JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH J Tissue Eng Regen Med (2012) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1572
ARTICLE
Autonomous isolation, long-term culture and differentiation potential of adult salivary glandderived stem/progenitor cells Hyunjung Baek, Yoo Hun Noh, Joo Hee Lee, Soo-In Yeon, Jaemin Jeong and Heechung Kwon* Division of Radiation Oncology, Korea Institute of Radiological and Medical Sciences, Seoul, South Korea
Abstract Salivary gland stem/progenitor cells belong to the endodermal lineage and may serve as good candidates to replace their dysfunctional counterparts. The objective of this study was to isolate large numbers of salivary gland tissue-derived stem cells (SGSCs) from adult rats in order to develop a clinically applicable method that does not involve sorting or stem cell induction by duct ligation. We analysed SGSCs isolated from normal rat salivary glands to determine whether they retained the major characteristics of stem cells, self-renewal and multipotency, especially with respect to the various endodermal cell types. SGSCs expressed high levels of integrin a6b1 and c-kit, which are surface markers of SGSCs. In particular, the integrin a6b1+/c-kit+ salivary gland cells maintained the morphology, proliferation activity and multipotency of stem cells for up to 92 passages in 12 months. Furthermore, we analysed the capacity of SGSCs to differentiate into endoderm lineage cell types, such as acinar-like and insulin-secreting cells. When cultured on growth factor reduced matrigel, the morphology of progenitor cells changed to acinar-like structures and these cells expressed the acinar cell-specific marker, a-amylase, and tight junction markers. Moreover, reverse transcription–polymerase chain reaction (RT–PCR) data showed increased expression of pancreatic cell markers, including insulin, Pdx1, pan polypeptide and neurogenin-3, when these cells formed pancreatic clusters in the presence of activin A, exendin-4 and retinoic acid. These data demonstrate that adult salivary stem/progenitor cells may serve as a potential source for cell therapy in salivary gland hypofunction and diabetes. Copyright © 2012 John Wiley & Sons, Ltd. Received 10 August 2011; Revised 7 May 2012; Accepted 12 June 2012
Keywords
salivary gland stem cell; differentiation; endodermal tissue
1. Introduction Identification, isolation, in vitro expansion and therapeutic use of stem cells from various tissues represent the greatest current challenges in tissue engineering and regenerative medicine. Recent studies have demonstrated that adult stem cells exist within several differentiated tissues (Apel et al., 2009; Baglioni et al., 2009; Docherty, 2009). The identification of adult stem/progenitor cells is important, not only to facilitate our understanding of developmental processes but also for cell therapy, since *Correspondence to: H. Kwon, Division of Radiation Oncology, Institute of Radiological and Medical Sciences, 215–4 Gongneung-Dong, Nowon-Gu, Seoul 139–706, South Korea. E-mail:
[email protected] Copyright © 2012 John Wiley & Sons, Ltd.
these cells are ideally suitable for cell transplantation. Previous reports regarding the treatment of endodermal tissues concentrated on the use of mesenchymal stem cells (MSCs), which originate from mesodermal tissues (Karnieli et al., 2007; Yan et al., 2009). The mesodermal origin of MSCs brings into question their ability to correctly transdifferentiate into endodermal tissues. Thus, attempts have been made to identify an appropriate source of endodermal cells within tissues such as the salivary glands, pancreas and liver (Suzuki et al., 2002; Okumura et al., 2003; Suzuki et al., 2004). Salivary glands originate from the endoderm, as do the pancreas and liver. They are very attractive candidates for obtaining adult stem cells because they can be easily extracted from patients. In particular, the ability of salivary gland stem cells (SGSCs) to differentiate into endodermal
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tissues may provide an effective therapeutic benefit for other damaged tissues of endodermal origin. The putative presence of SGSCs in salivary glands was suggested by Zajicek (1981). Recently, these SGSCs were isolated and expanded from rodent and human salivary glands and then differentiated into both salivary glands and endodermal progeny, including cell types that are never found in salivary glands, such as pancreatic b-cells and hepatocytes (Okumura et al., 2003; Hisatomi et al., 2004; Sato et al., 2007). Studies characterizing SGSCs have paved the way for their potential clinical use in the field of regenerative medicine and tissue transplantation. However, previous methods used to isolate human SGSCs, such as enrichment after ligation-induced atrophy or cell sorting by flow cytometry, have limitations, and these methods would not be suitable for the large-scale production of SGSCs necessary for clinical applications such as cell transplantation. While there are several putative SGSC markers, a really effective isolation procedure for clinically useful SGSCs has not yet been developed, and the relative efficiencies of the various methods for isolating adult stem cells from this tissue remain unknown. In addition, to our knowledge the development of an optimal medium for the efficient and specific differentiation of SGSCs into acinar cells, hepatocytes and insulinsecreting cells has not been reported. The present study shows that adult rat SGSCs can be isolated, amplified on a large scale and differentiated into various endodermal cell types. These combined steps form a complete protocol that could meet the requirements of future clinical applications. The first objective of this study was focused on developing techniques for the isolation and characterization of SGSCs derived from rat salivary gland tissue. The second objective was to differentiate the cells into specific endoderm-derived tissues. We demonstrate that SGSCs isolated by our autonomous system, without the use of sorting methods, are self-renewing and multipotent adult precursor cells, capable of generating salivary glands and endodermal progeny even after long-term expansion. In particular, this study describes the ability to promote the differentiation of SGSCs into the various cell types found in other endodermal tissues, such as acinar-like and insulinsecreting cells.
2. Materials and methods 2.1. Antibodies We used antibodies to identify the expression of genes specific to SGSCs. The antibodies and dilutions used were as follows: anti-integrin a6b1, 1:500 (Millipore, Billerica, MA, USA; MAB1410); anti-CD90/Thy1, 1:100 (BD Biosciences, San Diego, CA, USA; 554895); anti-c-kit, 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-5535); anti-laminin, 1:500 (Dako, Glostrup, Denmark; Copyright © 2012 John Wiley & Sons, Ltd.
Z0097); anti-CD44, 1:100 (BD Biosciences; 554869); anti-CD45, 1:100 (BD Biosciences; 554875); anti-nestin, 1:500 (Abcam, Cambridge, MA, USA; ab92391); antiOct4, 1:100 (Abcam; ab18976); anti-Met (c-met), 1:200 (Abcam; ab51067); anti-aquaporin 5 (AQP5), 1:200 (Millipore; AB15858); anti-amylase, 1:1000 (Santa Cruz Biotechnology; sc-46657); anti-claudin-1, 1:200 (Invitrogen, Carlsbad, CA, USA; 51–9000); anti-claudin-3, 1:200 (Invitrogen; 34–1700); anti-occludin, 1:200 (Invitrogen; 33–1500); anti-vimentin, 1:100 (BD Biosciences; 550513); anti-Nkx6.1, 1:200 (Santa Cruz Biotechnology; sc-15030); anti-insulin, 1:100 (Santa Cruz Biotechnology; sc-9168); anti-c-peptide, 1:100 (Cell Signalling Technology, Danvers, MA, USA; 4593); anti-CK18, 1:200 (Santa Cruz; sc-28264), and anti-CK19, 1:200 (Santa Cruz; sc-33119). For primary antibody detection, the following fluorescence-labelled secondary antibodies were used: AlexaFluor 488- and 594-labelled goat anti-mouse, anti-goat and anti-rabbit IgG H + L antibodies (1:500; Molecular Probes, Eugene, OR, USA).
2.2. Isolation and long-term culture of salivary gland-derived cells Wistar-ST Rats (males aged 10–18 weeks) were purchased from Japan SLC Inc. (Shizuoka Prefecture, Japan). All animal experiments were performed in accordance with the Korean National Institute of Health guidelines, as approved by the Institutional Animal Care and Use Committee of the Korea Institute of Radiological and Medical Sciences. The rats were anaesthetized by injecting 10 mg/kg lumpun and 40 mg/kg ketamine intraperitoneally. The rats were held in the supine position with the neck extended, a midline incision was performed and both submandibular glands were excised. The submandibular glands were dissected under the microscope in phosphate-buffered saline (PBS) and incubated in PBS containing 0.1% collagenase type I and type IV (Sigma, St. Louis, MO, USA) for 30 min at 37 C. Digestion was carried out by mechanical dissection and gentle pipetting. Triturated salivary gland cells were then passed through a 24-gauge needle and centrifuged at 1300 rpm. The salivary gland cells were divided and plated into eight separate T75 flasks coated with collagen type I. These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 1:1 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% insulin–transferrin–selenium supplement (ITS-X; Gibco), 10 mM nicotinamide, 1 10–7 M dexamethasone, 1 mM b-mercaptoethanol, 20 mg/l epidermal growth factor (EGF) and 20 mg/l hepatocyte growth factor (HGF). This standard medium was changed every 2 days. On day 7, cells were harvested using 1 trypsin/ethylenediamine tetra-acetic acid (EDTA) and plated onto newly prepared T75 flasks at a density of 4 103 cells/cm2. Primitive cells were plated at a density of 4 103 cells/cm2 until passage 3, and then were plated at a density of 1 104 cells/cm2. Cells of passages 20–30 were used for studies. J Tissue Eng Regen Med (2012) DOI: 10.1002/term
Proliferation and differentiation of salivary gland stem cells
2.3. Measurement of proliferative activity SGSCs and rat bone marrow MSCs were plated at 1 104 cells/well on 96-well tissue culture plates and were cultured for 48 h. The rat MSCs were isolated by flushing the femurs and tibias of 8 week-old Wistar-ST rats with 20 ml DMEM. The flushed marrow was centrifuged at 1000 rpm for 5 min. The cell pellet was washed with PBS and then resuspended in fresh medium (DMEM supplemented with 10% FBS and 1% penicillin/streptomycin) and seeded in a tissue culture flask. Non-adherent cells were removed by changing the culture medium after 48 h. The cultures were changed every 2 days. Cells of passages 3–5 were utilized for proliferation activity. The proliferation rate of salivary gland cells was determined by measuring the activity of mitochondrial dehydrogenase with a Quick Cell Proliferation Assay kit (Medical and Biological Laboratories Co., Ltd). The formazan dye produced in the reaction between mitochondrial dehydrogenase and the tetrazolium salt, WST-1, was quantified using a multi-well spectrophotometer, measuring the absorbance of the dye solution at 450 nm.
2.4. Flow-cytometry analysis Monolayer-cultured SGSCs were dissociated with trypsin/ EDTA (Gibco). The cells were then washed with ice-cold PBS and incubated with an unconjugated primary antibody and a fluorochrome-conjugated secondary antibody (Alexa 488 or Alexa 594). Controls consisted of samples treated with secondary antibodies only. The fluorescenceactivated cell sorting (FACS) analysis procedure was provided by Abcam (www.abcam.com/technical). Positive cells were quantified using a FACS Calibur Flow Cytometer (BD Biosciences) and data were analysed using the CellQuest software program (BD Biosciences).
(iNtRON Biotechnology Inc., Korea). Reverse transcriptions for preparing complementary DNA (cDNA) for the polymerase chain reaction (PCR) were performed as a one-step process using Maxime reverse transcription (RT)–PCR PreMix kits (iNtRON Biotechnology). The cDNA was amplified using SWT–MX–BLC-7 (Esco Micro Pte Ltd) with the following primer sets: Amy1, 50 -TCT GGG TGG TGA AGC AGT GT-30 , 50 -AGG TGG TCC AAC CCA GTC AT-30 ; Aquaporin 5, 50 -CAA TGC GCT GAA CAA CAA CAC-30 , 50 -TGG GGA GGG GTG CTT CAA ACT-30 ; b-actin, 50 -TGA TGG TGG GTA TGG GTC AG-30 , 50 -ATG CCA GTG GTA CGA CCA GA-30 ; Bmi1, 50 -CAG CAG AGA GAT GGA CTG ACC-30 , 50 -CCA GAC AGC GGT CAC AGT AA-30 ; C-Met, 50 -TTG GCA ACG AGA GCT GTA CC-30 , 50 -TCA GGT TCT TTC CAA TCC CC-30 ; CNN1, 50 -CTT GAA GGG TTT CTG GTT CTG G-30 , 50 -GGC ACC TGC CTA TAG GGT TAC A-30 ; Enc1, 50 -ATC TCA TGG AGA ACG TGG CA-30 , 50 -GGA ATG TCA GCC TTG GGA AT-30 ; Insulin, 50 -TGT CAA ACA GCA CCT TTG TGG TCC-30 , 50 -ACT GAT CCA CAA TGC CAC GCT TCT-30 ; Meis1, 50 -CTT CTT GCC TAG GAT TTC AGC C-30 , 50 -CGA TTT ACA TCT GTT CAG GCC-30 ; Notch1, 50 -AGT GCA ACC CCC TGT ATG AC-30 , 50 -TCG ATC TCC AGG TAG ACG ATG-30 ; Nestin, 50 -TCA CAA GTC CTT GGG GTC TC-30 , 50 -TCT TGG TCC TGT CCT GCT AGA-30 ; Ngn3, 50 -AGT TGG CAC TGA GCA AGC AG-30 , 50 -GCT TGG GAA ACT GGG GAG TA-30 ; Pdx-1, 50 -TGC CAC CAT GAA TAG TGA GGA GCA-30 , 50 -CGC GTG AGC TTT GGT GGA TTT CAT-30 ; PPy, 50 -ACT GCC TCT CCC TGT TTC TCC TAT-30 , 50 -CTG TGT CCT CAT CTC TCT TCC CAT-30 . PCR amplification conditions were: 5 min at 94 C, followed by 30 cycles at 94 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 30 s, and final polymerization for 10 min. PCR products were separated on 1.0% agarose gels.
2.5. Immunostaining
2.7. Culture conditions for cell differentiation
For immunocytochemistry, cells were cultured as a monolayer in polystyrene eight-well chamber slides or on collagen type 1-coated 13 mm diameter circular cover slips. The cells were fixed for 15 min in 4% paraformaldehyde and permeabilized for 30 min in PBS containing 0.1% Triton X-100 at room temperature. Non-specific binding was blocked for 1 h with PBS containing 3% bovine serum albumin (BSA). Immunocytochemical procedures were performed according to a general procedure provided by Abcam (www.abcam.com/technical) and the cells were viewed using an LSM 610 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). For negative controls, we omitted the primary antibody.
To assess the multipotent capacity of SGSCs, we induced differentiation of SGSCs under various conditions. To induce SGSCs into salivary acinar-like cells, SGSCs were grown on a six-well plate precoated with growth factor reduced (GFR) matrigel (BD Biosciences). GFR-matrigel was thawed overnight on ice and used to coat plates at 100 ml/cm2. The coated plates were incubated at 37 C for 1 h before cell seeding. We used a three-stage protocol to induce insulin-secreting cells from SGSCs: stage 1, cells seeded on a six-well plate at 5 103 cells/cm2 were cultured for 2 days in standard medium; stage 2, standard medium was removed and the cells were cultured in standard medium containing 5% FBS, 2 mM retinoic acid, 100 ng/ml EGF, 100 ng/ml Exendin-4, and 10 ng/ml Activin-A for 5 days. Stage 3: cells were cultured for an additional 16 days in standard medium containing 5% FBS, 17.51 mM glucose, 25 nM retinoic acid, 100 ng/ml EGF, 100 ng/ml Exendin-4 and 10 ng/ml Activin-A. Media changes were performed every 2–3 days.
2.6. Reverse transcriptase–polymerase chain reaction (RT–PCR) Total RNA was extracted from cultured cells at passages 0 and 29, using easy-spin Total RNA Extraction kits Copyright © 2012 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2012) DOI: 10.1002/term
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2.8. Immunohistochemistry of differentiated acinar-like cells For the detection of acinar-like cell types, matrigel-coated plates were treated with 1 mg/ml dispase (Invitrogen) for 2 h on days 1 and 3. The cells were washed twice with PBS and processed in paraffin following Histogel (Richard-Allan Scientific, Kalamazoo, MI, USA) embedding. The cell blocks were sectioned at 5 mm and then the sections were sequentially deparaffinized and hydrated. For immunofluorescence staining, sections were stained with antibodies, using the same methods as for immunostaining. For periodic acid–Schiff’s (PAS) staining, sections were incubated in 1% periodic acid solution (Sigma) for 5 min at room temperature (RT). After rinsing the slides with distilled water (DW), they were treated with Schiff’s reagent (Sigma) for 20 min at RT and rinsed in DW for 5 min. The nuclei were counterstained with haematoxylin and assessed under a light microscope.
2.9. Western blotting Cells were harvested into lysis buffer (50 mM Tris-Cl, 2 mM EDTA, 150 mM NaCl, 1% NP-40) containing 1 mM phenylmethylsulphonyl fluoride, 20 mg/ml aprotinin, 20 mg/ml leupeptin and 20 mg/ml pepstatin (Sigma). Protein samples (30 mg each) were electrophoresed on a 10%
polyacrylamide sodium dodecyl sulphate gel and blotted onto a Protran nitrocellulose membrane (Whatman GmbH, Dassel, Germany). The membrane was blocked with a solution of 3% skimmed milk in PBS/0.1% Tween20 and incubated for 1 h at RT with the anti-amylase antibody diluted in blocking buffer. The membrane was washed three times and treated with horseradish peroxideconjugated goat anti-mouse IgG (Sigma) for 1 h at RT. Peroxidase activity was detected using an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
3. Results and discussion 3.1. Isolation of salivary gland precursor cells In order to effectively isolate and amplify stem cells from rat salivary glands, we developed a culture system (lasting 4–5 weeks) that did not require any selection procedure. Single-cell suspensions, which were obtained by mechanical and enzymatic dissociation of a tissue mass taken from an adult rat salivary gland, were grown in our optimized culture media, as described in Materials and methods. The primary cells remaining in the plate were composed of different morphological cell types (Figure 1Aa). We repeated the subculture of single-cell suspensions several times at a low cell density (4 103 cells/cm2) and observed
Figure 1. Isolation and proliferation of SGSCs. (A) Various cultured primary cells from the salivary gland were maintained for up to 6 months (a–d). On day 1 (passage 0; a) and day 17 (passage 3; b) after primary culture, salivary gland cell cultures were composed of a mixture of mature and precursor cells. After 1 month (passage 5; c), other cell types (ductal and acinar cells, fibroblasts), excluding stem/progenitor cells, were eliminated naturally, and the remaining cells showed morphologically homogeneous features (d); scale bar = 200 mm. (B) The proliferative activity of SGSCs was investigated by comparing the activity of mitochondrial dehydrogenase with rat bone marrow-derived MSCs (rMSCs). The amount of mitochondrial dehydrogenase was quantified using a multiwell spectrophotometer, measuring the absorbance of the dye solution at 450 nm Copyright © 2012 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2012) DOI: 10.1002/term
Proliferation and differentiation of salivary gland stem cells
that non-SGSC primary salivary gland cell types were progressively eliminated from the dishes. Typically, after five passages the cultured cell population appeared homogeneous and the cells were morphologically similar to SGSCs. The cells had features common to immature cells, such as a round shape and small size, and grew to confluence after approximately 2 days of culture (Figure 1Aa–d). Moreover, the more homogeneous the cells became naturally, the more dramatically the doubling times of SGSCs was decreased. The first replating of the primitive salivary gland cells occurred after 6–7 days; however, the average doubling time of SGSCs purified to homogeneity after five to eight passages was only 17.3 h. We also investigated the proliferative activity of SGSCs by measuring mitochondrial dehydrogenase activity. As we observed for the SGSC doubling times, the mitochondrial dehydrogenase activity was higher for the purified SGSCs than for primitive cultured salivary gland cells (Figure 1B). Interestingly, the mitochondrial dehydrogenase activity for rat mesenchymal stem cells (bone marrow), which are representative of adult stem cells, was lower than for SGSCs. This proliferation activity was maintained in our culture system for 6 months (Figure 1B; 74 passages).
3.2. Formation of colonies from single SGSCs To characterize the SGSCs isolated by our culture system, we studied both self-renewal and multipotency, which are the two main characteristics of stem cells. First, we
performed a single-cell colony assay in a low-density culture system (200 cells/cm2) in order to test the selfrenewing potential of SGSCs. The single-cell colony assay is a well-known method of studying stem cell self-renewal (Friedenstein et al., 1974; Ema et al., 2006; Kishi et al., 2006). We observed cells every 24 h and noted that single SGSCs grew into large colonies after only 4 days (Figure 2A). We monitored colony formation by recording the total number of cells over time (Figure 2B). On day 3, these cells formed colonies consisting of > 50 cells, and by day 4 clonal colonies consisted of > 100 cells. The average cell number/colony on day 5 was 456.14 121.5 (standard deviation, SD). These results indicate that our SGSCs successfully formed colonies through the process of self-renewal.
3.3. Characterization of isolated SGSCs We performed flow-cytometry analysis on isolated SGSCs to characterize their stemness and differentiation status using expression markers. The choice of target markers was based on previously published literature suggesting that integrin a6b1 and c-kit are markers for somatic stem cells derived from adult glands (Petersen et al., 1998; Jiang et al., 2002). It is well known that integrin a6b1 is a receptor for laminin (Giancotti, 1996). As shown in Figure 3A, SGSCs were positive for integrin a6b1 (CD49f/ CD29), c-kit (CD117), CD44, laminin, nestin and c-met, but the cells were negative for CD34, CD45, Thy-1 (CD90) and CD133. These results were confirmed in over 20
Figure 2. Colony formation and generation of endospheres from single SGSCs. (A, B) SGSCs isolated at 12 weeks (passage 30) from the primary cell culture were seeded in a collagen-coated T75 plate and cultured in normal maintenance medium. The cells continued to divide and grew into large colonies in only 6 days. The doubling time of the cells was reduced after natural selection of the salivary gland precursor cells. These colony-forming cells showed a homogeneous morphology. Scale bar = 50 mm Copyright © 2012 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2012) DOI: 10.1002/term
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Figure 3. Characterization of SGSCs. (A) Results of flow-cytometry analysis of markers for stem cells, immature cells and mature cells in cultured primary cells derived from salivary glands. For FACS analysis, cells were cultured as monolayers in collagen type 1-coated T75 flasks. SGSCs were analysed by single-wavelength FACS analysis. (B) Various markers, indicative of the differentiation stage, were detected by RT–PCR in SGSCs after 0 and 29 passages. cDNAs, transcribed from RNAs by reverse transcriptase, were used as templates for PCR with primers for Bmi1, Notch1, Meis1, Amy1, c-met, CNN1, Enc1, aquaporin 5 and nestin; b-actin was used as the internal control
passages of SGSCs by both immunocytochemistry and FACS analysis. Immunocytochemistry analysis of cell markers revealed similar expression patterns to that of the FACS analysis. Isolated SGSCs were stained for integrin a6b1 and c-kit cell surface proteins (Figure 4). Interestingly, we observed expression of the aforementioned stemness markers, as well as Oct4 and nestin, which are important to the self-renewal of undifferentiated embryonic stem cells (Rosner et al., 1990; Tohyama et al., 1992). We observed expression of c-met+ cells in SGSC colonies. Further, we found no expression of insulin, Pdx1 and albumin (Nagy et al., 1994; Kubo et al., 2004), which are other endodermal lineage cell markers for pancreatic b-cells and hepatocytes (data not shown). Furthermore, semi-quantitative RT–PCR analyses were performed to genetically characterize the stem, progenitor and mature cell phenotypes of the isolated SGSCs (passage 29) (Figure 3B). Undifferentiated cells express Bmi1, Notch1 and Meis1 (Hisatomi et al., 2004; Conigliaro et al., 2008), while Enc1 and nestin are markers for ectoderm Copyright © 2012 John Wiley & Sons, Ltd.
precursor cells. Amy1 and AQP5 were chosen as acinar cell markers, while c-met was used as a marker for ductal cells and CNN1 was included as a marker for mesodermal cells (Conigliaro et al., 2008). Our RT–PCR analysis revealed strong expression for all stem cell markers, including Bmi1, Notch1 and Meis1, as well as the ductal cell marker, c-met, from the primitive cultured salivary cells, and this was maintained until the SGSCs were subsequently isolated (Figure 3B). At passage 29, the mesodermal and ectodermal markers (CNN1 and Nestin) showed moderately reduced expression during isolation of SGSCs. AQP5, which is highly expressed by acinar cells, disappeared by passage 29. These results suggest that isolated SGSCs are undifferentiated somatic stem cells.
3.4. Differentiation potential into endodermal lineage tissues Multipotency is a major characteristic of adult stem cells, and thus we tested whether SGSCs were able to J Tissue Eng Regen Med (2012) DOI: 10.1002/term
Proliferation and differentiation of salivary gland stem cells
Figure 4. Immunocytochemistry for expression of stem cell, immature cell and mature cell markers in cultured stem cells derived from salivary glands. For immunocytochemistry, cells were cultured as monolayers in polystyrene eight-chamber slides. Immunostaining was used on SGSCs at passage 29. Nuclei were counterstained with DAPI (blue). Scale bar = 20 mm
differentiate into various endoderm-related cell types. First, we induced the morphological and functional differentiation of SGSCs into acinar-like cell types using GFR-matrigel, a basement membrane extract. On the surface of GFR-matrigel, SGSCs formed acino-tubular structures within 24 h (Figure 5A, B). As shown in Figure 5B, some acinar-like formations (arrow) sprouted out from the tubular structure (arrowhead). The morphology of the acino-tubular structures was similar at days 2 and 3, whereas the cells on GFR-matrigel were detached after 5 days (data not shown). Amylase protein expression showed the cells on GFR-matrigel at days 1 and 3 by western blot analysis (Figure 5C). Stained sections of paraffin-embedded cultures showed expression of amylase-1, claudin-1, claudin-3, occludin and vimentin (Figure 5D). Cells cultured on a plastic dish (day 0) did not express these markers, but cells grown on GFR-matrigel showed a similar level of marker expression on days 1 and 3 (Figure 5D). We observed PAS staining, indicating the presence of mucin/mucopolysaccharide-containing acinar cells (data not shown). Taken together, these data suggest that isolated SGSCs are capable of in vitro differentiation towards acinar-like cell types. Next, we investigated whether SGSCs can differentiate into insulin-secreting cells. When SGSCs were grown in standard media containing Activin-A, Exendin-4 and retinoic acid, they formed clusters of islet-like structures (pancreatic clusters) after 5 days (Figure 6A). To determine whether SGSCs underwent pancreatic differentiation, gene expression for pancreatic cell differentiation markers was assessed using RT–PCR. As shown in Figure 6B, no expression of Pdx1, Ngn3, insulin or PPy was detected in undifferentiated SGSCs. When the cells were induced for 21 days, low levels of the pancreatic cell differentiation markers Pdx1 and Ngn3 were detected. Immunofluorescence staining revealed positive expression Copyright © 2012 John Wiley & Sons, Ltd.
of insulin, c-peptide and Nkx6.1 in the differentiated cells (Figure 6C). The pancreatic transcription factor Nkx6.1 was expressed during differentiation stage 2 but the expression was not detected on day 21. Insulin and c-peptide expression was detected at low levels in the differentiated cells. Cytokeratin 18 and 19 were strongly expressed on the surface of undifferentiated SGSCs, but the expression was decreased when the SGSCs differentiated into insulinsecreting cells. In our differentiation conditions, a low level of cells (1–10%) were positive for insulin. Taken together, these data provide support for the differentiation of SGSCs into islet-like cells under pancreatic induction, as well as acinar-like cells.
4. Discussion In this study, we established a feasible isolation method for the long-term culture of SGSCs from the salivary glands of normal adult rats. Using our autonomous isolation method, SGSC proliferation was maintained in vitro for more than 6 months. Isolated SGSCs had the capability of differentiating into other cell types of endodermal origin. The isolation and purification of SGSCs remains challenging, due to the low frequencies of adult stem cells in the salivary gland and also to the unwanted growth of non-SGSCs during culture. In order to isolate adult stem cells from the salivary gland, previous studies developed more artificial methods. Hisatomi et al. (2004) isolated adult stem cells induced in response to experimental injury of mouse salivary glands, using FACS analysis of CD117-labelled cells. Other research groups have also used FACS analysis to isolate stem cells (Suzuki et al., 2004; Sato et al., 2007); however, these methods of isolating and establishing SGSCs are not appropriate for J Tissue Eng Regen Med (2012) DOI: 10.1002/term
H. Baek et al.
Figure 5. Acinar-like differentiation of SGSCs. (A) Morphological changes of SGSCs on the surface of GFR-matrigel were taken using phase-contrast microscopy; scale bar = 200 mm. (B) Arrows depict the formation of acinar-like aggregates and the arrowhead shows the tubular structure; scale bar = 100 mm. (C) Western blot analysis of amylase expression in the cells grown on GFR-matrigel; b-actin protein was used as an internal control. (D) Immunofluorescence staining of claudin-1, claudin-3, occludin, vimentin and amylase of SGSCs cultured on GFR-matrigel at day 3. Nuclei were counterstained with DAPI (blue); scale bar = 20 mm
clinical applications because cells sorted by specific fluorescence markers cannot be used in patients. The objective of this study was to isolate SGSCs from rats and induce them to differentiate into endodermal cells, as the first step in developing a system for isolating and culturing human SGSCs for use in clinical applications. Our SGSC isolation method was based on the concept that stem cells possess the ability for self-renewal. The main feature of our isolation system is the autonomous deletion of adult cells that exist in primary cultures derived from salivary gland tissue. Our results showed that homogeneous stem cells were gradually and autonomously isolated from the primitive and heterogeneous cells present in the salivary gland. We hypothesized that the autonomous elimination of differentiated adult cells was caused by culturing them long-term at low cell density (4 103 cell/cm2), under which conditions other primitive cells (except SGSCs) were not present in sufficient numbers to stimulate cell proliferation and mitosis by the paracrine signalling of adjacent cells. Human SGSCs were isolated from adult human salivary glands by FACS, using anti-CD49f and anti-Thy-1 antibodies (Sato et al., 2007). In this study, cells of the CD49f+/ Thy-1+ fraction were 2 0.77% and the proportion of Copyright © 2012 John Wiley & Sons, Ltd.
cells that bound to the collagen-coated dish was < 5.0% of the sorted whole cells. CD49f+/Thy-1– cells constituted 50%, had a large cell size and attached to culture dishes. However, they mentioned that these cells did not proliferate under their culture conditions. Our results showed that homogeneous stem cells, CD49f+/Thy-1–, were autonomously isolated from the primitive and heterogeneous cells present in the salivary gland. In our culture system, > 100 colonies/1 106 primary single-cell suspensions were formed. We believe that EGF and HGF acted as activators for the proliferation and maintenance of SGSCs. It is worth noting that Teruki Kishi et al. (2006) revealed that SGSCs expanded more efficiently when EGF and HGF were added to the medium. This technique has generally been used to isolate adult stem cells from pancreas and bone marrow (Sun et al., 2003; Yamamoto et al., 2006). Specifically, these studies have shown that mature cells derived from pancreas, liver and other tissues gradually disappear from primitive cultured cells, while stem cells do not. Previous studies demonstrated that all cell types derived from salivary glands have some baseline proliferative capacity (Srinivasan and Chang, 1979); however, the culture of these salivary gland cells, including acinar cells, oxyphilic cells, J Tissue Eng Regen Med (2012) DOI: 10.1002/term
Proliferation and differentiation of salivary gland stem cells
Figure 6. Differentiation into insulin-secreting cells. SGSCs differentiated for 21 days. (A) Multiple clusters formed by day 5 in culture. Arrows indicate pancreatic clusters; scale bar = 100 mm. (B) The differentiated SGSCs expressed pancreatic cell specific markers such as Pdx-1, Ngn3, insulin and PPy by RT–PCR analysis. (C) Immunofluorescence staining of differentiated cells expressing c-peptide, Nkx6.1, insulin and CK18. UD, undifferentiated cells; D, differentiated cells in pancreatic differentiation medium; scale bar = 20 mm
intercalated duct cells and myoepithelial cells, was not maintained for a long period of time. Meanwhile, the SGSCs isolated by our method were morphologically homogeneous and their doubling time was significantly decreased to 17.3 h over five passages. We also observed higher levels of mitochondrial dehydrogenases in the autonomously isolated SGSCs compared to the primitive cultured cells. Therefore, we demonstrated that the maintenance of cultured primary cells from adult tissue containing stem cells is another method of isolating adult SGSCs. Our isolated SGSCs were morphologically identical and also had identical stem cell surface markers, such as integrin a6b1 and c-kit. These are well known surface markers of adult endodermal stem cells and have been found in adult stem cells derived from liver, pancreas and salivary gland, which originate from the endoderm. Several research groups have reported the isolation of SGSCs from rat, mouse, human and swine (Kishi et al., 2006; Sato et al., 2007; Rotter et al., 2008). Fumio Endo’s Copyright © 2012 John Wiley & Sons, Ltd.
laboratory demonstrated the existence of progenitor cells (SGP-1) from rat, mouse, human, and swine salivary glands. They reported that progenitor cells isolated from the injured salivary glands of rats, mice and swine express surface markers such as CD29 (integrin b1), CD44, CD49f (integrin a6), c-kit and Thy1 (Okumura et al., 2003; Hisatomi et al., 2004; Matsumoto et al., 2007). They isolated progenitor cells from intact human tissues and showed marker expression similar to rodent progenitor cells, except for the absence of c-kit expression. Our SGSCs did not express the Thy-1 antigen, although other stem/progenitor markers and morphology were similar to SGP-1. Thy-1 is a well-known stem cell marker that is expressed in fetal liver, umbilical cord blood and MSCs in human, mouse and rat (Masson et al., 2006; Dominici et al., 2006). Stevenson et al. (2009) reported Thy1.1positive and Thy1.1-negative pancreatic progenitor cell populations. In this report, the Thy1.1-positive population exhibited a fibroblast-like morphology, whereas the Thy1.1negative population exhibited an epithelial-like morphology. J Tissue Eng Regen Med (2012) DOI: 10.1002/term
H. Baek et al.
This morphological difference was similar in our Thy-1negative SGSC population that exhibited an epithelial-like morphology. Damage to the salivary glands is a common side-effect of radiation therapy for head and neck cancer patients. These patients suffer from conditions including dry mouth, severe dental caries and frequent mucosal infection (Fox, 1989; Vissink et al., 2003a, 2003b). There are no effective prevention treatments or therapies for patients with such irreversible salivary gland hypofunction caused by radiotherapy. Approximately 90% of the salivary gland is composed of acinar cells, which are highly sensitive to radiation (Baum, 1993; Konings et al., 2005). Maintenance of an acinar phenotype in primary salivary gland cell cultures has proved difficult (Redman and Quissell, 1993). Therefore, several approaches have been reported to restore the damaged salivary gland function by stem cell-based therapy (Tran et al., 2007; Lombaert et al., 2008). We induced acinar-like differentiation to verify the possibility of SGSCs as a source of cell therapy for salivary hypofunction. On the surface of GFR-matrigel, SGSCs formed acino-ductal structures and stained for amylase. During many biological processes, cell–cell contact through the formation of functional junctions or direct contact is important for communication with other cells to receive complex signals from their environment that can regulate development, homeostasis and even disease progression. Intra-islet interactions are known to be extremely important for the normal function of the islet insulin-secreting b cells, and disruption of islet architecture results in a reduction in glucose-induced insulin secretion (Wojtusciszyn et al., 2008; Kelly et al., 2010; Benninger et al., 2011). Recent work has identified that connexin36 is found constitutively in human pancreatic islets and regulates b cell gap junctions (Serre-Beinier et al., 2009). Connexin-dependent signalling is required for the regulation of the biosynthesis, storage and secretion of insulin (Kanno et al., 2002). Three-dimensional aggregates secrete more insulin as opposed to single layers of cells (Bereton et al., 2006). Numerous culture systems have evolved to promote cell–cell contact and culture cells in aggregate structures (Chandra et al., 2011; Ku et al., 2007). In the present study, rat SGSCs
were induced to differentiate into insulin-producing aggregates through stepwise culture in conditioned medium. Cell aggregates appeared to remain similar in size throughout the culture period 5 days after differentiation and expressed the pancreatic transcription factors Nkx6.1, insulin and c-peptide. SGSCs were cultured for 3 weeks in standard medium with Activin-A, EGF, Exendin-4 and retinoic acid. Activin-A is a member of the transforming growth factor-b (TGFb) superfamily, which regulates pancreatic development and endocrine determination (Demeterco et al., 2000). Similar to Activin-A, EGF and retinoic acid also expand the pancreatic endoderm (Huotari et al., 1998; Stafford and Prince, 2002). Previous studies using human fetal pancreas cultures and rat culture models demonstrated that the addition of Exendin-4 promotes these cells towards the endodermal lineage (Xu et al., 1999). Thus, in our experimental system, we observed that a combination of Activin-A, Exendin-4 and retinoic acid effectively induces SGSCs to differentiate into insulinproducing cells. Our report is the first to show that the treatment of SGSCs with various components specifically induces their differentiation into insulin-secreting cells. Our results showed that isolated SGSCs were able to differentiate into acinar-like cells and insulin-secreting cells. Thus, our SGSCs have the potential to differentiate morphologically, structurally and functionally into endodermal lineage cells. In summary, our study reports the differentiation of SGSCs into acinar cells and insulin-secreting cells in the presence of appropriate culture conditions. Furthermore, our data demonstrate that adult salivary stem/progenitor cells could be used in future clinical applications in xerostomia and diabetes. Future work will focus on the regenerative effect of SGSCs in a rat model and the establishment of SGSCs from the human salivary gland.
Acknowledgements This work was supported by grants to H.K. from the Korea Science and Engineering Foundation (KOSEF), funded by the Korean government (MEST; Grant Nos 20110093737 and 20110062246).
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