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发表于 2009-3-5 10:47 |只看该作者 |倒序浏览 |打印

Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands

Key Words. Human mesenchymal stem cells ? Gene transfer ? Adenoviral vector ? Tropism modification ? Human species B adenoviruses ? CD466 R- u/ O8 R9 i; _% S5 Z

Correspondence: S. Kna?n-Shanzer, Ph.D., Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Telephone: 31-71-5271966; Fax: 31-71-5276180; e-mail: s.knaan@lumc.nl
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ABSTRACT% a5 ]) \# D7 P3 w6 R" o! ]( Y
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Mesenchymal stem cells (MSCs) have attracted much attention in recent years as a source of multipotent precursor cells that can give rise to bone, cartilage, fat, ligament/tendon, muscle, and bone marrow (BM) stroma . In adults, the presence of MSCs was first demonstrated in the BM . Subsequent studies identified cells with similar properties in adipose , muscle , and synovial  tissues. Cells with MSC properties have also been found in amniotic fluid  and in human first-trimester fetal blood, liver, and BM .; ?4 H' M2 v2 y5 s7 ?7 L0 a3 ?  j
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In human BM samples, MSCs represent 0.01%–0.001% of all nucleated cells and are distinguishable from hematopoietic stem cells (HSCs) by their repertoire of cell surface antigens. In addition, MSCs can be easily separated from HSCs by their propensity to adhere to tissue culture plastics. As opposed to HSCs, MSCs are capable of extensive proliferation ex vivo while maintaining a normal karyotype and telomerase activity for several passages ., C  t5 |6 m+ |- S

The ability to generate a substantial MSC pool from a small BM aspirate, together with their multipotentiality, makes these cells an attractive cellular substrate for cell-based therapies entailing ex vivo genetic modification and autologous transplantation to replenish cells in diseased or damaged tissues. Genetically modified MSCs can also be used as cellular vehicles for the local or systemic delivery of therapeutic gene products . To supplement MSCs with exogenous genes, viral vectors are often used. The choice for a particular vector system is determined primarily by the requirements of a given treatment (e.g., permanent or short-term transgene expression) and by the entry receptors available at the surface of the target cells. Studies using vectors based on murine oncoretroviruses, lentiviruses, and adenoviruses (Ads) have shown that each of them is able to deliver genes into MSCs, albeit with a different efficiency.

Murine oncoretroviral vector–mediated transduction of MSCs is relatively inefficient . Improvement of transduction protocols and inclusion of selection procedures yielded human MSC (hMSC) preparations enriched for stably transduced cells . The lack of long-term transgene expression, often observed after murine oncoretroviral transduction, is probably due to promoter inactivation .

Lentiviral vectors based on the HIV pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) have also been used to transduce MSCs. Although very effective , they were found, in some cases, to be toxic for MSCs even at relatively low multiplicities of infection (MOIs). Cytotoxicity, however, could be overcome, without compromising transduction efficiency, by pseudotyping the vector particles with the envelope glycoprotein of the feline endogenous virus RD114 instead of VSV-G .3 v$ G) ~- }, e4 t" z
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Most of the Ad vectors that have been used to transduce hMSCs were based on human Ad serotype 5 (Ad5). Although hMSCs do not express the Coxsackie B-Ad receptor (CAR), which constitutes the primary attachment receptor for human species C Ads, some groups reported successful transduction of these cells with Ad5 vectors using very high MOIs . The entry of Ad5 vectors can be rendered independent of CAR by modification of their fiber proteins. We and others have shown that Ad5 vectors containing fiber shaft and knob domains of human species B Ads very efficiently transduce a variety of human cells, including early hematopoietic progenitor cells , committed and malignant hematopoietic cells , and various primary cells of other tissues . Likewise, hMSCs have been effectively transduced by Ad5 vectors carrying fiber domains of human Ad serotype 35 . However, a thorough comparison of gene transfer efficiencies into hMSCs by conventional and fiber-modified Ad5 vectors has not been performed. We therefore quantified enhanced green fluorescent protein (eGFP) gene transfer into hMSCs after incubation with Ad5 vectors or with Ad5 vectors displaying fiber shaft and knob domains of Ad serotypes 50, 35, and 16. Furthermore, the effects of Ad vector infection and eGFP expression on the growth characteristics and differentiation ability of hMSCs were studied.2 O! V* s! U# b, f8 a& N8 ^

MATERIALS AND METHODS
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Ad Vector–Mediated Gene Transfer into hMSCs5 v* J& U5 `5 m: w

The permissiveness of hMSCs to replication-deficient Ad5 vectors carrying fiber shaft and knob domains of species B Ads (Ad5FBs) was compared with that of an unmodified Ad5 vector. The Ad5FBs used were Ad5F50, Ad5F35, and Ad5F16 displaying fiber domains from Ad serotypes 50, 35, and 16, respectively . Gene delivery efficiency was determined 48 hours post-transduction using flow cytometry and fluorescence microscopy. The flow cytometry data are presented both as the frequency of transgene-expressing cells and as the average amount of eGFP per cell expressed as mean fluorescence intensity (MFI). The results of a representative experiment in which hMSCs were transduced with increasing doses of Ad5F50 are shown in Figure 2. The images depicted in the photomicrographs were all captured after a 100-millisecond exposure. With this moderate exposure time, no background signals due to autofluorescence were detected (see upper panel, MOI 0); it did, however, reduce the sensitivity of the assay. The flow cytometric analysis, on the contrary, enables a better signal-to-background ratio. Plotting the green (i.e., x-axis) against the red (i.e., y-axis) fluorescence permits visualization of autofluorescence-derived signals as a diagonal cluster (see upper dot plot; MOI 0). Genuine eGFP signals are located to the right of the diagonal threshold line. Hence, only the flow cytometry results are presented.6 i' I2 T& h% R

Figure 2. Fluorescence microscopy and flow cytometric analyses of hMSCs transduced with Ad5F50 vector at different MOIs. Cultured hMSCs were transduced with Ad5F50 vector at MOIs ranging from 1 to 300. Two days after transduction, eGFP expression was monitored using both fluorescence microscopy and flow cytometry. The photomicrographs (left panels) show representative fields of the different cultures, containing similar numbers of hMSCs. The dot plots (right panels) are based on the flow cytometric analysis of 10,000 events. The eGFP fluorescence (x-axis) is plotted against the signal from the FL-2 channel (y-axis). The gate in the dot plots defining the eGFP-positive population was set using mock-transduced cells. At the right side of each dot plot, the corresponding frequency of eGFP-expressing cells and the MFI of the eGFP-positive cells are specified. Abbreviations: eGFP, enhanced green fluorescent protein; hMSC, human mesenchymal stem cell; MFI, mean fluorescence intensity; MOI, multiplicity of infection.
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Transduction of hMSCs derived from BM of three donors with fiber-modified Ad5 vectors gave rise to a larger percentage of transduced cells and higher transgene expression levels than infection with the unmodified Ad5 vector (Fig. 3). Approximately 50% of the cells in the hMSC cultures expressed eGFP after transduction with any of the Ad5FBs at an MOI of 10, and 80%–100% of the cells became eGFP-positive at an MOI of 100. The MFI of eGFP-positive hMSCs infected with 100 IU of Ad5FB per cell was 4.3-fold that of cells transduced at an MOI of 10. With Ad5 vectors, the frequency of eGFP-positive cells reached approximately 25% at an MOI of 100, but the MFI was only 1.8-fold that of hMSCs transduced at an MOI of 10. Furthermore, at an MOI of 100, the MFI of Ad5FB-transduced cells was six- to eightfold higher than that of hMSCs transduced with Ad5 vectors.
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Figure 3. Transduction of bone marrow–derived hMSCs with conventional and fiber-modified Ad5 vectors. hMSCs derived from three donors that were kept in culture for 9 (left panel), 12 (middle panel), and 8 (right panel) passages were infected with the Ad5 vector or with the chimeric fiber-containing vectors Ad5F50, Ad5F35, or Ad5F16 at MOIs of 10 and 100 and subjected to flow cytometric analysis at 48 hours postinfection. Mock-transduced samples were used to determine background fluorescence. Abbreviations: eGFP, enhanced green fluorescent protein; hMSC, human mesenchymal stem cell; MFI, mean fluorescence intensity; MOI, multiplicity of infection.
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The correlation between Ad5FB vector dose and the intracellular levels of eGFP was even clearer in an experiment using a broader range of MOIs. As displayed in Figure 4 (upper panel), more than 90% of the hMSCs express eGFP after transduction with Ad5F50 or Ad5F35 at an MOI of 30. In the cell cultures treated with the Ad5 vector, no more than approximately 50% of the hMSCs were found to be eGFP-positive at a 10-fold higher MOI. Furthermore, in contrast to the situation in the Ad5 vector–modified cells, the reporter protein load of hMSCs transduced with Ad5F50 and Ad5F35 markedly increased with increasing vector doses and did not reach saturation even at an MOI of 300 (Fig. 4, lower panel). All hMSC lots tested shared a high permissiveness for Ad5FBs. Importantly, the variability in the frequency of eGFP-positive cells as well as in the average transgene expression level was limited between samples of different donors. The passage history of the cells did not affect their susceptibility to Ad5FBs (Table 1).
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Figure 4. Comparative analysis of the transduction of hMSCs by Ad5FBs and by the Ad5 vector. hMSCs were infected with 1–300 IU per cell of Ad5F50, Ad5F35, or the Ad5 vector and subjected to flow cytometric analysis at 48 hours postinfection. The results of a representative experiment are presented. Abbreviations: eGFP, enhanced green fluorescent protein; hMSC, human mesenchymal stem cell; MFI, mean fluorescence intensity; MOI, multiplicity of infection.3 a4 z2 t) E4 ]8 N2 H

Table 1. Transduction of human mesenchymal stem cells with Ad5F50

Longevity of Transgene Expression in Cultured hMSCs
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The persistence of eGFP in Ad5F50-transduced hMSCs was studied under standard culture conditions and in a setting of limited cell replication. To investigate eGFP persistence under conditions of restrained cell division, hMSCs were seeded at a high density (2 x 104 cells per well of 24-well plates) and transduced 24 hours later with various doses of Ad5F50. Flow cytometric analysis at different time points after transduction (Fig. 5A) revealed that the numbers of eGFP-positive hMSCs remained fairly constant in all samples for the first 3 weeks (apart from an early decrease in cell number in cultures transduced at an MOI of 3). After day 21, the numbers of eGFP-positive cells began to decline. The corresponding MFIs (Fig. 5B) at day 36 post-transduction were, however, very similar to the levels measured at 2 days after infection except for the hMSCs infected at an MOI of 100. In this sample, eGFP levels rose sharply during the first week to decline to half the initial levels at day 36. The reduction in MFI may in this particular case have been caused by the preferential loss of cells containing very high concentrations of the reporter protein. The total number of hMSCs in all cultures except for the one infected with 100 IU of Ad5F50 per cell was approximately 1.5 times the input value both at 2 weeks and at 5 weeks post-transduction (data not shown). This latter finding in combination with the absence of cell death in these cultures confirmed the very limited expansion of hMSCs in this experiment.

Figure 5. Longevity of eGFP expression in Ad5F50-transduced hMSCs. The eGFP expression in hMSCs transduced with a wide dose range of Ad5F50 was assessed under (A, B) conditions of restrained cell division (i.e., seeding the cells at high cell density and no passaging) and under (C–E) standard culture conditions (i.e., seeding the cells at a low cell density and subculturing at 70% confluency). For both culture types, the numbers of (A, D) eGFP-positive cells and the (B, E) MFI are displayed as a function of time. (C): hMSCs transduced with Ad5F50 at the second passage and cultured under standard culture conditions were used to calculate the cell doubling time during three consecutive passages (PN3 through PN5). Mock-transduced cells served as controls. Abbreviations: eGFP, enhanced green fluorescent protein; hMSC, human mesenchymal stem cell; MFI, mean fluorescence intensity; MOI, multiplicity of infection.
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To monitor the kinetics of transgene expression under conditions of unrestrained cell replication and to evaluate the effect of eGFP and vector-encoded adenoviral proteins on the division rate of hMSCs, cells at the second passage were plated in 25-cm2 flasks at a concentration of 3 x 103 cells per cm2 and transduced with increasing MOIs of Ad5F50. The cells were subcultured each time they reached 70% confluency. Parallel cultures, initiated in 24-well plates, served to determine the transduction efficiencies at 48 hours postinfection. At every passage, the DT of the cells and the eGFP expression levels were determined. As shown in Figure 5C, transduction of hMSCs with Ad5F50 at passage 2 and the consequent expression of heterologous genes resulted in an immediate (PN3), dose-dependent effect on cell DT. Whereas at the lowest MOIs (1 and 3) the DT equalled that of the mock-transduced cells, at the highest vector dose tested, it was six times as long. In subsequent passages (PN4 and PN5), the DT of cells in all samples, except for the one infected at an MOI of 100, regained control values. The vector dose of 100 IU per cell was found, under these conditions, to be detrimental to hMSC replication.
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The total number of eGFP-positive cells (calculated at each time point from the frequency of eGFP-positive cells and the total number of progeny derived from the inoculum) during the 30-day culture, reflects the replication kinetics of the cells in each group (Fig. 5D). The highest numbers of eGFP-positive hMSCs (three times the number of eGFP-positive cells measured at 48 hours) were present at 2 and 3 weeks post-transduction in the samples transduced at low (3 and 10) and moderate (30) MOIs, respectively. There was no significant increase in the number of eGFP-positive hMSCs in the cell culture that received the highest vector dose. Transgene expression levels in proliferating hMSCs gradually declined with time (Fig. 5E).

From these experiments, we conclude that foreign DNA delivered by Ad5FBs can be expressed at high to moderate levels in a large population of replicating hMSCs for approximately 3 weeks. Under conditions of limited hMSC replication and at moderate vector doses, the foreign protein persisted at high levels for at least 36 days.$ M+ O% t+ _0 D- W" b1 j

The Effect of Ad5F50 Transduction on hMSC Differentiation. I+ Z, d! h2 @# e7 }& r
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The multilineage differentiation potential of BM-derived hMSCs was assessed by culturing the cells in medium reported to induce adipogenesis and osteogenesis. For this purpose, we used hMSCs from different human adults at passages 4 through 6. Lipid vacuoles were readily detectable in all samples after incubation for 1 week in adipogenic differentiation medium. Three weeks after providing the adipogenic trigger, lipid vacuoles were visualized by staining of the cells with Oil Red O (Fig. 6A). Cells exposed to osteogenic differentiation medium showed, 3 weeks later, an abundance of calcium deposits after incubation with Alizarin Red S (Fig. 6B). hMSCs maintained in regular culture medium for the same time period were not stained with Oil Red O or Alizarin Red S (Figs. 6C and 6D, respectively).8 _, [' q' c/ z) T" O7 A! R( ^# L6 `

Figure 6. Adipogenic and osteogenic differentiation of Ad5F50-transduced human mesenchymal stem cells (hMSCs). hMSCs at the fifth passage were transduced with Ad5F50 at a multiplicity of infection of 30. Control cultures consisted of untransduced hMSCs cultured in differentiation medium promoting (A) adipogenesis or (B) osteogenesis or (C, D) on regular culture medium. At 48 hours postinfection, the culture medium was exchanged for (E, G) adipogenic or (F, H) osteogenic differentiation medium. Adipogenic and osteogenic differentiation was evaluated 3 weeks later by staining the cells of each culture with (A, C, E, G) Oil Red O or (B, D, F, H) Alizarin Red S. The photomicrographs were taken at magnifications of (A–F) x40 or (G, H) x100. Photomicrographs (E–H) are merges of bright field images with the corresponding fluorescence field images.
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To determine the effect of Ad5F50-mediated transduction on the differentiation capacity of hMSCs, the cells were transduced at an MOI of 30. Before exchanging the regular culture medium for the differentiation medium, approximately 70% of the hMSCs expressed eGFP. As with the untransduced cells, lipid vacuoles were detectable 1 week after exposure to adipogenic differentiation medium. Three weeks after induction of adipogenesis, most of the cells stained positive with Oil Red O (Figs. 6E, 6G). The majority of these cells also expressed eGFP. Likewise, Ad5F50 transduction did not inhibit osteogenic differentiation as was evident from the high percentage of cells that contained both eGFP and calcium deposits. (Figs. 6F, 6H).: j$ V, F9 s& g3 g' D$ K

Taken together, these results show that BM-derived hMSCs that have been transduced by Ad5F50 vectors retain intact their adipogenic and osteogenic differentiation capacity.' c/ |1 d3 ?, r/ \

CD46 May Not be the Sole Receptor for Species B Ads on hMSCs

Two recent studies have demonstrated that CD46 serves as a receptor for species B Ads . Surprisingly, although BM-derived hMSCs express relatively low levels of CD46 as detected by flow cytometry with MAb E4.3, they were highly susceptible to Ad5FBs. The E4.3 antibody was used as directed against the short consensus repeat 1 of CD46, which is present in all isoforms of the protein. To evaluate whether flow cytometry is an appropriate method to estimate the number of surface antigens on cells, we plotted for five different cell lines the MFI shift (i.e., the MFI after subtraction of background fluorescence) after labeling with E4.3 against the number of CD46 molecules quantified by Cho et al.  using a radioimmunoassay based on the same MAb. The results depicted in Figure 7A showed a linear correlation between the two measurements with a coefficient of determination (R2) of 0.9847. Analysis of 11 different hMSC samples yielded an MFI shift of 18.5 ± 12. Using the calibration curve, we extrapolated that the number of CD46 molecules per hMSC is less than 30,000. In accordance with the previous reports implicating CD46 as a cellular receptor for species B Ads , the permissiveness of HeLa and K-562 cells for Ad5F50 was directly proportional to the number of CD46 molecules present on their surface (compare Fig. 7A with Fig. 7B).. |2 t- U. @4 s- j4 K. Q, `

Figure 7. The low expression of CD46 at the surface of hMSCs does not correlate with their efficient transduction by Ad5FBs. (A): Correlation analysis between two different methods to evaluate CD46 levels at the surface of five different cell types. The x-axis indicates the average number of CD46 molecules on the surface of the specified cell types determined by radioimmunoassay , and the y-axis indicates the MFI shift (i.e., the difference in MFI between populations of the indicated cell types after labeling with the CD46-specific MAb E4.3 and with an isotope-matched control antibody). The MFI of the control samples was always less than 10. The slope was determined by linear regression analysis. The coefficient of determination (R2) of 0.9847 indicates a good correlation. The MFI shift of hMSCs is 18.5 ± 12 (n = 11), which corresponds to less than 30,000 CD46 molecules. (B): Susceptibility of HeLa and K-562 cells to Ad5F50. HeLa and K-562 cells, which express on average 102,707 and 58,902 CD46 molecules at their surface, respectively, were transduced with escalating doses of Ad5F50. The frequencies of eGFP-positive cells (upper panel) and the corresponding MFIs (lower panel) at 48 hours postinfection are presented. (C): Comparison of the susceptibility of HeLa, (102,707 CD46 molecules per cell), CHO (0 CD46 molecules per cell), and hMSCs (
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We next compared the susceptibility of hMSCs with Ad5F50 and Ad5F35 with that of HeLa and CHO cells, which express on average 102,707 and 0 CD46 molecules on their surface, respectively . With both vectors and at all MOIs tested, the frequency of eGFP-positive cells (Fig. 7C, upper panels), as well as the average amount of eGFP per cell (Fig. 7C, lower panels), was similar for hMSCs and HeLa cells. Transduction of CHO cells with Ad5F50 and Ad5F35 was very inefficient. To transduce approximately half of the CHO cells, an MOI of 1,000 was required (Fig. 7C, upper panels). However, the eGFP levels in these cells were extremely low (Fig. 7C, lower panels), confirming that CHO cells take up Ad5FBs very poorly.
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Taken together, our observations strongly suggest that species B Ads can use cell surface molecules other than CD46 to enter hMSCs.4 H0 H" J$ s$ R1 [3 n8 ]  E+ U

DISCUSSION
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The authors are indebted to Jeffrey Bergelson (Children’s Hospital of Philadelphia) for donating the CAR-specific MAb, Peter Bredenbeek (Department of Medical Microbiology, LUMC) for supplying the Hep-2 cells, and Rob Nelissen (Department of Orthopedic Surgery, LUMC) for providing surgical remnants. We also thank Dick van Bekkum (Crucell N.V., Leiden, the Netherlands) and Vered Raz (Department of Molecular Cell Biology, LUMC) for their comments on the manuscript.+ s& F- [+ o$ U  L; [9 _
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DISCLOSURES! S8 H+ V7 L- Q4 a( t; f
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D.V. owns stock in Crucell and within the past 2 years has acted as a consultant and served as an officer or member of the Board for Crucell.
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