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TGF-β stimulates glial-like differentiation in murine dental follicle precursor cells (mDFPCs)
Neuroscience Letters 471 (2010) 179–184
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
TGF- stimulates glial-like differentiation in murine dental follicle precursor cells (mDFPCs) Oliver Felthaus a,b , Wolfgang Ernst c , Oliver Driemel b , Torsten E. Reichert b , Gottfried Schmalz a , Christian Morsczeck a,c,∗ a b c
Department of Operative Dentistry and Periodontology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany Department of Oral and Maxillofacial Surgery, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
a r t i c l e
i n f o
Article history: Received 16 July 2009 Received in revised form 15 January 2010 Accepted 15 January 2010 Keywords: Dental follicle cells Glial differentiation Transforming growth factor  Retinal progenitor cells Glial fibrillar acidic protein
a b s t r a c t Dental stem cells such as dental follicle precursor cells (DFPCs) are capable of neural-like differentiation. However, compared to neuroectodermal progenitor cells such as murine retinal progenitor cells (mRPCs) they show only a limited capacity for glial cell differentiation. In this study we tested the influence of cell signaling on glial differentiation of mDFPCs. These cells were treated with inhibitors and activators of the Sonic hedgehog-, the Wnt/-Catenin-, and the TGF--pathway. After incubation only an activation of the TGF--pathway showed a remarkable glial-like cell differentiation. In contrast gene expression of neural cell markers was not regulated. In conclusion, TGF- improved glial-like, but not neural-like, differentiation of mDFPCs. © 2010 Elsevier Ireland Ltd. All rights reserved.
Dental stem cells or precursor cells such as dental follicle cells are easily accessible and an excellent source of somatic stem cells [9,10]. The dental follicle is a loose ectomesenchymally derived connective tissue that surrounds the unerupted tooth. It harbours progenitor cells that differentiate during tooth development into cementoblasts, periodontal ligament fibroblasts and alveolar bone osteoblasts. These cells surround and support the teeth. It has additionally been shown that dental follicle precursor cells can differentiate into multiple cell types such as adipocytes and chondrocytes [6]. Interestingly, the neural crest derived mDFPCs and neural stem cells (NSCs) have a common ontogeny and can both be successfully differentiated into neurons [16]. Recently, we compared murine dental follicle precursor cells (mDFPCs) with murine retinal progenitor cells (mRPCs) to evaluate their potential for differentiation into neural-like cells. Although markers for neural cells were expressed in the differentiated mDFPCs and mRPCs, the differentiation of the mDFPCs into glial-like cells and neuron-like cells was less complete. After differentiation 37% of mRPCs, but only 5% of DFPCs expressed the astroglia cell-marker GFAP [3]. Neurons and glial cells arise from a common neural precursor cell during central nervous system (CNS) development [5].
∗ Corresponding author at: Department of Operative Dentistry and Periodontology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. Tel.: +49 944 6161; fax: +49 944 6025. E-mail address: [email protected] (C. Morsczeck). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.01.037
Cell signaling pathways play important roles in neural development and cell specification. These activated pathways importantly include Sonic hedgehog (Shh), transforming growth factor- superfamily including bone morphogenic protein (TGF-/BMP), and Wnt/-Catenin [18]. Sonic hedgehog regulates early stages of glial development and is required for neural differentiation in murine embryonic stem cells (mESCs) [8]. Unlike Shh, Wnt promotes astroglial differentiation and inhibits oligodendrocyte differentiation that is mediated by BMP2/4 [5]. TGF-1 has a key role in several CNS developmental processes. The neurotransmitter glutamate triggers astrocyte differentiation through induction of the TGF-1 signaling pathway [12]. In order to improve glial-like differentiation of mDFPCs, we first analyzed the expression of genes that are associated with the Sonic hedgehog-, the Wnt/-Catenin-, and the TGF--pathway. Moreover, we tested the influence of activators and inhibitors of these pathways for their ability to induce the glial differentiation. For a fast evaluation of glial cell differentiation, mDFPCs were isolated from tooth germs of GFEA mice that express GFP under the control of a human GFAP promoter [11]. Here, the expression of GFAP and the differentiation into glial-like cells can be detected by green fluorescence. Isolation of mDFPCs has been described elsewhere [10]. Briefly, 6 C57BL/6 mice (postnatal day 9) were anesthetized and killed by cervical dislocation. The coronal parts of 18 teeth were dissected under a binocular. The developing molar teeth follicles were pooled in PBS and digested in a solution consisting
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of 1 mg/ml collagenase (Sigma–Aldrich, Taufkirchen, Germany), 2 mg/ml hyaluronidase (Sigma–Aldrich), 0.3 mg/ml DNAse I (Roche, Germany), and PBS (PAA, Pasching, Austria) for 40 min at 37 ◦ C. Subsequently, enzymatic digestion was stopped by adding cultivation medium (Mesenchym Stem Medium supplemented with 100 mg/ml penicillin/streptomycin (PAA)). To prepare a single cell suspension the solution was forced through a cell strainer (70 m). Cells were seeded into 25 cm2 (T25) cell culture flasks (Nunc, Wiesbaden, Germany) in cultivation medium. Isolation of murine retinal progenitor cells (mRPCs) was performed by a modified protocol of Angénieux et al. [2]. Briefly, 10 C57BL/6 mice (postnatal day 0) were anesthetized and killed by cervical dislocation. The ciliary marginal zone and the optic nerve head were surgically removed from the eyes. The tissue was digested in a solution of 1 mg/ml collagenase, 2 mg/ml hyaluronidase and 0.3 mg/ml DNAse I and PBS at 37 ◦ C for 40 min. Subsequently, enzymatic activity was stopped by adding cultivation medium. To prepare a single cell suspension the solution was forced through a cell strainer (70 m). The cells were washed three times in cultivation medium (D-MEM F12 Glutamax, N2 supplement (Invitrogen, Karlsruhe, Germany)). Cells were re-suspended in cultivation medium (D-MEM F12 Glutamax, N2 supplement, 20 ng/ml fibroblast growth factor (FGF)-2, 20 ng/ml epidermal growth factor (EGF) (Biomol, Hamburg, Germany) and 100 mg/ml penicillin/streptomycin) seeded in T25 cell culture flasks. The homogeneity of the single cell suspension was checked under the microscope. Cornea cells were isolated from transgenic GFEA mice that express GFP under the control of a human GFAP promoter. The GFEA mice were kindly provided by Dr. Frank Kirchhoff (MPG, Göttingen, Germany). Corneal discs including the epithelium, stroma and endothelium were prepared under the Leica S6D stereomicroscope (Leica, Germany), to avoid contamination with periocular or pericorneal tissue. The tissues were minced to fine pieces and collected in PBS (PAA, Austria). After centrifugation at 800 × g for 2 min the pellet was re-suspended in growth medium (D-MEM high glucose (4.5 g/l)) with l-glutamine (PAA, Austria) supplemented with 10% foetal bovine serum (PAA, Austria), and 1× penicillin/streptomycin (PAA, Austria)). The suspension was seeded in 25 cm2 culture flasks (Nunc, Denmark). 3–5 days after pieces of tissue attached on the cell culture surface an exodus of single cells (murine cornea cells, mCCs) could be observed. These cells were further sub-cultivated in growth medium in humified air (5% CO2 ) at 37 ◦ C. Medium was changed twice a week. For neural-like differentiation of mDFPCs (passage 8), cells were plated at a density of (1–5) × 104 cells/cm2 and cultivated under differentiation conditions (neural differentiation medium (NDM): D-MEM F12 Glutamax, N2 supplement, 20 ng/ml FGF2, 20 ng/ml EGF, 5 M retinoic acid (Sigma–Aldrich, Taufkirchen, Germany), 100 mg/ml penicillin/streptomycin) for 5 days. For neural-like differentiation of mRPCs, cells were plated at a density of (5–10) × 104 cells/cm2 and cultivated in medium with 0.5 M retinoic acid for 1 day. Subsequently, cells were cultured for 4 days in D-MEM F12 Glutamax, N2 supplement, and 0.5 M retinoic acid. After neural differentiation the expression of genes involved in the Sonic hedgehog-, the Wnt/-Catenin-, and the TGF--pathway was measured by real time RT-PCR. For further improvement of glial-like differentiation, mDFPCs were cultured with neural differentiation medium containing activators and inhibitors for the Sonic hedgehog-, the Wnt/-Catenin-, and the TGF--pathway. For comparison with other ectomesenchymally derived cells, we also tested cornea cells isolated from GFEA mice. We incubated the dental follicle cells and the cornea cells for 5 days with 5 M of the Sonic hedgehog-activator purmorphamine (Biozol Diagnostica, Eching, Germany), 2 M of the Sonic hedgehog-inhibitor cyclopamine (Sigma–Aldrich), 100 nM
of the Wnt-activator 6-bromoindirubin-3 -oxime (Sigma–Aldrich), 10 ng/ml of the Wnt-inhibitor Dickkopf-1 (R&D Systems, Minneapolis, USA), 5 ng/ml TGF- (BD Bioscience, Franklin Lakes, USA), or 10 M of the TGF- inhibitor SB431542 (Sigma–Aldrich). The GFP expression was observed by fluorescence microscopy. The expression of GFAP, -III tubulin, and neurofilament-M was measured by real time RT-PCR. We performed adipogenic, chondrogenic and osteogenic differentiation with the mDFPCs to demonstrate that multipotent cells were used in this study. Cells were incubated with the StemPro® Adipogenesis Differentiation Kit, the StemPro® Chondrogenesis Differentiation Kit, and the StemPro® Osteogenesis Differentiation Kit (all Gibco), respectively according to the manufacturer’s recommendations. Briefly, for adipogenic and osteogenic differentiation 10,000 cells/cm2 were seeded in 25 cm2 cell culture flasks and grown for 21 days in their respective StemPro® differentiation medium. For chondrogenic differentiation, 5 l droplets of a 1.6 × 107 cells/ml suspension (micromass culture) were seeded in 25 cm2 cell culture flasks and grown for 21 days in StemPro® Chondrogenesis differentiation medium. Media were changed three times a week. After 3 weeks of differentiation, adipogenic differentiated cells were investigated for lipid droplets, chondrogenic differentiated cells were stained with Alcian blue, and osteogenic differentiated cells were stained with Alizarin-Red-S (Sigma–Aldrich). In order to isolate RNA, mDFPCs, mCCs, and mRPCs were processed according to the RNA isolation kit NucleoSpin RNA II (Macherey-Nagel, Düren, Germany) manual. Reverse transcription of 1 g total RNA into cDNA was performed with the RevertAid First strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). For qRT-PCR the 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, USA) was used. Primers and the corresponding probes (Universal ProbeLibrary Roche) are listed in the supplemental material (oligonucleotides were purchased at Metabion (Martinsried, Germany)). Samples were measured in duplicates. The gene expression of ATPase, a housekeeper gene, was used for normalization. The Ct calculation method was used for relative quantification of gene expression [17] with undifferentiated mDFPCs used for calibration (relative gene expression = 1). In order to show that the mDFPCs and the mCCs express marker for undifferentiated progenitor cells, we performed an immunocytochemical staining using rat anti-mouse lymphocyte antigen 6 complex locus A (Sca1) (purified anti-mouse Ly-6A/E Clone E13161.7, Biolegend, USA) primary antibody. 5000 cells/cm2 were seeded in 4-well chamber slides (Nunc, Wiesbaden, Germany). The next day, cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) (Roth, Karlsruhe, Germany) for 30 min and incubated with the primary antibody for 1 h at room temperature. Subsequently, the Dako EnVisionTM + Dual Link System HRP (DakoCytomation, Denmark) was used for visualization according to the manufacturer’s recommendations. To show that the TGF- pathway is activated upon stimulation with TGF-, we detected the phosphorylisation of Smad2, an activated target of the TGF- signaling pathway. 5000 cells/cm2 were seeded in 10 cm cell culture dishes and grown in cultivation medium. The mDFPCs were incubated with 5 ng/ml TGF-, or 10 M SB431542 for 5 h. For control cells were grown without TGF-. Total proteins were isolated in a lysis buffer (1 mM Na3 VO4 , 150 mM NaCl, 1 mM EDTA, 1% NP-40, Roche complete mini) on ice for 30 min. Proteins were separated by precast 8–16% gradient Tris-glycine polyacrylamid gel electrophoresis (Novex Invitrogen). After the electrophoresis proteins were blotted on a nitrocellulose membrane. Staining of the membrane with Ponceau S validated equal amount of protein-loading. Subsequently, the membrane was washed with TBST and blocked with blocking buffer (5% non-fat dry milk in TBS) for 1 h. After washing the mem-
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brane was incubated with the primary antibody PhosphoDetectTM Anti-Smad2 (pSer465/467) Rabbit Calbiochem® pAb (Calbiochem) at 4 ◦ C over night. The membrane was rinsed again and incubated with the HRP-conjugated secondary antibody W401B anti rabbit (Promega Madison, WI, USA) (1 mg/ml 1:2000) for 1 h at room temperature. The detection of the horseradish peroxidise was carried out using ECL-solution and a subsequent exposition of Hyperfilm ECL autoradiography films (GE Healthcare Life Sciences). The isolated cells were successfully grown for >15 passages (mDFPCs). To evaluate the multipotency of mDFPCs, cells were positively stained for the murine stem cell-marker Sca1 (Figure S1D) and differentiated into adipogenic, chondrogenic and osteogenic cells. Here, adipogenic differentiated mDFPCs had intracellular
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lipid droplets (Figure S1A). Osteogenic differentiated cells formed Alizarin red positive mineralized clusters (Figure S1C) and chondrogenic differentiated micromass cultures of mDFPCs were positively stained with Alcian blue (Figure S1B). To evaluate differences between mDFPCs and mRPCs, which could be propagated for more than 18 passages, after neural-like differentiation, we compared gene expression profiles of marker genes of signaling pathways that are important for development and differentiation. In all three signaling pathways differences between the regulation of mDFPCs and mRPCs during neural-like differentiation were observed (Fig. 1). The secreted signaling protein Shh for example was strongly up-regulated in mRPCs during neural-like differentiation. It was neither expressed before nor
Fig. 1. Relative gene expression changes of pathway specific genes after neural-like differentiation in mRPCs and mDFPCs. The gene expression changes of differentiated mRPCs (blue) and of differentiated mDFPCs (red) are presented. The corresponding undifferentiated cells served as calibrators for the qRT-PCR analysis. The following pathways were investigated: (A) Sonic hedgehog; (B) Wnt/-Catenin and (C) TGF/BMP. Each bar represents the average of two experiments. The asterisk indicates that Sonic hedgehog (Shh) was not expressed in mDFPCs. Error bars denote the range between experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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after differentiation in mDFPCs. The transcription factors Gli1, Gli2, and Gli3 were down-regulated in mRPCs. Gli1 was down-regulated in mDFPCs, too, but Gli2 and Gli3 were up-regulated (Fig. 1A). Moreover, Wnt1 was strongly up-regulated in mRPCs but only weak in mDFPCs whereas Wnt5b was strongly down-regulated in mDFPCs but only weakly in mRPCs. Interestingly, gene expression changes were smaller in mDFPCs than in mRPCs in most genes associated with the WNT-signaling pathway (Fig. 1B). The growth factors BMP2 and BMP7 were strongly up-regulated in mDFPCS, whereas in mRPCs BMP2 was only weakly up-regulated and BMP7 was down-regulated. The Gene regulatory protein Smad1 and Smad ubiquitination regulatory factor 1 (Smurf1) were strongly upregulated in mRPCs, but showed no regulation in mDFPCs at all. Smad9 was up-regulated in mRPCs but down-regulated in mDFPCs, and the receptor TGFˇR3 was up-regulated in mDFPCs without a regulation in mRPCs (Fig. 1C). The green fluorescence of mDFPCs was evaluated after 4–5 days of incubation with neural differentiation medium containing compounds for the manipulation of cell signaling pathways. Only cells treated with TGF- showed a remarkable expression of GFP indicating the glial-like differentiation (Fig. 2E). Interestingly, the cells treated with the TGF- pathway inhibitor SB431542 showed a minor GFP expression, as well (Fig. 2F). In contrast, mDFPCs expressed no GFP after the manipulation of neither the Sonic hedgehog- nor the Wnt/-Catenin-pathway (data not shown). Incubation with the neural differentiation medium alone had no stimulating effect on GFAP/GFP expression (Fig. 2D). Fig. 2A–C show the corresponding phase contrast microscopy images. Real-time PCR analysis revealed that gene expression of endogenous GFAP was up-regulated in mDFPCs with an exogenous TGF- stimulus. In contrast, the GFAP expression was down-regulated in cells treated with the inhibitor SB431542 (Fig. 3A). Neither the early neural cell-marker gene -III tubulin (Fig. 3B) nor the gene for neurofilament-M (Fig. 3C) were up-regulated by TGF-.
To evaluate the activation of the TGF- signaling pathway the phosphorylation of Smad2 (pSmad2) was estimated by Westernblot. After stimulation with TGF- the amount of pSmad2 was increased in comparison to untreated cells. No pSmad2 was detected after inhibiting the TGF- pathway with SB431542 (Figure S2). We investigated the neural-like cell differentiation of mCCs to estimate the influence of TGF- on the differentiation of another type of undifferentiated ectomesenchymal cells. Like mDFPCs mCCs expressed the murine stem cell-marker Sca-1 (data not shown). Interestingly, mCCs differentially express neuronal cell markers after stimulation with TGF- (Figure S4). However, they did not express the glial cell-marker GFAP after any condition (Figure S3). The mDFPCs from transgenic GFEA mice are multipotent undifferentiated cells similar to follicle cells from our previous study [3]. These cells differentiate into neural-like cells, but have only a limited potential for glial cell differentiation [3]. The aim of this study was to analyze the cell signaling pathways driving glial-like cell differentiation of mDFPCs and to make comparisons with those of mRPCs. Gene expression profiles demonstrated that diverse signaling pathways were activated in mDFPCs and mRPCs during neural-like differentiation and are therefore likely involved in their differentiation. In rodents, Shh is critical for the development of oligodendrocytes. For example, Shh has been shown to be necessary and sufficient for the expression of the oligodendrocyte marker gene Olig in the mammalian embryonic brain [7]. We found a downregulation of Shh signaling pathway associated genes in neural-like differentiated mDFPCs. Moreover, Shh signaling is down-regulated during neural-like differentiation of mRPCs, which can be explained with the key role of the Shh signaling in the proliferation of retinal progenitor cells [1]. Although Shh regulates early stages of glial development, exogenous Shh has no effect on differentiation of mDFPCs. Neither the activation nor the inhibition of Shh
Fig. 2. Glial-like differentiation of mDFPCs under green fluorescence. Phase contrast microscopy (A–C) and fluorescence microscopy (D–F) of mDFPCs after differentiation with NDM (A and D) and with NDM supplemented with TGF- (B and E) or SB431542 (C and F). Original magnification: 200×.
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Fig. 3. Gene expression analyses of neural-like marker genes GFAP (A), -III tubulin (B), and neurofilament-M (C) in mDFPCs with qRT-PCR after differentiation. Calibrator was undifferentiated mDFPCs. Each bar represents the average of three experiments. Error bars denote the standard deviation. Abbreviation: GFAP: glial fibrillar acidic protein; Tubb3: -III tubulin; NF: neurofilament. The asterisk (*) indicates a significant difference (t-test p < 0.05) between control and TGF- stimulated mDFPCs.
resulted in an activation of the GFAP promoter. This is consistent with the finding that Shh promotes oligodendrocyte differentiation in rodents [14] that do not express-unlike astroglia cells – GFAP. Wnt such as Wnt5b is up-regulated in various tumor cell lines [13]. This indicates a role for Wnt signaling in cell proliferation and explains the strong down-regulation in differentiated mRPCs. In this context, it is interesting that gene expression of Wnt1 is increased during neural-like differentiation in mRPCs. This is consistent with the finding that Wnt1 promotes neuronal differentiation in P19 cells, a model cell line for neural differentiation [15]. Neural-like differentiation had a similar but much lower effect on the expression of Wnt signaling in mDFPCs (Fig. 1B). Although Wnt signaling has been shown to promote astroglial differentiation in Striatum cells [5], the treatment with Wnt pathway manipulating compounds did not activate the GFAP promoter in mDFPCs. This could be due to differences in the regulation of these cell types. Whereas effects of incubating cells under neural differentiation conditions on the Shh- and Wnt-pathways were similar but more pronounced in mRPCs than in mDFPCs, the latter population seem to specifically highly activate TGF- and to inhibit BMP signaling pathways. Only mDFPCs treated with TGF- showed a fluorescence signal, indicating the activation of the GFAP promoter. Here, the activation of the TGF- signaling pathway was proved by the phosphorylation of Smad2. This is consistent with the finding that TGF- induces cortical neurons to activate the GFAP gene promoter and induce astrocyte differentiation in vitro [4]. Since EGF and bFGF were not removed from culture conditions all through the differentiation process, the possibility that TGF- treatment might alternatively influence mDFPCs acquisition of a neural progenitorlike state instead of a glial-like one cannot be rule out. This would be consistent with the observation of that the GFAP expression level of differentiated mDFPCs is similar to the GFAP expression level of undifferentiated mRPCs [3]. Finally, there is also the possibility of that TGF- might only induce GFAP expression on mDFPCs independently on any acquisition of a neural-like phenotype. Further investigations have to be done to evaluate the effect of TGF- on glial-like differentiation of mDFPCs. Interestingly, in our culture conditions TGF- failed to induce neuronal-like differentiation of mDFPCs. However, after the induction with TGF- mCCs, which are also undifferentiated ectomesenchymal cells, differentiated into neuronal-like cells, but they did not express the glial cell-marker GFAP. This result demonstrates that TGF- is not a general inducer of glial cell differentiation in undifferentiated ectomesenchymal cells. Widera et al. [16] demonstrated that neurosphere-forming NSCs derived from the periodontal ligament have the capability to differentiate into both glial and neuronal cells. In contrast, mDFPCs could not be cultivated as neurospheres for unknown reason [3] and therefore they have probably a limited differentiation poten-
tial. However, Widera et al. [16] used ectomesenchymal cells from human tissues. Our study investigated for the first time the effects of TGF-, Shh- and Wnt-signaling pathways on glial-like cell differentiation of mDFPCs. From all signals analyzed, we found that only TGF- promoted a glial-like differentiation of mDFPCs.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.01.037.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
TGF- stimulates glial-like differentiation in murine dental follicle precursor cells (mDFPCs) Oliver Felthaus a,b , Wolfgang Ernst c , Oliver Driemel b , Torsten E. Reichert b , Gottfried Schmalz a , Christian Morsczeck a,c,∗ a b c
Department of Operative Dentistry and Periodontology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany Department of Oral and Maxillofacial Surgery, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
a r t i c l e
i n f o
Article history: Received 16 July 2009 Received in revised form 15 January 2010 Accepted 15 January 2010 Keywords: Dental follicle cells Glial differentiation Transforming growth factor  Retinal progenitor cells Glial fibrillar acidic protein
a b s t r a c t Dental stem cells such as dental follicle precursor cells (DFPCs) are capable of neural-like differentiation. However, compared to neuroectodermal progenitor cells such as murine retinal progenitor cells (mRPCs) they show only a limited capacity for glial cell differentiation. In this study we tested the influence of cell signaling on glial differentiation of mDFPCs. These cells were treated with inhibitors and activators of the Sonic hedgehog-, the Wnt/-Catenin-, and the TGF--pathway. After incubation only an activation of the TGF--pathway showed a remarkable glial-like cell differentiation. In contrast gene expression of neural cell markers was not regulated. In conclusion, TGF- improved glial-like, but not neural-like, differentiation of mDFPCs. © 2010 Elsevier Ireland Ltd. All rights reserved.
Dental stem cells or precursor cells such as dental follicle cells are easily accessible and an excellent source of somatic stem cells [9,10]. The dental follicle is a loose ectomesenchymally derived connective tissue that surrounds the unerupted tooth. It harbours progenitor cells that differentiate during tooth development into cementoblasts, periodontal ligament fibroblasts and alveolar bone osteoblasts. These cells surround and support the teeth. It has additionally been shown that dental follicle precursor cells can differentiate into multiple cell types such as adipocytes and chondrocytes [6]. Interestingly, the neural crest derived mDFPCs and neural stem cells (NSCs) have a common ontogeny and can both be successfully differentiated into neurons [16]. Recently, we compared murine dental follicle precursor cells (mDFPCs) with murine retinal progenitor cells (mRPCs) to evaluate their potential for differentiation into neural-like cells. Although markers for neural cells were expressed in the differentiated mDFPCs and mRPCs, the differentiation of the mDFPCs into glial-like cells and neuron-like cells was less complete. After differentiation 37% of mRPCs, but only 5% of DFPCs expressed the astroglia cell-marker GFAP [3]. Neurons and glial cells arise from a common neural precursor cell during central nervous system (CNS) development [5].
∗ Corresponding author at: Department of Operative Dentistry and Periodontology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. Tel.: +49 944 6161; fax: +49 944 6025. E-mail address: [email protected] (C. Morsczeck). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.01.037
Cell signaling pathways play important roles in neural development and cell specification. These activated pathways importantly include Sonic hedgehog (Shh), transforming growth factor- superfamily including bone morphogenic protein (TGF-/BMP), and Wnt/-Catenin [18]. Sonic hedgehog regulates early stages of glial development and is required for neural differentiation in murine embryonic stem cells (mESCs) [8]. Unlike Shh, Wnt promotes astroglial differentiation and inhibits oligodendrocyte differentiation that is mediated by BMP2/4 [5]. TGF-1 has a key role in several CNS developmental processes. The neurotransmitter glutamate triggers astrocyte differentiation through induction of the TGF-1 signaling pathway [12]. In order to improve glial-like differentiation of mDFPCs, we first analyzed the expression of genes that are associated with the Sonic hedgehog-, the Wnt/-Catenin-, and the TGF--pathway. Moreover, we tested the influence of activators and inhibitors of these pathways for their ability to induce the glial differentiation. For a fast evaluation of glial cell differentiation, mDFPCs were isolated from tooth germs of GFEA mice that express GFP under the control of a human GFAP promoter [11]. Here, the expression of GFAP and the differentiation into glial-like cells can be detected by green fluorescence. Isolation of mDFPCs has been described elsewhere [10]. Briefly, 6 C57BL/6 mice (postnatal day 9) were anesthetized and killed by cervical dislocation. The coronal parts of 18 teeth were dissected under a binocular. The developing molar teeth follicles were pooled in PBS and digested in a solution consisting
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of 1 mg/ml collagenase (Sigma–Aldrich, Taufkirchen, Germany), 2 mg/ml hyaluronidase (Sigma–Aldrich), 0.3 mg/ml DNAse I (Roche, Germany), and PBS (PAA, Pasching, Austria) for 40 min at 37 ◦ C. Subsequently, enzymatic digestion was stopped by adding cultivation medium (Mesenchym Stem Medium supplemented with 100 mg/ml penicillin/streptomycin (PAA)). To prepare a single cell suspension the solution was forced through a cell strainer (70 m). Cells were seeded into 25 cm2 (T25) cell culture flasks (Nunc, Wiesbaden, Germany) in cultivation medium. Isolation of murine retinal progenitor cells (mRPCs) was performed by a modified protocol of Angénieux et al. [2]. Briefly, 10 C57BL/6 mice (postnatal day 0) were anesthetized and killed by cervical dislocation. The ciliary marginal zone and the optic nerve head were surgically removed from the eyes. The tissue was digested in a solution of 1 mg/ml collagenase, 2 mg/ml hyaluronidase and 0.3 mg/ml DNAse I and PBS at 37 ◦ C for 40 min. Subsequently, enzymatic activity was stopped by adding cultivation medium. To prepare a single cell suspension the solution was forced through a cell strainer (70 m). The cells were washed three times in cultivation medium (D-MEM F12 Glutamax, N2 supplement (Invitrogen, Karlsruhe, Germany)). Cells were re-suspended in cultivation medium (D-MEM F12 Glutamax, N2 supplement, 20 ng/ml fibroblast growth factor (FGF)-2, 20 ng/ml epidermal growth factor (EGF) (Biomol, Hamburg, Germany) and 100 mg/ml penicillin/streptomycin) seeded in T25 cell culture flasks. The homogeneity of the single cell suspension was checked under the microscope. Cornea cells were isolated from transgenic GFEA mice that express GFP under the control of a human GFAP promoter. The GFEA mice were kindly provided by Dr. Frank Kirchhoff (MPG, Göttingen, Germany). Corneal discs including the epithelium, stroma and endothelium were prepared under the Leica S6D stereomicroscope (Leica, Germany), to avoid contamination with periocular or pericorneal tissue. The tissues were minced to fine pieces and collected in PBS (PAA, Austria). After centrifugation at 800 × g for 2 min the pellet was re-suspended in growth medium (D-MEM high glucose (4.5 g/l)) with l-glutamine (PAA, Austria) supplemented with 10% foetal bovine serum (PAA, Austria), and 1× penicillin/streptomycin (PAA, Austria)). The suspension was seeded in 25 cm2 culture flasks (Nunc, Denmark). 3–5 days after pieces of tissue attached on the cell culture surface an exodus of single cells (murine cornea cells, mCCs) could be observed. These cells were further sub-cultivated in growth medium in humified air (5% CO2 ) at 37 ◦ C. Medium was changed twice a week. For neural-like differentiation of mDFPCs (passage 8), cells were plated at a density of (1–5) × 104 cells/cm2 and cultivated under differentiation conditions (neural differentiation medium (NDM): D-MEM F12 Glutamax, N2 supplement, 20 ng/ml FGF2, 20 ng/ml EGF, 5 M retinoic acid (Sigma–Aldrich, Taufkirchen, Germany), 100 mg/ml penicillin/streptomycin) for 5 days. For neural-like differentiation of mRPCs, cells were plated at a density of (5–10) × 104 cells/cm2 and cultivated in medium with 0.5 M retinoic acid for 1 day. Subsequently, cells were cultured for 4 days in D-MEM F12 Glutamax, N2 supplement, and 0.5 M retinoic acid. After neural differentiation the expression of genes involved in the Sonic hedgehog-, the Wnt/-Catenin-, and the TGF--pathway was measured by real time RT-PCR. For further improvement of glial-like differentiation, mDFPCs were cultured with neural differentiation medium containing activators and inhibitors for the Sonic hedgehog-, the Wnt/-Catenin-, and the TGF--pathway. For comparison with other ectomesenchymally derived cells, we also tested cornea cells isolated from GFEA mice. We incubated the dental follicle cells and the cornea cells for 5 days with 5 M of the Sonic hedgehog-activator purmorphamine (Biozol Diagnostica, Eching, Germany), 2 M of the Sonic hedgehog-inhibitor cyclopamine (Sigma–Aldrich), 100 nM
of the Wnt-activator 6-bromoindirubin-3 -oxime (Sigma–Aldrich), 10 ng/ml of the Wnt-inhibitor Dickkopf-1 (R&D Systems, Minneapolis, USA), 5 ng/ml TGF- (BD Bioscience, Franklin Lakes, USA), or 10 M of the TGF- inhibitor SB431542 (Sigma–Aldrich). The GFP expression was observed by fluorescence microscopy. The expression of GFAP, -III tubulin, and neurofilament-M was measured by real time RT-PCR. We performed adipogenic, chondrogenic and osteogenic differentiation with the mDFPCs to demonstrate that multipotent cells were used in this study. Cells were incubated with the StemPro® Adipogenesis Differentiation Kit, the StemPro® Chondrogenesis Differentiation Kit, and the StemPro® Osteogenesis Differentiation Kit (all Gibco), respectively according to the manufacturer’s recommendations. Briefly, for adipogenic and osteogenic differentiation 10,000 cells/cm2 were seeded in 25 cm2 cell culture flasks and grown for 21 days in their respective StemPro® differentiation medium. For chondrogenic differentiation, 5 l droplets of a 1.6 × 107 cells/ml suspension (micromass culture) were seeded in 25 cm2 cell culture flasks and grown for 21 days in StemPro® Chondrogenesis differentiation medium. Media were changed three times a week. After 3 weeks of differentiation, adipogenic differentiated cells were investigated for lipid droplets, chondrogenic differentiated cells were stained with Alcian blue, and osteogenic differentiated cells were stained with Alizarin-Red-S (Sigma–Aldrich). In order to isolate RNA, mDFPCs, mCCs, and mRPCs were processed according to the RNA isolation kit NucleoSpin RNA II (Macherey-Nagel, Düren, Germany) manual. Reverse transcription of 1 g total RNA into cDNA was performed with the RevertAid First strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). For qRT-PCR the 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, USA) was used. Primers and the corresponding probes (Universal ProbeLibrary Roche) are listed in the supplemental material (oligonucleotides were purchased at Metabion (Martinsried, Germany)). Samples were measured in duplicates. The gene expression of ATPase, a housekeeper gene, was used for normalization. The Ct calculation method was used for relative quantification of gene expression [17] with undifferentiated mDFPCs used for calibration (relative gene expression = 1). In order to show that the mDFPCs and the mCCs express marker for undifferentiated progenitor cells, we performed an immunocytochemical staining using rat anti-mouse lymphocyte antigen 6 complex locus A (Sca1) (purified anti-mouse Ly-6A/E Clone E13161.7, Biolegend, USA) primary antibody. 5000 cells/cm2 were seeded in 4-well chamber slides (Nunc, Wiesbaden, Germany). The next day, cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) (Roth, Karlsruhe, Germany) for 30 min and incubated with the primary antibody for 1 h at room temperature. Subsequently, the Dako EnVisionTM + Dual Link System HRP (DakoCytomation, Denmark) was used for visualization according to the manufacturer’s recommendations. To show that the TGF- pathway is activated upon stimulation with TGF-, we detected the phosphorylisation of Smad2, an activated target of the TGF- signaling pathway. 5000 cells/cm2 were seeded in 10 cm cell culture dishes and grown in cultivation medium. The mDFPCs were incubated with 5 ng/ml TGF-, or 10 M SB431542 for 5 h. For control cells were grown without TGF-. Total proteins were isolated in a lysis buffer (1 mM Na3 VO4 , 150 mM NaCl, 1 mM EDTA, 1% NP-40, Roche complete mini) on ice for 30 min. Proteins were separated by precast 8–16% gradient Tris-glycine polyacrylamid gel electrophoresis (Novex Invitrogen). After the electrophoresis proteins were blotted on a nitrocellulose membrane. Staining of the membrane with Ponceau S validated equal amount of protein-loading. Subsequently, the membrane was washed with TBST and blocked with blocking buffer (5% non-fat dry milk in TBS) for 1 h. After washing the mem-
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brane was incubated with the primary antibody PhosphoDetectTM Anti-Smad2 (pSer465/467) Rabbit Calbiochem® pAb (Calbiochem) at 4 ◦ C over night. The membrane was rinsed again and incubated with the HRP-conjugated secondary antibody W401B anti rabbit (Promega Madison, WI, USA) (1 mg/ml 1:2000) for 1 h at room temperature. The detection of the horseradish peroxidise was carried out using ECL-solution and a subsequent exposition of Hyperfilm ECL autoradiography films (GE Healthcare Life Sciences). The isolated cells were successfully grown for >15 passages (mDFPCs). To evaluate the multipotency of mDFPCs, cells were positively stained for the murine stem cell-marker Sca1 (Figure S1D) and differentiated into adipogenic, chondrogenic and osteogenic cells. Here, adipogenic differentiated mDFPCs had intracellular
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lipid droplets (Figure S1A). Osteogenic differentiated cells formed Alizarin red positive mineralized clusters (Figure S1C) and chondrogenic differentiated micromass cultures of mDFPCs were positively stained with Alcian blue (Figure S1B). To evaluate differences between mDFPCs and mRPCs, which could be propagated for more than 18 passages, after neural-like differentiation, we compared gene expression profiles of marker genes of signaling pathways that are important for development and differentiation. In all three signaling pathways differences between the regulation of mDFPCs and mRPCs during neural-like differentiation were observed (Fig. 1). The secreted signaling protein Shh for example was strongly up-regulated in mRPCs during neural-like differentiation. It was neither expressed before nor
Fig. 1. Relative gene expression changes of pathway specific genes after neural-like differentiation in mRPCs and mDFPCs. The gene expression changes of differentiated mRPCs (blue) and of differentiated mDFPCs (red) are presented. The corresponding undifferentiated cells served as calibrators for the qRT-PCR analysis. The following pathways were investigated: (A) Sonic hedgehog; (B) Wnt/-Catenin and (C) TGF/BMP. Each bar represents the average of two experiments. The asterisk indicates that Sonic hedgehog (Shh) was not expressed in mDFPCs. Error bars denote the range between experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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after differentiation in mDFPCs. The transcription factors Gli1, Gli2, and Gli3 were down-regulated in mRPCs. Gli1 was down-regulated in mDFPCs, too, but Gli2 and Gli3 were up-regulated (Fig. 1A). Moreover, Wnt1 was strongly up-regulated in mRPCs but only weak in mDFPCs whereas Wnt5b was strongly down-regulated in mDFPCs but only weakly in mRPCs. Interestingly, gene expression changes were smaller in mDFPCs than in mRPCs in most genes associated with the WNT-signaling pathway (Fig. 1B). The growth factors BMP2 and BMP7 were strongly up-regulated in mDFPCS, whereas in mRPCs BMP2 was only weakly up-regulated and BMP7 was down-regulated. The Gene regulatory protein Smad1 and Smad ubiquitination regulatory factor 1 (Smurf1) were strongly upregulated in mRPCs, but showed no regulation in mDFPCs at all. Smad9 was up-regulated in mRPCs but down-regulated in mDFPCs, and the receptor TGFˇR3 was up-regulated in mDFPCs without a regulation in mRPCs (Fig. 1C). The green fluorescence of mDFPCs was evaluated after 4–5 days of incubation with neural differentiation medium containing compounds for the manipulation of cell signaling pathways. Only cells treated with TGF- showed a remarkable expression of GFP indicating the glial-like differentiation (Fig. 2E). Interestingly, the cells treated with the TGF- pathway inhibitor SB431542 showed a minor GFP expression, as well (Fig. 2F). In contrast, mDFPCs expressed no GFP after the manipulation of neither the Sonic hedgehog- nor the Wnt/-Catenin-pathway (data not shown). Incubation with the neural differentiation medium alone had no stimulating effect on GFAP/GFP expression (Fig. 2D). Fig. 2A–C show the corresponding phase contrast microscopy images. Real-time PCR analysis revealed that gene expression of endogenous GFAP was up-regulated in mDFPCs with an exogenous TGF- stimulus. In contrast, the GFAP expression was down-regulated in cells treated with the inhibitor SB431542 (Fig. 3A). Neither the early neural cell-marker gene -III tubulin (Fig. 3B) nor the gene for neurofilament-M (Fig. 3C) were up-regulated by TGF-.
To evaluate the activation of the TGF- signaling pathway the phosphorylation of Smad2 (pSmad2) was estimated by Westernblot. After stimulation with TGF- the amount of pSmad2 was increased in comparison to untreated cells. No pSmad2 was detected after inhibiting the TGF- pathway with SB431542 (Figure S2). We investigated the neural-like cell differentiation of mCCs to estimate the influence of TGF- on the differentiation of another type of undifferentiated ectomesenchymal cells. Like mDFPCs mCCs expressed the murine stem cell-marker Sca-1 (data not shown). Interestingly, mCCs differentially express neuronal cell markers after stimulation with TGF- (Figure S4). However, they did not express the glial cell-marker GFAP after any condition (Figure S3). The mDFPCs from transgenic GFEA mice are multipotent undifferentiated cells similar to follicle cells from our previous study [3]. These cells differentiate into neural-like cells, but have only a limited potential for glial cell differentiation [3]. The aim of this study was to analyze the cell signaling pathways driving glial-like cell differentiation of mDFPCs and to make comparisons with those of mRPCs. Gene expression profiles demonstrated that diverse signaling pathways were activated in mDFPCs and mRPCs during neural-like differentiation and are therefore likely involved in their differentiation. In rodents, Shh is critical for the development of oligodendrocytes. For example, Shh has been shown to be necessary and sufficient for the expression of the oligodendrocyte marker gene Olig in the mammalian embryonic brain [7]. We found a downregulation of Shh signaling pathway associated genes in neural-like differentiated mDFPCs. Moreover, Shh signaling is down-regulated during neural-like differentiation of mRPCs, which can be explained with the key role of the Shh signaling in the proliferation of retinal progenitor cells [1]. Although Shh regulates early stages of glial development, exogenous Shh has no effect on differentiation of mDFPCs. Neither the activation nor the inhibition of Shh
Fig. 2. Glial-like differentiation of mDFPCs under green fluorescence. Phase contrast microscopy (A–C) and fluorescence microscopy (D–F) of mDFPCs after differentiation with NDM (A and D) and with NDM supplemented with TGF- (B and E) or SB431542 (C and F). Original magnification: 200×.
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Fig. 3. Gene expression analyses of neural-like marker genes GFAP (A), -III tubulin (B), and neurofilament-M (C) in mDFPCs with qRT-PCR after differentiation. Calibrator was undifferentiated mDFPCs. Each bar represents the average of three experiments. Error bars denote the standard deviation. Abbreviation: GFAP: glial fibrillar acidic protein; Tubb3: -III tubulin; NF: neurofilament. The asterisk (*) indicates a significant difference (t-test p < 0.05) between control and TGF- stimulated mDFPCs.
resulted in an activation of the GFAP promoter. This is consistent with the finding that Shh promotes oligodendrocyte differentiation in rodents [14] that do not express-unlike astroglia cells – GFAP. Wnt such as Wnt5b is up-regulated in various tumor cell lines [13]. This indicates a role for Wnt signaling in cell proliferation and explains the strong down-regulation in differentiated mRPCs. In this context, it is interesting that gene expression of Wnt1 is increased during neural-like differentiation in mRPCs. This is consistent with the finding that Wnt1 promotes neuronal differentiation in P19 cells, a model cell line for neural differentiation [15]. Neural-like differentiation had a similar but much lower effect on the expression of Wnt signaling in mDFPCs (Fig. 1B). Although Wnt signaling has been shown to promote astroglial differentiation in Striatum cells [5], the treatment with Wnt pathway manipulating compounds did not activate the GFAP promoter in mDFPCs. This could be due to differences in the regulation of these cell types. Whereas effects of incubating cells under neural differentiation conditions on the Shh- and Wnt-pathways were similar but more pronounced in mRPCs than in mDFPCs, the latter population seem to specifically highly activate TGF- and to inhibit BMP signaling pathways. Only mDFPCs treated with TGF- showed a fluorescence signal, indicating the activation of the GFAP promoter. Here, the activation of the TGF- signaling pathway was proved by the phosphorylation of Smad2. This is consistent with the finding that TGF- induces cortical neurons to activate the GFAP gene promoter and induce astrocyte differentiation in vitro [4]. Since EGF and bFGF were not removed from culture conditions all through the differentiation process, the possibility that TGF- treatment might alternatively influence mDFPCs acquisition of a neural progenitorlike state instead of a glial-like one cannot be rule out. This would be consistent with the observation of that the GFAP expression level of differentiated mDFPCs is similar to the GFAP expression level of undifferentiated mRPCs [3]. Finally, there is also the possibility of that TGF- might only induce GFAP expression on mDFPCs independently on any acquisition of a neural-like phenotype. Further investigations have to be done to evaluate the effect of TGF- on glial-like differentiation of mDFPCs. Interestingly, in our culture conditions TGF- failed to induce neuronal-like differentiation of mDFPCs. However, after the induction with TGF- mCCs, which are also undifferentiated ectomesenchymal cells, differentiated into neuronal-like cells, but they did not express the glial cell-marker GFAP. This result demonstrates that TGF- is not a general inducer of glial cell differentiation in undifferentiated ectomesenchymal cells. Widera et al. [16] demonstrated that neurosphere-forming NSCs derived from the periodontal ligament have the capability to differentiate into both glial and neuronal cells. In contrast, mDFPCs could not be cultivated as neurospheres for unknown reason [3] and therefore they have probably a limited differentiation poten-
tial. However, Widera et al. [16] used ectomesenchymal cells from human tissues. Our study investigated for the first time the effects of TGF-, Shh- and Wnt-signaling pathways on glial-like cell differentiation of mDFPCs. From all signals analyzed, we found that only TGF- promoted a glial-like differentiation of mDFPCs.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.01.037.
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