International Journal of Neuroscience, 2014; Early Online: 1–8 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2014.950373
Neural crest stem cells from hair follicles and boundary cap have different effects on pancreatic islets in vitro Anastasiia Kosykh,2,∗ Anongnad Ngamjariyawat,1,∗ Svitlana Vasylovska,1,∗ Niclas Konig,1 Carl Trolle,1 Joey Lau,3 Arsen Mikaelyan,2 Michael Panchenko,1 Per-Ola Carlsson,3 Ekaterina Vorotelyak,2,∗ and Elena N Kozlova1
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Department of Neuroscience, Uppsala University Biomedical Center, Uppsala, Sweden; 2 Laboratory of Cell Proliferation, Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia, and 3 Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden Purpose: Neural crest stem cells derived from the boundary cap (bNCSCs), markedly promote survival, proliferation and function of insulin producing β-cells in vitro and in vivo after coculture/transplantation with pancreatic islets [1, 2]. Recently, we have shown that beneficial effects on β-cells require cadherin contacts between bNCSCs and β-cells [3, 4]. Here we investigated whether hair follicle (HF) NCSCs, a potential source for human allogeneic transplantation, exert similar positive effects on β-cells. Materials and Methods: We established cocultures of HF-NCSCs or bNCSCs from mice expressing enhanced green fluorescent protein together with pancreatic islets from DxRed expressing mice or NMRI mice and compared their migration towards islet cells and effect on proliferation of β-cells as well as intracellular relations between NCSCs and islets using qRT-PCR analysis and immunohistochemistry. Results: Whereas both types of NCSCs migrated extensively in the presence of islets, only bNCSCs demonstrated directed migration toward islets, induced β-cell proliferation and increased the presence of cadherin at the junctions between bNCSCs and β-cells. Even in direct contact between β-cells and HF-NCSCs, no cadherin expression was detected. Conclusions: These observations indicate that HF-NCSCs do not confer the same positive effect on β-cells as demonstrated for bNCSCs. Furthermore, these data suggest that induction of cadherin expression by HF-NCSCs may be useful for their ability to support β-cells in coculture and after transplantation. KEYWORDS: Diabetes, migration, cell culture, coculture, intercellular contacts
Introduction Pancreatic β-cells have a low capacity to proliferate [5]. Patients with Type-1 diabetes lose their β-cell mass and transplantation of pancreatic islets is an attractive option to cure this disease. However the survival of islets after transplantation is low and after 5 years only 10% of patients have a sufficient amount of functional β-cells [6]. Thus, methods to improve β-cell survival and function, as well as methods to expand an insufficient β-cell mass, are required. We recently discovered that mouse boundary cap derived neural crest stem cells (bNCSCs) have remarkable effects on β-cell proliferation, survival and funcReceived 7 April 2014; revised 18 July 2014; accepted 28 July 2014 ∗ These authors contributed equally to the paper Correspondence: Elena N Kozlova, Department of Neuroscience, Uppsala University Biomedical Center, Uppsala, Sweden. E-Mail: [email protected] Tel.: +46-18-4714968; Fax: +46-18-511540
tion [1–3]. These findings indicate that NCSCs, which have been shown to regulate β-cell mass during development [7], are potentially useful for coculture with βcells and for cotransplantation with pancreatic islets to induce proliferation of β-cells in vivo after transplantation and improve their survival and function. However, bNCSCs are present only transiently during early embryonic stages [8, 9], and other sources of NCSCs have to be found in order to translate the beneficial effects of bNCSCs to a viable treatment strategy. One of the attractive alternatives is NCSCs from the hair follicle (HF), which can be harvested from the patient and subsequently prepared for autologous transplantation. It was shown previously that the bulge area of HFs contains stem cells with neural crest characteristics [10]. In our in vitro experiments we found that direct contact between β-cells and bNCSCs is crucial for their positive effects on β-cells [3]. We showed that in transwell assays when β-cells and bNCSCs were separated, no β-cell proliferation occurred [3]. Subsequent 1
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experiments, which showed that bNCSCs can protect βcells from cytokine-induced apoptosis, confirmed that this effect requires direct contact between β-cells and bNCSCs and that this effect is mediated by cadherin [2, 11]. Furthermore, these contacts were shown to develop as a result of mutual migration by β-cells and bNCSCs [4]. Here we compare migration behavior of HF-NCSCs and bNCSCs in coculture with pancreatic islets as well as their effect on β-cell proliferation and cadherin expression. Our findings show that HF-NCSCs, despite their excellent capacity to migrate, do not have directed migration toward islets, do not develop direct contacts with islet cells, and hence do not induce β-cell proliferation. Furthermore, cadherin expression in the islet cells was reduced in the coculture with HF-NCSCs whereas this expression remained on a high level in the presence of bNCSCs.
Materials and methods Animals Transgenic heterozygous C57BL/6-b-actin enhanced green fluorescent protein (eGFP) mice (Jackson Laboratories, Bar Harbor, Maine, USA) were used for isolation of bNCSCs and HF-NCSCs. Pancreatic islets were isolated from C57BL/6 mice (M&B, Ry, Denmark) and transgenic heterozygous C57BL/6-b-actin DxRed fluorescent protein (RFP) mice (Jackson Laboratories, Bar Harbor, Maine, USA) and from C57BL mice. All procedures were approved by the Regional Ethical Committee for Research on Animals.
Preparation of bNCSC neurospheres Dorsal root ganglia (DRGs) from 11.5-day-old eGFP mouse embryos were isolated and used for setting up bNCSC cultures as described previously [3, 8, 9]. Briefly, the uterus was removed from the anaesthetized pregnant mouse and placed in cold phosphate-buffered saline (PBS). Embryos were separated, rinsed in PBS, placed in N2 medium and the DRGs were removed and collected in N2 medium under a dissection microscope. Collected DRGs were allowed to settle down before removing the supernatant and adding a Collagenase/Dispase (1 mg/ml) and DNase (0.5 mg/ml) solution in N2 and incubating for 20–30 min in a 37◦ C water bath, followed by rinsing in N2 medium with B27 (1:50) and plating ∼1-2×105 cells/well in a 24-well dish after dissociation. Cells were placed directly into 500μl of Propagation medium: DMEM F-12 medium (Invitrogen cat no. 31330-038) supplemented with B27 (Invitrogen cat no. 17504-044), N2 (Invitrogen cat
no. 17502-048), 20 ng/ml bFGF (Invitrogen cat no. 13256-029) and 20 ng/ml EGF (R&D system cat no. 236-EG). After 12 h, nonadherent cells were removed together with half of the medium before adding up to 250 μl of fresh medium. The medium was then changed every other day (50% of the medium replaced with fresh medium) before neurospheres began to form.
Preparation of HF-NCSCs HF-NCSCs cultures were prepared according to SieberBlum’s protocol [12] with slight modification. In brief, whiskers were dissected from the whisker pad and follicles were detached from connective tissue and rinsed in PBS. The follicle was cut at the level above the cavernous sinus and then below the skin. The capsule was cut longitudinally with a pair of eye scissors; the bulge was rolled out of the capsule, rinsed three times in PBS, and placed into 48-well plate with DMEM/F12 with 10% FBS and FGF (10 ng/ml). After two to three days explants had adhered to the well. After 6 days, when cells started to migrate from the explants, the cells were suspended by trypsinization for 10 min and bulge explants were removed. Cells were plated at 1.5–2×105 cells/ml. The cells adhered within one hour. After that cells were cultured for 1 additional week.
Islet isolation Pancreatic islets were isolated from C57BL/6-b-actin DxRed or from C57BL mice by collagenase digestion method, as described previously [13]. The islets were cultured free-floating for 3–5 days, with approximately 150 islets in each culture dish, in 5 ml culture medium, RPMI 1640 (Sigma-Aldrich, Irvine, UK) supplemented with L-glutamine (Sigma-Aldrich), benzylpenicillin (100 U/ml; Roche Diagnostics Scandinavia, Bromma, Sweden), streptomycin (0.1 mg/ml; Sigma-Aldrich) and 10% (v/v) fetal calf serum (SigmaAldrich). The medium was changed every day.
Coculture experiments with bNCSCs and HF-NCSCs Two types of cocultures were used: (i) islets harvested from RFP mice and bNCSCs (in neurosphere form) from eGFP mice, or HF-NCSCs from eGFP mice; (ii) islets harvested from C57BL mice and bNCSCs (eGFP) as single cell suspension or HF-NCSCs (eGFP) as single cell suspension. Groups used for the study were: (A) bNCSCs alone; (B) HF-NCSCs alone; (C) islets alone; (D) bNCSCs and islets together in equal proportions; and (E) HF-NCSCs and islets together in equal proportions. Groups A-D were seeded in 4-well International Journal of Neuroscience
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Table 1. Primary antibody
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Anti pan-cadherin antibody mouse monoclonal, Abcam 6528, 1:100 Anti-human Ki67 antibody mouse monoclonal, BD Pharmingen 556003, 1:200 Insulin antibody guinea pig polyclonal, Reactionlab Sverige AB, 20-IP30, 1:400
dishes on poly-D-Lysine (PDL) (50 μg/ml) and laminin (20 mg/ml) (Sigma-Aldrich) coated coverslips for seven days. Cells were cultured in Differentiation medium consisting of 50% DMEM/F-12 medium (Invitrogen cat no. 31330-038) and 50% Neurobasal (Invitrogen cat no. 10888022) supplemented with B27 and N2. In cocultures with islets (C57BL) when dissociated bNCSCs and HF-NCSCs were used, a drop containing 1×105 cells was placed at a distance of 700μm from the islets. To visualize live cells originating from eGFP NCSCs (green) and RFP (red) or C57BL islets the images were taken from day 1 to day 7 of the differentiation assay and the character of cell migration was analyzed. The distance between migrated cells (bNCSCs and HF-NCSCs) and the surface of cocultured islets during this period of time was measured and the data were analyzed using parametric one-way analysis of variance (ANOVA). In all analyses, p < 0.05 was selected as the threshold for statistical significance. In the end of the coculture experiment (one week) the coverslips were fixed with 4% phosphate buffered formalin, rinsed with PBS and processed for immunohistochemistry. Antibodies, which were used, are shown in Table 1. Ki67 cells were counted in islets cultured alone, or cocultured with bNCSCs, or with HF-NCSCs. For statistical analysis of possible differences in islet proliferation in different cocultures, the t test was applied.
RNA extraction and real-time PCR Total RNA was extracted from cell cultures using TRIzol reagent (Invitrogen, Life Technologies) and treated with TurboDNAfree (Ambion, Life Technologies) to remove any contaminating DNA. The first strand cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Ferments, Thermo Fisher Scientific) and oligo(dT)-oligonucleotides according to the manufacturer’s protocol. Real-time quantitative PCR (qPCR) was performed using the StepOnePlus Real-Time PCR Systems (Applied Biosystems, Life Technologies). qPCR was done using the SYBR Green PCR Master Mix (Evrogene) in a total volume of 20 μL. The following primers were used: E-Cadherin (108 bp), sense: 5 -GACGCTGAGCATGTGAAGAA-3 and antisense: 5 -GTCTAACAGGACCAGGAGAAGA-3 ; C
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Secondary antibody Alexa flour 594 goat anti-mouse, Invitrogen A11032, 1:1000 Cy3 donkey anti-mouse, Jackson ImmunoResearch, 715-165-151, 1:500 Alexa flour 633 goat anti-guinea pig, Invitrogen A21105, 1:1000
N-Cadherin (72 bp): sense: 5 -GGTGGAGGAGAAGA AGACCAG-3 and antisense: 5 -GGCATCAGGCTC CACAGT-3 ; GAPDH (123 bp): sense: 5 -AGG TCGGTGTGAACGGATTTG-3 and antisense: 5 TGTAGACCATGTAGTTGAGGTCA-3 . The following qPCR amplification protocols were used: first denature at 95◦ C for 5◦ min, subsequent 40 cycles – denature at 95◦ C for 15◦ s, anneal and extend at 60◦ C for 1 min. All samples were run in triplicate. The specificity of the amplification reactions was confirmed by melting curve analysis and agarose gel electrophoresis. The threshold cycle (Ct ) value for each gene was normalized to the Ct value for GAPDH.
Results Migration assay We first analyzed coculture where NCSCs and islets were harvested from fluorescent mice. bNCSCs and HF-NCSCs were placed as a drop of dissociated cells at a distance of 200◦ μm from the islets and NCSC migration was analyzed under a fluorescent microscope. We found directed migration of bNCSCs toward islets and detected bNCSCs in the vicinity of islets from the first experimental day (Figure 1A) whereas HF-NCSCs were randomly dispersed throughout the culture without migration towards islet cells (Figure 1B). After 2 days in coculture with bNCSCs, we registered mutual migration of bNCSCs and islet cells (Figure 1C). In HFNCSC-islet cocultures, despite active migration of both cell types, they did not display mutually directed migration (Figure 1D). During the following days the association between islet cells and bNCSCs increased and after one week in coculture, islets were completely surrounded by bNCSCs, whereas contacts between islet cells and HF-NCSCs remained rare (Figure 1E and F). Since HF-NCSCs do not form neurospheres we had to use dissociated HF-NCSCs in our islet coculture experiments. For comparative purposes, we therefore performed additional coculture migration assay experiments with dissociated bNCSCs (Figure 2). A drop of dissociated HF-NCSCs and bNCSCs was placed at a certain distance from the islets (Material and methods),
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our previous findings [1–4], whereas no proliferation was detected in islet alone-cultures or in HF-NCSCislet cocultures (Figure 3).
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Cadherin in cocultures After one week of coculture, the coverslips were stained with Hoechst and anti-pan cadherin antibodies and analyzed with a confocal microscope. We found expression of cadherin in islets cultured with bNCSCs (Figure 4A) and HF-NCSCs (Figure 4B), but specific cadherin connections were detected only between bNCSCs and islet cells (Figure 4A). Quantitative real-time PCR showed a high relative level of expression of E-cadherin in islets but not in bNCSCs or HF-NCSCs (Figure 4C). N-cadherin, on the other hand, was found in abundance in bNCSCs and their cocultures, but not in HFNCSCs nor in their islet cocultures (Figure 4D). The high expression of N-cadherin in bNCSC-islet cocultures correlated with increased expression of β-catenin in bNCSC-islet cocultures compared to HF-NCSCsislet cocultures (Figure 4E). Figure 1. One-day coculture of bNCSCs (A, green) and of HF-
NCSCs (B, green) with pancreatic islets (red). The bNCSCs reached the surface of islets (A, arrowheads), whereas the HFNCSCs settled irregularly on the coverslip. Two-days coculture of bNCSCs (C, green) and HF-NCSCs (D, green) with pancreatic islets (red). Extensive mutual migration of bNCSCs and islet cells but not of HF-NCSCs was seen. One-week coculture of bNCSCs (E, green) and HF-NCSCs (F, green) with pancreatic islets (red). The surface of islets in coculture with bNCSCs was completely covered with bNCSCs. In HF-NCSC-islet cocultures islet cells migrated extensively, but HF-NCSCs were rarely in close contact with islet cells. Scale bar = 100 μm.
the migration of NCSCs towards the islets was monitored in live cultures, and the distance between migrating cells and the islets was measured. We found that bNCSCs reached the islets in five days, whereas only occasional HF-NCSCs reached the islets at the end of the experiment (Graph 1). For each group three independent experiments were analyzed. To further analyze interaction between bNCSCs and islets, and between HF-NCSCs and islets, respectively, we placed bNCSCs or HF-NCSCs in direct vicinity to islets. After one week of coculture the coverslips were fixed as described above and processed for immunohistochemistry with cadherin antibodies (see below).
Proliferation assay After one week of coculture, coverslips were fixed and stained with Ki67 antibodies. In the presence of bNCSCs, β-cells proliferated extensively in accordance with
Discussion Numerous experimental studies have shown that transplanted stem cells are able to exert protection of diseased or injured tissues and thereby counteract cell loss and promote tissue repair. Thus, mesenchymal stem cells have been shown to harbor these properties and have emerged as an attractive source also because of their potential for clinical autologous transplantation. Our previous studies show that bNCSCs have unique properties in the context of stem cell based treatment of type 1 diabetes because of their combined ability to induce β-cell proliferation and promote β-cell survival and function in vitro [3] and in vivo [1] when cotransplanted with pancreatic islets and thus be of potential relevance for regenerative medicine. Our in vitro studies showed that these beneficial effects require direct contacts between bNCSCs and β-cells [3, 11], which develop as a result of mutual migration of both cell types [4]. However, generation of bNCSCs requires tissue collection from embryos which makes this source of stem cells unsuitable for human allogeneic transplantation. An attractive alternative source of NCSCs for autologous transplantation is HF-NCSCs. Since the beneficial effects of bNCSCs on β-cells were based on the ability of these cells to mutually migrate and establish direct contacts, we thus tested here if HF-NCSCs have a similar capacity to establish direct contacts with islet cells in vitro. We find that both HF-NCSCs and islet cells migrated extensively in coculture, but without mutual attraction and without formation of cadherin connections International Journal of Neuroscience
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Neural crest stem cells on pancreatic islets
Figure 2. Overview of migration assay in cocultures of bNCSCs with islets (left) and HF-NCSCs with islets (right). Drop of 1×104 NCSCs
and 10 islets were placed on different sides of the coverslips and images were taken during one week of coculture. The distance between cells was measured. After 5 days of coculture the bNCSCs reached islets (B, C) whereas HF-NCSCs did not. Scale bar = 100 μm. C
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Figure 3. Coculture of HF-NCSCs (HF) with islets did not induce proliferation of β-cells (A, C) whereas coculture of bNCSCs (bC) with islets induced β-cell proliferation (B, C). Insulin (yellow) visualizes β-cells, Ki67 (red) labels proliferating cells, and nuclei are stained with DAPI (blue). Scale bar = 50 μm.
Figure 4. Confocal images of labeling with Hoechst (blue) and pan-cadherin (red) reveals the presence of cadherin connections
between islet cells and cocultured bNCSCs (A, connections green; arrows), whereas such connections are absent between islets and cocultured HF-NCSC (B). Cadherin connections between islet cells were present in both types of cocultures (A, B). Scale bar = 25 μm. Quantitative RT-PCR of E-cadherin (C), N-cadherin (D) and β-catenin expression (E). Relative expression of E-cadherin is high in islets (Is) but low in both bNCSCs (bC) and HF-NCSCs (HF) (C). Relative expression of N-cadherin is high in bNCSCs, islets as well as in islet-bNCSC cocultures but is low in HF-NCSCs and islet-HF-NCSC co-cultures. Relative β-catenin expression (E) coincides with high expression of N-cadherin in bNCSCs alone and in bNCSC-islet cocultures (D, E).
International Journal of Neuroscience
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Neural crest stem cells on pancreatic islets
between each other. Specifically, N-cadherin is present in high levels in bNCSCs alone as well as in coculture, whereas it is nearly absent in HF-NCSCs and their cocultures with islets. The cadherin expression in bNCSCs and β-cell junctions as well as at the sites of their interactions [11], and its correlation with cadherin/catenin expression, indicate that cadherin/β-catenin signaling may be crucial for the positive effects of bNCSC induced proliferation of β-cells. This is also supported by our recent finding that β-cell protection from apoptosis in the presence of bNCSCs is eliminated when N-cadherin connections are interrupted [11]. N-cadherin belongs to the family of classical cadherins and mediates strong cell-cell adhesions and appears to be activated in part by the early neuronal transcription factor Sox2 [14]. Furthermore, N-cadherin also influences neural cell directional migration as well as maintaining progenitor pools [14]. Interestingly, Ncadherin is important for the functional dynamics of β-cells [15], as well as their viability [16]. Thus the N-cadherin expression patterns in this study indicate that islets cultured together with bNCSCs retain certain characteristics necessary for insulin secretion [15]. In contrast, HF-NCSCs, although having a related developmental origin, do not spontaneously display the same properties as bNCSCs and the expression level of Ncadherin in HF-NCSCs as well as HF-NCSC-islet cocultures remains low. We have also found increased expression level of βcatenin in islet-bNCSC cocultures in comparison to islet-only cultures. Interestingly, Wnt/β-catenin signaling has been implicated in both β-cell proliferation and growth [17]. The canonical Wnt-signaling through the Wnt/β-catenin-pathway is initiated after binding of the Wnt-ligand to the Frizzled-receptor. This leads to stabilization and subsequent nuclear relocalization of βcatenin, which allows β-catenin to influence different transcriptional processes. In contrast, without Frizzledactivation, β-catenin in Wnt-responsive cells is targeted for proteasomal degradation [18]. Possibly, the increased expression level of β-catenin mRNA could thus reflect an increased synthesis of β-catenin, suggesting activation of the Wnt/β-catenin pathway known to influence β-cell proliferation. Although our data show that untreated HF-NCSCs have no positive effect on β-cells, there are other sources of NCSCs which can be isolated from adult mammals, besides HFs. Thus NCSCs from the palate [19], the inferior turbine of the nasal cavity [20] and the periodontal ligament [21–23], are easily accessible in humans, and thus of particular potential for therapeutic purposes. It will be important to determine whether NCSCs from one or more of these sources are comparable with bNCSCs in terms of ability to induce proliferation and protection of β-cells. C
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In summary, our findings highlight the important role of cadherins in mediating contacts between pancreatic β-cells and NCSCs. The discovery that cadherin is present in bNCSCs and β-cell junctions as well as at the sites of their interactions [11], implies that cadherin signaling may be crucial for the positive effects of bNCSC-mediated protection and induced proliferation of β-cells. Thus, induction of the cadherin signaling pathway in HF-NCSCs may confer to these cells β-cell repair properties of translational applicability.
Conclusion We find that HF-NCSCs do not make the direct contacts with β-cells, which have been found to be crucial for the beneficial effects of bNCSCs on β-cells. Thus, unmodified HF-NCSCs appear to be inadequate for the potential translational purpose of β-cell repair. We propose that activation of cadherin expression in HFNCSC would enable these cells to make direct contacts with pancreatic islets and thereby acquire β-cell protective properties analogous to those of bNCSCs.
Acknowledgments We thank Ninnie Abrahamsson for help with bNCSC cultures.
Declaration of Interest No conflict of interest declared. The authors alone are responsible for the content and writing of this paper. This work was supported by Swedish Research Council, proj no. 20716, Stiftelsen Olle Engkvist Byggmastare, Signhild Engkvist’s Stiftelse and the Swedish Institute’s Visby program Dnr 00613/2011.
References 1. Olerud J, Kanaykina N, Vasylovska S, et al. Neural crest stem cells increase beta cell proliferation and improve islet function in co-transplanted murine pancreatic islets. Diabetologia 2009;52:2594–601. 2. Ngamjariyawat A, Turpaev K, Welsh N, Kozlova EN. Coculture of insulin producing RIN5AH cells with neural crest stem cells protects partially against cytokine induced cell death. Pancreas 2012;41:490–92. 3. Grouwels G, Vasylovska S, Olerud J, et al. Differentiating neural crest stem cells induce proliferation of cultured rodent islet beta cells. Diabetologia 2012;55:2016–25. 4. Kozlova EN, Jansson L. Differentiation and migration of neural crest stem cells are stimulated by pancreatic islets. Neuroreport 2009;20:833–8. 5. Kulkarni RN, Mizrachi E-B, Ocana AG, Stewart AF. Human βcell proliferation and intracellular signaling: driving in the dark without a road map. Diabetes 2012;61:2205–13.
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A. Kosykh et al. 6. Ryan EA, Bigam D, Shapiro AMJ. Current indications for pancreas or islet transplant. Diabetes Obes Metab 2006;8:1–7. 7. Nekrep N, Wang J, Miyatsuka T, German MS. Signals from the neural crest regulate beta-cell mass in the pancreas. Development 2008;135:2151–60. 8. Hjerling-Leffler J, Marmig`ere F, Heglind M, et al. The boundary cap: a source of neural crest stem cells that generate multiple sensory neuron subtypes. Development 2005;132: 2623–32. 9. Aldskogius H, Berens C, Kanaykina N, et al. Regulation of boundary cap neural crest stem cell differentiation after transplantation. Stem Cells 2009;27:1592–603. 10. Yu H, Kumar SM, Kossenkov AV, et al. Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles. J Invest Dermatol 2010;130: 1227–36. 11. Ngamjariyawat A, Turpaev K, Vasylovska S, et al. Co-culture of neural crest stem cells (NCSC) and insulin producing beta-TC6 cells results in cadherin junctions and protection against cytokine-induced beta-cell death. PlosOne 2013; 17:8 12. Sieber-Blum M, Grim M, Hu YF, Szeder V. Pluripotent neural crest stem cells in the adult hair follicle. Dev Dyn 2004;231:258–69. 13. Andersson, A. Isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. Diabetologia 1978;14:397–404. 14. Paulson AF, Prasad MS, Thuringer AH, Manzerra P. Regulation of cadherin expression in nervous system development. Cell Adh Migr 2013;8(1):18-28.
15. Johansson JK, Voss U, Kesavan G, et al. N-cadherin is dispensable for pancreas development but required for beta-cell granule turnover. Genesis 2010;48:374–81. 16. Parnaud G, Gonelle-Gispert C, Morel P, et al. Cadherin engagement protects human β-cells from apoptosis. Endocrinology 2011;152:4601–9. 17. Figeac F, Uzan B, Faro M, et al. Neonatal growth and regeneration of beta-cells are regulated by the Wnt/beta-catenin signalling in normal and diabetic rats. Am J Physiol Endocrinol Metab 2010;298:E245–56. 18. Gordon MD, Nusse R. Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem 2006;281:22429–33. 19. Widera D, Zander C, Heidbreder M, et al. Adult palatum as a novel source of neural crest-related stem cells. Stem Cells 2009;27:1899–910. 20. Hauser S, Widera D, Qunneis F, et al. Isolation of novel multipotent neural crest-derived stem cells from adult human inferior turbinate. Stem Cells Dev 2012;21:742–56. 21. Techawattanawisal W, Nakahama K, Komaki M, et al. Isolation of multipotent stem cells from adult rat periodontal ligament by neurosphere-forming culture system. Biochem Biophys Res Commun 2007;357:917–23. 22. Widera D, Grimm WD, Moebius JM, et al. Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery. Stem Cells Dev 2007;16:447–60. 23. Pelaez D, Huang CY, Cheung HS. Isolation of pluripotent neural crest-derived stem cells from adult human tissues by connexin-43 enrichment. Stem Cells Dev 2013; 22:2906–14.
International Journal of Neuroscience
Neural crest stem cells from hair follicles and boundary cap have different effects on pancreatic islets in vitro Anastasiia Kosykh,2,∗ Anongnad Ngamjariyawat,1,∗ Svitlana Vasylovska,1,∗ Niclas Konig,1 Carl Trolle,1 Joey Lau,3 Arsen Mikaelyan,2 Michael Panchenko,1 Per-Ola Carlsson,3 Ekaterina Vorotelyak,2,∗ and Elena N Kozlova1
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Department of Neuroscience, Uppsala University Biomedical Center, Uppsala, Sweden; 2 Laboratory of Cell Proliferation, Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia, and 3 Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden Purpose: Neural crest stem cells derived from the boundary cap (bNCSCs), markedly promote survival, proliferation and function of insulin producing β-cells in vitro and in vivo after coculture/transplantation with pancreatic islets [1, 2]. Recently, we have shown that beneficial effects on β-cells require cadherin contacts between bNCSCs and β-cells [3, 4]. Here we investigated whether hair follicle (HF) NCSCs, a potential source for human allogeneic transplantation, exert similar positive effects on β-cells. Materials and Methods: We established cocultures of HF-NCSCs or bNCSCs from mice expressing enhanced green fluorescent protein together with pancreatic islets from DxRed expressing mice or NMRI mice and compared their migration towards islet cells and effect on proliferation of β-cells as well as intracellular relations between NCSCs and islets using qRT-PCR analysis and immunohistochemistry. Results: Whereas both types of NCSCs migrated extensively in the presence of islets, only bNCSCs demonstrated directed migration toward islets, induced β-cell proliferation and increased the presence of cadherin at the junctions between bNCSCs and β-cells. Even in direct contact between β-cells and HF-NCSCs, no cadherin expression was detected. Conclusions: These observations indicate that HF-NCSCs do not confer the same positive effect on β-cells as demonstrated for bNCSCs. Furthermore, these data suggest that induction of cadherin expression by HF-NCSCs may be useful for their ability to support β-cells in coculture and after transplantation. KEYWORDS: Diabetes, migration, cell culture, coculture, intercellular contacts
Introduction Pancreatic β-cells have a low capacity to proliferate [5]. Patients with Type-1 diabetes lose their β-cell mass and transplantation of pancreatic islets is an attractive option to cure this disease. However the survival of islets after transplantation is low and after 5 years only 10% of patients have a sufficient amount of functional β-cells [6]. Thus, methods to improve β-cell survival and function, as well as methods to expand an insufficient β-cell mass, are required. We recently discovered that mouse boundary cap derived neural crest stem cells (bNCSCs) have remarkable effects on β-cell proliferation, survival and funcReceived 7 April 2014; revised 18 July 2014; accepted 28 July 2014 ∗ These authors contributed equally to the paper Correspondence: Elena N Kozlova, Department of Neuroscience, Uppsala University Biomedical Center, Uppsala, Sweden. E-Mail: [email protected] Tel.: +46-18-4714968; Fax: +46-18-511540
tion [1–3]. These findings indicate that NCSCs, which have been shown to regulate β-cell mass during development [7], are potentially useful for coculture with βcells and for cotransplantation with pancreatic islets to induce proliferation of β-cells in vivo after transplantation and improve their survival and function. However, bNCSCs are present only transiently during early embryonic stages [8, 9], and other sources of NCSCs have to be found in order to translate the beneficial effects of bNCSCs to a viable treatment strategy. One of the attractive alternatives is NCSCs from the hair follicle (HF), which can be harvested from the patient and subsequently prepared for autologous transplantation. It was shown previously that the bulge area of HFs contains stem cells with neural crest characteristics [10]. In our in vitro experiments we found that direct contact between β-cells and bNCSCs is crucial for their positive effects on β-cells [3]. We showed that in transwell assays when β-cells and bNCSCs were separated, no β-cell proliferation occurred [3]. Subsequent 1
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experiments, which showed that bNCSCs can protect βcells from cytokine-induced apoptosis, confirmed that this effect requires direct contact between β-cells and bNCSCs and that this effect is mediated by cadherin [2, 11]. Furthermore, these contacts were shown to develop as a result of mutual migration by β-cells and bNCSCs [4]. Here we compare migration behavior of HF-NCSCs and bNCSCs in coculture with pancreatic islets as well as their effect on β-cell proliferation and cadherin expression. Our findings show that HF-NCSCs, despite their excellent capacity to migrate, do not have directed migration toward islets, do not develop direct contacts with islet cells, and hence do not induce β-cell proliferation. Furthermore, cadherin expression in the islet cells was reduced in the coculture with HF-NCSCs whereas this expression remained on a high level in the presence of bNCSCs.
Materials and methods Animals Transgenic heterozygous C57BL/6-b-actin enhanced green fluorescent protein (eGFP) mice (Jackson Laboratories, Bar Harbor, Maine, USA) were used for isolation of bNCSCs and HF-NCSCs. Pancreatic islets were isolated from C57BL/6 mice (M&B, Ry, Denmark) and transgenic heterozygous C57BL/6-b-actin DxRed fluorescent protein (RFP) mice (Jackson Laboratories, Bar Harbor, Maine, USA) and from C57BL mice. All procedures were approved by the Regional Ethical Committee for Research on Animals.
Preparation of bNCSC neurospheres Dorsal root ganglia (DRGs) from 11.5-day-old eGFP mouse embryos were isolated and used for setting up bNCSC cultures as described previously [3, 8, 9]. Briefly, the uterus was removed from the anaesthetized pregnant mouse and placed in cold phosphate-buffered saline (PBS). Embryos were separated, rinsed in PBS, placed in N2 medium and the DRGs were removed and collected in N2 medium under a dissection microscope. Collected DRGs were allowed to settle down before removing the supernatant and adding a Collagenase/Dispase (1 mg/ml) and DNase (0.5 mg/ml) solution in N2 and incubating for 20–30 min in a 37◦ C water bath, followed by rinsing in N2 medium with B27 (1:50) and plating ∼1-2×105 cells/well in a 24-well dish after dissociation. Cells were placed directly into 500μl of Propagation medium: DMEM F-12 medium (Invitrogen cat no. 31330-038) supplemented with B27 (Invitrogen cat no. 17504-044), N2 (Invitrogen cat
no. 17502-048), 20 ng/ml bFGF (Invitrogen cat no. 13256-029) and 20 ng/ml EGF (R&D system cat no. 236-EG). After 12 h, nonadherent cells were removed together with half of the medium before adding up to 250 μl of fresh medium. The medium was then changed every other day (50% of the medium replaced with fresh medium) before neurospheres began to form.
Preparation of HF-NCSCs HF-NCSCs cultures were prepared according to SieberBlum’s protocol [12] with slight modification. In brief, whiskers were dissected from the whisker pad and follicles were detached from connective tissue and rinsed in PBS. The follicle was cut at the level above the cavernous sinus and then below the skin. The capsule was cut longitudinally with a pair of eye scissors; the bulge was rolled out of the capsule, rinsed three times in PBS, and placed into 48-well plate with DMEM/F12 with 10% FBS and FGF (10 ng/ml). After two to three days explants had adhered to the well. After 6 days, when cells started to migrate from the explants, the cells were suspended by trypsinization for 10 min and bulge explants were removed. Cells were plated at 1.5–2×105 cells/ml. The cells adhered within one hour. After that cells were cultured for 1 additional week.
Islet isolation Pancreatic islets were isolated from C57BL/6-b-actin DxRed or from C57BL mice by collagenase digestion method, as described previously [13]. The islets were cultured free-floating for 3–5 days, with approximately 150 islets in each culture dish, in 5 ml culture medium, RPMI 1640 (Sigma-Aldrich, Irvine, UK) supplemented with L-glutamine (Sigma-Aldrich), benzylpenicillin (100 U/ml; Roche Diagnostics Scandinavia, Bromma, Sweden), streptomycin (0.1 mg/ml; Sigma-Aldrich) and 10% (v/v) fetal calf serum (SigmaAldrich). The medium was changed every day.
Coculture experiments with bNCSCs and HF-NCSCs Two types of cocultures were used: (i) islets harvested from RFP mice and bNCSCs (in neurosphere form) from eGFP mice, or HF-NCSCs from eGFP mice; (ii) islets harvested from C57BL mice and bNCSCs (eGFP) as single cell suspension or HF-NCSCs (eGFP) as single cell suspension. Groups used for the study were: (A) bNCSCs alone; (B) HF-NCSCs alone; (C) islets alone; (D) bNCSCs and islets together in equal proportions; and (E) HF-NCSCs and islets together in equal proportions. Groups A-D were seeded in 4-well International Journal of Neuroscience
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Table 1. Primary antibody
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Anti pan-cadherin antibody mouse monoclonal, Abcam 6528, 1:100 Anti-human Ki67 antibody mouse monoclonal, BD Pharmingen 556003, 1:200 Insulin antibody guinea pig polyclonal, Reactionlab Sverige AB, 20-IP30, 1:400
dishes on poly-D-Lysine (PDL) (50 μg/ml) and laminin (20 mg/ml) (Sigma-Aldrich) coated coverslips for seven days. Cells were cultured in Differentiation medium consisting of 50% DMEM/F-12 medium (Invitrogen cat no. 31330-038) and 50% Neurobasal (Invitrogen cat no. 10888022) supplemented with B27 and N2. In cocultures with islets (C57BL) when dissociated bNCSCs and HF-NCSCs were used, a drop containing 1×105 cells was placed at a distance of 700μm from the islets. To visualize live cells originating from eGFP NCSCs (green) and RFP (red) or C57BL islets the images were taken from day 1 to day 7 of the differentiation assay and the character of cell migration was analyzed. The distance between migrated cells (bNCSCs and HF-NCSCs) and the surface of cocultured islets during this period of time was measured and the data were analyzed using parametric one-way analysis of variance (ANOVA). In all analyses, p < 0.05 was selected as the threshold for statistical significance. In the end of the coculture experiment (one week) the coverslips were fixed with 4% phosphate buffered formalin, rinsed with PBS and processed for immunohistochemistry. Antibodies, which were used, are shown in Table 1. Ki67 cells were counted in islets cultured alone, or cocultured with bNCSCs, or with HF-NCSCs. For statistical analysis of possible differences in islet proliferation in different cocultures, the t test was applied.
RNA extraction and real-time PCR Total RNA was extracted from cell cultures using TRIzol reagent (Invitrogen, Life Technologies) and treated with TurboDNAfree (Ambion, Life Technologies) to remove any contaminating DNA. The first strand cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Ferments, Thermo Fisher Scientific) and oligo(dT)-oligonucleotides according to the manufacturer’s protocol. Real-time quantitative PCR (qPCR) was performed using the StepOnePlus Real-Time PCR Systems (Applied Biosystems, Life Technologies). qPCR was done using the SYBR Green PCR Master Mix (Evrogene) in a total volume of 20 μL. The following primers were used: E-Cadherin (108 bp), sense: 5 -GACGCTGAGCATGTGAAGAA-3 and antisense: 5 -GTCTAACAGGACCAGGAGAAGA-3 ; C
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Secondary antibody Alexa flour 594 goat anti-mouse, Invitrogen A11032, 1:1000 Cy3 donkey anti-mouse, Jackson ImmunoResearch, 715-165-151, 1:500 Alexa flour 633 goat anti-guinea pig, Invitrogen A21105, 1:1000
N-Cadherin (72 bp): sense: 5 -GGTGGAGGAGAAGA AGACCAG-3 and antisense: 5 -GGCATCAGGCTC CACAGT-3 ; GAPDH (123 bp): sense: 5 -AGG TCGGTGTGAACGGATTTG-3 and antisense: 5 TGTAGACCATGTAGTTGAGGTCA-3 . The following qPCR amplification protocols were used: first denature at 95◦ C for 5◦ min, subsequent 40 cycles – denature at 95◦ C for 15◦ s, anneal and extend at 60◦ C for 1 min. All samples were run in triplicate. The specificity of the amplification reactions was confirmed by melting curve analysis and agarose gel electrophoresis. The threshold cycle (Ct ) value for each gene was normalized to the Ct value for GAPDH.
Results Migration assay We first analyzed coculture where NCSCs and islets were harvested from fluorescent mice. bNCSCs and HF-NCSCs were placed as a drop of dissociated cells at a distance of 200◦ μm from the islets and NCSC migration was analyzed under a fluorescent microscope. We found directed migration of bNCSCs toward islets and detected bNCSCs in the vicinity of islets from the first experimental day (Figure 1A) whereas HF-NCSCs were randomly dispersed throughout the culture without migration towards islet cells (Figure 1B). After 2 days in coculture with bNCSCs, we registered mutual migration of bNCSCs and islet cells (Figure 1C). In HFNCSC-islet cocultures, despite active migration of both cell types, they did not display mutually directed migration (Figure 1D). During the following days the association between islet cells and bNCSCs increased and after one week in coculture, islets were completely surrounded by bNCSCs, whereas contacts between islet cells and HF-NCSCs remained rare (Figure 1E and F). Since HF-NCSCs do not form neurospheres we had to use dissociated HF-NCSCs in our islet coculture experiments. For comparative purposes, we therefore performed additional coculture migration assay experiments with dissociated bNCSCs (Figure 2). A drop of dissociated HF-NCSCs and bNCSCs was placed at a certain distance from the islets (Material and methods),
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our previous findings [1–4], whereas no proliferation was detected in islet alone-cultures or in HF-NCSCislet cocultures (Figure 3).
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Cadherin in cocultures After one week of coculture, the coverslips were stained with Hoechst and anti-pan cadherin antibodies and analyzed with a confocal microscope. We found expression of cadherin in islets cultured with bNCSCs (Figure 4A) and HF-NCSCs (Figure 4B), but specific cadherin connections were detected only between bNCSCs and islet cells (Figure 4A). Quantitative real-time PCR showed a high relative level of expression of E-cadherin in islets but not in bNCSCs or HF-NCSCs (Figure 4C). N-cadherin, on the other hand, was found in abundance in bNCSCs and their cocultures, but not in HFNCSCs nor in their islet cocultures (Figure 4D). The high expression of N-cadherin in bNCSC-islet cocultures correlated with increased expression of β-catenin in bNCSC-islet cocultures compared to HF-NCSCsislet cocultures (Figure 4E). Figure 1. One-day coculture of bNCSCs (A, green) and of HF-
NCSCs (B, green) with pancreatic islets (red). The bNCSCs reached the surface of islets (A, arrowheads), whereas the HFNCSCs settled irregularly on the coverslip. Two-days coculture of bNCSCs (C, green) and HF-NCSCs (D, green) with pancreatic islets (red). Extensive mutual migration of bNCSCs and islet cells but not of HF-NCSCs was seen. One-week coculture of bNCSCs (E, green) and HF-NCSCs (F, green) with pancreatic islets (red). The surface of islets in coculture with bNCSCs was completely covered with bNCSCs. In HF-NCSC-islet cocultures islet cells migrated extensively, but HF-NCSCs were rarely in close contact with islet cells. Scale bar = 100 μm.
the migration of NCSCs towards the islets was monitored in live cultures, and the distance between migrating cells and the islets was measured. We found that bNCSCs reached the islets in five days, whereas only occasional HF-NCSCs reached the islets at the end of the experiment (Graph 1). For each group three independent experiments were analyzed. To further analyze interaction between bNCSCs and islets, and between HF-NCSCs and islets, respectively, we placed bNCSCs or HF-NCSCs in direct vicinity to islets. After one week of coculture the coverslips were fixed as described above and processed for immunohistochemistry with cadherin antibodies (see below).
Proliferation assay After one week of coculture, coverslips were fixed and stained with Ki67 antibodies. In the presence of bNCSCs, β-cells proliferated extensively in accordance with
Discussion Numerous experimental studies have shown that transplanted stem cells are able to exert protection of diseased or injured tissues and thereby counteract cell loss and promote tissue repair. Thus, mesenchymal stem cells have been shown to harbor these properties and have emerged as an attractive source also because of their potential for clinical autologous transplantation. Our previous studies show that bNCSCs have unique properties in the context of stem cell based treatment of type 1 diabetes because of their combined ability to induce β-cell proliferation and promote β-cell survival and function in vitro [3] and in vivo [1] when cotransplanted with pancreatic islets and thus be of potential relevance for regenerative medicine. Our in vitro studies showed that these beneficial effects require direct contacts between bNCSCs and β-cells [3, 11], which develop as a result of mutual migration of both cell types [4]. However, generation of bNCSCs requires tissue collection from embryos which makes this source of stem cells unsuitable for human allogeneic transplantation. An attractive alternative source of NCSCs for autologous transplantation is HF-NCSCs. Since the beneficial effects of bNCSCs on β-cells were based on the ability of these cells to mutually migrate and establish direct contacts, we thus tested here if HF-NCSCs have a similar capacity to establish direct contacts with islet cells in vitro. We find that both HF-NCSCs and islet cells migrated extensively in coculture, but without mutual attraction and without formation of cadherin connections International Journal of Neuroscience
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Figure 2. Overview of migration assay in cocultures of bNCSCs with islets (left) and HF-NCSCs with islets (right). Drop of 1×104 NCSCs
and 10 islets were placed on different sides of the coverslips and images were taken during one week of coculture. The distance between cells was measured. After 5 days of coculture the bNCSCs reached islets (B, C) whereas HF-NCSCs did not. Scale bar = 100 μm. C
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Figure 3. Coculture of HF-NCSCs (HF) with islets did not induce proliferation of β-cells (A, C) whereas coculture of bNCSCs (bC) with islets induced β-cell proliferation (B, C). Insulin (yellow) visualizes β-cells, Ki67 (red) labels proliferating cells, and nuclei are stained with DAPI (blue). Scale bar = 50 μm.
Figure 4. Confocal images of labeling with Hoechst (blue) and pan-cadherin (red) reveals the presence of cadherin connections
between islet cells and cocultured bNCSCs (A, connections green; arrows), whereas such connections are absent between islets and cocultured HF-NCSC (B). Cadherin connections between islet cells were present in both types of cocultures (A, B). Scale bar = 25 μm. Quantitative RT-PCR of E-cadherin (C), N-cadherin (D) and β-catenin expression (E). Relative expression of E-cadherin is high in islets (Is) but low in both bNCSCs (bC) and HF-NCSCs (HF) (C). Relative expression of N-cadherin is high in bNCSCs, islets as well as in islet-bNCSC cocultures but is low in HF-NCSCs and islet-HF-NCSC co-cultures. Relative β-catenin expression (E) coincides with high expression of N-cadherin in bNCSCs alone and in bNCSC-islet cocultures (D, E).
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between each other. Specifically, N-cadherin is present in high levels in bNCSCs alone as well as in coculture, whereas it is nearly absent in HF-NCSCs and their cocultures with islets. The cadherin expression in bNCSCs and β-cell junctions as well as at the sites of their interactions [11], and its correlation with cadherin/catenin expression, indicate that cadherin/β-catenin signaling may be crucial for the positive effects of bNCSC induced proliferation of β-cells. This is also supported by our recent finding that β-cell protection from apoptosis in the presence of bNCSCs is eliminated when N-cadherin connections are interrupted [11]. N-cadherin belongs to the family of classical cadherins and mediates strong cell-cell adhesions and appears to be activated in part by the early neuronal transcription factor Sox2 [14]. Furthermore, N-cadherin also influences neural cell directional migration as well as maintaining progenitor pools [14]. Interestingly, Ncadherin is important for the functional dynamics of β-cells [15], as well as their viability [16]. Thus the N-cadherin expression patterns in this study indicate that islets cultured together with bNCSCs retain certain characteristics necessary for insulin secretion [15]. In contrast, HF-NCSCs, although having a related developmental origin, do not spontaneously display the same properties as bNCSCs and the expression level of Ncadherin in HF-NCSCs as well as HF-NCSC-islet cocultures remains low. We have also found increased expression level of βcatenin in islet-bNCSC cocultures in comparison to islet-only cultures. Interestingly, Wnt/β-catenin signaling has been implicated in both β-cell proliferation and growth [17]. The canonical Wnt-signaling through the Wnt/β-catenin-pathway is initiated after binding of the Wnt-ligand to the Frizzled-receptor. This leads to stabilization and subsequent nuclear relocalization of βcatenin, which allows β-catenin to influence different transcriptional processes. In contrast, without Frizzledactivation, β-catenin in Wnt-responsive cells is targeted for proteasomal degradation [18]. Possibly, the increased expression level of β-catenin mRNA could thus reflect an increased synthesis of β-catenin, suggesting activation of the Wnt/β-catenin pathway known to influence β-cell proliferation. Although our data show that untreated HF-NCSCs have no positive effect on β-cells, there are other sources of NCSCs which can be isolated from adult mammals, besides HFs. Thus NCSCs from the palate [19], the inferior turbine of the nasal cavity [20] and the periodontal ligament [21–23], are easily accessible in humans, and thus of particular potential for therapeutic purposes. It will be important to determine whether NCSCs from one or more of these sources are comparable with bNCSCs in terms of ability to induce proliferation and protection of β-cells. C
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In summary, our findings highlight the important role of cadherins in mediating contacts between pancreatic β-cells and NCSCs. The discovery that cadherin is present in bNCSCs and β-cell junctions as well as at the sites of their interactions [11], implies that cadherin signaling may be crucial for the positive effects of bNCSC-mediated protection and induced proliferation of β-cells. Thus, induction of the cadherin signaling pathway in HF-NCSCs may confer to these cells β-cell repair properties of translational applicability.
Conclusion We find that HF-NCSCs do not make the direct contacts with β-cells, which have been found to be crucial for the beneficial effects of bNCSCs on β-cells. Thus, unmodified HF-NCSCs appear to be inadequate for the potential translational purpose of β-cell repair. We propose that activation of cadherin expression in HFNCSC would enable these cells to make direct contacts with pancreatic islets and thereby acquire β-cell protective properties analogous to those of bNCSCs.
Acknowledgments We thank Ninnie Abrahamsson for help with bNCSC cultures.
Declaration of Interest No conflict of interest declared. The authors alone are responsible for the content and writing of this paper. This work was supported by Swedish Research Council, proj no. 20716, Stiftelsen Olle Engkvist Byggmastare, Signhild Engkvist’s Stiftelse and the Swedish Institute’s Visby program Dnr 00613/2011.
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