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Skeletal Myogenic Differentiation of Mesenchymal Stem Cells Isolated from Human Umbilical Cord Blood

Research Institute of Biotechnology, Histostem Co. Kangdong-gu, Seoul, Korea

Key Words. Human umbilical cord blood • Mesenchymal stem cells • Immunophenotyping • Myogenic differentiation

Correspondence: Hoeon Kim, Ph.D., Research Institute of Biotechnology, Histostem Co. 518-4 Taijul Bldg, Doonchundong, Kangdong-gu, Seoul 134-060, Korea. Telephone: 82-2-470-9773; Fax: 82-2-470-6342; e-mail: hoeonkim{at}seoulcord.co.kr

	ABSTRACT

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Human umbilical cord blood (UCB) has been regarded as an alternative source for cell transplantation and cell therapy because of its hematopoietic and nonhematopoietic (mesenchymal) potential. Although there has been debate about whether mesenchymal stem cells (MSCs) are invariably present in UCB, several reports showed that MSC-like cells could be consistently derived from human UCB and, moreover, could differentiate into various cells of a mesodermal origin. However, it remains unclear whether these UCB-derived MSCs are also capable of differentiating into skeletal muscle cells. In this study, we isolated MSCs from human UCB and induced them to differentiate into skeletal muscle cells. During cell culture expansion, UCB-derived mononuclear cells gave rise to adherent layers of fibroblast-like cells expressing MSC-related antigens such as SH2, SH3, -smooth muscle actin, CD13, CD29, and CD49e. More important, when these UCB-derived MSCs were incubated in promyogenic conditions for up to 6 weeks, they expressed myogenic markers in accordance with myogenic differentiation pattern. Both flow cytometric and reverse transcriptase–polymerase reaction analyses showed that two early myogenic markers, MyoD and myogenin, were expressed after 3 days of incubation but not after 2 weeks. At week 6, more than half of UCB-derived MSCs expressed myosin heavy chain, a late myogenic marker. Our results demonstrate that UCB-derived MSCs possess a potential of skeletal myogenic differentiation and also imply that these cells could be a suitable source for skeletal muscle repair and a useful tool of muscle-related tissue engineering.

	INTRODUCTION

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Bone marrow (BM) has been regarded as a good source of both hematopoietic stem/progenitor cells and mesenchymal stem cells (MSCs) [1–5]. These stem cells have the capacity for self-renewal and differentiating into cells of multiple lineages. MSCs derived from BM are capable of not only supporting hematopoiesis but also differentiating into mesodermal layer cells such as osteoblasts, chondrocytes, adipocytes, and myoblasts [6–8]. However, the process to collect BM is invasive to donors and can cause complications such as infection, bleeding, and chronic pain, thereby limiting a wide application of BM-derived MSCs in tissue engineering and cell therapy.

In recent decades, human umbilical cord blood (UCB) has been explored as an alternative source to BM for cell transplantation and cell therapy because of its hematopoietic and nonhematopoietic (mesenchymal) components. In contrast to BM aspiration, human UCB is obtained by a simple, safe, and painless procedure when the baby is delivered. Since the late 1980s, UCB has become an indispensable source of hematopoietic stem/progenitor cells for transplantation of hematopoietic stem cells to treat some hematological disorders [9–14]. However, human UCB has been controversial for the presence of MSCs; some researchers successfully isolated MSCs from UCB, whereas others have not [15–18]. Nevertheless, several groups reported that the UCB-derived MSCs could proliferate ex vivo and differentiate, at least, into osteoblasts and adipocytes [17–19]. No evidence has shown yet that UCB-derived MSCs differentiate into skeletal myoblasts, but they are believed to have such a potential.

Myogenic differentiation is regulated by a family of myogenic regulatory factors (MRFs), including Myf5, MyoD, myogenin, and MRF4; MyoD and Myf5 are required for the determination of skeletal myogenic lineages, whereas myogenin and MRF4 are thought to regulate cell fusion and terminal differentiation [20–23]. In postnatal life, the satellite cells located between muscle fiber sarcolemma and basal lamina are quiescent myoblasts, but they are fully determined to myogenic phenotype so that, once activated, they are capable of terminal differentiation [24–27]. The quiescent satellite cells do not express transcription factors of a MRF family, whereas the activated ones exhibit a battery of molecular markers of Myf5, MyoD, and, to a lesser extent, myogenin. These satellite cells were once regarded as an ideal source for muscle regeneration and repair, but it turned out that they were few in injured muscle and that they were exhausted immediately during healing processes. A search for an alternative source with equivalent myogenic potential yielded MSCs not long ago when the BM-derived MSCs were shown to expand in vitro and differentiated successfully into myoblasts [8].

In this paper, we report that fibroblast-like cells from human UCB, exhibiting mesenchymal phenotypes, are also able to differentiate into cells that express several skeletal muscle–specific genes. Our findings implicate that UCB is a potential source of MSCs for therapy of degenerative muscular diseases or muscle damage/loss from trauma.

	MATERIALS AND METHODS

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Human UCB Harvest and Preparation of MSCs
Human UCB samples were harvested from term or preterm deliveries at the time of birth with the mother’s consent. Blood samples were processed within 24 hours of collection. The mononuclear cells were separated from UCB using Ficoll-Paque^TM PLUS (Amersham Biosciences, Uppsala, Sweden, http://www.amershambiosciences.com) and were suspended in culture medium (low-glucose Dulbecco’s modified Eagle’s medium [DMEM; GIBCO, Grand Island, NY, http://www.lifetech.com] containing 15% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate). The cells were then seeded at a density of 1 x 10⁶ cells/cm² in culture flasks. After 5 days of culture, suspended cells were removed and adherent cells were additionally cultured. Cultures were maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide, with a change of culture medium every 5 days. Approximately 50%–60% of confluent cells were detached with 0.1% trypsin–EDTA and replated at a density of 2 x 10³/cm² in culture flasks.

Immunophenotyping of MSCs
To detect surface antigens, cells were detached and washed with phosphate-buffered saline (PBS; Jeil Biotechservices Inc; Daegu, Korea, http://www.jbilife.com) and incubated at 4°C for 30 minutes with the following cell-specific antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (Becton Dickinson, San Jose, CA, http://www.bdbiosciences.com): SH2 (CD105, endoglin), SH3 (CD73), CD13, CD29 (ß 1 integrin), CD44, CD49e (₅ integrin), CD54 (ICAM-1), CD90 (Thy-1), CD14, CD34, CD45, CD31, CD49d (₄ integrin), CD106 (VCAM-1), HLA-ABC, and HLA-DR. For staining with monoclonal mouse anti-human -smooth muscle actin (ASMA; Sigma, St. Louis, http://www.sigmaaldrich.com) antibody, cells were first permeabilized with cold methanol/PBS for 2 minutes at –20°C. After wash with cold PBS, cells were incubated with mouse anti-ASMA antibody at 4°C for 30 minutes followed by staining with a secondary antibody, anti-mouse-IgG-FITC (Becton Dickinson), for an additional 20 minutes. Labeled cells were analyzed by flow cytometry (Beckman Coulter Epics XL; Miami, http://www.beckmancoulter.com).

Proliferation Studies and Cell-Cycle Analysis
For proliferation studies, cells were detached and replated at a density of 1 x 10⁴/ml in culture medium. Viable cells were counted by trypan blue exclusion. As for cell-cycle analysis, cells were detached and washed with cold PBS and fixed with 70% ethanol/PBS overnight at 4°C and centrifuged. The pellets were resuspended in 500 µl PBS in the presence of propidium iodide (50 µg/ml) and DNase-free RNase A (1 mg/ml) and incubated in the dark for 30 minutes at room temperature. Cell-cycle status was determined using fluorescence-activated cell sorting (FACS) flow cytometry and analyzed with MultiCycle software for the proportions of cells in G₁, S, and G₂/M phase.

FACS Analysis of Skeletal Myogenic Differentiation
Skeletal myogenic differentiation was induced by culturing MSCs in myogenic medium (culture medium supplemented with 5% horse serum, 0.1 µM dexamethasone, and 50 µM hydrocortisone) for up to 6 weeks, as described previously by Zuk and colleagues [28–30]. Myogenic differentiation was analyzed with FACS for MyoD1, myogenin, and myosin heavy chain (MyHC). For FACS, cells were detached and stained sequentially with primary antibodies (mouse-anti MyoD and anti-myogenin antibodies; Becton Dickinson) and FITC-conjugated secondary antibodies (FITC-rat anti-mouse IgG1; Becton Dickinson). Cells were fixed with 2% formaldehyde until analysis with FACS. For detection of an intracellular protein MyHC, cells were permeabilized with cold methanol/PBS for 2 minutes at –20°C before staining with primary mouse anti-myosin (fast, Sigma) and FITC-conjugated secondary antibody.

Immunocytochemical Staining
Cells were plated at a density of 5 x 10³/well onto cover slides in six-well plates overnight and induced in myogenic medium for up to 6 weeks. For immunochemical staining, cells were washed twice with cold PBS and fixed with 4% paraformaldehyde/PBS for 20 minutes at 4°C. Staining was performed using mouse ABC Staining System (Santa Cruz Biotechnology; Santa Cruz, CA, http://www.scbt.com) according to the manufacturer’s protocol. Briefly, cells on slides were incubated with mouse-anti MyoD and anti-myogenin antibodies, respectively, overnight at 4°C after blocking with 1.5% blocking serum in PBS. Slides were washed and incubated with biotinylated secondary antibody at 1 µg/ml for 30 minutes at room temperature. The secondary antibody was detected by avidin and biotinylated horseradish peroxidase for 30 minutes followed by incubating in peroxidase substrate until desired stain intensity developed. The slides were counterstained with hematoxylin (Sigma) for 10 seconds and observed under a light microscope.

mRNA Detection by Reverse Transcription–Polymerase Chain Reaction
To detect mRNA levels of MyoD, myogenin, and myosin, cells were harvested and washed once in PBS. Total RNA was extracted using RNeasy Mini kit (QIAGEN, Hilden, Germany, http://www.qiagen.com) according to the manufacturer’s protocol. The first-strand complementary DNA (cDNA) was synthesized using RNA polymerase chain reaction (PCR) kit (Takara Bio Inc; Shiga, Japan, http://www.takara-bio.co.jp). The PCR mixture was amplified using DNA Engine Dyad^TM Peltier Thermal Cycler (MJ Research^TM, Inc; Waltham, MA, http://www.mjr.com) with initial denaturation at 95°C for 5 minutes, followed by 35 cycles at 95°C for 1 minute, 60°C for 1 minute, 72°C for 1.5 minutes, and, finally, 72°C for 7 minutes. The sense and antisense primers used in this study were as follows: MyoD, sense 5'-AAG CGC CAT CTC TTG AGG TA-3' and antisense 5'-GCG CCT TTA TTT TGA CC-3' (PCR product, 500 bp) [29]; myogenin, sense 5'-TAA GGT GTG TAA GAG GAA GTC G-3' and antisense 5'-CCA CAG ACA CAT CTT CCA CTG T-3' (PCR product, 438 bp) [30]; and myosin heavy chain, sense 5'-TGT GAA TGC CAA ATG TGC TT-3' and antisense 5'- GTG GAG CTG GGT ATC CTT GA-3' (PCR product, 750 bp) [31]. Identical PCR conditions were used to amplify GAPDH cDNA that was used as an internal control; the sense primer for GAPDH was 5'-CCC ATC ACC ATC TTC CAG GA-3', and the antisense primer was 5'-TTG TCA TAC CAG GAA ATG AGC -3' (PCR product, 731 bp) [32]. The amplified cDNAs (10 µl) were separated on 1.2% agarose gels, and the bands were visualized by ethidium bromide and photographed with Chemi Doc XRS (Bio-Rad Laboratories, Hercules, CA, http://www.bio-rad.com).

Immunoblotting
Cells cultured for the indicated times were washed with cold PBS and lysed in 400 ml of an ice-cold Tris lysis buffer (0.01 M Tris, pH 7.5, 0.1 M NaCl, 1% Triton X-100, 0.5% sodium deoxychloate, and 0.1% sodium dodecyl sulfate) with added protease inhibitors. Cell lysates were centrifuged at 12,000 rpm for 10 minutes after sonication for 30 seconds. Protein concentration was measured using the BioRad protein assay kit (BioRad) after boiling for 5 minutes. Forty micrograms of protein was mixed with x 1 SDS-sample buffer (62.5 mM Tris-HCl, pH 6.7, 2% SDS, 10% glycerol, 50 mM 2-mercap-toethanol, 0.1% bromophenol blue), boiled for 5 minutes, and then subjected to 5% SDS-polyacrylamide gel before being electrotransferred to a Hybond^TM-C extra nitrocellu-lose membrane (Amersham Biosciences). The membrane was blocked with 5% nonfat dry milk in x 1 TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature, and the blots were incubated with mouse anti-MyoD monoclonal antibody, mouse anti-myogenin monoclonal antibody, mouse anti-skeletal myosin, and mouse anti–-actin monoclonal antibody. This was followed by incubation with anti-mouse horseradish peroxidase–conjugated IgG (Amersham Biosciences). Protein visualization was performed using the enhanced chemiluminescence detection system (Amersham Biosciences) according to the manufacturer’s protocol.

	RESULTS

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Characteristics of UCB-Derived Adherent Cells
The mononuclear cells were obtained from UCB by Ficoll-Paque density gradient centrifugation and plated in the culture flasks. After 5 days of culture, nonadherent cells were removed by medium change. The adherent cells were small and rounded in shape. These cells grew larger and seemed to be comprised of heterogeneous cells, as judged by their appearance. The elongated cells began to appear among rounded cells between 8 and 15 days of culture, and they continued to grow to become fibroblast-like cells. By two or three passages of culture, the adherent cells became a population comprised mainly of bipolar fibroblast-like cells and could grow to confluency.

We examined the proliferation characteristics of the fibroblast-like cells at the fourth passage. The population-doubling time of cells is approximately 60 hours, as determined by viable counting. FACS analysis showed that 86% of cells were in the phase of G₀/G₁.

Immunophenotyping of UCB-Derived Adherent Cells
To characterize the adherent cell population derived from UCB, expression of a variety of CD markers and intracellular antigens like ASMA was examined by flow cytometry. Those adherent cells expressed CD13, CD29 (ß 1 integrin), CD44, CD49e (5 integrin), CD54 (ICAM-1), CD90 (Thy-1), ASMA, CD105/SH2/endoglin, and CD73/SH3 (Fig. 1). Among these, SH2 and SH3 are well known as MSC-specific antigens. They expressed neither hematopoietic lineage markers such as CD34 nor monocyte-macrophage antigens such as CD14 and CD45 (Fig. 1). The lack of expression of CD14, CD34, and CD45 suggests that cell cultures were depleted of hematopoietic cells during subcultivation.

View larger version (27K): [in this window] [in a new window]   Figure 1. Immunophenotyping of umbilical cord blood–derived mesenchymal stem cells. Mesenchymal stem cells were detached, labeled with FITC- or phycoerythrin-conjugated monoclonal antibodies, and detected by flow cytometry. Relative number cells (counts) versus fluorescence intensity are presented. Abbreviations: ASMA, -smooth muscle actin; FITC, fluorescein isothiocyanate.

FACS and Reverse Transcription–PCR Analyses of Myogenic Differentiation
A potential of UCB-derived MSCs differentiating into osteoblasts, chondrocytes, and adipocytes was demonstrated elsewhere [17–19 and unpublished data]. To investigate whether UCB-derived MSCs show a potential to differentiate into skeletal muscle cells, MSCs were cultured for up to 6 weeks in myogenic medium containing dexamethasone and hydrocortisone. At different time intervals, treated cells were observed by phase-contrast microscopy and then analyzed by flow cytometry with monoclonal antibodies against two muscle-specific transcription factors, MyoD and myogenin, as well as a skeletal protein, fast-twitch myosin. At week 1, MyoD and myogenin were expressed in approximately 8.7% and 90% of the treated cells, respectively, whereas non-treated cells remained unstained against anti-MyoD and anti-myogennin antibodies (Fig. 2A). However, the expression of MyoD and myogenin quickly vanished from week 2. This result is consistent with the fact that the two factors are involved in early myogenesis.

View larger version (28K): [in this window] [in a new window]   Figure 2. Myogenic differentiation of MSCs isolated from human umbilical cord blood. (A): After 1 and 2 weeks of induction in myogenic medium, cultures were analyzed by FACS for MyoD1 and myogenin. (B): Cells were incubated with myogenic medium for up to 6 weeks and were then analyzed by FACS for fast-twitch myosin. (C): MyoD1, myogenin, and myosin heavy chain mRNA levels were measured by reverse transcription–polymerase chain reaction during MSC differentiation in myogenic medium. For corresponding controls, cells were cultured in control medium (culture medium with no addition of dexamethasone and hydrocortisone). Abbreviations: FACS, fluorescence-activated cell sorting; MSC, mesenchymal stem cell.

Skeletal myoblast differentiation of UCB-derived MSCs was also analyzed by semiquantitative reverse transcription (RT)-PCR of MyoD, myogenin, and MyHC. None of these factors were significantly expressed in the cells treated with nonmyogenic medium. In the case of the cells treated with myogenic medium, however, the mRNA levels of both MyoD and myogenin were significantly increased after 3 days (Fig. 2C). At week 1, the mRNA level of myogenin was highly increased to reach a presumed peak, whereas that of MyoD subsided quickly. The mRNA levels of both factors were almost abolished after 2 weeks (Fig. 2C). The mRNA of MyHC, on the other hand, appeared after 3 weeks of induction, and its expression steadily increased until the sixth week (Fig. 2C).

Immunocytochemical Analysis of Myogenic Differentiation
To further confirm myogenic differentiation of UCB-derived MSCs, cells were examined immunocytochemically with monoclonal anti-MyoD, anti-myogenin, and anti-skeletal myosin antibodies. Figure 3A shows nuclear staining of MyoD and myogenin in treated cells. Consistent with our previous RT-PCR and FACS results, expression of myogenin was much higher than that of MyoD at 1 week of induction (Fig. 3A). Western blot analysis indicated that fast-twitch myosin, which appeared as a 200,000-dalton protein band, was highly expressed in the cells incubated for 6 weeks (Fig. 3B). Taken together with the data above, it is very likely that human UCB-derived MSCs are able to differentiate into skeletal myoblasts.

View larger version (46K): [in this window] [in a new window]   Figure 3. Immunocytochemistry and Western blotting of umbilical cord blood–derived mesenchymal stem cells cultured in myogenic medium. (A): Immunocytochemical staining of the cells cultured in myogenic medium for 1 week. The cells were stained with MyoD or myogenin antibodies followed by a horseradish peroxidase (HRP)–conjugated secondary antibody and were colorized with the substrates. The control was stained only with the secondary antibody. (B): Western blot of the protein extract from the cells cultured in myogenic medium for 6 weeks. The blot was stained with a fast-twitch antibody followed by an HRP–conjugated secondary antibody. The level of -actin protein was used as a loading control.

	DISCUSSION

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UCB-derived MSCs have been demonstrated to be capable of differentiating into osteoblasts and adipocytes [17–19]. We observed that MSCs prepared in our laboratory also possess osteogenic, adipogenic, and neurogenic potential in vitro (data not shown). This multipotent feature of UCB-derived MSCs is further reinforced by the myogenic potential that is proven in this work.

Skeletal myogenesis is a developmental cascade that involves the regulatory MyoD gene family that determines the progress of multipotential mesodermal stem/progenitor cells into myogenic lineage. The MyoD family is one of the basic helix-loop-helix transcription factors that directly regulate myocyte cell specification and differentiation [33]. MyoD expression occurs at the early stage of myogenic differentiation, whereas myogenin is expressed later at the time related to cell fusion and differentiation [20,33]. Similar to muscle development, the two muscle-specific transcription factors are expressed during myogenic differentiation of BM-derived MSCs [28–30]. According to FACS and RT-PCR results of this study, these two factors are also expressed in UCB-derived MSCs during myogenic induction by myogenic medium. MyoD expression was first detected from day 3 of induction and decreased thereafter so that there was no measurable level of MyoD mRNA and protein at week 1. In contrast, expression of myogenin peaked at 1 week and diminished considerably by 2 weeks (Fig. 2C). Mizuno et al. [29], however, reported that MyoD1 expression is highest during the first 3 weeks of myogenic induction and remains at high levels even after 6 weeks of induction. This difference seems to originate from their use of processed lipoaspirate cells that were isolated from raw human lipoaspirates displaying multilineage mesodermal potential in vitro.

On the other hand, it is known that myosin is expressed in myogenic precursors undergoing terminal differentiation. Likewise, we observed weak myosin expression in 3-week cultures after myogenic induction and significantly enhanced expression at 6 weeks, in which approximately 55.7% of UCB-derived MSCs are myosin-positive in flow cytometric analysis (Fig. 2). All observations by immunohistochemical staining, Western blot, RT-PCR, and FACS analyses indicate that the myosin was formed from 6 weeks of induction (Fig. 3B). These data agree well with the study of a time-dependent myosin expression that showed the highest number of myosin-positive cells after 6 weeks of myogenic induction in processed lipoaspirate cells [29].

Our findings in this study suggest that human UCB-derived MSCs could be alternative cell sources to treat muscle injury or chronic muscular disease. For clinical application, however, the functional capacity of these cells needs be further studied in more complex in vitro and animal models.

	ACKNOWLEDGMENTS

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This research was supported in part by a grant (SC13032) from Stem Cell Research Center of the 21^st Century Frontier Research Program funded by the Ministry of Science and Technology and a grant (01-PJ10-PG8-01EC01-0015) of Korea Health 21 R&D Project funded by the Ministry of Health and Welfare, Republic of Korea.

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