Skeletal Myogenic Differentiation of Mesenchymal Stem Cells Isolated from Human - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:37 |只看该作者 |倒序浏览 |打印

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

Key Words. Human umbilical cord blood ? Mesenchymal stem cells ? Immunophenotyping ? Myogenic differentiation* ~5 u! Z7 M7 V9 ]0 }! z; g& i
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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@seoulcord.co.kr

ABSTRACT# Y: q# B" r* R  D2 z( v

Bone marrow (BM) has been regarded as a good source of both hematopoietic stem/progenitor cells and mesenchymal stem cells (MSCs) . 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 . 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.3 u6 N! T' U3 O
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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 . However, human UCB has been controversial for the presence of MSCs; some researchers successfully isolated MSCs from UCB, whereas others have not . Nevertheless, several groups reported that the UCB-derived MSCs could proliferate ex vivo and differentiate, at least, into osteoblasts and adipocytes . No evidence has shown yet that UCB-derived MSCs differentiate into skeletal myoblasts, but they are believed to have such a potential.
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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 . 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 . 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 .

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.
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MATERIALS AND METHODS. A$ r/ q8 r, z! l+ f9 E" H

Characteristics of UCB-Derived Adherent Cells/ {+ u; T# r  O4 L5 f
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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.( G! o7 O" i! ?6 g. o3 j9 d* Q* ^
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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.
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Immunophenotyping of UCB-Derived Adherent Cells: Z% o" Q! V, f$ @$ G7 m

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 (? 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.

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.

The adherent cells were also negative for expression of CD49d (4 integrin), CD106 (VCAM-1), and CD31 (an endothelial-related antigen) (Fig. 1). Similar to BM-derived MSCs, the cell population was positive for HLA class I but not for HLA DR (Fig. 1). All data above indicate that the adherent cells derived from UCB exhibit the phenotype of MSCs.
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FACS and Reverse Transcription–PCR Analyses of Myogenic Differentiation. K8 ]3 o4 d$ |8 |7 h# q3 J2 S; T
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A potential of UCB-derived MSCs differentiating into osteoblasts, chondrocytes, and adipocytes was demonstrated elsewhere . 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.

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./ z, M$ j# L+ R. x6 ~

On the other hand, fast-twitch myosin began to express only after 3 weeks of induction, and approximately 55.7% of treated MSCs at week 6 were visibly stained with monoclonal anti-skeletal myosin antibodies (Fig. 2B). This finding is also not surprising when considering that myosin is an element of skeletal muscle fibers that appears in late myogenesis.0 F& c2 l5 f5 J: q6 K, E

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) [! G  g9 t$ j8 [& t) }5 o
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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., `) W- i7 J8 W) P' i+ T

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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This research was supported in part by a grant (SC13032) from Stem Cell Research Center of the 21st 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.$ y5 U7 `. q6 [' E! v: ?
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