashton B.A, Allen T.D, Howlett C.R, et al. Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin Orthop (1980) 151:294–307.[Medline]
  33. Johnstone B, Hering T.M, Caplan A.I, Goldberg V.M, Yoo J.U. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res (1998) 238:265–272.[CrossRef][Web of Science][Medline]
  34. Umezawa A, Maruyama T, Segawa K, et al. Multipotent marrow stromal cell line is able to induce hematopoiesis in vivo. J Cell Physiol (1992) 151:197–205.[CrossRef][Web of Science][Medline]
  35. Petersen B.E, Bowen W.C, Patrene K.D, et al. Bone marrow as a potential source of hepatic oval cells. Science (1999) 248:1168–1170.
  36. Kopen G.C, Prockop D.J, Phinney D.G. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brain. Proc Natl Acad Sci USA (1999) 96:10711–10766.[Abstract/Free Full Text]
  37. Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci (2000) 113(Pt 7):1161–1166.[Abstract]
  38. Fukuda A. Reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes. CR Biol. (2002) 325:1027–1038.
  39. Chiu C.P, Blau H.M. 5-Azacytidine permits gene activation in a previously noninducible cell type. Cell (1985) 40:417–424.[CrossRef][Web of Science][Medline]
  40. Taylor S.M, Jones P.A. Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell (1979) 17:771–779.[CrossRef][Web of Science][Medline]
  41. Creusot F, Acs G, Christman J.K. Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2'-deoxycytidine. J Biol Chem (1982) 257:2041–2048.[Abstract/Free Full Text]
  42. Ley T.J, DeSimone J, Anagnou N.P, et al. 5-Azacytidine selectively increases {gamma}-globin synthesis in a patient with β+ thalassemia. New Eng J Med (1982) 307:1469–1475.[Abstract]
  43. Papayannopoulou T, Torrealba deRon A, Veith R. Arabinosylcytosine induces fetal hemoglobin in baboons by perturbing erythroid cell differentiation kinetics. Science (1984) 224:617–619.[Abstract/Free Full Text]
  44. Jones P.A, Taylor S.M. Cellular differentiation, cytidine analogs and DNA methylation. Cell (1980) 20:85–93.[CrossRef][Web of Science][Medline]
  45. Clough D.W, Kunkel L.M, Davidson R.L. 5-Azacytidine-induced reactivation of a herpes simplex thymidine kinase gene. Science (1982) 216:70–73.[Abstract/Free Full Text]

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