Communication pubs.acs.org/molecularpharmaceutics
Up-Regulation of Gene Expression by Transfection to Hepatocyte Spheroids Kennichi Hazama, Shoichiro Asayama,* and Hiroyoshi Kawakami* Department of Applied Chemistry, Tokyo Metropolitan University, Minami-Osawa, Hachioji, Tokyo 192-0397, Japan on the mechanism of the up-regulation of the gene expression are outside the scope of the present study. We have already reported that the spheroids are formed on a rubbed polyimide surface.7,8 In this study, the porcine primary hepatocytes were cultured on a rubbed polyimide surface for 3 days, resulting in the spheroid formation (Figure 1A). [A
ABSTRACT: The gene expression is up-regulated by transfection to spheroids rather than monolayered cells. Monolayered hepatocytes form spheroids on rubbed polyimide membrane after 3 day incubation. The transfection of a rhodamine-labeled plasmid DNA or a green fluorescent protein (GFP) reporter gene to monolayered hepatocytes makes the spheroids exhibiting the fluorescence from the rhodamine or GFP inside the resulting spheroids. On the other hand, the transfection to hepatocyte spheroids makes the spheroids exhibit the fluorescence only outside the resulting spheroids. However, the whole gene expression of the luciferase reporter plasmid DNA from the lysate of the transfected spheroids is up-regulated in early incubation time, as compared to that of the transfected monolayered hepatocytes. Furthermore, the secretion of vascular endothelial growth factor (VEGF) from the VEGF-transfected spheroids is higher than that from the transfected monolayered hepatocytes. These results suggest that the up-regulation of exogenous gene expression is achieved by the control of differentiation and proliferation of hepatocytes via spheroid formation. KEYWORDS: hepatocyte, gene transfer, cell morphology, gene expression, growth factors
Figure 1. Phase-contrast micrograph of the hepatocyte morphology on the polyimide membrane surface. (A) No transfection, (B) transfection to monolayer cells, and (C) transfection to spheroids, corresponding to Scheme 1, are carried out by use of plasmid DNA encoding vascular endothelial growth factor (VEGF). Each hepatocyte morphology after 1, 3, 5, and 7 day of incubation is presented.
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ere we have reported that gene expression is up-regulated by transfection to spheroids rather than monolayered cells. Proliferation and differentiation are controlled by cell morphology such as spread and round shapes.1 Cell spheroids, which are a spherical mass consisting of many cells with a round shape, have been used in the research areas of tissue engineering and regenerative medicine, because the spheroids mimic physiological cellular functions as well as the morphology in living tissue and organs, unlike the monolayered cells with a spread shape.2−6 The spheroids are well-known to maintain high levels of cellular functions, as compared with the monolayered cells. Namely, the cells in the spheroids are considered to be more differentiated than the monolayered cells. In the present study, we have compared the gene expression level of the transfected spheroids with that of the transfected monolayered cells. If the transfected spheroids enhance the gene expression owing to cell differentiation, as compared with the transfected monolayered cells, the resulting spheroids would be promising for cell transplantation. This paper describes the morphology, plasmid DNA distribution, reporter gene expression, and vascular endothelial growth factor (VEGF) secretion of the transfected spheroids; detailed studies © 2012 American Chemical Society
typical procedure is as follows: On a rubbed polyimide membrane (1.5 cm × 1.5 cm) in a well of a 12-well plate, primary porcine hepatocytes, which were kindly provided by Dr. Shin Enosawa (Department of Innovative Surgery, National Research Institute for Child Health and Development, Tokyo, Japan), were seeded at 2.5 × 105 cells/well (1 mL of William’s E medium). Then, the resulting hepatocytes were observed with phase-contrast microscope after 1, 3, 5, and 7 day incubation as described in Scheme 1.] The resulting spheroids of the porcine hepatocytes were transfected, as shown in Scheme 1C, at fourth day from seeding of the hepatocytes on the polyimide surface. As a control, the monolayered Received: Revised: Accepted: Published: 3602
September 14, 2012 October 18, 2012 October 25, 2012 October 25, 2012 dx.doi.org/10.1021/mp300519x | Mol. Pharmaceutics 2012, 9, 3602−3605
Molecular Pharmaceutics
Communication
Scheme 1. Timing of the Transfection in This Studya
rubbed polyimide membrane in a 12-well plate were transfected by the addition of 1 μg of rhodamine-labeled (LabelIT CXrhodamine labeling kit, Mirus Bio LLC) plasmid DNA (pGL3Control Vector, from Promega Co.) complexed with lipofectin reagent (Invitrogen) at positive/negative charge ratio of 1 as described in Scheme 1. Then, the resulting hepatocytes were observed with confocal laser-scanning and differential interference microscope after 1, 3, 5, and 7 day incubation.] The rhodamine fluorescence was almost wholly observed inside the spheroids from transfected monolayered hepatocytes (Figure 2, upper panels). In case of the transfected spheroids, on the other hand, the fluorescence was observed only around the spheroids (Figure 2, lower panels). These results suggest that the transfected plasmid DNA after spheroid formation failed to enter the inside of the spheroids. Namely, the plasmid DNA transfected after spheroid formation is considered to exist in the surface of the resulting spheroids. To examine the gene expression of plasmid DNA, as shown in Figure 3, we used the reporter plasmid DNA encoding green
a
The arrow indicates the day when the transfection was carried out; namely, day 1 (B) means 1 day incubation after transfection to monolayered cells, and day 5 (C) means 1 day incubation after transfection to spheroids.
hepatocytes were transfected immediately after seeding (Scheme 1B). As shown in Figure 1, in spite of the transfection, no significant difference of the hepatocyte morphology was observed. The transfected monolayered hepatocytes formed spheroids at the third day from seeding of the hepatocytes (Figure 1B) as well as the hepatocyte with no transfection (Figure 1A). The transfected spheroids maintained the spheroid formation at the seventh day from seeding of the hepatocytes (Figure 1C) as well as other controls (Figure 1A and B). However, it should be noted that the distribution of the transfected plasmid DNA inside the spheroids depended on the timing of the transfection. Figure 2 shows the distribution of rhodamine-labeled plasmid DNA inside the resulting spheroids. [A typical procedure is as follows: The hepatocytes seeded on a
Figure 3. Confocal laser-scanning and differential interference microscope images of the hepatocyte morphology after transfection with plasmid DNA encoding green fluorescent protein (GFP). “Transfection to Mono-layered Cells” and “Transfection to Spheroids” correspond to “B” and “C”, respectively, in Scheme 1. Each hepatocyte morphology after 1, 3, 5, and 7 day of incubation is presented.
fluorescent protein (GFP) instead of rhodamine-labeled plasmid DNA. [A typical procedure is the same as Figure 2 except for use of plasmid DNA encoding green fluorescent protein (GFP, from Promega Co.).] The GFP was almost wholly observed inside the spheroid from transfected monolayered hepatocytes (Figure 3, upper panels), whereas the green fluorescence was observed in the surface of the transfected spheroids (Figure 3, lower panels). These results suggest that the gene expression was consistent with the
Figure 2. Confocal laser-scanning and differential interference microscope images of the hepatocyte morphology after transfection with rhodamine-labeled pGL3 plasmid DNA. “Transfection to Monolayered Cells” and “Transfection to Spheroids” correspond to “B” and “C”, respectively, in Scheme 1. Each hepatocyte morphology after 1, 3, 5, and 7 day of incubation is presented. 3603
dx.doi.org/10.1021/mp300519x | Mol. Pharmaceutics 2012, 9, 3602−3605
Molecular Pharmaceutics
Communication
transfected spheroids was up-regulated in early incubation time (1 day), as compared to that of the transfected monolayered hepatocytes. Finally, we carried out the transfection by use of a practical gene encoding VEGF. Figure 4B shows VEGF secretion from VEGF-transfected monolayered hepatocytes or transfected spheroids. [A typical procedure is the same as Figures 2 and 3 except for use of plasmid DNA encoding vascular endothelial growth factor A (VEGF A, from Open Biosystems). The VEGF A secretion was determined by enzyme-linked immunosorbent assay (ELISA) kit (Bender MedSystems). The resulting secretion was normalized by the number of cells counted after trypsinization.] As well as luciferase expression, the VEGF secretion of the transfected spheroids for 1 day incubation after transfection, that is, total incubation time of 5 day (black bar), was higher than that of the transfected monolayered hepatocytes for 1 day incubation (gray bar). Furthermore, the VEGF secretion of the transfected spheroids for 3 day incubation, that is, total incubation time of 7 days (black bar), increased as compared with that of the transfected spheroids for 1 day incubation, that is, total incubation time of 5 days (black bar). On the other hands, the transfected monolayered hepatocytes secreted no significant VEGF for 3 day incubation (gray bar). Moreover, it is worth noting that the VEGF secretion of the transfected spheroids for 3 day incubation (total 7 day, black bar) was higher than that of the transfected monolayered hepatocytes for 7 day as well as 5 day incubation (gray bars). These results support that the VEGF secretion was enhanced by the spheroid formation to up-regulate gene expression. It is reported that the 3H-thymidine uptake of cultured hepatocytes with a spread shape was higher than that with a round shape.1 In the report, on the other hand, bile acid release as a typical differentiated function of hepatocytes was maintained at higher levels in hepatocytes with a round shape. Therefore, monolayered hepatocytes, being spread shape, are considered to exhibit more proliferation phase, whereas hepatocytes forming spheroids, being round shape, are considered to exhibit more differentiation phase. Taking these results into account, the transfection to more differentiated hepatocytes is considered to enhance the gene expression. Consequently, the control of differentiation and proliferation of hepatocytes via spheroid formation suggests the up-regulation of exogenous gene expression such as VEGF expression. In general, VEGF promotes the proliferation and migration of endothelial cells, so that it is well-known as a point inducer of angiogenesis defined as the formation of new blood vessels from a pre-existing microvascular bud.9 The up-regulation of VEGF gene expression in the surface of the transfected spheroids is therefore considered to be advantageous to VEGF secretion outside the resulting spheroids for surrounding endothelial cells. The resulting methodology to establish the spheroids with more secretion of VEGF in this study can be applied for cell transplantation in vivo by angiogenesis.
distribution of the rhodamine-labeled plasmid DNA. By transfection after spheroid formation, therefore, the transfected gene is considered to be expressed in the surface of the spheroid, but not inside the spheroid. To determine the gene expression of plasmid DNA, we used the reporter plasmid DNA encoding luciferase instead of GFP. Figure 4A shows the whole gene expression of the luciferase
Figure 4. (A) Effect of the luciferase gene expression from luciferasetransfected hepatocytes on the incubation time of hepatocytes. (B) Effect of VEGF secretion from VEGF-transfected hepatocytes on the incubation time of hepatocytes. White bars, gray bars, and black bars correspond to A (“No Transfection”), B (“Transfection to Monolayered Cells”), and C (“Transfection to Spheroids”), respectively, in Scheme 1.
reporter plasmid DNA from the lysate of the transfected monolayered hepatocytes or transfected spheroids. [A typical procedure is the same as Figures 2 and 3 except for use of plasmid DNA encoding modified firefly luciferase (pGL3Control Vector). Then, the cells were subjected to the luciferase assay (Promega kit) according to the manufacturer’s instructions. Luciferase activities were normalized by protein concentrations and are presented as relative light units (RLU). Protein concentrations were determined by the BCA protein assay kit (Pierce) according to the manufacturer’s instructions.] The gene expression of the transfected spheroids for 1 day incubation after transfection, that is, total incubation time of 5 day (black bar), was higher than that of the transfected monolayered hepatocytes for 1 day incubation (gray bar). From the results of Figures 2 and 3, the number of the hepatocytes in the transfected spheroids is considered to be less than that of the transfected monolayered hepatocytes because only the hepatocytes in the surface of the spheroids were transfected. Therefore, these results suggest that the gene expression of the
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AUTHOR INFORMATION
Corresponding Author
*Tokyo Metropolitan University, Department of Applied Chemistry, Minami-Osawa, Hachioji, Tokyo 192-0397, Japan. Telephone: +81-42-677-1111 (ext.) 4976; Fax: +81-42-6772821 (S. Asayama). E-mail:
[email protected] (S. Asayama),
[email protected] (H. Kawakami). 3604
dx.doi.org/10.1021/mp300519x | Mol. Pharmaceutics 2012, 9, 3602−3605
Molecular Pharmaceutics
Communication
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Dr. Shin Enosawa of the Department of Innovative Surgery, National Research Institute for Child Health and Development, for the supply of primary porcine hepatocytes and use of the confocal laser-scanning and differential interference microscope with his helpful assistance.
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REFERENCES
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