Lineage Differentiation-Associated Loss of Adenoviral Susceptibility and Coxsackie-Adenovirus Receptor Expression in Human Mesenchymal Stem
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《干细胞学杂志》
a Departments of Orthopedics and Traumatology and
b Medical Research and Education, Veterans General Hospital-Taipei, Taipei, Taiwan, Republic of China;
c Departments of Surgery and
d Internal Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China;
e Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan, Republic of China;
f Department of Orthopedic Surgery, Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan, Republic of China
Key Words. Human mesenchymal stem cells ? Adenoviral vectors ? Coxsackie-adenovirus receptor ? Green fluorescent protein ? Gene transfer
Correspondence: Shih-Chieh Hung, M.D, Ph.D., Department of Orthopedics and Traumatology, Veterans General Hospital-Taipei, 201, Sec. 2, Shih-Pai Road, Taipei, 11217, Taiwan. Telephone: 886-2-28757557, ext. 118; Fax: 886-2-28265164; e-mail: hungsc@vghtpe.gov.tw
ABSTRACT
Adenoviral vectors (AdVs) are well-known for their stability, ease of handling, and high uptake by many cell types, including noncycling cells. In addition, AdV can be concentrated to extremely high titers as well . Recent advances in AdV modifications offer a less immunogenic strain (defective recombinants) and an increase in the recombinant genome insert size . Therefore, the application of AdV is more relevant for human gene therapy than other means.
Because the efficacy of virus entry is restricted by interaction of viral fibers and cell-surface receptors, the abundance and the variety of viral-associated surface molecules expressed in cells are particularly important. The entry pathway for AdV consists of initial binding to the cells, which is mediated by the association of the adenoviral fiber protein and a 46-kDa membrane protein known as Coxsackie-adenovirus receptor (CAR) , followed by internalization, which is through an interaction of viral penton arginine-glycine-aspartate sequence to the v?3 and v?5 integrins .
The CAR is a primary passage, mediating the uptake of adenovirus in the 293 cell line, HeLa cell line, and cells derived from airways. However, cellular function of CAR is not clear, and CAR is only expressed in some types of cells and tissues. It has been demonstrated on oropharyngeal epithelial cells that the superficial layer (more developed) had less CAR expression than the basal layer (less developed), suggesting CAR expression correlated reciprocally with the status of differentiation . In contrast, CAR expression was only demonstrated in a small subset of CD34+ bone marrow (BM) and mobilized blood cells , whereas AdV transduction efficiency and CAR expression increase as the cells differentiate into erythroid and myeloid hematopoietic cells . CAR is expressed in human mesenchymal stem cells (hMSCs) but not expressed in certain kinds of mesenchymal cells such as primary human fibroblast .
Because hMSCs can be induced to differentiate into certain kinds of mesenchymal and nonmesenchymal tissues, they provide a model to study the effects of differentiation on AdV transduction and CAR expression. Therefore, we investigated AdV transduction efficiency and CAR expression in hMSCs and their differentiated progeny. Efforts were also made to characterize the transduced hMSCs in the differentiation potential and to know the persistence of transgene expression into hMSC progeny.
MATERIALS AND METHODS
InVitro AdV-Mediated Gene Transfer into hMSCs
For these studies, gene transfer was demonstrated by AdV transduction with the EGFP gene. The hMSC cultures were infected with Ad-PGK-EGFP, and 2 days later, transduction was assessed by transgene expression. As seen in Figure 1, after exposure to Ad-PGK-EGFP for 2 days, almost all cells were transduced as detected under both fluorescent microscope (Figs. 1A, 1B) and by flow cytometry analysis (Fig. 1C), displaying high fluorescent signal for GFP. At this time, transduced cells encoding GFP fluorescence were found to have a good viability and no disturbance in adherence. A strong green fluorescence in the infected hMSCs indicated a high efficiency of virus particles entering in cells. The efficiency of AdV-mediated gene transfer into hMSCs as analyzed by flow cytometry was 91.7 ± 6.6% (Table 1). High AdV transduction efficiency was also attained by the use of Ad-CMV-EGFP, a vector directing the expression of EGFP under the control of a cytomegalovirus (CMV) promoter (data not shown). These cells were expanded in normal growth medium with transgene retained and expressed in a small fraction of hMSC progeny even up to 4 weeks after infection (Figs. 1E, 1F). The cells carrying transgene adopted a picture of senescence, with a greater size and a broader shape, suggesting the transgene expression in the case of adenoviral infection is transient in origin and cells tend to lose transgene expression during several cycles of proliferation.
Figure 1. The hMSC culture after infection with Ad-PGK-EGFP adenoviral vectors. (A): Phase microscopic appearance of hMSCs. (B): Transduced cells encoding GFP. Original magnification x100. (C): Percentage and GFP fluorescence intensity of transduced cells. (D): Expression of AdV attachment receptor CAR in hMSCs. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; CAR, Coxsackie-adenovirus receptor; hMSC, human mesenchymal stem cell.
Table 1. The average percentage of green fluorescent protein–positive cells after adenoviral vector infection in respective cell category (the culture duration of lineage-committed induction)
Expression and Participation of CAR in AdV-Mediated Gene Transfer into hMSCs
Conget and Minguell have reported that the attachment of CAR, but not integrins v?3 and v?5, is required for AdV-mediated gene transfer into hMSCs. Therefore, we investigated by flow cytometry whether these cells express CAR. As shown in Figure 1D, CAR expression was demonstrated in hMSCs using CAR-specific monoclonal antibody RmcB. The pattern of CAR expression was homogeneous, and nearly half of the cells had moderate expression of CAR. To determine the participation of CAR in AdV infection, hMSCs were infected with Ad-PGK-EGFP in either the presence or absence of monoclonal antibody RmcB for competing CAR. As shown in Figure 2, incubating specific monoclonal antibody RmcB against CAR immediately before AdV infection allowed AdV infection to be inhibited by nearly 40%. Moreover, the effect of CAR competition by specific monoclonal antibody to inhibit AdV infection in hMSCs was dose dependent.
Figure 2. Effect of inhibition of Coxsackie-adenovirus receptor on adenoviral vector–mediated transduction of hMSCs. The hMSC suspension was incubated in the absence (control) or presence of control mouse IgG or the RmcB antibody in ascites fluid at selected dilutions (x250, x50, x10, and x2). Cells were then infected with Ad-PGK-EGFP (48 hours), and the percentage of transduced cells was scored by flow cytometry and calculated with respect to control, which was set to 1. An inverse relationship of percentage of transduced cells to the concentration of RmcB antibody is demonstrated. Data shown represent the mean ± standard deviation. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; hMSC, human mesenchymal stem cell; IgG, immunoglobulin G.
Reduced AdV Transduction Efficiency and CAR Expression in Lineage-Differentiated hMSCs
The hMSC culture was induced to differentiate along the osteogenic, adipogenic, or neurogenic lineage, as previously described . As shown in Figure 3 and Table 1, hMSCs had a relatively low efficiency of AdV transduction, with 31%, 14%, and 59% at 10 days, 17 days, and 23 days, respectively, after osteogenic induction. The low efficiency of AdV transduction in the differentiated cells was correlated with a decrease in CAR expression after 10 days of osteogenic induction. The level of CAR expression was undetectable at 17 days after induction, but a regain of CAR was observed in 23-day induced cells.
Figure 3. The hMSC culture following infection with Ad-PGK-GFP adenoviral vectors after treatment with osteogenic (A), adipogenic (B), and neurogenic medium (C) for selected time periods. Histogram shows GFP fluorescence intensity data for transduced cells and expression of adenoviral vector attachment receptor CAR in hMSCs. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; CAR, Coxsackie-adenovirus receptor; hMSC, human mesenchymal stem cell.
An inverse correlation was also observed between the maturation of adipogenic differentiation and the efficiency of adenoviral transduction. The observed efficiency was 44%, 30%, and 19% at 3 days, 7 days, and 10 days, respectively, after adipogenic induction. CAR expression was only observed in a subpopulation of adipogenic-differentiated hMSCs, equal to 5% or less at 3 days after adipogenic induction, but CAR expression was undetectable if the induction was continued for more than 7 days.
Unlike osteogenic and adipogenic inductions, neurogenic induction of hMSCs had only minor influence on AdV transduction efficiency. The efficiency corresponding to 5 hours and 5 days after neurogenic induction was 93% and 89%, respectively. However, the prolonged neurogenic culture for 5 days had a lower fluorescent intensity compared with those that had neurogenic induction for 5 hours. In addition, hMSCs that underwent neurogenic induction had less CAR expression in total population compared with the undifferentiated status.
Differentiation Potentials of Transduced hMSCs and Persistence of Transgene Expression into hMSC Progeny
Because the adenoviral transduction is more efficient in the undifferentiated hMSCs than the fate-determined hMSC progeny, we have additionally investigated the plasticity of transduced hMSCs and the persistence of transgene expression into hMSC progeny after lineage-specific differentiation. After AdV infection, the hMSC culture was induced to differentiate along the osteogenic, adipogenic, or neurogenic lineage, and the persistence of the transgene into differentiated cells was studied under fluorescent microscope and by flow cytometry. As shown in Figure 4, AdV-transduced hMSCs that expressed GFP fluorescence and had the lipid vesicles synthesized and stained red by Oil red O dye were clearly identified at 10 days after adipogenic induction. The transduced cells loose in attachment were prone to retract from the culture surface. Moreover, our data have also demonstrated that AdV-transduced hMSCs maintained the potential to differentiate along the osteogenic lineage. Osteogenic differentiation was identified by the bone alkaline phosphatase (AP) stain, and fluorescence was proven on day 14 after initial induction. AP stain marked osteogenic progeny red to dark purple, and a thickening and mesh network of cytoskeleton was observed in the osteogenic culture (Fig. 5). In addition, indirect immunofluorescent staining for neuron-specific protein, ?-tubulin III, was used to identify neural differentiation of transduced hMSCs after induction with neural medium for 2 days. Those cells exhibit neuron maker primarily in cytoplasm but not nucleus, whereas GFP was concentrated in nucleus and some was dispersed in cytoplasm (Fig. 6).
Figure 4. Adipogenic differentiation of transduced hMSCs. The hMSC culture after infection with Ad-PGK-EGFP was treated with adipogenic medium for 10 days. (A): Transduced cells encoding GFP. (B): Oil red O staining cells. (C): The merged picture of (A, B). The same field exhibiting green fluorescence and stained red indicates the transduced cells have been differentiated into the adipogenic lineage. Original magnification x150. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; hMSC, human mesenchymal stem cell.
Figure 5. Osteogenic differentiation of transduced hMSCs. The hMSC culture after infection with Ad-PGK-EGFP was treated with osteogenic medium for 14 days. (A): Transduced cells encoding GFP. (B): Alkaline phosphatase staining cells. (C): The merged picture of (A, B). The same field exhibiting green fluorescence and stained red indicates the transduced cells have been differentiated into the osteogenic lineage. Original magnification x150. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; hMSC, human mesenchymal stem cell.
Figure 6. Neurogenic differentiation of transduced hMSCs. The hMSC culture after infection with Ad-PGK-EGFP was treated with neurogenic medium for 2 days. (A): Confocal image of transduced cells encoding GFP. (B): Some corresponding cells are positive for neuron marker, ?-tubulin III, as evidenced by immunofluorescence staining. Original magnification x300.Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; hMSC, human mesenchymal stem cell.
DISCUSSION
We thank R. Finberg (Harvard Medical School, Boston) for the gift of monoclonal antibody RmcB. We also thank MingLing Hsu for excellent flow cytometry technical support and Bor-Chun Weng for his assistance in preparing the manuscript. This work was supported in part by grant No. 92-376-1 from Veteran General Hospital-Taipei and by NSC 91-2321-B-002-005 from the National Science Council, Taipei, Taiwan.
REFERENCES
Crystal RG. Transfer of genes to humans: early lessons and obstacles to success. Science 1995;270:404–410.
Shenk T. Adenoviridae: the viruses and their replication. In: Fields BN, Knipe DM, Howley PM, eds. Fields Virology. Philadelphia: Lippincott-Raven Publishers, 1996:2112–2137.
Wilson JM. Adenoviruses as gene-delivery vehicles. N Engl J Med 1996;334:1185–1187.
Lee MG, Abina MA, Haddada H et al. The constitutive expression of the immunomodulatory gp19k protein in E1-, E3- adenoviral vectors strongly reduces the host cytotoxic T cell response against the vector. Gene Ther 1995;2:256–262.
Kochanek S, Clemens PR, Mitani K et al. A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase. Proc Natl Acad Sci U S A 1996;93:5731–5736.
Bergelson JM, Cunningham JA, Droguett G et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997;275:1320–1323.
Wickham TJ, Mathias P, Cheresh DA et al. Integrins V?3 and V?5 promote adenovirus internalization but not virus attachment. Cell 1993;73:309–319.
Hutchin ME, Pickles RJ, Yarbrough WG. Efficiency of adenovirus-mediated gene transfer to oropharyngeal epithelial cells correlates with cellular differentiation and human cox-sackie and adenovirus receptor expression. Hum Gene Ther 2000;11:2365–2375.
Neering SJ, Hardy SF, Minamoto D et al. Transduction of primitive human hematopoietic cells with recombinant adenovirus vectors. Blood 1996;88:1147–1155.
Bregni M, Shammah S, Malaffo F et al. Adenovirus vector for gene transduction into mobilized blood CD34+ cells. Gene Ther 1998;5:465–472.
Rebel VI, Hartnett S, Denham J et al. Maturation and lineage-specific expression of the coxsackie and adenovirus receptor in hematopoietic cells. STEM CELLS 2000;18:176–182.
Conget PA, Minguell JJ. Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells. Exp Hematol 2000;28:382–390.
Hidaka C, Milano E, Leopold PL et al. CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts. J Clin Invest 1999;103:579–587.
Hung SC, Chen NJ, Hsieh SL et al. Isolation and characterization of size-sieved stem cells from human bone marrow. STEM CELLS 2002;20:249–258.
Shyue SK, Tsai MJ, Liou JY et al. Selective augmentation of prostacyclin production by combined prostacyclin synthase and cyclooxygenase-1 gene transfer. Circulation 2001;103: 2090–2095.
Hung SC, Cheng H, Pan CY et al. In vitro differentiation of size-sieved stem cells into electrically active neural cells. STEM CELLS 2002;20:522–529.
Ma HL, Hung SC, Lin SY et al. Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads. J Biomed Mat Res 2003;64:273–281.
Oyama M, Tatlock A, Fukuta S et al. Retrovirally transduced bone marrow stromal cells isolated from a mouse model of human osteogenesis imperfecta (oim) persist in bone and retain the ability to form cartilage and bone after extended passaging. Gene Ther 1999;6:321–329.
Studeny M, Marini FC, Champlin RE et al. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-? delivery into tumors. Cancer Res 2002;62:3603–3608.
Olmsted-Davis EA, Gugala Z, Gannon FH et al. Use of a chimeric adenovirus vector enhances BMP2 production and bone formation. Hum Gene Ther 2002;13:1337–1347.
Partridge K, Yang X, Clarke NM et al. Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds. Biochem Biophys Res Commun 2002;292:144–152.(Shih-Chieh Hunga,b,c, Che)
b Medical Research and Education, Veterans General Hospital-Taipei, Taipei, Taiwan, Republic of China;
c Departments of Surgery and
d Internal Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China;
e Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan, Republic of China;
f Department of Orthopedic Surgery, Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan, Republic of China
Key Words. Human mesenchymal stem cells ? Adenoviral vectors ? Coxsackie-adenovirus receptor ? Green fluorescent protein ? Gene transfer
Correspondence: Shih-Chieh Hung, M.D, Ph.D., Department of Orthopedics and Traumatology, Veterans General Hospital-Taipei, 201, Sec. 2, Shih-Pai Road, Taipei, 11217, Taiwan. Telephone: 886-2-28757557, ext. 118; Fax: 886-2-28265164; e-mail: hungsc@vghtpe.gov.tw
ABSTRACT
Adenoviral vectors (AdVs) are well-known for their stability, ease of handling, and high uptake by many cell types, including noncycling cells. In addition, AdV can be concentrated to extremely high titers as well . Recent advances in AdV modifications offer a less immunogenic strain (defective recombinants) and an increase in the recombinant genome insert size . Therefore, the application of AdV is more relevant for human gene therapy than other means.
Because the efficacy of virus entry is restricted by interaction of viral fibers and cell-surface receptors, the abundance and the variety of viral-associated surface molecules expressed in cells are particularly important. The entry pathway for AdV consists of initial binding to the cells, which is mediated by the association of the adenoviral fiber protein and a 46-kDa membrane protein known as Coxsackie-adenovirus receptor (CAR) , followed by internalization, which is through an interaction of viral penton arginine-glycine-aspartate sequence to the v?3 and v?5 integrins .
The CAR is a primary passage, mediating the uptake of adenovirus in the 293 cell line, HeLa cell line, and cells derived from airways. However, cellular function of CAR is not clear, and CAR is only expressed in some types of cells and tissues. It has been demonstrated on oropharyngeal epithelial cells that the superficial layer (more developed) had less CAR expression than the basal layer (less developed), suggesting CAR expression correlated reciprocally with the status of differentiation . In contrast, CAR expression was only demonstrated in a small subset of CD34+ bone marrow (BM) and mobilized blood cells , whereas AdV transduction efficiency and CAR expression increase as the cells differentiate into erythroid and myeloid hematopoietic cells . CAR is expressed in human mesenchymal stem cells (hMSCs) but not expressed in certain kinds of mesenchymal cells such as primary human fibroblast .
Because hMSCs can be induced to differentiate into certain kinds of mesenchymal and nonmesenchymal tissues, they provide a model to study the effects of differentiation on AdV transduction and CAR expression. Therefore, we investigated AdV transduction efficiency and CAR expression in hMSCs and their differentiated progeny. Efforts were also made to characterize the transduced hMSCs in the differentiation potential and to know the persistence of transgene expression into hMSC progeny.
MATERIALS AND METHODS
InVitro AdV-Mediated Gene Transfer into hMSCs
For these studies, gene transfer was demonstrated by AdV transduction with the EGFP gene. The hMSC cultures were infected with Ad-PGK-EGFP, and 2 days later, transduction was assessed by transgene expression. As seen in Figure 1, after exposure to Ad-PGK-EGFP for 2 days, almost all cells were transduced as detected under both fluorescent microscope (Figs. 1A, 1B) and by flow cytometry analysis (Fig. 1C), displaying high fluorescent signal for GFP. At this time, transduced cells encoding GFP fluorescence were found to have a good viability and no disturbance in adherence. A strong green fluorescence in the infected hMSCs indicated a high efficiency of virus particles entering in cells. The efficiency of AdV-mediated gene transfer into hMSCs as analyzed by flow cytometry was 91.7 ± 6.6% (Table 1). High AdV transduction efficiency was also attained by the use of Ad-CMV-EGFP, a vector directing the expression of EGFP under the control of a cytomegalovirus (CMV) promoter (data not shown). These cells were expanded in normal growth medium with transgene retained and expressed in a small fraction of hMSC progeny even up to 4 weeks after infection (Figs. 1E, 1F). The cells carrying transgene adopted a picture of senescence, with a greater size and a broader shape, suggesting the transgene expression in the case of adenoviral infection is transient in origin and cells tend to lose transgene expression during several cycles of proliferation.
Figure 1. The hMSC culture after infection with Ad-PGK-EGFP adenoviral vectors. (A): Phase microscopic appearance of hMSCs. (B): Transduced cells encoding GFP. Original magnification x100. (C): Percentage and GFP fluorescence intensity of transduced cells. (D): Expression of AdV attachment receptor CAR in hMSCs. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; CAR, Coxsackie-adenovirus receptor; hMSC, human mesenchymal stem cell.
Table 1. The average percentage of green fluorescent protein–positive cells after adenoviral vector infection in respective cell category (the culture duration of lineage-committed induction)
Expression and Participation of CAR in AdV-Mediated Gene Transfer into hMSCs
Conget and Minguell have reported that the attachment of CAR, but not integrins v?3 and v?5, is required for AdV-mediated gene transfer into hMSCs. Therefore, we investigated by flow cytometry whether these cells express CAR. As shown in Figure 1D, CAR expression was demonstrated in hMSCs using CAR-specific monoclonal antibody RmcB. The pattern of CAR expression was homogeneous, and nearly half of the cells had moderate expression of CAR. To determine the participation of CAR in AdV infection, hMSCs were infected with Ad-PGK-EGFP in either the presence or absence of monoclonal antibody RmcB for competing CAR. As shown in Figure 2, incubating specific monoclonal antibody RmcB against CAR immediately before AdV infection allowed AdV infection to be inhibited by nearly 40%. Moreover, the effect of CAR competition by specific monoclonal antibody to inhibit AdV infection in hMSCs was dose dependent.
Figure 2. Effect of inhibition of Coxsackie-adenovirus receptor on adenoviral vector–mediated transduction of hMSCs. The hMSC suspension was incubated in the absence (control) or presence of control mouse IgG or the RmcB antibody in ascites fluid at selected dilutions (x250, x50, x10, and x2). Cells were then infected with Ad-PGK-EGFP (48 hours), and the percentage of transduced cells was scored by flow cytometry and calculated with respect to control, which was set to 1. An inverse relationship of percentage of transduced cells to the concentration of RmcB antibody is demonstrated. Data shown represent the mean ± standard deviation. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; hMSC, human mesenchymal stem cell; IgG, immunoglobulin G.
Reduced AdV Transduction Efficiency and CAR Expression in Lineage-Differentiated hMSCs
The hMSC culture was induced to differentiate along the osteogenic, adipogenic, or neurogenic lineage, as previously described . As shown in Figure 3 and Table 1, hMSCs had a relatively low efficiency of AdV transduction, with 31%, 14%, and 59% at 10 days, 17 days, and 23 days, respectively, after osteogenic induction. The low efficiency of AdV transduction in the differentiated cells was correlated with a decrease in CAR expression after 10 days of osteogenic induction. The level of CAR expression was undetectable at 17 days after induction, but a regain of CAR was observed in 23-day induced cells.
Figure 3. The hMSC culture following infection with Ad-PGK-GFP adenoviral vectors after treatment with osteogenic (A), adipogenic (B), and neurogenic medium (C) for selected time periods. Histogram shows GFP fluorescence intensity data for transduced cells and expression of adenoviral vector attachment receptor CAR in hMSCs. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; CAR, Coxsackie-adenovirus receptor; hMSC, human mesenchymal stem cell.
An inverse correlation was also observed between the maturation of adipogenic differentiation and the efficiency of adenoviral transduction. The observed efficiency was 44%, 30%, and 19% at 3 days, 7 days, and 10 days, respectively, after adipogenic induction. CAR expression was only observed in a subpopulation of adipogenic-differentiated hMSCs, equal to 5% or less at 3 days after adipogenic induction, but CAR expression was undetectable if the induction was continued for more than 7 days.
Unlike osteogenic and adipogenic inductions, neurogenic induction of hMSCs had only minor influence on AdV transduction efficiency. The efficiency corresponding to 5 hours and 5 days after neurogenic induction was 93% and 89%, respectively. However, the prolonged neurogenic culture for 5 days had a lower fluorescent intensity compared with those that had neurogenic induction for 5 hours. In addition, hMSCs that underwent neurogenic induction had less CAR expression in total population compared with the undifferentiated status.
Differentiation Potentials of Transduced hMSCs and Persistence of Transgene Expression into hMSC Progeny
Because the adenoviral transduction is more efficient in the undifferentiated hMSCs than the fate-determined hMSC progeny, we have additionally investigated the plasticity of transduced hMSCs and the persistence of transgene expression into hMSC progeny after lineage-specific differentiation. After AdV infection, the hMSC culture was induced to differentiate along the osteogenic, adipogenic, or neurogenic lineage, and the persistence of the transgene into differentiated cells was studied under fluorescent microscope and by flow cytometry. As shown in Figure 4, AdV-transduced hMSCs that expressed GFP fluorescence and had the lipid vesicles synthesized and stained red by Oil red O dye were clearly identified at 10 days after adipogenic induction. The transduced cells loose in attachment were prone to retract from the culture surface. Moreover, our data have also demonstrated that AdV-transduced hMSCs maintained the potential to differentiate along the osteogenic lineage. Osteogenic differentiation was identified by the bone alkaline phosphatase (AP) stain, and fluorescence was proven on day 14 after initial induction. AP stain marked osteogenic progeny red to dark purple, and a thickening and mesh network of cytoskeleton was observed in the osteogenic culture (Fig. 5). In addition, indirect immunofluorescent staining for neuron-specific protein, ?-tubulin III, was used to identify neural differentiation of transduced hMSCs after induction with neural medium for 2 days. Those cells exhibit neuron maker primarily in cytoplasm but not nucleus, whereas GFP was concentrated in nucleus and some was dispersed in cytoplasm (Fig. 6).
Figure 4. Adipogenic differentiation of transduced hMSCs. The hMSC culture after infection with Ad-PGK-EGFP was treated with adipogenic medium for 10 days. (A): Transduced cells encoding GFP. (B): Oil red O staining cells. (C): The merged picture of (A, B). The same field exhibiting green fluorescence and stained red indicates the transduced cells have been differentiated into the adipogenic lineage. Original magnification x150. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; hMSC, human mesenchymal stem cell.
Figure 5. Osteogenic differentiation of transduced hMSCs. The hMSC culture after infection with Ad-PGK-EGFP was treated with osteogenic medium for 14 days. (A): Transduced cells encoding GFP. (B): Alkaline phosphatase staining cells. (C): The merged picture of (A, B). The same field exhibiting green fluorescence and stained red indicates the transduced cells have been differentiated into the osteogenic lineage. Original magnification x150. Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; hMSC, human mesenchymal stem cell.
Figure 6. Neurogenic differentiation of transduced hMSCs. The hMSC culture after infection with Ad-PGK-EGFP was treated with neurogenic medium for 2 days. (A): Confocal image of transduced cells encoding GFP. (B): Some corresponding cells are positive for neuron marker, ?-tubulin III, as evidenced by immunofluorescence staining. Original magnification x300.Abbreviations: Ad-PGK-EGFP, adenoviral phosphoglycerate kinase enhanced green fluorescent protein; hMSC, human mesenchymal stem cell.
DISCUSSION
We thank R. Finberg (Harvard Medical School, Boston) for the gift of monoclonal antibody RmcB. We also thank MingLing Hsu for excellent flow cytometry technical support and Bor-Chun Weng for his assistance in preparing the manuscript. This work was supported in part by grant No. 92-376-1 from Veteran General Hospital-Taipei and by NSC 91-2321-B-002-005 from the National Science Council, Taipei, Taiwan.
REFERENCES
Crystal RG. Transfer of genes to humans: early lessons and obstacles to success. Science 1995;270:404–410.
Shenk T. Adenoviridae: the viruses and their replication. In: Fields BN, Knipe DM, Howley PM, eds. Fields Virology. Philadelphia: Lippincott-Raven Publishers, 1996:2112–2137.
Wilson JM. Adenoviruses as gene-delivery vehicles. N Engl J Med 1996;334:1185–1187.
Lee MG, Abina MA, Haddada H et al. The constitutive expression of the immunomodulatory gp19k protein in E1-, E3- adenoviral vectors strongly reduces the host cytotoxic T cell response against the vector. Gene Ther 1995;2:256–262.
Kochanek S, Clemens PR, Mitani K et al. A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase. Proc Natl Acad Sci U S A 1996;93:5731–5736.
Bergelson JM, Cunningham JA, Droguett G et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997;275:1320–1323.
Wickham TJ, Mathias P, Cheresh DA et al. Integrins V?3 and V?5 promote adenovirus internalization but not virus attachment. Cell 1993;73:309–319.
Hutchin ME, Pickles RJ, Yarbrough WG. Efficiency of adenovirus-mediated gene transfer to oropharyngeal epithelial cells correlates with cellular differentiation and human cox-sackie and adenovirus receptor expression. Hum Gene Ther 2000;11:2365–2375.
Neering SJ, Hardy SF, Minamoto D et al. Transduction of primitive human hematopoietic cells with recombinant adenovirus vectors. Blood 1996;88:1147–1155.
Bregni M, Shammah S, Malaffo F et al. Adenovirus vector for gene transduction into mobilized blood CD34+ cells. Gene Ther 1998;5:465–472.
Rebel VI, Hartnett S, Denham J et al. Maturation and lineage-specific expression of the coxsackie and adenovirus receptor in hematopoietic cells. STEM CELLS 2000;18:176–182.
Conget PA, Minguell JJ. Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells. Exp Hematol 2000;28:382–390.
Hidaka C, Milano E, Leopold PL et al. CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts. J Clin Invest 1999;103:579–587.
Hung SC, Chen NJ, Hsieh SL et al. Isolation and characterization of size-sieved stem cells from human bone marrow. STEM CELLS 2002;20:249–258.
Shyue SK, Tsai MJ, Liou JY et al. Selective augmentation of prostacyclin production by combined prostacyclin synthase and cyclooxygenase-1 gene transfer. Circulation 2001;103: 2090–2095.
Hung SC, Cheng H, Pan CY et al. In vitro differentiation of size-sieved stem cells into electrically active neural cells. STEM CELLS 2002;20:522–529.
Ma HL, Hung SC, Lin SY et al. Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads. J Biomed Mat Res 2003;64:273–281.
Oyama M, Tatlock A, Fukuta S et al. Retrovirally transduced bone marrow stromal cells isolated from a mouse model of human osteogenesis imperfecta (oim) persist in bone and retain the ability to form cartilage and bone after extended passaging. Gene Ther 1999;6:321–329.
Studeny M, Marini FC, Champlin RE et al. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-? delivery into tumors. Cancer Res 2002;62:3603–3608.
Olmsted-Davis EA, Gugala Z, Gannon FH et al. Use of a chimeric adenovirus vector enhances BMP2 production and bone formation. Hum Gene Ther 2002;13:1337–1347.
Partridge K, Yang X, Clarke NM et al. Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds. Biochem Biophys Res Commun 2002;292:144–152.(Shih-Chieh Hunga,b,c, Che)