Naohiro
Takemoto
a,
Yuji
Teramura
bc and
Hiroo
Iwata
*a
aDepartment of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-Cho, Shogoin, Sakyo-Ku, Kyoto 606-8507, Japan. E-mail: iwata@frontier.kyoto-u.ac.jp; Tel: +81-75-751-4119
bRadioisotope Research Center, Kyoto University, Yoshida-Konoe-Cho, Sakyo-Ku, Kyoto 606-8501, Japan
cDepartment of Immunology, Genetics and Pathology (IGP), Uppsala University, Dag Hammarskjölds väg 20, 751 85, Uppsala, Sweden
First published on 28th November 2012
Sertoli cells play a crucial role in creating the immunoprivileged environment of the testis. We examined the survival of islets of Langerhans after co-transplantation with Sertoli cells. Sertoli cells near islets should protect the graft from rejection. In this study, conjugates of single stranded oligonucleotides, poly(ethylene glycol) and phospholipids (ssDNA-PEG-DPPE) were used to immobilize Sertoli cells on islets. The 20-mer of deoxyadenylic acid (oligo(dA)20) and 20-mer of deoxythymidylic acid (oligo(dT)20) were presented as ssDNAs on the surfaces of Sertoli cells and islets, respectively, through the hydrophobic interaction between a lipid unit of the conjugates and the cell membrane. The Sertoli cells were immobilized on the islets through hybridization between oligo(dA)20 and oligo(dT)20. When Sertoli cell-immobilized islets were infused into the liver of mice through the portal vein, the Sertoli cells remained around the islets.
Sertoli cells play a crucial role in creating the immunoprivileged environment of the testis9 by secreting immunoprotective factors10,11 and forming a barrier to inhibit infiltrating lymphocytes.10,12,13 Some groups reported long term survival of islets when co-transplanted with Sertoli cells7,8 into the subcapsular site of the kidney for co-localization. However, the co-localization of Sertoli cells with islets is difficult in the liver because for clinical islet transplantation islets and Sertoli cells are to be transfused into the portal vein together. The islets congregate in larger veins in the liver, but Sertoli cells are usually carried to more peripheral smaller veins by the blood flow.
It has been reported that conjugates of single stranded oligonucleotides (ssDNA) and long alkyl chains can be used to attach cells to other cells without deterioration of cell survival and functions.14–18 In our previous studies, we synthesized conjugates of ssDNA and poly(ethylene glycol)-conjugated phospholipid (ssDNA-PEG-lipid) and employed them to immobilize cells on an islet19 for improvement of graft survival.
In this study, ssDNA-PEG-lipid was applied to immobilize Sertoli cells on the islet surface, as shown in Scheme 1. The surface of Sertoli cells and islets were modified with 20-mer of deoxyadenylic acid (oligo(dA)20) and 20-mer of complementary deoxythymidylic acid (oligo(dT)20), respectively, conjugated to PEG-lipids. Sertoli cells were immobilized on islets through DNA hybridization between oligo(dA)20 and oligo(dT)20. The cell composites of Sertoli cells and islets were infused into the mouse portal vein to examine the co-localization of Sertoli cells with islets.
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Scheme 1 Immobilization of Sertoli cells onto an islet using ssDNA-PEG-DPPEs. (a) Oligo(dA)20 and oligo(dT)20 were used as ssDNA conjugated to PEG-DPPE. (b) A schematic illustration of the anchoring of ssDNA-PEG-DPPE to a lipid bilayer of the cell membrane and immobilization of Sertoli cells onto an islet through hybridization between oligo(dA)20 and oligo(dT)20. |
To identify Sertoli cells, the isolated cells were immunohistochemically stained for GATA-4, which is a transcription factor and known to be abundantly expressed in the nuclei of Sertoli cells.20,21 Cells were fixed in 4% formalin solution at 37 °C for 15 min, permeabilized in 0.2% Triton X-100 in PBS at room temperature (rt) for 15 min, treated with Blocking One for 1 h to block nonspecific binding of antibodies and then washed with PBS. The cells were immersed in 2% polyclonal goat anti-mouse GATA-4 in Blocking One and left for 1 h at rt, followed by washing with PBS. The cells were incubated with fluorescent labeled secondary antibody, 0.2% Alexa 488-labeled rabbit anti-goat IgG in Blocking One at rt for 1 h, followed by washing with PBS containing 0.05% Tween 20. Cell nuclei were counterstained with Hoechst 33258. The stained cells were observed under a fluorescence microscope (IX71, Olympus Optical, Co., Ltd, Tokyo, Japan). Population of GATA-4 positive cells was elucidated from 300 cells counts.
Islets were isolated from BALB/c mice (6 week old males, Japan SLC, Inc., Shizuoka, Japan) by the collagenase digestion method.22 Briefly, the pancreas was collected from mice, chopped into paste with scissors, and digested for 18 min at 37 °C in a collagenase solution (0.5 mg mL−1 in HBSS). Islets were isolated from the digest by Ficoll density gradient purification, and cultured for 3 days in Medium 199 supplemented with 10% FBS, 8.8 mM HEPES buffer, 100 units mL−1 penicillin, 100 μg mL−1 streptomycin, and 8.8 units mL−1 heparin to remove cells damaged by the isolation procedure.
Sertoli cell pellets (2 × 106 cells) were collected by centrifugation (180g, 5 min at 25 °C) after treatment with trypsin. The cells were washed with HBSS to remove the medium. After the addition of oligo(dA)20-PEG-DPPE solution (100 μL, 500 μg mL−1 in PBS) to the cell pellet, the cell suspension was incubated for 30 min with gentle agitation at rt. After washing with HBSS, oligo(dA)20-PEG-DPPE-modified cells were obtained by centrifugation (180g, 5 min, 25 °C, twice). To examine the presence of oligo(dA)20 on Sertoli cells, the cells were mixed with a solution of FITC-oligo(dT)20 (100 μL, 500 μg mL−1 in PBS), washed with HBSS and observed under a confocal laser scanning microscope.
Oligo(dA)20-PEG-DPPE-modified Sertoli cells (2 × 106) and oligo(dT)20-PEG-DPPE-modified islets (200 islets) were mixed in serum-free medium (200 μL). The mixture was incubated for 60 min with gentle agitation at rt. The Sertoli cell-immobilized islets were obtained after washing with culture medium. The Sertoli cell-immobilized islets were immunohistochemically stained for GATA-4 and were observed under a confocal laser scanning microscope. In addition, Sertoli cells stained by CellTracker™ were also employed for easy finding of Sertoli cells after their transplantation.
Sertoli cell-immobilized islets (500 islets/100 μL) without CellTracker™ staining were also used for transplantation. As a control experiment, 100 μL of a mixed suspension of Sertoli cells (5 × 106 cells) and islets (500 islets) was infused into the liver through a portal vein. Immediately after transplantation, mice were sacrificed and paraffin-embedded liver specimens were sliced into sections 4-μm thick. After deparaffinization, the sections were permeabilized with 0.2% Triton X-100 in PBS at rt for 15 min. The samples were then treated with Blocking One for 1 h, followed by washing with PBS. The sections were treated with 1% polyclonal guinea pig anti-swine insulin and 2% polyclonal goat anti-mouse GATA-4 in Blocking One for 1 h at rt. The sections were then incubated with fluorescent labeled secondary antibody, 2% TRITC-labeled rabbit anti-guinea pig IgG and 0.2% Alexa 488-labeled rabbit anti-goat IgG in Blocking One, at rt for 1 h followed by washing with PBS containing 0.05% Tween 20. The stained sections were observed under a fluorescence microscope.
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Fig. 1 Immunohistochemical examination of Sertoli cells isolated from the testis of male BALB/c mice. (a) GATA-4 (green fluorescence), (b) GATA-4 (green fluorescence) and Hoechst 33258 (blue fluorescence), and (c) phase contrast microscopic image. Scale bar: 100 μm. |
After washing with HBSS and serum-free medium, Sertoli cells and islets were treated with oligo(dA)20-PEG-DPPE and oligo(dT)20-PEG-DPPE, respectively. Each was then mixed with a solution containing the corresponding complementary FITC-ssDNA. They were then observed under a confocal laser microscope (see Fig. 2(a-1) and (b-1)). Bright fluorescent rings were found at the periphery of the islets and the Sertoli cells. When the Sertoli cells and islets were mixed with mismatched ssDNA, that is, FITC-oligo(dA)20 and FITC-oligo(dT)20 solutions, respectively, no fluorescent rings were seen (see Fig. 2(a-2) and (b-2)). These results indicate that FITC was immobilized on Sertoli cell and islet surface through hybridization between oligo(dA)20 and oligo(dT)20. Oligo(dA)20- and oligo(dT)20-PEG-DPPEs were present on Sertoli cells and islets, respectively, and maintained the ability to hybridize with complementary ssDNA, that is, FITC-oligo(dT)20 and FITC-oligo(dA)20.
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Fig. 2 Surface modification of islets and Sertoli cells with ssDNA-PEG-DPPE. Sertoli cells and islets were observed under a confocal laser scanning fluorescent microscope. (a-1) FITC-oligo(dA)20 fixed on islets modified with oligo(dT)20-PEG-DPPE. (a-2) Islets were modified with oligo(dT)20-PEG-DPPE and then mixed with FITC-oligo(dT)20. (b-1) FITC-oligo(dT)20 fixed on Sertoli cells modified with oligo(dA)20-PEG-DPPE. (b-2) Sertoli cells were modified with oligo(dA)20-PEG-DPPE and then FITC-oligo(dA)20. |
Fig. 3 shows that Sertoli cells were immobilized on islets through hybridization between oligo(dA)20 and oligo(dT)20, and contains images of the aggregates observed under a confocal florescence microscope. Sertoli cells and islets were treated with oligo(dA)20-PEG-DPPE and oligo(dT)20-PEG-DPPE, respectively. After washing with HBSS and serum-free medium, suspensions of each were mixed together and left for 60 min. Existence of Sertoli cells was examined by immunohistochemical staining of GATA-4 which was specifically expressed in Sertoli cells.20,21 GATA-4 positive cells were found on the islet surface as shown in Fig. 3(a). Sertoli cells stained with CellTracker™ were also used to see location of Sertoli cells in the aggregates. Sertoli cells were clearly observed at the periphery of the islets immediately after the immobilization (see Fig. 3(b)). However, few cells were found at the periphery of the islets when oligo(dT)20-PEG-DPPE modified-islets were mixed with naïve Sertoli cells, that were observed under a phase contrast microscope (see Fig. 3(c)). When the aggregate prepared via hybridization were cultured for 4 days, some of Sertoli cells migrated into the inside of islets (see Fig. 3(d)). These results clearly demonstrate that Sertoli cells could be immobilized on islets through hybridization between oligo(dT)20 on the islets and oligo(dA)20 on the Sertoli cells.
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Fig. 3 Immobilization of Sertoli cells onto islets. (a) Immunohistochemical staining of GATA-4 of Sertoli cells immobilized on an islet with the ssDNA-PEG-DPPE method. (b) Sertoli cells (stained with CellTracker™) immobilized on an islet with the ssDNA-PEG-DPPE method just after immobilization. (c) A phase contrast microscopic image of a mixture of oligo(dT)20-PEG-DPPE-modified islets and naïve Sertoli cells. (d) Sertoli cells (stained with CellTracker™) immobilized on an islet after 4 days of culture. Insets: phase contrast microscopic image. |
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Fig. 4 The functional evaluation of Sertoli cell-immobilized islets in vitro. (a) Glucose stimulation test of Sertoli cell-immobilized islets and naïve islets. Islets were sequentially exposed to 0.1, 0.3 and 0.1 g dL−1 glucose solutions in Krebs–Ringer buffer (KRB) at 37 °C for 1 h each. The amounts of released insulin are expressed as mean ± SD for n = 3. (b) Activin A release from Sertoli cell-immobilized islets and naïve islets. The islets were incubated in serum-free medium at 37 °C for 24 h. The amounts of released Activin A are expressed as mean ± SD for n = 5. |
Activin A is secreted by Sertoli cells24 and was examined in Sertoli cells on islets to determine if they were active. Activin A was secreted from Sertoli cell-immobilized islets; whereas, no secretion was seen from naïve islets (see Fig. 4(b)). These results indicate immobilization of Sertoli cells on the islet surface using ssDNA-PEG-DPPE did not affect the ability of Sertoli cells to secrete Activin A. Sertoli cells could be immobilized onto islets without loss of function of islets or Sertoli cells.
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Fig. 5 Histochemical analyses of Sertoli cells and islets in liver veins. (a) Sertoli cell-immobilized islets in the mouse liver: (a-1) CellTracker™ (green fluorescence) and (a-2) phase contrast microscopic image. (b) Sertoli cell-immobilized islets in the mouse liver: (b-1) GATA-4 (green fluorescence) and (b-2) Merged images of GATA-4 (green fluorescence) and insulin (red fluorescence). (c) Naïve islets in the mouse liver: (c-1) GATA-4 (green fluorescence) and (c-2) merged images of GATA-4 (green fluorescence) and insulin (red fluorescence). Scale bar: 100 μm. |
In this method, bioactive substances are immobilized on the cell through the polymers. The avidin–biotin complex is most often used.27,28 It can form stable bonding under physiological conditions and is suitable for in vitro examination of modified cells. Various avidins are commercially available, but are derived from albumen or bacteria. These avidins are hard to use for in vivo experiments, particularly in the clinical setting, because they are expected to induce unfavorable immune reactions. As an alternative, we employed DNA duplex formation. Oligo(dA)20 and oligo(dT)20 can form a stable complex under physiological conditions and the immunogenicity of DNA is much lower than that of avidin.
We were able to immobilize Sertoli cells on islets without loss of functions of either using amphiphilic ssDNA-PEG-DPPEs, as shown in Fig. 4. The aggregates of Sertoli cells and islets could be co-transplanted into the mouse liver though the portal vein without detachment of Sertoli cells from the islet surface as shown in Fig. 5. Further studies are necessary to determine if our method is clinically useful for the co-transplantation of Sertoli cells and islets in the liver. Factors to be examined include the stability of the cell composite, life span of Sertoli cells in the liver and the blood compatibility of Sertoli cells. However, cell immobilization using ssDNA-PEG-DPPEs is a versatile method to immobilize any kind of cells, such as regulatory T cells and endothelial cells, on islets without exerting harmful effects on the cells. We expect this method will increase the success rate of islet transplantations.
Although we have mainly focused on the application of ssDNA-PEG-lipid to prolong graft survival of islets, this technology has many potential applications in modification of surface of cells and tissues with bioactive substances, study of cell–cell interaction and three dimensional tissue fabrication from dissociated single cells as discussed previously.16,19
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