Biotechnol Lett DOI 10.1007/s10529-015-1809-1
ORIGINAL RESEARCH PAPER
Transdifferentiation of bone marrow-derived mesenchymal stem cells into salivary gland-like cells using a novel culture method Liang Liang • Jun Wang • Yuming Zhang • Zhiyuan Shen • Jun Zheng Jianhu Li • Zhongping Su • Juan Cai • Wei Jiang • Moyi Sun
•
Received: 6 November 2014 / Accepted: 24 February 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objectives To investigate the transdifferentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into salivary gland-like cells via a novel culture method employing induction culture medium collected from salivary gland cells. Results Primary salivary gland cells were cultured, and after the first passage, the culture medium was collected for use as induction medium. BMSCs (passage 3) were cultured in either induction medium Liang Liang and Jun Wang have contributed equally to this work.
(induction group) or DMEM/F12 medium with 10 % (v/v) fetal bovine serum (control group) before seeding on three-dimensional collagen/chitosan scaffolds and subcutaneous transplantation into nude mice. The in vitro and in vivo transdifferentiation of BMSCs into salivary gland-like cells was evaluated by immunocytochemical analysis of a-amylase and cytokeratin-8 (CK-8) expression. Salivary gland-like cells cultured using this novel method maintained excellent biostability and exhibited relatively stable expression of a-amylase and CK-8 in vitro and in vivo. Conclusion This novel culture method is feasible for inducing the transdifferentiation of BMSCs into salivary gland-like cells.
L. Liang Z. Shen J. Zheng J. Li Z. Su J. Cai W. Jiang M. Sun (&) State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military University, Xi’an 710032, People’s Republic of China e-mail: [email protected]
Keywords Bone marrow-derived mesenchymal stem cells (BMSCs) Collagen/chitosan scaffolds Novel culture methods Salivary gland-like cells Salivary gland repair Transdifferentiation Transplantation
L. Liang Department of Stomatology, Peace Hospital Attached to ChangZhi Medical College, Changzhi, People’s Republic of China
Introduction
J. Wang Department of Anesthesiology, Shannxi Tumor Hospital, Xi’an, People’s Republic of China Y. Zhang Department of Physiology and Pathophysiology, Xi’an Jiaotong University School of Medicine, Xi’an, People’s Republic of China
Radiation therapy for head and neck cancer results in atrophy, fibrosis, and degeneration salivary gland tissue, leading to salivary gland hypofunction, which increases patients’ risks of tooth decay, oral and mucosal infections, and gastrointestinal conditions as well as increases susceptibility of the mucosa to ulcers
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(Vissink et al. 2003). Unfortunately, no effective treatments are available for irreversible salivary gland damage. A previous study applied muscarinic agonist medications, such as pilocarpine and cevimeline, to induce salivary secretion from the residual functional salivary gland tissue (Fox 2004). However, this method only had a small effect on the recovery of damaged tissue, and such an approach would be ineffective if all or most of the salivary gland cells are already lost. Therefore, developing an adequate treatment by using alternative strategies is required. Recently, approaches for treating injured salivary glands based on tissue engineering (Kaigler and Mooney 2001) and stem cell therapy have drawn much attention (Gersh et al. 2009). Bone marrow-derived mesenchymal stem cells (BMSCs) can self-renew, expand rapidly, and be induced down several cellular lineages for eventual differentiation into osteoblasts, chondrocytes, and adipocytes among other cell types (Pittenger et al. 1999; Prockop 1997). In addition, BMSCs offer advantages such as easy availability, minimal ethical concerns, and low immunogenicity (Prockop et al. 2000). These properties make BMSCs an ideal ‘‘seed cell’’ for tissue engineering and stem cell therapies. The use of BMSCs was effective in both preventing loss of saliva secretion and reducing lymphocytic influx in salivary glands (Khalili et al. 2012). However, no consensus has been reached on the potential biohazard of BMSCs for in vivo transplantation or injection. A method to establish an in vitro culture system for the transdifferentiation of BMSCs into salivary gland cells is urgently needed. An indirect-contact model for inducing BMSC transdifferentiation into acinar cells was developed using a double chamber co-culture system (Lin et al. 2007). Based on the concept of an indirect-contact model, we hypothesized that a novel culture method employing induction culture medium collected from salivary gland cells could effectively induce the BMSC transdifferentiation into salivaryglad like cells. To confirm this hypothesis, we prepared conditioned induction medium from cultures of salivary gland cells and investigated BMSC transdifferentiation in this medium. We found that BMSCs could be induced to transdifferentiate into salivary gland-like cells that may be valuable in the clinical treatment of salivary gland damage.
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Materials and methods Animals Two-week-old Sprague–Dawley (SD) rats and 5-week-old nude mice were obtained from Fourth Military Medical University Animal Center, Xi’an, China. Donor rats were kept under clean conventional conditions (25 °C, 44–47 % relative humidity, and 12-h light/dark cycle) and allowed access to food and water ad libitum. All procedures were conducted in accordance with the animal experiment guidelines of the ethics committee of the Fourth Military Medical University Animal Center (Permit Number: SCXK2012-0007). The experiment was approved by the Committee on the Ethics of Animal Experiments of the Fourth Military Medical University Animal Center (Permit Number: SCXK2012-0007). Isolation and culture of rat submandibular gland cells SD rats submandibular gland were cut into pieces of approx. 1 mm3. Following the protocol of Lin et al. (2007), the explants were placed in D-MEM/F-12 (Gibco) supplemented with 20 % (v/v) fetal bovine serum (FBS), 100 U penicillin G/ml, 100 lg streptomycin sulfate/ml, and 0.25 lg amphotericin B/ml. Isolation and culture of rat BMSCs The femura and tibiae of SD rats were removed, washed with phosphate-buffered saline (PBS), and placed in DMEM/F12 supplemented with 10 % (v/v) FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin. The bone marrow was collected and cultured in flasks. At approx. 80 % confluence after 1 week, the cells were passaged at 1:3, and thereafter, the cells were passaged every 3 days. We used three generations of cells for subsequent experiments. Characterization of BMSCs Osteogenic and adipogenic differentiation of BMSCs SD rat BMSCs (passage 3) were induced to differentiate along the osteogenic pathway following the protocol of Prockop (1997). BMSCs were induced to
Biotechnol Lett
differentiate along the adipogenic pathway following the protocol of Pittenger et al. (1999).
containing collagen/chitosan scaffolds and cultured in an incubator for 5 days.
Flow cytometric analysis of BMSC phenotype
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)
The expression of specific cell surface markers was analyzed in rat BMSCs (passage 3). Aliquots of BMSCs (107 cells/sample) were washed in PBS and incubated with fluorescent-labeled anti-mouse CD29PE, CD31-PE, CD34-PE, CD44-PE, CD45-PE, and CD90-CP CY5.5 (BD Biosciences, San Jose, CA, USA) for 1 h at 4 °C. The labeled cells in each group were analyzed using flow cytometry. Differentiation of BMSCs into salivary gland-like cells The medium from cultures of primary salivary gland cells was collected every 48 h, filtered through a 0.22 lm filter, adjusted to pH 7.2, and refrigerated at 4 °C until use as an induction medium. BMSCs (passage 3) were cultured in either induction medium (induction group) or DMEM/F12 medium containing 10 % (v/v) FBS (control group). For both groups, the medium was changed every 2 days. Hereafter, the cells in the induction group are referred to as salivary gland-like cells, and the cells in the control group are referred to as control cells. Preparation of three-dimensional (3D) collagen/chitosan scaffolds Collagen was extracted from rat tails and dissolved in 2 % (v/v) acetic acid. Chitosan was dissolved in 2 % (v/v) acetic acid at 0.5 %. The collage and chitosan solutions were mixed at 7:3 (v/v), maintained in a constant temperature oscillator to ensure complete mixing and then freeze-dried at -55 °C for 72 h. Seeding of bromodeoxyuridine (BrdU)-labeled cells onto scaffolds BrdU (Sigma-Aldrich) was dissolved in the induction medium and normal culture medium at 10 lM. After 4 weeks in culture, cells in both the induction and control groups were cultured in BrdU-containing medium in a incubator for 24 h. BrdU-labeled gland-like and control cells were seeded at 106 cells/well into the wells of 24-well plates
Passage 1 salivary gland cells, passage 3 BMSCs, cells in the induction and control groups, and cells seeded onto the scaffolds were examined using scanning electron microscopy and transmission electron microscopy. Transplantation study Two groups of scaffolds seeded with either salivary gland-like or control cells were implanted into the nude mice, with a scaffold from each group implanted into opposing sides of the same mouse. Transplantations of scaffolds in the induction group were on the left side, and those of the control group were on the right side. The implantation sites were marked, and scaffolds were removed at weekly intervals. An immunofluorescence assay was used to evaluate aamylase expression by the transplanted cells within the collage/chitosan scaffolds. Sections of explanted tissue samples also were subjected to double immunofluorescence labeling for a-amylase and BrdU.
Results Characterization of salivary gland cells and tissue Using an inverted microscope, we observed that salivary gland cells appeared after about 5 and 10 days. Purified salivary gland cells grew well with a uniform shape in a pavestone arrangement (Fig. 1a, b). Characterization of BMSCs The morphology of BMSCs (passage 3) exhibited a uniform fusiform shape and appeared to be fibroblastlike cells (Fig. 2a). After 3 weeks, BMSCs culture in osteogenic medium showed many red mineralized nodules (Fig. 2b). Oil red ‘O’ staining of third generation BMSCs cultured in adipogenic medium demonstrated visible red lipid droplets in the cytoplasm of cells (Fig. 2c). BMSCs could also be
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Biotechnol Lett Fig. 1 Isolation of primary salivary gland acinar cells. a Salivary gland tissue in culture with surrounding cells migrating away from the specimen at 5 days. b Purification of salivary gland cells at 10 days
characterized by the expression of nonspecific surface antigens. According to flow cytometric analysis, high percentages of BMSCs expressed CD29 (Fig. 2d), CD44 (Fig. 2g), and CD90 (Fig. 2i) and low levels of CD31 (Fig. 2e), CD34 (Fig. 2f), and CD45 (Fig. 2h). The blank control group showed no antigen staining (Fig. 2j). BMSC differentiation into salivary gland-like cells For differentiation into salivary gland-like cells, BMSCs were cultured in induction medium (induction group), whereas the control group was cultured in DMEM/F-12. After 2 weeks, the induction group included a small number of BMSCs with a polygonal shape instead of the long spindle shape. At the same time, BMSCs in the control group retained a uniform spindle shape. After 4 weeks, cells in the induction group exhibited morphological changes, with an arrangement similar to duct-like structures, and a number of cells in clusters surrounding a round hollow structure, whereas no changes were observed in the morphology or arrangement of the control cells. The morphological characteristics of salivary gland cells and induced BMSCs were compared upon observation by SEM. Passage 1 salivary gland cells exhibited centrally located nuclei with dark coloring and an uneven surface as well as many long processes (Fig. 3a). Passage 3 BMSCs were overlapping as well as polygonal and irregular in shape. Pseudopodia were obvious and of different thicknesses and lengths, and nuclei were large and obvious with deep color and a round or oval shape (Fig. 3b). After 4 weeks, the shape of BMSCs in the induction group had changed to round or oval with an uneven cell surface and choppy pseudopodia (Fig. 3c). At the same time point,
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BMSCs in the control group were polygonal in shape with large, round nuclei (Fig. 3d). The same groups of cells at the same passage numbers and times were also examined by TEM. The salivary gland cells showed a tapered morphology with rounded ends, and the cell surface showed only a few visible short microvilli. Their nuclei were round or oval and clear, containing nucleoli (Fig. 3e). BMSCs developed pseudopodia-like structures with microvilli of different lengths, and their nuclei were large. The cells had few organelles (Fig. 3f). After 4 weeks, cells in the induction group were round or oval. Microvilli were evident on the cell surface and showed varying lengths and bending. Their nuclei were large with visible heterochromatin. Compared to control cells, the number of organelles was increased in induced BMSCs. The cells contained a round element resembling secretory granules in the cytoplasm (Fig. 3g). BMSCs in the control group did not show any morphological changes after 3 weeks (Fig. 3h).
Cell seeding on collagen/chitosan scaffolds Figure 4a presents a micrograph of BMSCs seeded on collagen/chitosan scaffolds, and Fig. 4b shows an SEM image of the 3D collagen/chitosan scaffold structure. TEM images of BMSCs in the induction group cultured on collagen/chitosan scaffolds demonstrated that the cells were round with uneven surfaces and pseudopodia. The cells were embedded within large pockets of collagen (Fig. 4c). BMSCs in the control group on collagen/chitosan scaffolds were flat and polygonal, with smooth surfaces and visible connections to the scaffold material (Fig. 4d).
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Fig. 2 BMSC characterization according to adipocytic and osteogenic differentiation. a Passage 3 BMSCs exhibited cell growth. b Alizarin red staining in BMSCs cultured in osteogenic differentiation medium. c Oil red ‘O’ staining in BMSCs cultured in adipocytic differentiation medium. Surface antigen
expression on passage 3 BMSCs was analyzed by flow cytometry. j Control cells exhibited no staining. BMSCs stained positively for d CD29, g CD44, and i CD90, but were negative for e CD31, f CD34, and h CD45
Transplantation of BMSCs on collagen/chitosan scaffolds into nude mice
and 2 weeks were analyzed by immunofluorescence staining for a-amylase along with BrdU (red) and DAPI (40 ,6-diamidino-2-phenylindole) staining (blue). In the induction group, many of the explanted cells stained positively for a-amylase. Void spaces
After transplantation, BMSCs on scaffold materials exhibited good cell growth. Scaffolds removed after 1
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Fig. 3 SEM images of a first generation primary salivary gland cells; b third generation BMSCs; c induced BMSCs after 4 weeks; and d control BMSCs after 4 weeks. TEM images of:
e first generation primary salivary gland cells; f third generation BMSCs; g induced BMSCs after 4 weeks; and h control BMSCs after 4 weeks
were visible in the scaffold structure in superimposed images of staining with two antibodies (Fig. 5a–d). After 2 weeks, BMSCs in the induction group expressed a-amylase and BrdU, and the stents appeared to have been mostly resorbed (Fig. 5e–h). BMSCs in the control group at 1 and 2 weeks stained positively for BrdU only, without expression of a-amylase (Fig. 5i–p). After 2 weeks, the scaffold material had been absorbed.
and stem cell therapy. In the current study, our data for the characterization of BMSCs were consistent with the above description. Both pre-clinical and clinical studies regarding the restoration of hyposalivation offer dramatic examples demonstrating the therapeutic value of BMSCs. Lombaert et al. (2006) reported that BMSCs can be mobilized and targetted to damaged salivary glands after irradiation and that they ameliorate radiation-induced complications and induce limited repair of the function and morphology in the submandibular gland. Although the therapeutic testing of these cells has progressed well, the mechanisms underlying these therapeutic effects remain to be determined. It is speculated that BMSCs could play a better role in such therapies via in vitro transdifferentiation into target cells before transplantation or injection. Based on this concept, Lin et al. (2007) established a doublechamber system to promote the transdifferentiation of BMSCs into acinar cells. In another study (Lv et al. 2011), medium containing submandibular gland cell lysate was used to induce BMSC transdifferentiation. However, these strategies are not only associated with high costs but also require large numbers of primary salivary gland cells; thus, whether these methods can satisfy the requirements for clinical application needs to be explored.
Discussion Salivary gland cells can be permanently destroyed by radiotherapy, leading to reduced salivary function and disastrous consequences for oral health (Jensen et al. 2014). Current treatment strategies are focused on the minimization of radiation damage by parotid-sparing radiation delivery or traditional care based on the use of salivary substitutes and sialogogues (Lim et al. 2013a, b). However, the ideal therapeutic strategy for hyposalivation remains poorly defined. Thus, the beneficial effects of tissue engineering and stem cell therapy have been advocated. BMSCs have generated a great deal of interest because of their potential value in tissue engineering
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Biotechnol Lett Fig. 4 Morphological comparison of salivary gland cells, induced BMSCs, and control BMSCs using scanning and transmission electron microscopy. a Photograph of a collagen/chitosan scaffold. b SEM image of three-dimensional collagen/chitosan scaffold structure. c SEM image of induced BMSCs after 4 weeks on a collagen/chitosan scaffold. d SEM image of control BMSCs after 4 weeks on a collagen/chitosan scaffold
In the present study, we established a novel indirect-contact model for inducing transdifferentiation of BMSCs into salivary gland-like cells (which are functionally or phenotypically similar to salivary gland cells) using relative few salivary gland cells in vitro. The data suggest that the novel technique simplifies the culture process and can generate large numbers of salivary gland-like cells that distinctively secreted a-amylase. However, it is unclear whether the salivary gland-like cells also function similarly to real salivary gland cells. Another objective of our current study was to observe the activity of salivary gland-like cells in vivo. Salivary gland-like cells were seeded onto 3D collagen/chitosan (7:3) scaffolds and transplanted into nude mice, as described previously (Zhu et al. 2009). Collagen is a natural cell substrate that allows for the direct formation of cellular networks and is conducive to cell spreading and extracellular matrix formation (Yang et al. 2004). However, because of the short
degradation time and poor mechanical properties, chitosan was added. Chitosan can delay the absorption of collagen and increase the hardness of the scaffold. However, it is unclear whether this scaffold is suitable for salivary gland-like cell culture. The data in present study indicate that salivary gland-like cells seeded on the 3D collagen/chitosan scaffold exhibited relatively stable a-amylase secretion and were optimally distributed and differentiated. However, the precise underlying mechanisms require further investigation. Our study has several limitations. First, salivary glands comprise several cell types: acinar cells, ductal cells, and myoepithelial cells. Thus, we refer to the transdifferentiated BMSCs as salivary gland-like cells, and further studies are needed to determine whether these cells have similar function and phenotype to acinar cells or another cell type. Second, the complex mechanisms and signal transduction processes involved in the transdifferentiation of
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Fig. 5 Immunofluorescence staining of induced and control BMSCs in collagen/chitosan scaffolds after transplantation into nude mice. Sections of explanted scaffolds were stained with BrdU (red), antibodies for a-amylase expression (green), and DAPI (blue). a BrdU staining, b a-amylase staining, and c DAPI staining of induced BMSCs after 1 week; and d images in a– c overlaid. e BrdU staining, f a-amylase staining, and g DAPI
staining of induced BMSCs after 2 weeks; and h images in e, f overlaid. i BrdU staining, j a-amylase staining, and k DAPI staining of control BMSCs after 1 week; and l images in i– k overlaid. m BrdU staining, n a-amylase staining, and o DAPI staining of control BMSCs after 2 weeks; and p images in m– o overlaid
BMSCs into salivary gland-like cells warrant further investigation. Nevertheless, this transdifferentiation might be attributable to host and donor cell fusion in vitro (Terada et al. 2002). Third, experiments in which 3D collagen/chitosan scaffolds are seeded with
salivary gland-like cells and transplanted into an animal model of salivary gland hypofunction will offer more information. Thus, the efficacy of salivary gland-like cells in treating salivary gland hypofunction requires further study.
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In summary, Transdifferentiation of BMSCs into salivary gland-like cells was achieved using a novel culture method. BMSC-derived salivary gland-like cells maintained relatively stable bioactivity in terms of secretion of unique functional a-amylase. The potential of BMSC-derived salivary gland-like cells for clinical application should be confirmed in future investigations. Acknowledgments We thank Medjaden Bioscience Limited for assisting in the preparation of this manuscript. This study was supported by Natural Science Foundation of China 81072230 and 30772428. Conflict of interest The authors have declared that no competing interests exist.
References Fox PC (2004) Salivary enhancement therapies. Caries Res 38:241–246 Gersh BJ, Simari RD, Behfar A et al (2009) Cardiac cell repair therapy: a clinical perspective. Mayo Clin Proc 84:876–892 Henson BS, Eisbruch A, D’Hondt E et al (1999) Two-year longitudinal study of parotid salivary flow rates in head and neck cancer patients receiving unilateral neck parotid-sparing radiotherapy treatment. Oral Oncol 35:234–241 Jensen DH, Oliveri RS, Trojahn Kolle SF et al (2014) Mesenchymal stem cell therapy for salivary gland dysfunction and xerostomia: a systematic review of preclinical studies. Oral Surg Oral Med Oral Pathol Oral Radiol 117(335–342):e331 Kaigler D, Mooney D (2001) Tissue engineering’s impact on dentistry. J Dent Educ 65:456–462 Khalili S, Liu Y, Kornete M et al (2012) Mesenchymal stromal cells improve salivary function and reduce lymphocytic infiltrates in mice with Sjogren’s-like disease. PLoS ONE 7:e38615 Kok MR, Yamano S, Lodde BM et al (2003) Local adenoassociated virus-mediated interleukin 10 gene transfer has disease-modifying effects in a murine model of Sjogren’s syndrome. Hum Gene Ther 14:1605–1618 Lim JY, Ra JC, Shin IS et al (2013a) Systemic transplantation of human adipose tissue-derived mesenchymal stem cells for the regeneration of irradiation-induced salivary gland damage. PLoS ONE 8:e71167
Lim JY, Yi T, Choi JS et al (2013b) Intraglandular transplantation of bone marrow-derived clonal mesenchymal stem cells for amelioration of post-irradiation salivary gland damage. Oral Oncol 49:136–143 Lin CY, Lee BS, Liao CC et al (2007) Transdifferentiation of bone marrow stem cells into acinar cells using a double chamber system. J Formos Med Assoc 106:1–7 Lodde BM, Mineshiba F, Wang J et al (2006) Effect of human vasoactive intestinal peptide gene transfer in a murine model of Sjogren’s syndrome. Ann Rheum Dis 65:195–200 Lombaert IM, Wierenga PK, Kok T et al (2006) Mobilization of bone marrow stem cells by granulocyte colony-stimulating factor ameliorates radiation-induced damage to salivary glands. Clin Cancer Res 12:1804–1812 Lv H-X, Jiang J-L, Yang Z-A et al (2011) Transdifferentiation of bone marrow mesenchymal stem cells into salivary gland cells under the induction with submandubular gland cell lysate. J Oral Sci Res 27:376–379 Pittenger MF, Mackay AM, Beck SC et al (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147 Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71–74 Prockop DJ, Azizi SA, Colter D et al (2000) Potential use of stem cells from bone marrow to repair the extracellular matrix and the central nervous system. Biochem Soc Trans 28:341–345 Robar JL, Day A, Clancey J et al (2007) Spatial and dosimetric variability of organs at risk in head-and-neck intensitymodulated radiotherapy. Int J Radiat Oncol Biol Phys 68:1121–1130 Stocum DL (2001) Stem cells in regenerative biology and medicine. Wound Repair Regen 9:429–442 Vissink A, Burlage FR, Spijkervet FK et al (2003) Prevention and treatment of the consequences of head and neck radiotherapy. Crit Rev Oral Biol Med 14:213–225 Wang JA, Fan YQ, Li CL et al (2005) Human bone marrowderived mesenchymal stem cells transplanted into damaged rabbit heart to improve heart function. J Zhejiang Univ Sci B 6:242–248 Yang S, Leong KF, Du Z et al (2001) The design of scaffolds for use in tissue engineering. Part I. Traditional factors. Tissue Eng 7:679–689 Yang C, Hillas PJ, Baez JA et al (2004) The application of recombinant human collagen in tissue engineering. BioDrugs 18:103–119 Zhu Y, Liu T, Song K et al (2009) Collagen-chitosan polymer as a scaffold for the proliferation of human adipose tissuederived stem cells. J Mater Sci Mater Med 20:799–808
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ORIGINAL RESEARCH PAPER
Transdifferentiation of bone marrow-derived mesenchymal stem cells into salivary gland-like cells using a novel culture method Liang Liang • Jun Wang • Yuming Zhang • Zhiyuan Shen • Jun Zheng Jianhu Li • Zhongping Su • Juan Cai • Wei Jiang • Moyi Sun
•
Received: 6 November 2014 / Accepted: 24 February 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objectives To investigate the transdifferentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into salivary gland-like cells via a novel culture method employing induction culture medium collected from salivary gland cells. Results Primary salivary gland cells were cultured, and after the first passage, the culture medium was collected for use as induction medium. BMSCs (passage 3) were cultured in either induction medium Liang Liang and Jun Wang have contributed equally to this work.
(induction group) or DMEM/F12 medium with 10 % (v/v) fetal bovine serum (control group) before seeding on three-dimensional collagen/chitosan scaffolds and subcutaneous transplantation into nude mice. The in vitro and in vivo transdifferentiation of BMSCs into salivary gland-like cells was evaluated by immunocytochemical analysis of a-amylase and cytokeratin-8 (CK-8) expression. Salivary gland-like cells cultured using this novel method maintained excellent biostability and exhibited relatively stable expression of a-amylase and CK-8 in vitro and in vivo. Conclusion This novel culture method is feasible for inducing the transdifferentiation of BMSCs into salivary gland-like cells.
L. Liang Z. Shen J. Zheng J. Li Z. Su J. Cai W. Jiang M. Sun (&) State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military University, Xi’an 710032, People’s Republic of China e-mail: [email protected]
Keywords Bone marrow-derived mesenchymal stem cells (BMSCs) Collagen/chitosan scaffolds Novel culture methods Salivary gland-like cells Salivary gland repair Transdifferentiation Transplantation
L. Liang Department of Stomatology, Peace Hospital Attached to ChangZhi Medical College, Changzhi, People’s Republic of China
Introduction
J. Wang Department of Anesthesiology, Shannxi Tumor Hospital, Xi’an, People’s Republic of China Y. Zhang Department of Physiology and Pathophysiology, Xi’an Jiaotong University School of Medicine, Xi’an, People’s Republic of China
Radiation therapy for head and neck cancer results in atrophy, fibrosis, and degeneration salivary gland tissue, leading to salivary gland hypofunction, which increases patients’ risks of tooth decay, oral and mucosal infections, and gastrointestinal conditions as well as increases susceptibility of the mucosa to ulcers
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Biotechnol Lett
(Vissink et al. 2003). Unfortunately, no effective treatments are available for irreversible salivary gland damage. A previous study applied muscarinic agonist medications, such as pilocarpine and cevimeline, to induce salivary secretion from the residual functional salivary gland tissue (Fox 2004). However, this method only had a small effect on the recovery of damaged tissue, and such an approach would be ineffective if all or most of the salivary gland cells are already lost. Therefore, developing an adequate treatment by using alternative strategies is required. Recently, approaches for treating injured salivary glands based on tissue engineering (Kaigler and Mooney 2001) and stem cell therapy have drawn much attention (Gersh et al. 2009). Bone marrow-derived mesenchymal stem cells (BMSCs) can self-renew, expand rapidly, and be induced down several cellular lineages for eventual differentiation into osteoblasts, chondrocytes, and adipocytes among other cell types (Pittenger et al. 1999; Prockop 1997). In addition, BMSCs offer advantages such as easy availability, minimal ethical concerns, and low immunogenicity (Prockop et al. 2000). These properties make BMSCs an ideal ‘‘seed cell’’ for tissue engineering and stem cell therapies. The use of BMSCs was effective in both preventing loss of saliva secretion and reducing lymphocytic influx in salivary glands (Khalili et al. 2012). However, no consensus has been reached on the potential biohazard of BMSCs for in vivo transplantation or injection. A method to establish an in vitro culture system for the transdifferentiation of BMSCs into salivary gland cells is urgently needed. An indirect-contact model for inducing BMSC transdifferentiation into acinar cells was developed using a double chamber co-culture system (Lin et al. 2007). Based on the concept of an indirect-contact model, we hypothesized that a novel culture method employing induction culture medium collected from salivary gland cells could effectively induce the BMSC transdifferentiation into salivaryglad like cells. To confirm this hypothesis, we prepared conditioned induction medium from cultures of salivary gland cells and investigated BMSC transdifferentiation in this medium. We found that BMSCs could be induced to transdifferentiate into salivary gland-like cells that may be valuable in the clinical treatment of salivary gland damage.
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Materials and methods Animals Two-week-old Sprague–Dawley (SD) rats and 5-week-old nude mice were obtained from Fourth Military Medical University Animal Center, Xi’an, China. Donor rats were kept under clean conventional conditions (25 °C, 44–47 % relative humidity, and 12-h light/dark cycle) and allowed access to food and water ad libitum. All procedures were conducted in accordance with the animal experiment guidelines of the ethics committee of the Fourth Military Medical University Animal Center (Permit Number: SCXK2012-0007). The experiment was approved by the Committee on the Ethics of Animal Experiments of the Fourth Military Medical University Animal Center (Permit Number: SCXK2012-0007). Isolation and culture of rat submandibular gland cells SD rats submandibular gland were cut into pieces of approx. 1 mm3. Following the protocol of Lin et al. (2007), the explants were placed in D-MEM/F-12 (Gibco) supplemented with 20 % (v/v) fetal bovine serum (FBS), 100 U penicillin G/ml, 100 lg streptomycin sulfate/ml, and 0.25 lg amphotericin B/ml. Isolation and culture of rat BMSCs The femura and tibiae of SD rats were removed, washed with phosphate-buffered saline (PBS), and placed in DMEM/F12 supplemented with 10 % (v/v) FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin. The bone marrow was collected and cultured in flasks. At approx. 80 % confluence after 1 week, the cells were passaged at 1:3, and thereafter, the cells were passaged every 3 days. We used three generations of cells for subsequent experiments. Characterization of BMSCs Osteogenic and adipogenic differentiation of BMSCs SD rat BMSCs (passage 3) were induced to differentiate along the osteogenic pathway following the protocol of Prockop (1997). BMSCs were induced to
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differentiate along the adipogenic pathway following the protocol of Pittenger et al. (1999).
containing collagen/chitosan scaffolds and cultured in an incubator for 5 days.
Flow cytometric analysis of BMSC phenotype
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)
The expression of specific cell surface markers was analyzed in rat BMSCs (passage 3). Aliquots of BMSCs (107 cells/sample) were washed in PBS and incubated with fluorescent-labeled anti-mouse CD29PE, CD31-PE, CD34-PE, CD44-PE, CD45-PE, and CD90-CP CY5.5 (BD Biosciences, San Jose, CA, USA) for 1 h at 4 °C. The labeled cells in each group were analyzed using flow cytometry. Differentiation of BMSCs into salivary gland-like cells The medium from cultures of primary salivary gland cells was collected every 48 h, filtered through a 0.22 lm filter, adjusted to pH 7.2, and refrigerated at 4 °C until use as an induction medium. BMSCs (passage 3) were cultured in either induction medium (induction group) or DMEM/F12 medium containing 10 % (v/v) FBS (control group). For both groups, the medium was changed every 2 days. Hereafter, the cells in the induction group are referred to as salivary gland-like cells, and the cells in the control group are referred to as control cells. Preparation of three-dimensional (3D) collagen/chitosan scaffolds Collagen was extracted from rat tails and dissolved in 2 % (v/v) acetic acid. Chitosan was dissolved in 2 % (v/v) acetic acid at 0.5 %. The collage and chitosan solutions were mixed at 7:3 (v/v), maintained in a constant temperature oscillator to ensure complete mixing and then freeze-dried at -55 °C for 72 h. Seeding of bromodeoxyuridine (BrdU)-labeled cells onto scaffolds BrdU (Sigma-Aldrich) was dissolved in the induction medium and normal culture medium at 10 lM. After 4 weeks in culture, cells in both the induction and control groups were cultured in BrdU-containing medium in a incubator for 24 h. BrdU-labeled gland-like and control cells were seeded at 106 cells/well into the wells of 24-well plates
Passage 1 salivary gland cells, passage 3 BMSCs, cells in the induction and control groups, and cells seeded onto the scaffolds were examined using scanning electron microscopy and transmission electron microscopy. Transplantation study Two groups of scaffolds seeded with either salivary gland-like or control cells were implanted into the nude mice, with a scaffold from each group implanted into opposing sides of the same mouse. Transplantations of scaffolds in the induction group were on the left side, and those of the control group were on the right side. The implantation sites were marked, and scaffolds were removed at weekly intervals. An immunofluorescence assay was used to evaluate aamylase expression by the transplanted cells within the collage/chitosan scaffolds. Sections of explanted tissue samples also were subjected to double immunofluorescence labeling for a-amylase and BrdU.
Results Characterization of salivary gland cells and tissue Using an inverted microscope, we observed that salivary gland cells appeared after about 5 and 10 days. Purified salivary gland cells grew well with a uniform shape in a pavestone arrangement (Fig. 1a, b). Characterization of BMSCs The morphology of BMSCs (passage 3) exhibited a uniform fusiform shape and appeared to be fibroblastlike cells (Fig. 2a). After 3 weeks, BMSCs culture in osteogenic medium showed many red mineralized nodules (Fig. 2b). Oil red ‘O’ staining of third generation BMSCs cultured in adipogenic medium demonstrated visible red lipid droplets in the cytoplasm of cells (Fig. 2c). BMSCs could also be
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Biotechnol Lett Fig. 1 Isolation of primary salivary gland acinar cells. a Salivary gland tissue in culture with surrounding cells migrating away from the specimen at 5 days. b Purification of salivary gland cells at 10 days
characterized by the expression of nonspecific surface antigens. According to flow cytometric analysis, high percentages of BMSCs expressed CD29 (Fig. 2d), CD44 (Fig. 2g), and CD90 (Fig. 2i) and low levels of CD31 (Fig. 2e), CD34 (Fig. 2f), and CD45 (Fig. 2h). The blank control group showed no antigen staining (Fig. 2j). BMSC differentiation into salivary gland-like cells For differentiation into salivary gland-like cells, BMSCs were cultured in induction medium (induction group), whereas the control group was cultured in DMEM/F-12. After 2 weeks, the induction group included a small number of BMSCs with a polygonal shape instead of the long spindle shape. At the same time, BMSCs in the control group retained a uniform spindle shape. After 4 weeks, cells in the induction group exhibited morphological changes, with an arrangement similar to duct-like structures, and a number of cells in clusters surrounding a round hollow structure, whereas no changes were observed in the morphology or arrangement of the control cells. The morphological characteristics of salivary gland cells and induced BMSCs were compared upon observation by SEM. Passage 1 salivary gland cells exhibited centrally located nuclei with dark coloring and an uneven surface as well as many long processes (Fig. 3a). Passage 3 BMSCs were overlapping as well as polygonal and irregular in shape. Pseudopodia were obvious and of different thicknesses and lengths, and nuclei were large and obvious with deep color and a round or oval shape (Fig. 3b). After 4 weeks, the shape of BMSCs in the induction group had changed to round or oval with an uneven cell surface and choppy pseudopodia (Fig. 3c). At the same time point,
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BMSCs in the control group were polygonal in shape with large, round nuclei (Fig. 3d). The same groups of cells at the same passage numbers and times were also examined by TEM. The salivary gland cells showed a tapered morphology with rounded ends, and the cell surface showed only a few visible short microvilli. Their nuclei were round or oval and clear, containing nucleoli (Fig. 3e). BMSCs developed pseudopodia-like structures with microvilli of different lengths, and their nuclei were large. The cells had few organelles (Fig. 3f). After 4 weeks, cells in the induction group were round or oval. Microvilli were evident on the cell surface and showed varying lengths and bending. Their nuclei were large with visible heterochromatin. Compared to control cells, the number of organelles was increased in induced BMSCs. The cells contained a round element resembling secretory granules in the cytoplasm (Fig. 3g). BMSCs in the control group did not show any morphological changes after 3 weeks (Fig. 3h).
Cell seeding on collagen/chitosan scaffolds Figure 4a presents a micrograph of BMSCs seeded on collagen/chitosan scaffolds, and Fig. 4b shows an SEM image of the 3D collagen/chitosan scaffold structure. TEM images of BMSCs in the induction group cultured on collagen/chitosan scaffolds demonstrated that the cells were round with uneven surfaces and pseudopodia. The cells were embedded within large pockets of collagen (Fig. 4c). BMSCs in the control group on collagen/chitosan scaffolds were flat and polygonal, with smooth surfaces and visible connections to the scaffold material (Fig. 4d).
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Fig. 2 BMSC characterization according to adipocytic and osteogenic differentiation. a Passage 3 BMSCs exhibited cell growth. b Alizarin red staining in BMSCs cultured in osteogenic differentiation medium. c Oil red ‘O’ staining in BMSCs cultured in adipocytic differentiation medium. Surface antigen
expression on passage 3 BMSCs was analyzed by flow cytometry. j Control cells exhibited no staining. BMSCs stained positively for d CD29, g CD44, and i CD90, but were negative for e CD31, f CD34, and h CD45
Transplantation of BMSCs on collagen/chitosan scaffolds into nude mice
and 2 weeks were analyzed by immunofluorescence staining for a-amylase along with BrdU (red) and DAPI (40 ,6-diamidino-2-phenylindole) staining (blue). In the induction group, many of the explanted cells stained positively for a-amylase. Void spaces
After transplantation, BMSCs on scaffold materials exhibited good cell growth. Scaffolds removed after 1
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Fig. 3 SEM images of a first generation primary salivary gland cells; b third generation BMSCs; c induced BMSCs after 4 weeks; and d control BMSCs after 4 weeks. TEM images of:
e first generation primary salivary gland cells; f third generation BMSCs; g induced BMSCs after 4 weeks; and h control BMSCs after 4 weeks
were visible in the scaffold structure in superimposed images of staining with two antibodies (Fig. 5a–d). After 2 weeks, BMSCs in the induction group expressed a-amylase and BrdU, and the stents appeared to have been mostly resorbed (Fig. 5e–h). BMSCs in the control group at 1 and 2 weeks stained positively for BrdU only, without expression of a-amylase (Fig. 5i–p). After 2 weeks, the scaffold material had been absorbed.
and stem cell therapy. In the current study, our data for the characterization of BMSCs were consistent with the above description. Both pre-clinical and clinical studies regarding the restoration of hyposalivation offer dramatic examples demonstrating the therapeutic value of BMSCs. Lombaert et al. (2006) reported that BMSCs can be mobilized and targetted to damaged salivary glands after irradiation and that they ameliorate radiation-induced complications and induce limited repair of the function and morphology in the submandibular gland. Although the therapeutic testing of these cells has progressed well, the mechanisms underlying these therapeutic effects remain to be determined. It is speculated that BMSCs could play a better role in such therapies via in vitro transdifferentiation into target cells before transplantation or injection. Based on this concept, Lin et al. (2007) established a doublechamber system to promote the transdifferentiation of BMSCs into acinar cells. In another study (Lv et al. 2011), medium containing submandibular gland cell lysate was used to induce BMSC transdifferentiation. However, these strategies are not only associated with high costs but also require large numbers of primary salivary gland cells; thus, whether these methods can satisfy the requirements for clinical application needs to be explored.
Discussion Salivary gland cells can be permanently destroyed by radiotherapy, leading to reduced salivary function and disastrous consequences for oral health (Jensen et al. 2014). Current treatment strategies are focused on the minimization of radiation damage by parotid-sparing radiation delivery or traditional care based on the use of salivary substitutes and sialogogues (Lim et al. 2013a, b). However, the ideal therapeutic strategy for hyposalivation remains poorly defined. Thus, the beneficial effects of tissue engineering and stem cell therapy have been advocated. BMSCs have generated a great deal of interest because of their potential value in tissue engineering
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Biotechnol Lett Fig. 4 Morphological comparison of salivary gland cells, induced BMSCs, and control BMSCs using scanning and transmission electron microscopy. a Photograph of a collagen/chitosan scaffold. b SEM image of three-dimensional collagen/chitosan scaffold structure. c SEM image of induced BMSCs after 4 weeks on a collagen/chitosan scaffold. d SEM image of control BMSCs after 4 weeks on a collagen/chitosan scaffold
In the present study, we established a novel indirect-contact model for inducing transdifferentiation of BMSCs into salivary gland-like cells (which are functionally or phenotypically similar to salivary gland cells) using relative few salivary gland cells in vitro. The data suggest that the novel technique simplifies the culture process and can generate large numbers of salivary gland-like cells that distinctively secreted a-amylase. However, it is unclear whether the salivary gland-like cells also function similarly to real salivary gland cells. Another objective of our current study was to observe the activity of salivary gland-like cells in vivo. Salivary gland-like cells were seeded onto 3D collagen/chitosan (7:3) scaffolds and transplanted into nude mice, as described previously (Zhu et al. 2009). Collagen is a natural cell substrate that allows for the direct formation of cellular networks and is conducive to cell spreading and extracellular matrix formation (Yang et al. 2004). However, because of the short
degradation time and poor mechanical properties, chitosan was added. Chitosan can delay the absorption of collagen and increase the hardness of the scaffold. However, it is unclear whether this scaffold is suitable for salivary gland-like cell culture. The data in present study indicate that salivary gland-like cells seeded on the 3D collagen/chitosan scaffold exhibited relatively stable a-amylase secretion and were optimally distributed and differentiated. However, the precise underlying mechanisms require further investigation. Our study has several limitations. First, salivary glands comprise several cell types: acinar cells, ductal cells, and myoepithelial cells. Thus, we refer to the transdifferentiated BMSCs as salivary gland-like cells, and further studies are needed to determine whether these cells have similar function and phenotype to acinar cells or another cell type. Second, the complex mechanisms and signal transduction processes involved in the transdifferentiation of
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Fig. 5 Immunofluorescence staining of induced and control BMSCs in collagen/chitosan scaffolds after transplantation into nude mice. Sections of explanted scaffolds were stained with BrdU (red), antibodies for a-amylase expression (green), and DAPI (blue). a BrdU staining, b a-amylase staining, and c DAPI staining of induced BMSCs after 1 week; and d images in a– c overlaid. e BrdU staining, f a-amylase staining, and g DAPI
staining of induced BMSCs after 2 weeks; and h images in e, f overlaid. i BrdU staining, j a-amylase staining, and k DAPI staining of control BMSCs after 1 week; and l images in i– k overlaid. m BrdU staining, n a-amylase staining, and o DAPI staining of control BMSCs after 2 weeks; and p images in m– o overlaid
BMSCs into salivary gland-like cells warrant further investigation. Nevertheless, this transdifferentiation might be attributable to host and donor cell fusion in vitro (Terada et al. 2002). Third, experiments in which 3D collagen/chitosan scaffolds are seeded with
salivary gland-like cells and transplanted into an animal model of salivary gland hypofunction will offer more information. Thus, the efficacy of salivary gland-like cells in treating salivary gland hypofunction requires further study.
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In summary, Transdifferentiation of BMSCs into salivary gland-like cells was achieved using a novel culture method. BMSC-derived salivary gland-like cells maintained relatively stable bioactivity in terms of secretion of unique functional a-amylase. The potential of BMSC-derived salivary gland-like cells for clinical application should be confirmed in future investigations. Acknowledgments We thank Medjaden Bioscience Limited for assisting in the preparation of this manuscript. This study was supported by Natural Science Foundation of China 81072230 and 30772428. Conflict of interest The authors have declared that no competing interests exist.
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