- Email: [email protected]
Transdifferentiation of Bone Marrow Stem Cells into Acinar Cells Using a Double Chamber System
ORIGINAL ARTICLE
Transdifferentiation of Bone Marrow Stem Cells into Acinar Cells Using a Double Chamber System Chia-Yung Lin,1,2 Bor-Shiunn Lee,1,2 Chih-Chen Liao,3 Wei-Jhih Cheng,3 Feng-Ming Chang,1,2 Min-Huey Chen1,2* Background/Purpose: Hypofunction of the salivary glands can substantially affect quality of life. Current treatments for salivary hypofunction are of limited effectiveness. Although the implantation of functional salivary gland tissue from autologous glandular cells represents a possible physiologic solution to this problem, tissue engineering of salivary glands would require the generation of a great number of acinar cells (ACs). The purpose of this study was to investigate the feasibility of transdifferentiation of bone marrow stem cells (BMSCs) into functional ACs using a co-culture system. Methods: BMSCs were isolated from adult rats and co-cultured with rat parotid ACs using a double chamber system. The transdifferentiation of BMSCs was evaluated by immunocytochemical analysis of α-amylase, which has unique functional expression in ACs. Results: Expression of α-amylase, indicating successful transdifferentiation of BMSCs into ACs, was found in 30% of BMSCs after co-culturing for 1 week, and in 50% after co-culturing for 2 and 3 weeks. Conclusion: This study has demonstrated the potential of rat BMSCs to transdifferentiate into ACs, and support the feasibility of application of BMSCs in salivary gland tissue engineering. [J Formos Med Assoc 2007;106(1):1–7] Key Words: cell transdifferentiation, salivary gland, tissue engineering
complication of radiation therapy for head and neck cancer,6 and may lead to persistent salivary hypofunction with marked reduction in salivary output.7 Currently, there is no effective therapy for this condition. Tissue engineering is an interdisciplinary field that applies the principles of biology and engineering toward the development of functional substitutes that maintain or restore tissue function.8 Production of an artificial salivary gland involves culturing salivary ACs on biomaterial, which can function in an exocrine capacity after transplantation. To develop an
Salivary glands are multifunctional and are able to mediate oral microbial colonization, mucosal repair, dental remineralization, food bolus formation and translocation, lubrication, and digestion.1 Patients with salivary gland hypofunction often suffer from conditions including rampant caries, frequent mucosal infection, dysphagia, as well as considerable pain and discomfort.2 Up to approximately 90% of the glandular volume is constituted by acinar cells (ACs),3,4 which are highly sensitive to irradiation.5 Thus, irreversible damage to the salivary glands is a common
©2007 Elsevier & Formosan Medical Association .
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Graduate Institute of Clinical Dentistry and School of Dentistry, 3Graduate Institute of Biomedical Engineering, College of Medicine, National Taiwan University, and 2Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan. Received: April 18, 2006 Revised: May 22, 2006 Accepted: August 1, 2006
*Correspondence to: Dr Min-Huey Chen, Department of Dentistry, National Taiwan University Hospital, 1 Chang-Te Street, Taipei 100, Taiwan. E-mail: [email protected]
J Formos Med Assoc | 2007 • Vol 106 • No 1
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artificial salivary gland by tissue engineering, a great number of salivary ACs are needed.9 Bone marrow stem cells (BMSCs), like all stem cells, share at least two characteristics. Firstly, they can make identical copies of themselves for long periods of time, a process described as long-term self-renewal. Secondly, they can give rise to mature cell types that have characteristic morphologies and specialized functions.10 Besides, stem cells in adults can generate the specialized cell types of another tissue from which they normally reside— either a tissue of the same embryonic germ layer or a different germ layer.11 This characteristic of stem cells is termed plasticity or transdifferentiation.12 For example, a BMSC, which is a mesodermal derivative, may differentiate into another mesodermally derived tissue such as skeletal muscle13 or ectodermally derived tissue such as neural tissue.14 Mesenchymal stem cells (MSCs) in bone marrow are distinct from hematopoietic stem cells, and may differentiate into osteoblasts, chondroblasts, adipocytes, or hematopoietic supporting stroma.15–17 They are readily isolated from patients by bone marrow aspiration and expand in culture by up to 1 billion-fold in 8 weeks.18 These appealing features make MSCs a good candidate for potential therapeutic applications. Bone marrow-derived MSCs can differentiate into cardiomyocytes following treatment with chemicals,19 growth factors,20 and microenvironments in vivo.21 These findings suggest the potential for use of BMSCs as a cell source for salivary gland tissue engineering. In this study, we hypothesized that BMSCs could transdifferentiate into parotid ACs by co-culturing with ACs. We isolated rat parotid ACs and BMSCs. By using a double chamber co-culture system, the transdifferentiation of BMSCs was evaluated.
Methods All animal studies were performed according to a protocol approved by the review board of the College of Medicine, National Taiwan University. 2
Isolation and culture of ACs Parotid ACs were obtained from 7-day-old Wistar rats by the explant outgrowth technique without collagenase treatment as previously described.9,22 Briefly, using sterile techniques, the thin fascia covering the parotid gland was carefully removed to expose the gland tissue, and then the parotid gland was cut into small fragments of about 1 mm3. The tissue fragments were then cultured at 37°C in a 5% CO2 atmosphere in a humidified incubator with M199 culture medium (Biochrom AG, Berlin, Germany) supplemented with 20% fetal bovine serum (FBS) (Biological Industries, Beit Haemek, Israel) and antibiotic-antimycotic (penicillin G sodium 100 U/mL, streptomycin sulfate 100 μg/mL, amphotericin B 0.25 μg/mL; Gibco BRL, Grand Island, NY, USA). Cells released from the tissues grew to confluence in approximately 6–8 days. At approximately 90% confluence, tissue fragments in the suspension were removed with the medium and the adherent cells were subcultured on two 100 mm culture dishes (Corning Inc., Corning, NY, USA) for another 4 days. Under these conditions, cell division and growth continued to yield enough cells for further identification and co-culture of the cells. The second passage of ACs was used.
Identification of ACs ACs were identified by immunocytochemical analysis before co-culture. A portion of the cultured cells were plated on the cover glass for fixation with 2% paraformaldehyde-0.1% Triton X-100 (Sigma, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 30 minutes, washed with PBS five times, and blocked with 2.5% nonfat milk for 30 minutes. The primary antibody was polyclonal anti-α-amylase antibody (Sigma), and rhodamineconjugated secondary antibodies (Chemicon International Inc., Temecula, CA, USA) were allowed to react with the cells for 30 minutes at room temperature to visualize the signal. Immunostained ACs were identified by indirect fluorescence under a fluorescence microscope (Axiovert 200 M; Carl Zeiss AG, Hallbergmoos, Germany), and the cultured cells were then prepared for co-culturing with BMSCs. J Formos Med Assoc | 2007 • Vol 106 • No 1
Transdifferentiation of bone marrow stem cells
Isolation and culture of BMSCs Bone marrow cells were harvested from the femur and tibia of 7-week-old male Wistar rats. Animals were anesthetized by an overdose of ethyl ether. Tissue was removed and the ends of the bone were cut. Bone marrow was collected by a syringe with a 21-gauge needle. Marrow was mixed (1:9, v/v) with a buffer containing 0.8% ammonium chloride and 10 μM EDTA (Sigma) for 10 minutes to lyse red blood cells. After 24 hours, the adherent cells were kept for culture and spindleshaped large cell colonies that had been identified as MSCs were observed under the microscope in these adherent cells. However, there were still some small stem cells in the non-adherent cells that required a longer time to adhere. Therefore, the non-adherent cells of the supernatant were also collected by centrifugation at 2000 rpm for 5 minutes and plated on the 100 mm culture dish containing Dulbecco’s Modified Eagle’s Medium (DMEM; Biochrom AG) supplemented with 20% FBS (Biological Industries) and antibioticantimycotic (penicillin G sodium 100 U/mL, streptomycin sulfate 100 μg/mL, amphotericin B 0.25 μg/mL; Gibco BRL) and incubated at 37°C with 5% humidified CO2. The small MSCs adhered to the culture dish after 24–48 hours and were collected by treating with 2 mM EDTA/ PBS for 5 minutes.23 Both the spindle-shaped large cells collected first and the small cells collected later were re-suspended for culture and identification of BMSCs as well as prepared for co-culturing with ACs.
supplemented with 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mM nonessential amino acid with adipogenetic stimulants (AD) consisting of 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 1 μM dexamethasone, 5 μg/mL insulin, and 50 μM indomethacin. The culture was maintained at 37°C in a 5% CO2 atmosphere in a humidified incubator with the medium changed twice per week. After 3 weeks, cells were fixed in 4% paraformaldehyde for 12 hours and stained for 45 minutes with a filtered 60% Oil Red-O solution in PBS, made from a stock solution of 0.70 g Oil Red-O powder (Sigma) in 200 mL of isopropanol (Sigma). Cells were then washed with PBS to remove background Oil Red-O stain, and photomicrographs were taken with a Nikon Eclipse E600 light microscope. BMSCs were identified and prepared for co-culturing with ACs.
Co-culture system BMSCs were co-cultured with parotid ACs in a double chamber system by plating ACs on the upper inserts and BMSCs at the lower culture plate area (Figure 1). The ratio of cell density was 1:4 for BMSCs and ACs. BMSCs were treated with 1% EDTA (Sigma) and re-suspended with M199 culture medium at a density of 2 × 104 cells/well in a 12-well plate (BD FalconTM; BD Biosciences, Franklin Lakes, NJ, USA). Culture inserts (Costar, Corning Inc.), each containing 8 × 104 ACs, were placed in nine of the 12 wells, one insert for each well. The other three wells containing only BMSCs Control group
Co-culture group Acinar cells
Identification of BMSCs BMSCs were identified by adipogenic induction and observed by Oil Red-O staining.24 Before induction, the cells were labeled with Oil Red-O stain to ensure the absence of adipocytes. The isolated cells were then plated on the glass cover slip of a 60 mm culture dish (Nunclon; Nalge Nunc International, Roskilde, Denmark) at 104 cells/mL for 3 days, and then subjected to adipogenetic differentiation medium for 3 weeks. Adipogenetic differentiation medium consisted of DMEM J Formos Med Assoc | 2007 • Vol 106 • No 1
BMSCs
BMSCs
Figure 1. Illustration of the seeding of cells in the double chamber co-culture system. The upper part of the co-culture system comprised 12 cell culture inserts with a 0.4 μm pore size to avoid direct cell-to-cell interaction. The lower part was a 12-well plate. Co-culturing was performed by seeding acinar cells (ACs) on the upper part and plating bone marrow stem cells in the lower well. M199 culture medium facilitated communication of the two parts via insert pores. Empty upper inserts that did not contain ACs served as controls.
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served as controls. An insert membrane with a pore size of 0.4 μm in diameter was used to allow only the transmission of soluble factors and prevent direct interaction between BMSCs and ACs.
Evaluation of the effects of the co-culture system Every 1 week after co-culture, BMSCs were immunofluorescence stained to identify expression of the AC phenotype. After removing the medium and the inserts, the BMSCs in the lower part of the culture plate were examined by immunocytochemical analysis for ACs as described previously. The cell nuclei of the BMSCs were also stained with 4′6-diamidino-2-phenylindole-2HCl (DAPI; Sigma). Immunostained cells were visualized by indirect fluorescence under a fluorescence microscope (Axiovert 200 M; Carl Zeiss AG). Both the number of cells that were immunopositive for α-amylase antibody and DAPI were counted in five visual fields (10–30 cells per field) in three independent cultures. Morphologic changes of BMSCs before and after transdifferentiation were also investigated and compared with isolated ACs.
Statistical analysis The ratio of BMSCs immunopositive for α-amylase antibody to that for DAPI was calculated and analyzed by Student’s t test. Data are presented as mean ± SEM, and statistical significance defined as p < 0.05.
Results Identification of ACs The explant outgrowth technique and M199 medium were used to culture parotid ACs from 7-day-old Wistar rats. Cells were subcultured for 4 days to about 90% confluence to obtain sufficient cells. By using Triton X-100, cell membranes could be perforated and easily stained. The expression of α-amylase in the cells was determined by immunocytochemical staining. Production of α-amylase is one of the most characteristic 4
Figure 2. Oil Red-O staining of adipocytes observed under light microscope (Nikon Eclipse E600) after induction of collected bone marrow stem cells with adipogenic culture medium for 3 weeks.
functions of ACs. Use of the explant outgrowth technique and M199 medium preserved the typical phenotype of ACs.
Identification of BMSCs Bone marrow cells were aspirated from 7-week-old male Wistar rats. To assess the plasticity of the collected cells, we examined their ability to differentiate into adipocytes. Initially, the cells did not stain positive with Oil Red-O. After culture with adipogenic inducers for 3 weeks, however, cytoplasmic accumulation of oil droplets stained with Oil Red-O (Figure 2) were found in the inducted cells, indicating that the cells obtained from bone marrow were indeed stem cells.
Evaluation of the effects of the co-culture system Immunocytochemical analysis was performed every week after the start of co-culture to determine the expression of the AC functional protein, α-amylase, to determine whether BMSCs could transdifferentiate into ACs. Figures 3 and 4 show the expression of α-amylase, indicating the transdifferentiation of BMSCs after co-culture for 1–3 weeks. Nuclear staining with DAPI was used to determine the total number of cells and the results allowed easy calculation of the percentage of transdifferentiation of BMSCs into ACs. The ratio of BMSCs expressing α-amylase was calculated. At weekly intervals after the start of co-culture, three wells of BMSCs were examined and then three J Formos Med Assoc | 2007 • Vol 106 • No 1
Transdifferentiation of bone marrow stem cells
A
B
Figure 3. (A) Fluorescence microscope image of the transdifferentiation of bone marrow stem cells (BMSCs) into acinar cells (ACs) after co-culturing with ACs for 1 week. Cells were stained with rhodamine-conjugated anti-α-amylase antibody (red). DAPI was used for staining nuclei (blue) (original magnification, 200×). (B) The control group, BMSCs alone, showed expression of nuclei only (blue) and not α-amylase (red) after 1 week (original magnification, 200×). A
B
Figure 4. (A) Phase-contrast image of the transdifferentiated bone marrow stem cells after co-culturing with acinar cells for 3 weeks (original magnification, 200×). (B) The same field observed under fluorescence microscope. Blue spots indicate cell nuclei, while red areas indicate the expression of α-amylase in the cytoplasm (original magnification, 200×).
additional wells were examined under the same conditions, i.e. six wells of BMSCs (n = 6) were examined in order to calculate the ratio. Expression of α-amylase was found in approximately 30 ± 4.6% of cells after 1 week of co-culture, 50 ± 4.5% after 2 weeks, and 50 ± 2.3% after 3 weeks. None of the control group experiments, which involved BMSCs alone, showed expression of α-amylase. There was a significant difference in expression of α-amylase every week after co-culture between the experimental and control groups (p < 0.001).
Morphologic investigation of cells Isolated ACs were shown to be spindle shaped (Figure 5A). Before transdifferentiation, the BMSCs were either of similar morphology—large spindle shaped, or small round shaped (Figure 5B). Figure 5C shows that the morphology of the transdifferentiated cells was like that of ACs. J Formos Med Assoc | 2007 • Vol 106 • No 1
Discussion Development of an effective implantable artificial fluid-secreting device for the treatment of patients who lose functional salivary epithelium because of irradiation for head and neck cancer is an urgent need. Currently, there is no effective preventive treatment or therapy for patients whose salivary function has been seriously damaged. Considering the immune reaction, if an allograft is used, it will lead to a stronger immune response. This problem can be circumvented by using autologous healthy ACs. In this study, we successfully established a model of transdifferentiation of BMSCs into ACs by using a double chamber system, by using an explant outgrowth procedure without tissue digestion to successfully maintain the growth and phenotypic expression of primary cultures of rat parotid ACs. 5
C.Y. Lin, et al
A
B
C
Figure 5. (A) Phase-contrast image of acinar cells (ACs). (B) Bone marrow stem cells (BMSCs) before co-culture. (C) BMSCs after co-culturing with ACs for 2 weeks. These transdifferentiated cells had similar morphology to ACs.
In this study, we examined the ability of rat BMSCs to differentiate by adipogenic induction. Small rapidly-renewing cells were previously found in bone marrow and were identified as small MSCs.25 These small cells (≤ 5 μm) take more time to adhere to the dish and therefore should be collected more carefully. Both the spindle-shaped large cells and small cells were used in our experiments. Oil Red-O staining indicated that these cells may differentiate into adipocytes after induction. The identified BMSCs were co-cultured with ACs. M199 medium with 20% FBS was used because of its greater suitability for AC growth.9 We found that the longer the time of co-culture, the higher the rate of transdifferentiation of BMSCs. However, transdifferentiation was limited to up to about 50%. After 1 week, approximately 30% of BMSCs could express the functional protein of ACs. This ratio increased to 50% at 2 and 3 weeks. Expression of α-amylase in the cytoplasm increased and was more easily observed with time. The morphologic changes in the BMSCs after coculturing with ACs also showed increased similarity to ACs as shown in Figure 5. 6
BMSCs were seeded at a density of 2 × 104 cells/well in a 12-well plate and proliferated to 2 × 105 in each well after 2 weeks of co-culture. The ratio of transdifferentiation after 2 weeks was 50%, which meant that about 105 acinar-like cells would be obtained. Co-culture of BMSCs with respiratory epithelial cells26 and cardiomyocytes27 by direct cell contact has been shown to induce transdifferentiation, but not without direct cell contact. These studies indicated that an indirect method would be more difficult than a direct contact co-culture system. This was a novel study that showed the possibility of transdifferentiation of BMSCs into ACs. The results showed the effect of an indirect co-culture method for transdifferentiation of BMSCs. However, further studies are needed to test the function of these acinar-like cells and their phenotypic similarity to ACs, which will determine their potential to serve as an unlimited cell source for the development of an artificial salivary gland for the treatment of patients with salivary hypofunction. Further studies are necessary for investigating the signal transduction and mechanisms involved in the transdifferentiation of BMSCs into ACs. J Formos Med Assoc | 2007 • Vol 106 • No 1
Transdifferentiation of bone marrow stem cells
Different co-culture systems will also be compared to find the best method for transdifferentiation of BMSCs into ACs. Animal experiments could then be established for the tissue engineering of salivary glands.
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Acknowledgments The authors would like to thank the Core Facility of the Medical Research Center, National Taiwan University Hospital. This study was funded by the National Taiwan University Hospital (NTUH 95-525) and the National Science Council (NSC 93-2314-B-002-182), Taiwan.
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References 1. Mandel ID. The role of saliva in maintaining oral homeostasis. J Am Dent Assoc 1989;119:289–304. 2. Fox PC. Acquired salivary dysfunction: drugs and radiation. Ann NY Acad Sci 1998;842:132. 3. Williams MA, Cope GH, Pattern MK. A system for the study of zymogen granule genesis and discharge in rabbit parotid gland tissue in vitro. J Anat 1973;115:141. 4. Baum BJ. Principles of saliva secretion. Ann NY Acad Sci 1993;694:17. 5. Kashima HK, Kirkham JR, Andrews JR, et al. Post-irradiation sialadenitis: a study of the clinical features, histopathologic changes and serum enzyme variations following irradiation of human salivary glands. Am J Roentgenol Radium Ther Nucl Med 1965;94:77. 6. Frazen L, Funegard U, Ericson T, et al. Parotid gland function during and following radiotherapy of malignancies in the head and neck. A consecutive study of salivary flow and patient discomfort. Eur J Cancer 1992;28:457. 7. Valdez IH, Atkinson JC, Ship JA, et al. Major salivary gland function in patients with radiation induced xerostomia: flow rates and sialochemistry. Int J Radiat Oncol Biol Phys 1993;25:41. 8. Langer R, Vacanti JP. Tissue engineering. Science 1993; 260:920–6. 9. Chen MH, Chen RS, Hsu YH, et al. Proliferation and phenotype preservation of rat parotid acinar cells. Tissue Eng 2005;11:526–34. 10. Department of Health and Human Services. Stem Cells: Scientific Progress and Future Research Directions. 2001, Chapter 4. Available at: http://stemcells.nih.gov/info/ scireport 11. Department of Health and Human Services. Stem Cells: Scientific Progress and Future Research Directions. 2001,
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Chapter 1. Available at: http://stemcells.nih.gov/info/ scireport Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000;290:1775–9. Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279:1528–30. Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;29:1779–82. Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV. Osteogenesis in transplants of bone marrow cells. J Embryol Exp Morphol 1966;16:381–90. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea pig bone marrow and spleen cells. Cell Tissue Kinet 1970;3:393–403. Owen M. Marrow derived stromal stem cells. J Cell Science Supp 1988;10:63–76. Colter DC, Class R, DiGirolamo CM, et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA 2000;97:3213–8. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103:697–705. Lough J, Barron M, Brogley M, et al. Combined BMP-2 and FGF-4, but neither factor alone, induces cardiogenesis in non-precardiac embryonic mesoderm. Dev Biol 1996;178: 198–202. Wang JS, Shum-Tim D, Galipeau J, et al. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J Thorac Cardiovasc Surg 2000;120: 999–1006. Dimitriou ID, Kapsogeorgou EK, Abu-Helu RF, et al. Establishment of a convenient system for the long-term culture and study of non-neoplastic human salivary gland epithelium cells. Eur J Oral Sci 2002;110:21–30. Chen MH, Lin SM, Hsieh CH, et al. Identification and initial characterization of small cells in adult cartilage and bone marrow. J Formos Med Assoc 2004;103:264–5. Mark FP, Alastair MM, Stephen CB, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–6. Colter DC, Sejiya I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA 2001;98:7841–5. Le Visage C, Dunham B, Flint P, Leong KW. Coculture of mesenchymal stem cells and respiratory epithelial cells to engineer a human composite respiratory mucosa. Tissue Eng 2004;9:1426–35. Garbade J, Schubert A, Rastan AJ, et al. Fusion of bone marrow-derived stem cells with cardiomyocytes in a heterologous in vitro model. Eur J Cardiothorac Surg 2005;28:685–91.
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Transdifferentiation of Bone Marrow Stem Cells into Acinar Cells Using a Double Chamber System Chia-Yung Lin,1,2 Bor-Shiunn Lee,1,2 Chih-Chen Liao,3 Wei-Jhih Cheng,3 Feng-Ming Chang,1,2 Min-Huey Chen1,2* Background/Purpose: Hypofunction of the salivary glands can substantially affect quality of life. Current treatments for salivary hypofunction are of limited effectiveness. Although the implantation of functional salivary gland tissue from autologous glandular cells represents a possible physiologic solution to this problem, tissue engineering of salivary glands would require the generation of a great number of acinar cells (ACs). The purpose of this study was to investigate the feasibility of transdifferentiation of bone marrow stem cells (BMSCs) into functional ACs using a co-culture system. Methods: BMSCs were isolated from adult rats and co-cultured with rat parotid ACs using a double chamber system. The transdifferentiation of BMSCs was evaluated by immunocytochemical analysis of α-amylase, which has unique functional expression in ACs. Results: Expression of α-amylase, indicating successful transdifferentiation of BMSCs into ACs, was found in 30% of BMSCs after co-culturing for 1 week, and in 50% after co-culturing for 2 and 3 weeks. Conclusion: This study has demonstrated the potential of rat BMSCs to transdifferentiate into ACs, and support the feasibility of application of BMSCs in salivary gland tissue engineering. [J Formos Med Assoc 2007;106(1):1–7] Key Words: cell transdifferentiation, salivary gland, tissue engineering
complication of radiation therapy for head and neck cancer,6 and may lead to persistent salivary hypofunction with marked reduction in salivary output.7 Currently, there is no effective therapy for this condition. Tissue engineering is an interdisciplinary field that applies the principles of biology and engineering toward the development of functional substitutes that maintain or restore tissue function.8 Production of an artificial salivary gland involves culturing salivary ACs on biomaterial, which can function in an exocrine capacity after transplantation. To develop an
Salivary glands are multifunctional and are able to mediate oral microbial colonization, mucosal repair, dental remineralization, food bolus formation and translocation, lubrication, and digestion.1 Patients with salivary gland hypofunction often suffer from conditions including rampant caries, frequent mucosal infection, dysphagia, as well as considerable pain and discomfort.2 Up to approximately 90% of the glandular volume is constituted by acinar cells (ACs),3,4 which are highly sensitive to irradiation.5 Thus, irreversible damage to the salivary glands is a common
©2007 Elsevier & Formosan Medical Association .
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Graduate Institute of Clinical Dentistry and School of Dentistry, 3Graduate Institute of Biomedical Engineering, College of Medicine, National Taiwan University, and 2Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan. Received: April 18, 2006 Revised: May 22, 2006 Accepted: August 1, 2006
*Correspondence to: Dr Min-Huey Chen, Department of Dentistry, National Taiwan University Hospital, 1 Chang-Te Street, Taipei 100, Taiwan. E-mail: [email protected]
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artificial salivary gland by tissue engineering, a great number of salivary ACs are needed.9 Bone marrow stem cells (BMSCs), like all stem cells, share at least two characteristics. Firstly, they can make identical copies of themselves for long periods of time, a process described as long-term self-renewal. Secondly, they can give rise to mature cell types that have characteristic morphologies and specialized functions.10 Besides, stem cells in adults can generate the specialized cell types of another tissue from which they normally reside— either a tissue of the same embryonic germ layer or a different germ layer.11 This characteristic of stem cells is termed plasticity or transdifferentiation.12 For example, a BMSC, which is a mesodermal derivative, may differentiate into another mesodermally derived tissue such as skeletal muscle13 or ectodermally derived tissue such as neural tissue.14 Mesenchymal stem cells (MSCs) in bone marrow are distinct from hematopoietic stem cells, and may differentiate into osteoblasts, chondroblasts, adipocytes, or hematopoietic supporting stroma.15–17 They are readily isolated from patients by bone marrow aspiration and expand in culture by up to 1 billion-fold in 8 weeks.18 These appealing features make MSCs a good candidate for potential therapeutic applications. Bone marrow-derived MSCs can differentiate into cardiomyocytes following treatment with chemicals,19 growth factors,20 and microenvironments in vivo.21 These findings suggest the potential for use of BMSCs as a cell source for salivary gland tissue engineering. In this study, we hypothesized that BMSCs could transdifferentiate into parotid ACs by co-culturing with ACs. We isolated rat parotid ACs and BMSCs. By using a double chamber co-culture system, the transdifferentiation of BMSCs was evaluated.
Methods All animal studies were performed according to a protocol approved by the review board of the College of Medicine, National Taiwan University. 2
Isolation and culture of ACs Parotid ACs were obtained from 7-day-old Wistar rats by the explant outgrowth technique without collagenase treatment as previously described.9,22 Briefly, using sterile techniques, the thin fascia covering the parotid gland was carefully removed to expose the gland tissue, and then the parotid gland was cut into small fragments of about 1 mm3. The tissue fragments were then cultured at 37°C in a 5% CO2 atmosphere in a humidified incubator with M199 culture medium (Biochrom AG, Berlin, Germany) supplemented with 20% fetal bovine serum (FBS) (Biological Industries, Beit Haemek, Israel) and antibiotic-antimycotic (penicillin G sodium 100 U/mL, streptomycin sulfate 100 μg/mL, amphotericin B 0.25 μg/mL; Gibco BRL, Grand Island, NY, USA). Cells released from the tissues grew to confluence in approximately 6–8 days. At approximately 90% confluence, tissue fragments in the suspension were removed with the medium and the adherent cells were subcultured on two 100 mm culture dishes (Corning Inc., Corning, NY, USA) for another 4 days. Under these conditions, cell division and growth continued to yield enough cells for further identification and co-culture of the cells. The second passage of ACs was used.
Identification of ACs ACs were identified by immunocytochemical analysis before co-culture. A portion of the cultured cells were plated on the cover glass for fixation with 2% paraformaldehyde-0.1% Triton X-100 (Sigma, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 30 minutes, washed with PBS five times, and blocked with 2.5% nonfat milk for 30 minutes. The primary antibody was polyclonal anti-α-amylase antibody (Sigma), and rhodamineconjugated secondary antibodies (Chemicon International Inc., Temecula, CA, USA) were allowed to react with the cells for 30 minutes at room temperature to visualize the signal. Immunostained ACs were identified by indirect fluorescence under a fluorescence microscope (Axiovert 200 M; Carl Zeiss AG, Hallbergmoos, Germany), and the cultured cells were then prepared for co-culturing with BMSCs. J Formos Med Assoc | 2007 • Vol 106 • No 1
Transdifferentiation of bone marrow stem cells
Isolation and culture of BMSCs Bone marrow cells were harvested from the femur and tibia of 7-week-old male Wistar rats. Animals were anesthetized by an overdose of ethyl ether. Tissue was removed and the ends of the bone were cut. Bone marrow was collected by a syringe with a 21-gauge needle. Marrow was mixed (1:9, v/v) with a buffer containing 0.8% ammonium chloride and 10 μM EDTA (Sigma) for 10 minutes to lyse red blood cells. After 24 hours, the adherent cells were kept for culture and spindleshaped large cell colonies that had been identified as MSCs were observed under the microscope in these adherent cells. However, there were still some small stem cells in the non-adherent cells that required a longer time to adhere. Therefore, the non-adherent cells of the supernatant were also collected by centrifugation at 2000 rpm for 5 minutes and plated on the 100 mm culture dish containing Dulbecco’s Modified Eagle’s Medium (DMEM; Biochrom AG) supplemented with 20% FBS (Biological Industries) and antibioticantimycotic (penicillin G sodium 100 U/mL, streptomycin sulfate 100 μg/mL, amphotericin B 0.25 μg/mL; Gibco BRL) and incubated at 37°C with 5% humidified CO2. The small MSCs adhered to the culture dish after 24–48 hours and were collected by treating with 2 mM EDTA/ PBS for 5 minutes.23 Both the spindle-shaped large cells collected first and the small cells collected later were re-suspended for culture and identification of BMSCs as well as prepared for co-culturing with ACs.
supplemented with 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mM nonessential amino acid with adipogenetic stimulants (AD) consisting of 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 1 μM dexamethasone, 5 μg/mL insulin, and 50 μM indomethacin. The culture was maintained at 37°C in a 5% CO2 atmosphere in a humidified incubator with the medium changed twice per week. After 3 weeks, cells were fixed in 4% paraformaldehyde for 12 hours and stained for 45 minutes with a filtered 60% Oil Red-O solution in PBS, made from a stock solution of 0.70 g Oil Red-O powder (Sigma) in 200 mL of isopropanol (Sigma). Cells were then washed with PBS to remove background Oil Red-O stain, and photomicrographs were taken with a Nikon Eclipse E600 light microscope. BMSCs were identified and prepared for co-culturing with ACs.
Co-culture system BMSCs were co-cultured with parotid ACs in a double chamber system by plating ACs on the upper inserts and BMSCs at the lower culture plate area (Figure 1). The ratio of cell density was 1:4 for BMSCs and ACs. BMSCs were treated with 1% EDTA (Sigma) and re-suspended with M199 culture medium at a density of 2 × 104 cells/well in a 12-well plate (BD FalconTM; BD Biosciences, Franklin Lakes, NJ, USA). Culture inserts (Costar, Corning Inc.), each containing 8 × 104 ACs, were placed in nine of the 12 wells, one insert for each well. The other three wells containing only BMSCs Control group
Co-culture group Acinar cells
Identification of BMSCs BMSCs were identified by adipogenic induction and observed by Oil Red-O staining.24 Before induction, the cells were labeled with Oil Red-O stain to ensure the absence of adipocytes. The isolated cells were then plated on the glass cover slip of a 60 mm culture dish (Nunclon; Nalge Nunc International, Roskilde, Denmark) at 104 cells/mL for 3 days, and then subjected to adipogenetic differentiation medium for 3 weeks. Adipogenetic differentiation medium consisted of DMEM J Formos Med Assoc | 2007 • Vol 106 • No 1
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Figure 1. Illustration of the seeding of cells in the double chamber co-culture system. The upper part of the co-culture system comprised 12 cell culture inserts with a 0.4 μm pore size to avoid direct cell-to-cell interaction. The lower part was a 12-well plate. Co-culturing was performed by seeding acinar cells (ACs) on the upper part and plating bone marrow stem cells in the lower well. M199 culture medium facilitated communication of the two parts via insert pores. Empty upper inserts that did not contain ACs served as controls.
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served as controls. An insert membrane with a pore size of 0.4 μm in diameter was used to allow only the transmission of soluble factors and prevent direct interaction between BMSCs and ACs.
Evaluation of the effects of the co-culture system Every 1 week after co-culture, BMSCs were immunofluorescence stained to identify expression of the AC phenotype. After removing the medium and the inserts, the BMSCs in the lower part of the culture plate were examined by immunocytochemical analysis for ACs as described previously. The cell nuclei of the BMSCs were also stained with 4′6-diamidino-2-phenylindole-2HCl (DAPI; Sigma). Immunostained cells were visualized by indirect fluorescence under a fluorescence microscope (Axiovert 200 M; Carl Zeiss AG). Both the number of cells that were immunopositive for α-amylase antibody and DAPI were counted in five visual fields (10–30 cells per field) in three independent cultures. Morphologic changes of BMSCs before and after transdifferentiation were also investigated and compared with isolated ACs.
Statistical analysis The ratio of BMSCs immunopositive for α-amylase antibody to that for DAPI was calculated and analyzed by Student’s t test. Data are presented as mean ± SEM, and statistical significance defined as p < 0.05.
Results Identification of ACs The explant outgrowth technique and M199 medium were used to culture parotid ACs from 7-day-old Wistar rats. Cells were subcultured for 4 days to about 90% confluence to obtain sufficient cells. By using Triton X-100, cell membranes could be perforated and easily stained. The expression of α-amylase in the cells was determined by immunocytochemical staining. Production of α-amylase is one of the most characteristic 4
Figure 2. Oil Red-O staining of adipocytes observed under light microscope (Nikon Eclipse E600) after induction of collected bone marrow stem cells with adipogenic culture medium for 3 weeks.
functions of ACs. Use of the explant outgrowth technique and M199 medium preserved the typical phenotype of ACs.
Identification of BMSCs Bone marrow cells were aspirated from 7-week-old male Wistar rats. To assess the plasticity of the collected cells, we examined their ability to differentiate into adipocytes. Initially, the cells did not stain positive with Oil Red-O. After culture with adipogenic inducers for 3 weeks, however, cytoplasmic accumulation of oil droplets stained with Oil Red-O (Figure 2) were found in the inducted cells, indicating that the cells obtained from bone marrow were indeed stem cells.
Evaluation of the effects of the co-culture system Immunocytochemical analysis was performed every week after the start of co-culture to determine the expression of the AC functional protein, α-amylase, to determine whether BMSCs could transdifferentiate into ACs. Figures 3 and 4 show the expression of α-amylase, indicating the transdifferentiation of BMSCs after co-culture for 1–3 weeks. Nuclear staining with DAPI was used to determine the total number of cells and the results allowed easy calculation of the percentage of transdifferentiation of BMSCs into ACs. The ratio of BMSCs expressing α-amylase was calculated. At weekly intervals after the start of co-culture, three wells of BMSCs were examined and then three J Formos Med Assoc | 2007 • Vol 106 • No 1
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Figure 3. (A) Fluorescence microscope image of the transdifferentiation of bone marrow stem cells (BMSCs) into acinar cells (ACs) after co-culturing with ACs for 1 week. Cells were stained with rhodamine-conjugated anti-α-amylase antibody (red). DAPI was used for staining nuclei (blue) (original magnification, 200×). (B) The control group, BMSCs alone, showed expression of nuclei only (blue) and not α-amylase (red) after 1 week (original magnification, 200×). A
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Figure 4. (A) Phase-contrast image of the transdifferentiated bone marrow stem cells after co-culturing with acinar cells for 3 weeks (original magnification, 200×). (B) The same field observed under fluorescence microscope. Blue spots indicate cell nuclei, while red areas indicate the expression of α-amylase in the cytoplasm (original magnification, 200×).
additional wells were examined under the same conditions, i.e. six wells of BMSCs (n = 6) were examined in order to calculate the ratio. Expression of α-amylase was found in approximately 30 ± 4.6% of cells after 1 week of co-culture, 50 ± 4.5% after 2 weeks, and 50 ± 2.3% after 3 weeks. None of the control group experiments, which involved BMSCs alone, showed expression of α-amylase. There was a significant difference in expression of α-amylase every week after co-culture between the experimental and control groups (p < 0.001).
Morphologic investigation of cells Isolated ACs were shown to be spindle shaped (Figure 5A). Before transdifferentiation, the BMSCs were either of similar morphology—large spindle shaped, or small round shaped (Figure 5B). Figure 5C shows that the morphology of the transdifferentiated cells was like that of ACs. J Formos Med Assoc | 2007 • Vol 106 • No 1
Discussion Development of an effective implantable artificial fluid-secreting device for the treatment of patients who lose functional salivary epithelium because of irradiation for head and neck cancer is an urgent need. Currently, there is no effective preventive treatment or therapy for patients whose salivary function has been seriously damaged. Considering the immune reaction, if an allograft is used, it will lead to a stronger immune response. This problem can be circumvented by using autologous healthy ACs. In this study, we successfully established a model of transdifferentiation of BMSCs into ACs by using a double chamber system, by using an explant outgrowth procedure without tissue digestion to successfully maintain the growth and phenotypic expression of primary cultures of rat parotid ACs. 5
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Figure 5. (A) Phase-contrast image of acinar cells (ACs). (B) Bone marrow stem cells (BMSCs) before co-culture. (C) BMSCs after co-culturing with ACs for 2 weeks. These transdifferentiated cells had similar morphology to ACs.
In this study, we examined the ability of rat BMSCs to differentiate by adipogenic induction. Small rapidly-renewing cells were previously found in bone marrow and were identified as small MSCs.25 These small cells (≤ 5 μm) take more time to adhere to the dish and therefore should be collected more carefully. Both the spindle-shaped large cells and small cells were used in our experiments. Oil Red-O staining indicated that these cells may differentiate into adipocytes after induction. The identified BMSCs were co-cultured with ACs. M199 medium with 20% FBS was used because of its greater suitability for AC growth.9 We found that the longer the time of co-culture, the higher the rate of transdifferentiation of BMSCs. However, transdifferentiation was limited to up to about 50%. After 1 week, approximately 30% of BMSCs could express the functional protein of ACs. This ratio increased to 50% at 2 and 3 weeks. Expression of α-amylase in the cytoplasm increased and was more easily observed with time. The morphologic changes in the BMSCs after coculturing with ACs also showed increased similarity to ACs as shown in Figure 5. 6
BMSCs were seeded at a density of 2 × 104 cells/well in a 12-well plate and proliferated to 2 × 105 in each well after 2 weeks of co-culture. The ratio of transdifferentiation after 2 weeks was 50%, which meant that about 105 acinar-like cells would be obtained. Co-culture of BMSCs with respiratory epithelial cells26 and cardiomyocytes27 by direct cell contact has been shown to induce transdifferentiation, but not without direct cell contact. These studies indicated that an indirect method would be more difficult than a direct contact co-culture system. This was a novel study that showed the possibility of transdifferentiation of BMSCs into ACs. The results showed the effect of an indirect co-culture method for transdifferentiation of BMSCs. However, further studies are needed to test the function of these acinar-like cells and their phenotypic similarity to ACs, which will determine their potential to serve as an unlimited cell source for the development of an artificial salivary gland for the treatment of patients with salivary hypofunction. Further studies are necessary for investigating the signal transduction and mechanisms involved in the transdifferentiation of BMSCs into ACs. J Formos Med Assoc | 2007 • Vol 106 • No 1
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Different co-culture systems will also be compared to find the best method for transdifferentiation of BMSCs into ACs. Animal experiments could then be established for the tissue engineering of salivary glands.
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Acknowledgments The authors would like to thank the Core Facility of the Medical Research Center, National Taiwan University Hospital. This study was funded by the National Taiwan University Hospital (NTUH 95-525) and the National Science Council (NSC 93-2314-B-002-182), Taiwan.
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