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Stem Cells 2004;22:972-980 www.StemCells.com
© 2004 AlphaMed Press

Immortalized Fibroblast-Like Cells Derived from Human Embryonic Stem Cells Support Undifferentiated Cell Growth

Chunhui Xua, Jianjie Jianga, Virginie Sottileb, Jim McWhirb, Jane Lebkowskia, Melissa K. Carpentera

a Geron Corporation, Menlo Park, California, USA;
b Department of Gene Expression and Development, Roslin Institute, Roslin Midlothian, United Kingdom

Key Words. Human embryonic stem cells • Human telomerase reverse transcriptase Telomerase • Immortalization • Differentiation

Correspondence: Chunhui Xu, Ph.D., Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025, USA. Telephone: 650-473-7795; Fax: 650-473-7750; e-mail: cxu{at}geron.com


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human embryonic stem cells (hESCs) have the potential to generate multiple cell types and hold promise for future therapeutic applications. Although undifferentiated hESCs can proliferate indefinitely, hESC derivatives significantly downregulate telomerase and have limited replication potential. In this study we examine whether the replicative lifespan of hESC derivatives can be extended by ectopic expression of human telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex. To this end, we have derived HEF1 cells, a fibroblast-like cell type, differentiated from hESCs. Infection of HEF1 cells with a retrovirus expressing hTERT extends their replicative capacity, resulting in immortal human HEF1-hTERT cells. HEF1-hTERT cells can be used to produce conditioned medium (CM) capable of supporting hESC growth under feeder-free conditions. Cultures maintained in HEF1-CM show characteristics similar to mouse embryonic fibroblast CM control cultures, including morphology, surface marker and transcription factor expression, telomerase activity, differentiation, and karyotypic stability. In addition, HEF1-hTERT cells have the capacity to differentiate into cells of the osteogenic lineage. These results suggest that immortalized cell lines can be generated from hESCs and that cells derived from hESCs can be used to support their own growth, creating a genotypically homogeneous system for the culture of hESCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Stem cells may provide the starting material for cell replacement in tissues that are damaged as a result of disease, infection, or congenital abnormalities. Several types of stem cells have the capacity to differentiate into subsets of mature cells that can carry out the unique functions of particular tissues when placed into an appropriate environment. However, many somatic stem cells show limited replicative capacity. In contrast, embryonic stem (ES) cells demonstrate both remarkable proliferative capacity as well as pluripotent differentiative potential and may thus prove to be an optimal source for cell replacement therapy. Human ES cells (hESCs) have been successfully cultured on a layer of mouse embryonic fibroblast (MEF) or human feeders [1–4] or under feeder-free conditions, in which the cells are maintained on Matrigel or laminin in MEF-conditioned medium (MEF-CM) [5–7] or are maintained on fibronectin with media supplemented with transforming growth factor-ß, leukemia inhibitory factor, and basic fibroblast growth factor [8]. Quantitative analysis of hESCs cultured either on feeders or on Matrigel with MEF-CM demonstrates that hESCs maintained in either condition show similar expression of SSEA-4, TRA-1-81, OCT4, and hTERT and are capable of long-term proliferation in vitro while retaining a normal karyotype [6–11]. These cells can differentiate into several derivatives, including neural progenitors, cardiomyocytes, trophoblasts, endothelial cells, hematopoietic lineages, hepatocytes, osteoblasts, and insulin-expressing cells, which may have the potential to repair damaged tissues [12–26].

However, differentiated cells derived from hESCs have limited proliferative capacity, as commonly observed for other mammalian somatic cells in culture. Because somatic cell senescence is known to be caused by telomere shortening, it is also possible that senescence of the hESC derivatives is related to reduction of telomere length during each round of cell division. Indeed, significant downregulation of hTERT, the catalytic component of human telomerase, has been observed when hESCs differentiate [6,20]. Therefore, it is predicted that hESC derivatives may be immortalized through overexpression of hTERT. Ectopic expression of hTERT has previously been demonstrated to extend the lifespan of many somatic cell types in vitro, including fibroblasts, retinal pigment epithelial cells, bone marrow stromal cells, and endothelial cells [27–32]. Although the hESC derivatives may also be immortalized by other means, such as over-expression of oncogenes, hTERT-mediated immortalization is particularly desirable for cell therapies and drug discovery applications, because it is not associated with neoplastic transformation [27–30].

In this report, we demonstrate that telomerase-immortalized cells can be generated from hESCs. Fibroblast-like cells, named HEF1, were obtained by differentiating hESCs in vitro and were subsequently immortalized by infection with a retroviral vector expressing hTERT. These immortal hESC-derived cells produced conditioned medium that supported growth of hESCs under feeder-free conditions. In addition, we found that these cells respond to inductive factors to produce at least one fully differentiated cell type, osteoblasts.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of hESCs
hESC lines H1, H7, and H9 [1] (NIH registry WA01, WA07, and WA09) were initially maintained on feeders and later maintained using feeder-free conditions, as described previously [6]. Detailed culture protocols are posted on the Geron Corporation website (http://www.geron.com). To evaluate whether HEF1 cells (generated as described below) can support hESC growth, hESCs maintained in MEF-CM were passaged in CM produced from HEF1 cells (HEF1-CM) on Matrigel-coated plates.

Derivation of HEF1 Cells
H1 hESCs (passage 57) maintained on feeders were harvested after incubation with collagenase IV (200 U/ml) at 37°C for 10 minutes. The cells were dissociated into small clusters and cultured in nonadherent cell culture plates (Corning Inc., Corning, NY) to form aggregates in differentiation medium comprised of 80% knockout Dulbecco’s modified Eagle’s medium (KO-DMEM) (Invitrogen, Carlsbad, CA), 1 mM L-glutamine, 0.1 mM ß-merca-ptoethanol, 20% fetal bovine serum (FBS) (Hyclone, Logan, UT), and 1% nonessential amino acids. After 4 days in suspension, the aggregates were transferred into gelatin-coated plates and cultured for an additional 9 days. The outgrowth culture was passaged by incubation in 2 mg/ml collagenase type II in phosphate-buffered saline (PBS) for 30 minutes at 37°C. The dissociated cells were subsequently resuspended in differentiation medium and plated. The cells proliferated and were serially passaged into HEF1 medium using trypsin/EDTA (Invitrogen). HEF1 medium is comprised of 90% KO-DMEM (Invitrogen), 2 mM L-glutamine, 10% heat-inactivated FBS (Hyclone), and 1% nonessential amino acids. Most of the cells in culture appeared fibroblast-like after two passages and maintained a similar morphology after transduction (see below) (Fig. 1Go). HEF1 cells were seeded at 2 x 104/cm2 and split when cultures were confluent. In all procedures, cells were fed every 2–3 days.



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  		Figure 1. Derivation and characterization of HEF1 cells. (A): Schematic diagram showing the procedure used to derive and immortalize HEF1 cells. (B): Phase-contrast image showing morphology of HEF1-hTERT cells. Bar = 300 µm. Abbreviations: EB, embryoid body; hTERT, human telomerase reverse transcriptase; HEF1, fibroblast-like cells derived from hESCs; pBABE-hTERT, a retrovirus expressing hTERT.

 
Transduction of HEF1 Cells with hTERT-Retrovirus
HEF1 cells were seeded onto six-well plates at 1 x 105 cells per well and infected the next day with pBABEhTERT retro-virus (a kind gift from Dr. Woody Wright, University of Texas Southwestern Medical Center, Dallas) or pBABE, a control virus, overnight. The viral supernatant was supplemented with 4 µg/ml polybrene. After overnight infection, the cells were cultured in HEF1 medium for 2 additional days and selected with 1.6 µg/ml puromycin for 6 days. The cells were harvested, reseeded in HEF1 medium containing 1.6 µg/ml puromycin for 4 days, and finally cultured in HEF1 medium without puromycin. HEF1 cells were counted at each passage after selection, and the population-doubling time was calculated. The HEF1-hTERT cells were cryopreserved in HEF1 medium supplemented with 10% dimethyl-sulfoxide and 45% FBS.

Senescence-Associated ß-Galactosidase ActivityAssay
Senescence-associated ß-galactosidase activity was measured as described previously [33]. Briefly, cells grown on chamber slides were fixed for 2 minutes in 0.2% glutaraldehyde in PBS, washed with PBS, and incubated overnight at 37°C in solution containing 1 mg/ml 5 bromo-4-chloro-3-indolyl-D-galactosidase (X-gal), 5 mM potassium ferro-cyanide, 150 mM NaCl, and 2 ml MgCl2 buffered at pH 6 with 40 mM citric acid/sodium phosphate.

TelomeraseActivityAssay
Telomerase activity was determined by radioactive telomeric repeat amplification protocol (TRAP) assay, as described previously [34,35], using cell lysates equivalent to approximately 5,000, 1,000 cells or heat-inactivated samples and polymerase chain reaction (PCR) amplification for 27 cycles. The PCR products were separated in nondenaturing polyacrylamide gels by electrophoresis.

CytogeneticAnalysis
Karyotype analysis was performed by the Medical Genetics Cytogenetics Laboratory at the Children’s Hospital in Oakland, California. Twenty cells were assessed in each culture.

Quantitative Reverse Transcription–PCR of OCT-4 and hTERT, Immunocytochemistry of Surface Markers and Detection of Alkaline Phosphatase on hESCs, and Flow Cytometry Analysis of Surface Markers on Human Mesenchymal Stem Cells and HEF1-hTERT
Detailed methods are described in the online supplementary materials.

Osteogenic Differentiation
To induce osteogenic differentiation, HEF1-hTERT cells were cultured in EBM medium described previously [13] supplemented with osteogenic supplement (OS) including 100 nM dexamethasone, 50 µM ascorbic acid phosphate, and 10 m ß-glycerophosphate (Sigma) for up to 3 weeks. Human mesenchymal stem cells (hMSCs) were used as a positive control, and HEK293, a human embryonic kidney epithelium cell, was used for a negative control. Methods for culturing of these cells are described in the online supplementary materials. Staining was performed on cells after two PBS (calcium- and magnesium-free) washes and fixation in 95% methanol. Mineralized nodules were stained after 10 minutes in a 1% alizarin red S (Sigma) solution and two washes in water. Cell matrix–associated calcium deposition was determined by colorimetric assay performed in 96-well plates using the Sigma calcium assay kit (Sigma) [13]. Alkaline phosphatase (AP) activity was determined in 96-well plates by measuring the dephosphorylation of nitrophenyl-phosphate as previously described [36]. The protein content of cell cultures was determined in parallel wells with Pierce BCA Protein Assay kit (Perbio, Tattenhall, U.K.).

Adipogenic and Chondrogenic Differentiation
Detailed methods are described in the online supplementary materials.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Derivation of Fibroblast-Like Cells from hESCs
H1 hESCs were induced to differentiate using embryoid body formation followed by culture on gelatin-coated plates. Initially, the cultures contained a heterogeneous population of cells that became more homogeneous with subsequent passaging (three passages). Most cells showed a fibroblast-like or mesenchymal-like morphology, and the cultures were designated as human embryonic fibroblast-like cells (HEF1 cells) (Fig. 1Go). At passage 5, the cells were infected with a retrovirus, pBABE-hTERT, containing the hTERT and puromycin resistance genes. Parallel control cultures were infected with a retrovirus pBABE containing only the puromycin resistance gene. Telomerase activity was assessed before and after infection in HEF1-hTERT and control cultures using TRAP analysis. No telomerase activity was detected before infection of the HEF1 cells (Fig. 2AGo). Twenty days (~ eight population doublings) after infection, the HEF1- hTERT cultures showed strong telomerase activity, whereas the control reference cultures did not (Fig. 2AGo). The HEF1-hTERT cells retained telomerase activity for at least 65 days (~30 population doublings, the latest time point examined), showing that hTERT was stably introduced into the HEF1 cells.



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  		Figure 2. Immortalization of HEF1 cells. (A): Telomerase activity of HEF1 cells before and 20 days (~8 population doublings) or 65 days (~30 population doublings) after virus infection, as determined by telomeric repeat amplification protocol assay. An aliquot of each sample equal to approximately 1,000 cells (left lanes under each condition) or 5,000 cells (middle lane under each condition) was assayed. As a control, heat-inactivated samples were also assayed (right lane under each condition). (B): Growth kinetics of HEF1 cells infected with a retrovirus expressing hTERT or a control virus. (C): Analysis of senescence-associated ß-galactosidase in HEF1-hTERT and HEF1-control cells 65 days after virus infection. Abbreviation: HEFI, fibroblast-like cells derived from hESCs; hTERT, human telomerase reverse transcriptase.

 
Introduction of hTERT into the HEF1 cells extended the lifespan of the culture. As shown in Figure 2CGo, HEF1-hTERT cells had very low or undetectable senescence-associated ß-galactosidase activity, an established biomarker associated with cellular aging [33]. In contrast, HEF1 control cells were strongly positive for ß-galactosidase activity (Fig. 2CGo), indicating that these populations were undergoing senescence. After selection with puromycin, HEF1 cells infected with the hTERT retrovirus (HEF1-hTERT) cells proliferated with an approximate doubling time of 54 hours (Fig. 2BGo). The HEF1-hTERT cells continued to expand and maintained a consistent morphology and doubling time even after cryopreservation. In contrast, cells infected with the control virus (HEF1-control) stopped proliferating 38 days after infection (Fig. 2AGo). These data indicate that the introduction of hTERT extended the lifespan of HEF1 cells.

Feeder-Free hESC Culture Using Medium Conditioned by HEF1-hTERT Cells
We have previously shown that undifferentiated hESCs can be maintained on Matrigel in media conditioned by cells such as MEF-CM [6]. To examine whether the HEF1-hTERT cells can provide critical factors for hESC growth, hESC medium was conditioned using mitotically inactivated HEF1-hTERT cells. The HEF1-CM was then tested for its ability to support growth of two separate hESC lines (H7 and H9) grown on Matrigel. Similar to parallel cultures maintained in MEF-CM, cells seeded onto Matrigel in HEF1-CM gave rise to many colonies of undifferentiated hESCs with differentiated stroma-like cells between the colonies. Over the next few days, these colonies increased in size and appeared indistinguishable from hESCs maintained on Matrigel in MEF-CM. As shown in Figure 3AGo, H9 hESCs maintained in these conditions for four passages using standard passaging techniques continued to display undifferentiated hESC morphology. Similar morphology was observed in H7 hESCs after being maintained in HEF1-CM for at least14 passages (98 days). The H7 cells maintained in HEF1-CM retained expression of undifferentiated cell markers, such as OCT-4 and hTERT, as examined by TaqMan real-time reverse transcriptase–PCR analysis (Fig. 3BGo), and telomerase activity, as measured by the TRAP assay (Fig. 3CGo). Furthermore, hESCs maintained in HEF1-CM or MEF-CM expressed surface markers for undifferentiated hESCs, such as SSEA-4 (Figs. 3D–3GGo), TRA-1-60 (data not shown), and TRA-1-81 (Figs. 3H, 3IGo), predominantly in the colonies but not in the differentiated stroma-like cells. Like the parallel cultures in MEF-CM, the colonies of hESCs maintained in HEF1-CM had alkaline phosphatase activity (Figs. 3J, 3KGo) and expressed connexin 43, a gap junction protein (Figs. 3H, 3IGo). Last, after 13 passages in HEF1-CM, H7 hESCs maintained a normal female karyotype, as analyzed by G banding (data not shown).



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  		Figure 3. Cultures of human embryoinic stem cells using CM produced by HEF1-hTERT. (A): Morphology of H9 cells maintained on Matrigel in MEF-CM (passage 29) or HEF1-CM for four passages (passage 25 + 4). Bar = 300 µm. (B): Real-time reverse transcription–polymerase chain reaction analysis of relative levels of OCT-4 and hTERT expression in H7 cells (passage 29) maintained in HEF1-CM for six passages (passage 29 + 6) compared with MEF-CM control cells (passage 35). (C): Telomerase activity of the H9 cells (passage 29) maintained in MEF-CM or HEF1-CM for four passages. Three lanes were run for each condition. The left lane represents approximately 1,000 cells, the middle lane represents 5,000 cells, and the right lane is the heat-inactivated control. (D–I): Detection of surface markers in H7 cells maintained in MEF-CM (passage 34) (D, F, H, J) or HEF1-CM for five passages (passage 29 + 5) (E, G, I, K). SSEA-4 and connexin 43 were detected by fluorescein isothiocyanate, and TRA-1-81 was detected by Texas red. Bar = 200 µm for D, E, J, and K. Bar = 20 µm for F, G, H, and I. Abbreviations:AP, alkaline phosphatase; HEFI-CM, conditioned medium from hESC-derived immortal fibroblast-like cells; hTERT, human telomerase reverse transcriptase; MEF-CM, mouse embryonic fibroblast conditioned medium.

 
To evaluate the differentiation capacity of the cells maintained in HEF1-CM, H7 cells maintained in the medium for 12 passages were induced to differentiate using EB formation and subsequent culture on a gelatin-coated surface. Like cells cultured in MEF-CM, hESCs maintained in HEF1-CM differentiated into a population of cells with heterogeneous morphologies, including beating cells. We then evaluated the differentiation by immunocytochemical analysis using antibodies specific for cardiac troponin I (cTnI), alpha feto-protein (AFP), and ß-tubulin III. cTnI+ cells with striations characteristic of the sarcomeric structures of muscle cells, AFP+ cells with a flat morphology, and ß-tubulin III+ cells with neuron morphology were detected in these cultures (Fig. 4Go).



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  		Figure 4. Differentiation of human embryonic stem cells maintained in HEF1-CM. H7 cells maintained in HEF1-CM for 12 passages (passage 29 + 12) were induced to differentiate through EB formation. After 4 days in suspension, EBs were plated on gelatin-coated chamber slides. The cultures were fixed after an additional 7 days and subjected to immunocytochemical analysis for expression of AFP, ß-tubulin III, and cTnI. Bar = 50 µm. Abbreviations:AFP, alpha fetoprotein; cTnI, cardiac troponin I; EB, embryoid body; HEFI-CM, conditioned medium from hESC-derived fibroblast-like cells.

 
Taken together, these results indicate that cells maintained in HEF1-CM possess characteristics of undifferentiated hESCs, including morphology, surface marker and transcription factor expression, telomerase activity, karyotypic stability, and differentiation capacity.

Characterization of HEF1-hTERT Cells
Because the HEF1-hTERT cells are useful for maintaining undifferentiated hESCs, we additionally characterized these cells. Morphologically, HEF1-hTERT cells seem to be similar to hMSCs. Flow cytometry analysis showed that the HEF1-hTERT cells expressed surface markers [37] CD29, CD44, CD71, and CD90 at similar levels to hMSCs. CD106 was expressed at lower levels than observed in hMSC, whereas no CD45 or CD14 expression was observed in either HEF1-hTERT or hMSC (online supplementary Fig. 1Go). When treated with OS factors, HEF1-hTERT cells and hMSCs differentiated into distinct nodules that stained positive with alizarin red and showed increased calcium deposition and alkaline phosphatase activity (Fig. 5Go). In contrast, HEK293 cells, a nonosteogenic cell line used as negative control, failed to respond to the same conditions. When treated with an adipogenic or chondrogenic culture conditions promoting hMSC differentiation, HEF1 did not seem to differentiate (online supplementary Fig. 2Go). These results indicate that HEF1-hTERT cells respond to osteogenic factors in a comparable way to hMSC cells but do not exhibit in vitro adipogenic or chondrogenic potential using the conditions tested.



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  		Figure 5. Differentiation of HEF1-hTERT cells. (A): alizarin red staining of cultures after 14 days in control (upper panel) or OS-supplemented medium (lower panel). (B): Time course of calcium deposition (upper panel) and ALP activity (lower panel) after the treatment. Results are presented as mean ± standard error of the mean (n = 4). Bar = 50 µm. Abbreviations: ALP, alkaline phosphatase; hMSC, human mesenchymal stem cell; hTERT, human telomerase reverse transcriptase; OS, osteogenic supplement.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
hESCs possess extended replicative capacity and express high levels of telomerase, indicating that these cells are functionally immortal. However, upon differentiation, these cells downregulate telomerase expression, resulting in cell populations with limited lifespan, as seen in normal somatic cells. Replicative senescence of somatic cells commences due to critical shortening of telomeres in the absence of telomerase expression [32, 38, 39]. Ectopic expression of hTERT in several cell lines has been shown to restore functional telomerase activity and to extend the replicative lifespan of the cells [27–32].

The data presented here show that ectopic expression of hTERT in HEF1 cells leads to sustained telomerase activity and continued proliferative expansion beyond senescence. It is expected that this technique can be applied to other types of hESC derivatives as well. Because various cell types can be generated from hESCs, immortalization of these cells broadens the potential applications of these cells for tissue regeneration, drug screening, or basic biological study. In addition, the introduction of telomerase may offer other advantages. For example, telomerase expression in hMSCs has been shown to enhance their osteogenic potential [40]. Similarly, ectopic expression of hTERT enhances regenerative activities of endothelial progenitor cells [41]. Furthermore, expression of TERT provides protection from apoptosis of cardiac muscle cells [42,43] and neurons [44–47]. Therefore, expression of hTERT in hESC derivatives may be valuable for multiple applications.

We have found that HEF1-hTERT cells can serve as feeders to support the growth of undifferentiated hESCs (data not shown). However, because the feeder-free system is a much more promising system for scale-up production of hESCs, our study focused on characterization of cells maintained in the feeder-free culture system using HEF1-hTERT cell CM. Cells in HEF1-CM maintained the morphology of typical undifferentiated hESC cultures. These cells also maintain expression of markers for undifferentiated hESCs, including SSEA-4, TRA-1-81,AP, OCT-4, and hTERT. After several months in culture, the cells retain a normal karyotype and the ability to differentiate in vitro into many cell types, including those with neuron and contracting muscle cell morphologies. Immunocytochemical analysis indicated the presence of cTnI+,APF+, and ß-tubulin III+ cells in differentiated cultures, although more studies are required to further confirm that these cells are functional differentiated cells. In addition, further investigation is desired to assess the capacity of teratoma formation from the hESCs maintained in the HEF1-CM.

Fibroblast-like cells similar to HEF1 cells can also be isolated from other hESC lines. For instance, we isolated fibrob-last-like cells from EB outgrowths of another cell line, H9. hTERT was introduced into these cells using the same methods we have described here, resulting in H9 cell–derived fibroblast-like cells. These cells showed a similar morphology to HEF1-hTERT and were also able to produce CM sustaining the growth of undifferentiated hESCs (data not shown). Using hES derivatives to maintain undifferentiated hESCs creates a genotypically homogeneous system for the culture of hESCs. It should be noted, however, that an isogenic culture system could not be used for derivation of new hESC lines, because the HEF1 fibroblast-like cells can only be differentiated from established hESC lines.

hESC culture has historically relied on mouse embryonic feeder cells [1,2] or medium conditioned by MEF [6], which can complicate their production for human therapy. CM from qualified human cells would facilitate the safe growth of hESCs for clinical applications. However, previous studies from our laboratory showed that CM from several human cell lines, such as a human foreskin fibroblast cell line, BJ5ta, and retinal epithelial cells, does not support the robust hESC growth over long-term culture [6]. Recently, it has been reported that human fetal and adult fibroblasts [3] and human adult marrow cells [48] can be used as feeders to grow hESCs; however, it was reported that these cells are unable to provide CM for hESCs. Thus, the HEF1-hTERT cells are uniquely suitable for generating growth medium to grow undifferentiated hESCs. Immortalization of CM–producing cells adds strong advantages for this type of application.

In addition to directly supplying CM for hESC culture, the system may be further exploited to identify and characterize novel factors that support hESC proliferation and pluripotency. This may ultimately lead to a completely defined medium that is compatible with therapeutic applications without the pitfalls associated with xeno components in the current hESC culture systems.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Dr. Calvin Harley for insightful discussions and critical review of the manuscript. This work is supported by the Geron Corporation.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received December 5, 2003; accepted for publication May 27, 2004.



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