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Vol. 16, Issue 3, 1491-1499, March 2005
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* Department of Reproductive Biology and Pathology, Tokyo 157-8535, Japan;
Department of Innovative Surgery, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan;
Virology Division, National Cancer Center Research Institute, Tokyo, Japan; and
Department of Pathology, Keio University School of Medicine, Tokyo 160-8582, Japan
Submitted July 31, 2004;
Accepted December 20, 2004
Monitoring Editor: Lawrence Goldstein
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Prockop, 1997; Caplan and Bruder, 2001). hMSCs have been studied extensively over the past 3 decades, and numerous independent research groups have successfully isolated hMSCs from a variety of sources, most commonly, from the bone marrow (Owen, 1988; Umezawa et al., 1992; Jaiswal et al., 1997; Makino et al., 1999; Pittenger et al., 1999; Sekiya et al., 2004). Umbilical cord blood (UCB) contains circulating stem/progenitor cells, and the cells contained in UCB are known to be distinct from those contained in bone marrow and adult peripheral blood (Mayani and Lansdorp, 1998). Isolation, characterization, and differentiation of clonally expanded hMSCs derived from UCB (UCBMSCs) have been reported (Goodwin et al., 2001; Lee et al., 2004), and UCBMSCs have been found to have multipotency, and the immunophenotype of the clonally expanded cells is consistent with that reported for bone marrow mesenchymal stem cells. Even now, most UCB is regarded as medical waste in the delivery rooms. Aspirating bone marrow from patients is, however, an invasive procedure, and the proliferation and differentiation capacity of hMSCs decreases with the donor age (D'Ippolito et al., 1999). Therefore, the applications of UCB should be further expanded.
UCBMSCs will be useful sources for cell transplantation, however, it is difficult to study and apply them because of their limited life span. One of the reasons for this is that normal human cells undergo a limited number of cell division in culture and then enter a nondividing state called "senescence" (Hayflick, 1976; Campisi, 1997). Human cells reach senescence or cease to divide after a limited number of cell replications, and the average number of hMSC population doublings (PDs) has been found to be 
40 (Takeda et al., 2004), implying that it would be difficult to obtain enough cells to restore the function of a failing human organ. Large numbers of cells must be injected into damaged tissues to restore function in humans, and cells sometimes need to be injected throughout entire organs.
To resolve these problems, the life span of hMSCs from bone marrow can be extended by retroviral transduction of human telomerase reverse transcriptase (hTERT) (Blackburn, 2000a,b, 2001) and human papillomavirus type 16 (HPV16) E6 and/or E7 (Sekiguchi et al., 1999; Burk et al., 2003; Takeda et al., 2004). Both p16INK4a/RB inactivation with E7 and telomerase activation with E6 are required to extend the life span of human mammary epithelial cells (Kiyono et al., 1998). E6 also accelerates degradation of p53, which induces the cdk inhibitor p21 (Sekiguchi and Hunter, 1998). This system in which p16INK4a/RB is inhibited and telomerase is activated is highly efficient in extending the life span of hMSCs (Okamoto et al., 2002).
In the present study, we investigated the growth regulatory mechanism of UCBMSCs and attempted to establish UCBMSCs with hTERT (UCBTERTs) to overcome their limited life span. Introduction of hTERT alone was sufficient to extend the life span of UCBMSCs in vitro, and this technique for prolonging the life span of UCBMSCs will be a useful tool. UCBTERTs with the extended life span provide a powerful model for further study of cellular senescence and application to transplantation therapy in the future.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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4 x 105 cells/well in a 12-well plate. MSCGM was used in all culture procedures after harvesting the colony. The cells were passaged at a density of 1 x 105 cells/100-mm dish (1:4), and the original cells were regarded as being PD 0 (day 0). When the cultures reached subconfluence, the cells were harvested with 0.25% trypsin and 1 mM EDTA and replated with one-half of the harvested cells. Cells were allowed to adhere overnight, and nonadherent cells were washed out with medium changes. Medium changes were carried out twice weekly thereafter. The cells were cultured for further experiments under the approval (approval nos. 7 and 55) of the Ethics Committee of National Research Institute for Child Health and Development, Tokyo.
Infection with Recombinant Retroviruses
The cells were prepared for infection with recombinant retroviruses expressing the E6, E7, and hTERT, as described previously (Takeda et al., 2004). Stably transduced cells with an expanded life span were designated UCBE6E7-20 and UCBTERT-21 cells.
Senescence-associated-
-gal (SA--gal) Staining
The SA--gal assay was performed as described previously (Dimri et al., 1995). Cells were washed in phosphate-buffered saline (PBS), fixed for 3–5 min at room temperature in 2% formaldehyde/0.2% glutaraldehyde (or 3% formaldehyde), washed, and incubated at 37°C with fresh SA--gal stain solution: 1 mg of 5-bromo-4-chloro-3-indolyl -D-galactosidase per milliliter (stock is 20 mg of dimethylformamide/ml), 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2. Staining was evident in 2–4 h and maximal in 12–16 h.
Cell Transplantation
Freshly collected confluent cells (106 cells) were subcutaneously and intramuscularly injected into BALB/c nu/nu mice (Sankyo Laboratory, Hamamatsu, Japan). Animals were monitored for malignant transformation of the injected cells for 3 mo after inoculation and then killed by cervical location.
Flow Cytometric Analysis
Cells were stained for 30 min at 4°C with primary antibodies and immunofluorescent secondary antibodies. The cells were then analyzed on a FACScan (BD Biosciences), and the data were analyzed with the CELLQUEST software (BD Biosciences). Antibodies against human CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD50, CD55, CD59, CD90, CD117, and CD133 were purchased from Beckman Coulter (Fullerton, CA), Immunotech (Marseille, France), Cytotech (Hellebaek, Denmark), and BD Biosciences PharMingen (San Diego, CA).
Western Blot Analysis
Cells were seeded at a density of 3 x 105 cells/100-mm culture dish and harvested at subconfluence. Cell lysates were prepared by sonication by using ultrasonic homogenizer VP-5S in WE16th lysis buffer (Gewin et al., 2004). Equal amounts of protein (20 µg) were loaded on SDS-polyacrylamide gels and blotted on Immobilon-P membranes (Millipore, Bedford, MA) by using a semidry transfer system (Atto, Tokyo, Japan). The primary antibodies used were as follows: G3-245 for retinoblastoma (RB) protein and G175-405 for p16INK4a (BD Biosciences PharMingen), DO-1 for p53 (Oncogene Science, Cambridge, MA), F-5 for p21 and I-19 for actin (Santa Cruz Biotechnology, Santa Cruz, CA), affinity-purified anti-phospho-ataxia telangiectasia mutated kinase (p-ATM) (Ser1981) (600-401-400; Rockland, Gilbertsville, PA), and phospho-p53 (p-p53) (Ser15) antibody (9284; Cell Signaling Technology, Beverly, MA). Blots were probed with horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), anti-rabbit IgG (New England Biolabs, Beverly, MA), or donkey anti-goat IgG (Santa Cruz Biotechnology), and visualized using an enhanced chemiluminescence detection kit (Roche Diagnostics, Indianapolis, IN).
Telomere Length Assay
Total genomic DNA was isolated from cultured cells by proteinase K digestion. The lengths of telomere in each sample were determined by Southern blot analysis as described previously (Vaziri et al., 1994). Briefly, 1 µg of genomic DNA extracted from each sample was digested with both Hinf I and RsaI and electrophoresed in 0.8% agarose gels for 16 h, transferred onto a Hybond N membrane (Amersham Biosciences), and hybridized with digoxigenin (DIG)-labeled (TTAGGG)3 probe. The membrane was incubated with anti-DIG alkaline phosphatase (ALP) antibody, and detection was performed with chemiluminescence solution.
Telomerase Activity
Telomerase activity in each sample was detected by the telomeric repeat amplification protocol (TRAP) assay by using the TRAPeze kit (Intergen, Purchase, NY) according to the manufacturer's instruction.
Karyotype Analysis
Fixation and chromosome preparation were performed according to the standard procedure described previously (Sasaki, 1975). For each sample, >50 cells were scored for their chromosome number.
Differentiation-Induction Experiments
The multidirectional differentiation potential of each cell line was assessed by the differentiation-induction protocols described below.
Histochemical Staining
After 21 d of culture, cells were rinsed twice with PBS and then fixed with 10% buffered formalin for 10 min at room temperature. The fixed cells were stained with 0.3% Oil-Red-O (Nakarai Tesque, Kyoto, Japan) for the adipogenic differentiation assay and with 5% silver nitrate (Nakarai Tesque) for von Kossa staining in the osteogenic differentiation assay (Tsuchiya et al., 2004).
Osteogenic Differentiation
Cells were seeded at a density of 5 x 104 cells/cm2 in tissue culture dishes and cultured with MSCGM containing 100 nM dexamethasone, 50 µM ascorbic, acid 2-phosphate, and -glycerophosphate. The cultures were maintained for 4 wk, and the cultured medium was replaced every 3 d.
Adipogenic Differentiation
Cells were seeded at a density of 3 x 104 cells/cm2 in tissue culture dishes. When the cells were confluent, the adipogenic differentiation was initiated by three cycles of induction/maintenance culture. Each cycle consists of 3 d of culture in the induction medium (DMEM with 10% FBS, 1 µM dexamethasone, 0.2 mM indomethacin, 10 µg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine) followed by 2 d of culture in the maintenance medium (DMEM with 10% FBS and 10 µg/ml insulin).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and multipotent adult progenitor stem cells (Jiang et al., 2002) (Figure 1A). The cells from PD 9 to PD 31 rapidly proliferated in culture, and propagated continuously (Figure 1, B and C). No SA--gal activity was detected histoenzymologically in the UCBMSCs in the growth phase on day 59. The UCBMSCs stopped replicating, became broad and flat, and exhibited SA--gal activity as indicated by blue staining of their cytoplasm at PD 32 or day 98, indicating that they had entered senescence (Figure 1, B and C). The morphological changes and SA--gal activity of UCBMSCs are PD dependent. To extend the cells' life span and obtain a large number of cells, two different types of cells were obtained by transferring a combination of HPV16 E6 and E7 or hTERT at 29 PDs or 12 passages (Figure 1C, indicated as an arrow). UCBMSCs transduced with a combination of E6 and E7 were designated UCBE6E7-20 cells, and UCBMSCs transduced with hTERT were named UCBTERT-21 cells. UCBTERT-21 cells successfully proliferated >120 PDs, and continued to grow. The cells were found to have an extended life span (Figure 1C, UCBTERT-21 cells, blue triangles). The UCBE6E7-20 cells, which had been transduced with E6 and E7, had a prolonged cell life span in culture, and underwent global cell death at 38 PDs, when the cells entered a "crisis" period. This implies that the E6 and E7 are capable of prolonging cell life span but that their effect is limited (Figure 1C, UCBE6E7-20 cells, pink squares). Mock infection (polybrene treatment alone) did not extend cell life span, and the cells reached senescence or cessation of growth at PD 32 (Figure 1C, UCBMock, yellow circles). SA--gal staining was performed to determine the proportions of cells that had entered senescence, and positive staining was observed in 0% of the UCBTERT-21 cells at PD 41, 2% of the UCBE6E7-20 cells at PD 37, and 100% of the UCBMock cells at PD 32 (Figure 1D). The low percentage of SA--gal–positive UCBE6E7-20 cells at PD 37 or day 98 is probably attributable to global cell death by crisis (Figure 1D, middle column). UCBMock cells exhibited a typical senescence-associated morphology, i.e., they were broad and flat and exhibited strong SA--gal activity enzyme cytochemically at PD 32 (Figure 1D, right column).
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The cells did not undergo malignant transformation. They stopped dividing after reaching confluence, and they did not form any foci after confluence in vitro. Nor did the cells grafted into the subcutaneous and muscle tissue of nude mice (n = 6) produce tumors, at least during the monitoring period (>100 d). Injected UCBTERT-21 cells survived but did not proliferate at the injection sites.
Unchanged Surface Markers of UCBMSCs after Prolongation of Their Life Span
Expression of UCBMSCs and UCBTERT-21 cell surface markers was evaluated by flow cytometric analysis (Figure 2). The results showed that both cells were positive for CD29 (integrin 1), CD44 (Pgp-1/ly-24), CD55, and CD59, and negative for CD13, CD14 (a marker for macrophages and dendritic cells), CD31 (platelet-endothelial cell adhesion molecule-1, PECAM-1), CD34, CD45 (leukocyte common antigen), CD50 (intercellular adhesion molecule-1, ICAM-1), CD90 (Thy-1), CD117 (c-kit), and CD133. Primary UCBMSCs displayed the same pattern of surface markers as UCBTERT-21 cells, implying that the surface marker expression was unaffected by the exogenously expressed hTERT.
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Absence of p16INK4a in Parental UCBMSCs
Expression of p16INK4a/RB premature senescence-associated proteins (Figure 3A) and telomere/p53 replicative senescence-associated proteins (Figure 3B) was analyzed in UCBMSCs, UCBTERT-21, and UCBE6E7-20 cells. p16INK4a was not detected in the UCBMSC lanes until the senescence stage; p16INK4a was not detected until PD 53 and started to be expressed in UCBTERT-21 cells at a low level at PD 69, and p16INK4a was detected in UCBE6E7-20 cells at PD 37, immediately before the crisis stage. The protein levels of p53 and p21 in UCBMSCs became up-regulated as the number of PDs increased, but the protein levels of p53 and p21 became down-regulated in UCBE6E7-20 cells, implying that exogenously introduced E6 targets p53 for proteolytic degradation. ATM in UCBE6E7-20 cells was phosphorylated, probably because of DNA damage or telomere length shortening (Figure 3B, lane 8). p53, phosphorylated p53, and p21 were induced by H2O2, a physiological stressor, in UCBTERT-21 cells (Figure 3B, lane 9). The hypophosphorylated forms of RB became dominant and the hyperphosphorylated forms decreased with passage of UCBMSCs (Figure 3A, lanes 1–3), correlating to the increase in p53 and p21 (Figure 3B, lanes 1–3) and to the decrease in cell growth. Transduction of hTERT transiently and markedly increased the hyperphosphorylated form (Figure 3A, lane 4), corresponding to the sudden recovery in proliferation and to a shorter doubling time. Finally, both hyper- and hypophosphorylated forms of RB remained at steady-state levels (Figure 3A, lanes 5–7), although the hypophosphorylated form seemed dominant at PD 53 (Figure 3A, lane 6), perhaps due to the higher cell density at collection of cell lysate. The protein level of RB was down-regulated in E7-overexpressing UCBE6E7-20 cells (Figure 3A, lane 8), probably as a result of enhanced proteolysis by E7.
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Increase in Telomerase Activity and Maintenance of Telomere Length in Cells Transduced with the hTERT
Telomerase activity is revealed by the characteristic six base pair ladder of bands detected by TRAP assay (Figure 4A). No telomerase activity was detected in UCBMSCs at any PDs tested, UCBE6E7-20 cells, UCBMSCs infected with the vector-alone or CHAPS buffer, or mock infected. By contrast, the cells transduced with the hTERT exhibited significant levels of telomerase activity, comparable to HeLa cells as a positive control and to TSR8, which is a synthetic template of eight telomeric repeats used as a polymerase chain reaction (PCR)-positive control.
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Average telomere length was longer in the UCBTERT-21 cells than in UCBMSCs. Telomere length in UCBMSCs decreased with the number of PDs, whereas it remained the same in UCBTERT-21 cells, regardless of the number of PDs. The telomere length of UCBE6E7-20 cells was shorter than that of the parental UCBMSCs at senescence.
Normal Diploid Karyotypes with XY Sex Chromosomes in UCBMSCs and UCBTERT-21 Cells
Karyotypic analyses of UCBMSCs were performed at PD 5 (2 passages) and of UCBTERT-21 cells at PD 32 (14 passages). UCBMSCs and UCBTERT-21 cells were found to be diploid and not to exhibit any significant chromosomal abnormalities (Figure 5, A and B). The chromosome number of both UCBMSCs at PD 5 and UCBTERT-21 cells at PD 35 was 46, except for one UCBTERT-21 cell, which contained 47 chromosomes (Figure 5C). No UCBTERT-21 cells containing abnormal numbers of chromosome were found on further analysis. The sex chromosomes were found to be XY, indicating that the cells were of fetal origin.
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Osteogenic and Adipogenic Differentiation Potentials of UCBMSCs and UCBTERT-21 Cells
The multipotency of UCBMSCs and UCBTERT-21 cells was assessed by conventional protocols. The osteogenic differentiation potential of UCBMSCs and UCBTERT-21 cells was assessed based on their morphology and von Kossa staining after 3 wk of induction (Figure 6). Multiple small Oil-Red-O–positive fat droplets had accumulated in UCBMSCs and UCBTERT-21 cells after 3 and 2 wk, respectively, of adipogenic induction. Adipocyte differentiation was estimated by counting 2000 cells per dish. The results of triplicate experiments showed that 5.0 and 5.4% of the UCBMSC and UCBTERT-21 cells became positive for fat droplets with Oil-Red stain as a result of adipogenic induction and >90% of the cells were positive on ALP staining after osteogenic induction. We also induced these cells to differentiate into multiple lineages by the methods for neural (Kohyama et al., 2001), cardiomyogenic (Makino et al., 1999; Takeda et al., 2004), and chondrogenic (Imabayashi et al., 2003) lineages; however, the UCBMSC and UCBTERT-21 cells were not induced to differentiate into these lineages in vitro.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Is Successful Prolongation of UCBMSCs Life Span without Inhibition of the p16INK4a/RB Pathway Attributable to a Lack of Ex Vivo Culture Stress?
In contrast to our previous study by using bone marrow-derived cells (Takeda et al., 2004), surprisingly, the successful prolongation of the life span of the UCB-derived fetal cells obtained in this study did not require inhibition of the p16INK4a/RB pathway or premature senescence-associated pathway. Immortalization of some human cell types requires inhibition of the p16INK4a/RB pathway in addition to activation of telomerase (Kiyono et al., 1998; Ishikawa, 2003). Human mammary epithelial cells, endometrial glandular cells, skin keratinocytes, and marrow-derived cells require inhibition of the p16INK4a/RB pathway for immortalization, but foreskin fibroblasts do not. Activation of telomerase alone is sufficient for immortalization of human foreskin fibroblasts. HPV16 E6 and E7 have been used to inhibit p53 and RB, respectively, to prolong the life span of marrow-derived MSCs (Okamoto et al., 2002; Takeda et al., 2004), endometrial gland cells (Kyo et al., 2003), mammary epithelial cells, and keratinocytes (Kiyono et al., 1998). Bmi-1 also has been used to inhibit p16INK4a transcription to prolong the life span of marrow-derived MSCs. One function of this p16INK4a protein is to maintain pRB in a hypophosphorylated active form, which inhibits cell cycle progression.
Our present findings that UCB-derived cells can be immortalized without inhibition of the p16INK4a/RB pathway is consistent with the results in regard to foreskin fibroblasts. This successful immortalization of UCB-cells by hTERT alone can be explained by lack of ex vivo culture stress under the culture condition used in this study. Alternatively, only cells insensitive to ex vivo culture stress or lacking p16INK4a induction may be expanded by hTERT alone. Primary UCB-derived cell culture succeeded in 94% of the attempts (15 of 16 trials), and the cells were passaged only two or three times before reaching premature senescence (13 of 15 primary UCB-derived cell cultures); however, only two cell strains (UCBMSCs) were established from them (2 of 15 primary UCB-derived cell cultures; see Materials and Methods. "Isolation and Cell Culture of UCBMSCs"). Based on the results of this study by using one of the two cell strains, the establishment of these strains (UCBMSCs) can be explained by 1) lack of p16INK4a in primary cultured UCB-derived cells or 2) selection of cells that do not express p16INK4a from a heterogeneous population. We cannot exclude either possibilities, and we did observe two different types of cells, i.e., rapidly growing cells and quiescent cells in the primary culture of cord blood cells. If the alternative explanation is true, these quiescent cells, in which p16INK4a may be expressed at a high level, can be efficiently expanded by introduction of E7, the inhibitor of RB, or Bmi-1, the down-regulator of p16INK4a. We also performed additional experiments by using newly obtained specimens from umbilical cord to determine whether infection of the primary or first passage cells generates long-term strains routinely and efficiently. We generated other cells, UCB408 cells, and found that generation of long-term strains was reproducible (Supplementary Figure A). The UCBE6E7-31 and UCBE7-32 cells proliferated for >30 PDs and exhibited persistent growth. The UCBTERT-30 cells exhibited a prolonged cell life span in culture and reached PD 19, but they failed to be immortalized. The success of immortalization of UCBMSCs may still be low, probably due to expression of p16INK4a premature senescence-associated proteins in the early passage of the UCB408 cells. Because the 5' CpG island of the p16INK4a promoter based on published genome sequences (GenBank accession no. AF022809
[GenBank]
, U12818
[GenBank]
, and AC000048
[GenBank]
) has been found to be methylation-free by the bisulfite method (Supplementary Figures B and C), the lack of p16INK4a expression in rapidly growing cells is not due to methylation of the p16INK4a promoter, unlike human mammary epithelial cells (Umezawa et al., 1997; Foster et al., 1998; Wong et al., 1999).
UCB-derived Cells Are of Mesenchymal Origin
The differentiation capacity of UCB-derived cells was unaffected during establishment of a plate-adhering population of cells from UCB. The cells established from UCB can be extensively and clonally expanded in vitro while retaining their potential to differentiate into osteoblasts that produce mineralized matrices and adipocytes that accumulate lipid vacuoles under in vitro conditions. This differentiation potential of the UCB-derived cells is the same as that reported for bone marrow MSCs (Goodwin et al., 2001; Lee et al., 2004).
The surface markers of the UCB-derived cells examined in this study are exactly the same as those of previously reported UCB-derived cells (Lee et al., 2004). Most of the surface markers are the same as those detected in their bone marrow counterparts (Takeda et al., 2004), with both UCB- and bone marrow-derived cells being positive for CD29, CD44, CD55, and CD59, and negative for CD34 and CD117. The CD90 and CD133 markers, on the other hand, can be used to distinguish UCB-derived cells from bone marrow-derived cells, because they are both expressed in multipotent marrow-derived cells (Takeda et al., 2004) and not in UCB-derived cells (Figure 2) (Lee et al., 2004).
This technique allows the applications of UCB to be further extended and permits it to be used as an alternative to bone marrow as a source of hMSCs; however, this study puts the controversy to rest and substantiates that UCB does contain hMSCs. We believe that the "UCBMSCs" are mesenchymal stem cells derived from UCB, as designated. However, it is difficult to exclude the possibility that the UCBMSCs were derived from mesenchymal cells embedded in the Wharton's jelly of the umbilical cord during insertion of the needle into the vessels through the umbilical cord.
Can primary UCB-derived cell "culture" contribute to cell-based therapy or regenerative medicine? The problems involved in cell-based therapy with human UCB-derived cells are the finite cell life span of the cells and the difficulty of obtaining a large enough number of cells. The technique that allows human cells to escape senescence used in this study may be used to obtain a large number of cells and to overcome these problems of a short life span.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Akihiro Umezawa (umezawa{at}1985.jukuin.keio.ac.jp).
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