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a Institute of Anatomy and Cell Biology and
b Department of Surgery, School of Medicine, Yang-Ming University, Taipei, Taiwan, Republic of China;
c Department of Orthopedics and Traumatology, Veterans General Hospital-Taipei, Taipei, Taiwan, Republic of China
Key Words. Mesenchymal stem cells • Wharton’s jelly • Umbilical cord • Multipotent cells
Correspondence: Hwai-Shi Wang, Ph.D., Department of Anatomy, Yang-Ming University, 155, Sec. 2, Li-Nung Street, Taipei, Taiwan, 112. Telephone: 886-2-28267035; Fax: 886-2-28283212; e-mail: hswang{at}ym.edu.tw
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ABSTRACT |
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INTRODUCTION |
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In this study, we demonstrate that mesenchymal cells from the umbilical cord, when expanded in culture, express adhesion molecules (CD44, CD105), integrin markers (CD29, CD51), and mesenchymal stem cell (MSC) markers (SH2, SH3) but not markers of hematopoietic differentiation (CD34, CD45). After exposure of these cells to cardiomyocyte-conditioned medium or 5-azacytidine, they expressed cardiac troponin-I and N-cadherin, indicating differentiation into cardiomyocytes. Under suitable culture conditions, these cells could also differentiate into osteogenic and adipogenic cells. Thus, human umbilical cord mesenchymal cells can be expanded in culture and induced to form several different types of cells. They may therefore prove to be a new source of cells for cell therapy, including targets such as stromal tissue and cardiac muscle. This will help to avoid several ethical and technical issues.
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MATERIALS AND METHODS |
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Induction of Cardiogenic Differentiation
For 5-azacytidine treatment, the mesenchymal cells were incubated for 24 hours in serum-free DMEM containing 3 µM 5-azacytidine. The medium was then changed to DMEM/10% FBS to prevent cell death due to prolonged exposure to 5-azacytidine, and the cells were maintained in DMEM/10% FBS for 3 days to 5 weeks. For treatment with cardiomyocyte-conditioned medium, conditioned medium was prepared by culturing cardiomyocytes, obtained from the hearts of 7-day-old rats, in DMEM supplemented with glucose (4.5 g/l) and 10% FBS for approximately 72 hours. The cell culture medium was then harvested and centrifuged at approximately 800 g for 10 minutes at room temperature, and, finally, the supernatant was filtered for use as conditioned medium for the mesenchymal cells.
Flow Cytometry
After the mesenchymal cells were extracted from the umbilical cord, they were cultured in DMEM supplemented with glucose (4.5 g/l) and 10% FBS for 5–7 days. Next, the cells were trypsinized and suspended in DMEM at a concentration of 5 x106/ml, and then a 1-ml sample was incubated for 45 minutes at 4°C with 150 µl of various nonlabeled mouse anti-human antibodies followed by fluorescein isothiocyanate (FITC)–conjugated anti-mouse immunoglobulin G (Ig G) antibodies (10 mg/ml) for 1 hour at room temperature. Finally, they were washed twice with phosphate-buffered saline (PBS), pH 7.4; centrifuged; and fixed in 1.5 ml of 4% paraformaldehyde. Control samples were incubated with PBS instead of primary antibody. A FACScan machine (Bec-ton, Dickinson, Franklin Lakes, NJ) was used to analyze antibody binding.
Induction of Adipogenic, Chondrogenic, or Osteogenic Differentiation
Cultured cells at passage 2 were incubated in adipogenic differentiation medium (DMEM and 1 g/ml glucose [DMEM-LG] containing 10% FBS, 50 µg/ml of ascorbate-1 phosphate, 10–7 M dexamethasone, and 50 µg/ml indomethacin [Sigma, St. Louis]) in chondrogenic differentiation medium (as cell pellets) (serum-free DMEM-LG containing insulin-transferrin-selenium (ITS) + premix [GIBCO, Carlsbad, CA] and 10 ng/ml transforming growth factor [TGF]-ß 1 [Pepro Tech, Rocky Hill, NJ]), in osteogenic differentiation medium (DMEM-LG containing 10% FBS, 50 µg/ml ascorbate-2 phosphate, 10–8 M dexamethasone, and 10 mM ß-glycerophosphate), or in DMEM-LG supplemented with 10% FBS as a control. The medium was changed every 3 days, and the cells, after completion of differentiation had been established by morphology, were used for the histochemical staining and immunohistochemistry studies.
Immunocytochemistry
After the mesenchymal cells were extracted from the umbilical cord, they were cultured in DMEM supplemented with glucose (4.5 g/l) and 10% FBS for 5–7 days, and then various cell-differentiating factors were added for 3 days to 5 weeks. Staining was performed on fixed, nonpermeabilized monolayers of mesenchymal cells grown on coverslips. The cells were washed with PBS, fixed for 30 minutes at 37°C in 0.05% glutaraldehyde, and incubated for 25 minutes at room temperature with a 1:9 dilution of normal goat serum in PBS to block nonspecific binding of the primary antibody. The slides were then incubated for 75 minutes at 4°C with various nonlabeled mouse anti-human antibodies (10 µg/ml) (Biosource International, Camarillo, CA) followed by FITC-coupled anti-mouse IgG antibodies (Vector, Burlingame, CA) for 1 hour at room temperature. The coverslips were mounted with mounting medium (Vector) and viewed with a confocal laser-scanning microscope (Leica) using a 100/1.30 oil-immersion objective lens and an appropriate filter. In the negative controls, in which the primary antibodies were omitted, negligible immunofluorescence was seen.
Western Blotting
For immunoblotting on polyvinylidene difluoride (PVDF) membranes, cells from two 100-mm dishes per treatment group were pooled, rinsed briefly with PBS, and then lysed at room temperature for 10 minutes in 1 ml of PBS containing 1% sodium dodecyl sulfate, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 10 µg/ml soybean trypsin inhibitor, and 0.5 mM dithiothreitol. After centrifugation for 20 minutes at 1,300 g and 4°C, the supernatant was removed and centrifuged at 6,000 g for 1 hour at 4°C. The final supernatant was used as the cytosolic fraction, and the pellet was used as the membrane fraction. Equal amounts (50 mg of protein) of the membrane fractions or cytosolic fractions were run on 12% polyacrylamide gels and transferred to PVDF membrane in transfer buffer (four parts 25-mM Tris/200 mM buffer, pH 8.0, and one part methanol). The membranes were then blocked for 2 hours at room temperature with 50 mM Tris HCl, 150 mM NaCl, 0.05% Tween-20 (tris-buffered saline with Tween-20 [TBST]), pH 7.0, containing 5% nonfat dry milk and then incubated overnight at 4°C with rabbit anti-human cardiac troponin I monoclonal antibody (1:500) (Serotec, Oxford, U.K.) or mouse anti-N-cadherin antibody (1:500) (Zymed, San Francisco) in TBST. After three washes in TBST, the membranes were incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibody at a 1:1,000 dilution (Vector, Burlingame, CA), and bound antibody was detected by enhanced chemiluminescence (Amersham Life Sciences, Uppsala, Sweden).
Histochemical Staining
The medium was removed and the cells were washed twice with PBS, fixed for 10 minutes at room temperature in 3.7% paraformaldehyde, and washed twice with PBS. Cells treated with the adipogenic and chondrogenic formulas were stained with Oil red O or Toluidine blue to show adipogenic or chondrogenic differentiation, respectively. Immunohisto-chemical staining for human type II collagen was also used to demonstrate chondrogenic differentiation of the treated cells. The mesenchymal cells treated by the osteogenic formula were stained with alkaline phosphatase staining and von Kossa staining to reveal osteogenic differentiation.
RNA Extraction and Reverse Transcriptase Polymerase Chain Reaction Analysis
Total RNA was extracted from untreated (control) and treated cells using RNeasy Purification Reagent (Qiagen, Valencia, CA), and then a sample (1 µg) was reverse transcribed with Mmlv reverse transcriptase (RT) for 30 minutes at 42°C in the presence of oligo-dT primer. Polymerase chain reaction (PCR) was performed using specific primers designed from the published sequence of each cDNA as follows: peroxisome proliferator–activated receptor-
2 (PPAR2) (595 bp), antisense 5'-CCTATTGACCCAGA AAGCGATTC-3' and sense 5'-GCATTATGAGACATCCC-CACTGC-3'; osteopontin (330 bp), antisense 5'-CAGTGA CCAGTTCATCAGATTCATC-3' and sense 5'-CTAGGC ATCACCTGTGCCATACC-3'; GAPDH (352 bp), antisense 5'-TCACGCCACAAGTTTCCCGGA-3' and sense 5'-CAC CATCTTCCAGGAGCGAG-3'. PCR was performed for 30 to 35 cycles, with each cycle consisting of denaturation at 95°C for 30 seconds, annealing at 55°C to 63°C for 30 seconds, and elongation at 72°C for 1 minute, with an additional 10-minute incubation at 72°C after completion of the last cycle. To exclude the possibility of contaminating genomic DNA, PCRs were also run without RT. The amplified cDNA was separated by electrophoresis through a 1% agarose gel, stained, and photographed under ultraviolet light.
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RESULTS |
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). Flow cytometric analysis showed that the cells expressed high levels of matrix markers (CD44, CD105), integrin markers (CD29, CD51), and MSC markers (SH2, SH3) but did not express hematopoietic lineage markers (CD34, CD45) (Fig. 1B).
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, cardiac troponin I was clearly more strongly expressed in the differentiated cells than in untreated cells. When umbilical cord mesenchymal cells were maintained in conditioned medium from primary rat cardiomyocyte cultures, the presence of N-cadherin expression was detected by staining at the cell–cell junctions (Fig. 2F). Moreover, Western blots showed that newly extracted cells did not express N-cadherin or cardiac troponin I but that both were expressed in cells grown in conditioned medium. Cells grown in DMEM with 10% FBS for 5 weeks expressed lower amounts of N-cadherin and cardiac troponin I (Fig. 2G).
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). When total RNA was isolated and analyzed by RT-PCR, expression of NADPH, used as an internal control, was the same in undifferentiated and differentiated cells, whereas the adipocyte marker PPAR2 was not expressed in untreated cells but was expressed in adipogenic formula-treated cells (Fig. 3D).
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), and a type II collagen-rich extracellular matrix was demonstrated immunohistochemically (Fig. 4B). However, the control groups were also positive for Alcine blue and type II collagen staining (data not shown). Osteogenic differentiation of mesenchymal cells was seen as the formation of alkaline phosphatase–positive aggregates and von Kossa stain–positive nodules (Fig. 4C), and this was achieved after 28 days of culture in DMEM-LG containing 50 µg/ml of ascorbate-2 phosphate, 10–8 M dexamethasone, and 10 mM ß-glycerophosphate. When total RNA was isolated and analyzed by RT-PCR, expression of NADPH, used as an internal control, was the same in undifferentiated and differentiated cells, whereas osteopontin, an osteogenic marker, was not expressed in newly isolated cells but was expressed in osteogenic formula-treated cells (Fig. 4D).
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DISCUSSION |
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). These results suggest that stroma cells from Wharton’s jelly are similar to MSCs.
We investigated the potential of these cells to differentiate into cardiomyocytes by treating them with 5-azacytidine or maintaining them in cardiomyocyte-conditioned medium and found that both treatments resulted in expression of the cardiomyocyte markers N-cadherin and cardiac troponin I (Fig. 2). Other cardiomyocyte-related markers, such as con-nexin 43, 
-actinin, and desmin, were also expressed after 5-azacytidine treatment for 3 weeks (data not shown). Troponin I, the inhibitory subunit of the troponin complex, is involved in cardiac muscle contraction, whereas F-actin is an essential cell cytoskeletal protein that maintains the structure of the cardiac cell. Under confocal laser microscopy, cardiac troponin I was clearly more strongly expressed in the differentiated cells than in untreated cells (Fig. 2), suggesting that the cells differentiated into cardiomyocytes rather than skeletal cells, whereas F-actin expression in the differentiated cells confirmed that the differentiated cells exhibited a specific cell-attachment pattern similar to that of cardiomyocytes.
Undifferentiated cells can respond to signals from their host tissue microenvironment and differentiate to produce progeny with the phenotypic characteristics of the mature cells of that tissue. N-cadherin is localized in the adherens junction and is important for the assembly of the adherens junction in cardiomyocytes [28, 29]. When umbilical cord mesenchymal cells were maintained in conditioned medium from primary rat cardiomyocyte cultures, Western blots showed that the newly extracted cells did not express N-cadherin or cardiac troponin I but that both markers were expressed in cells grown in conditioned medium and, to a lesser extent, those grown in 10% FBS (Fig. 2E). This suggests that FBS provides some differentiating factors, but not to the same extent as cardiomyocyte-conditioned medium. However, it should be noted that there has been no report up to the present that has described MSCs from adult bone marrow and adipose tissue as able to spontaneously become cardiomyocytes in the control medium. It is possible that MSCs from Wharton’s jelly are earlier-stage cells than MSCs from adult bone marrow and adipose tissue. Thus, there may be a fundamental difference between these cells from Wharton’s jelly and the adult MSCs from bone marrow and adipose tissue.
It has been found that after 5-azacytidine treatment for 2 weeks, murine bone marrow stromal cells can be differentiated into cardiomyocytes, which formed myotube-like structures and began spontaneously beating [30]. However, in our study, after 5-azacytidine treatment or culture in cardiomyocyte-conditioned media, the cells connected with adjoining cells but did not form myotubes or start spontaneous contraction. The differentiation differences may possibly be explained by the fact that the cells used in this study are derived from a different species; namely, they are human rather than mouse.
Under various culture conditions, these cells were able to also differentiate into the chondrogenic, osteogenic, and adipogenic lineages. Adipogenic differentiation was seen as Oil red O–positive cells and was also analyzed by RT-PCR, in which the adipocyte marker PPAR2 was expressed in the adipogenic formula–treated cells (Fig. 3). Chondrogenic differentiation of umbilical cord mesenchymal cells was achieved by pelleting the mesenchymal cells and then incubating them with serum-free medium containing chondrogenic induction factors. The cell pellets expressed chondrogenic characteristics both in the control and after treatment with chondrogenic induction formula. RT-PCR analysis of chondrogenic markers Col2a1 and Col10a1 showed that these were also expressed in the newly extracted mesenchymal cells (data not shown). Because umbilical cord is a supporting structure that contains an abundant amount of hyaluronan, the mesenchymal cells from Wharton’s jelly might have early chondrogenic characteristics in order to give stiffness to the cord. Osteogenic differentiation of mesenchymal cells was seen as the formation of alkaline phosphatase–positive aggregates and mineral deposition staining (Fig. 4). Osteopontin, an osteogenic marker, although not expressed in newly isolated cells, was expressed in chondrogenic or osteogenic formula–treated cells (Fig. 4E). Taking all these results together, the evidence suggests that multipotent cells exist in the umbilical cord.
The Wharton’s jelly contains colony-stimulating activity [31] and high levels of insulin-like growth factor (IGF)-I and IGF-1–binding proteins [32], epidermal growth factor, TGF-, and their common receptor genes [33]. Cultured human MSCs spontaneously produce, or can be induced to produce, cytokines for the support of hematopoietic cells [34, 35]. Thus, umbilical cord mesenchymal cells may have the characteristic ability of bone marrow MSCs to synthesize these same cytokines for the support of hematopoietic cells. However, the possible existence of hematopoietic cells in the umbilical cord needs to be investigated.
After exposure of these cells to 5-azacytidine or to car-diomyocyte-conditioned medium, the cells expressed both cardiac troponin I and N-cadherin. These changes were demonstrated by both immunofluorescent labeling and Western immunoblotting for cardiomyocyte markers. Because umbilical cord mesenchymal cells have been used in cardiovascular tissue engineering [36], umbilical cord mesenchymal cells may be a good source for such use in cardiomyogenic differentiation. Our results also show that these cells can be easily isolated and expanded in culture and induced to differentiate, and this can result in the expression of phenotypes from a variety of lineages. These cells may therefore prove to be a new source of cells for cellular therapies involving stromal tissue and, potentially, cardiac muscle repair. This will avoid the ethical and technical issues involved in the use of cells from other origins. These findings may have a significant impact on the study of cell therapy, early human cardiac differentiation, functional genomics, pharmacological testing, and tissue engineering and potentially help to avoid worrying ethical issues.
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ACKNOWLEDGMENTS |
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REFERENCES |
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