JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH J Tissue Eng Regen Med (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1927

ARTICLE

Differentiation of P19 embryonal carcinoma stem cells into insulin-producing cells promoted by pancreas-conditioned medium Akram Mansouri1, Fariba Esmaeili2,3*, Azadeh Nejatpour4, Fariba Houshmand5, Leila Shabani1,2 and Esmaeil Ebrahimie6,7 1

Department of Biology, Faculty of Basic Sciences, Shahrekord University, Iran Research Institute of Biotechnology, Shahrekord University, Iran 3 Department of Biology, Faculty of Basic Sciences, University of Isfahan, Iran 4 Shahrekord Branch, Islamic Azad University, Shahrekord, Iran 5 Department of Physiology, Faculty of Medical Sciences, Shahrekord University of Medical Sciences, Iran 6 Institute of Biotechnology, Shiraz University, Shiraz, Iran 7 School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, Australia 2

Abstract The ability of embryonal carcinoma )EC (stem cells to generate insulin-producing cells (IPCs) is still unknown. We examined the trophic effects of pancreas-conditioned medium (PCM) on in vitro production of IPCs. Initially, P19 EC cells were characterized by the expression of stem cell markers, Oct3/4, Sox-2 and Nanog. To direct differentiation, P19-derived embryoid bodies (EBs) were induced by selection of nestin-positive cells and treatment with different concentrations of PCM. Morphological studies documented the presence of islet-like cell IPCs clusters. The differentiated cells were immunoreactive for β cell-specific proteins, including insulin, proinsulin, C-peptide and insulin receptor-β. The expression of genes related to pancreatic β cell development and function (PDX-1, INS1, INS2, EP300 and CREB1) was confirmed by qPCR. During differentiation, the expression of EP300 and CREB1 increased by 2.5 and 3.1 times, respectively. In contrast, a sharp decrease in the expression of Oct3/4, Sox-2 and Nanog by 4, 1.5 and 1.5 times, respectively, was observed. The differentiated cells were functionally active, synthesizing and secreting insulin in a glucose-regulated manner. Network prediction highlighted crosstalk between PDX-1 transcription factor and INS2 ligand in IPC generation and revealed positive regulatory effects of EP300, CREB1, PPARA, EGR, KIT, GLP1R, and PKT2 on activation of PDX-1 and INS2. This is the first report of the induction of IPC differentiation from EC cells by using neonate mouse PCM. Since P19 EC cells are widely available, easily cultured without feeders and do not require special growth conditions, they would provide a valuable tool for studying pancreatic β cell differentiation and development. Copyright © 2014 John Wiley & Sons, Ltd. Received 25 September 2013; Revised 25 April 2014; Accepted 5 May 2014

Keywords conditioned medium; embryonal carcinoma cells; insulin-producing cells; pancreatic β cell differentiation; PDX-1; regulatory network

1. Introduction Impaired function of pancreatic islets is the major factor mediating the progression of type 1 and type 2 diabetes.

*Correspondence to: Fariba Esmaeili, Department of Biology, Faculty of Basic Sciences, University of Isfahan, PO Box 8174673441, Isfahan, Iran. E-mail: [email protected] Copyright © 2014 John Wiley & Sons, Ltd.

Production of differentiated functional cells from stem cells holds great promise for treatment of various celldegenerative diseases, including diabetes. Studies have shown that embryonic stem (ES) cells (Hori et al., 2002), pancreatic ductal cells (Bonner-Weir et al., 2000), hepatic stem cells (Liu et al., 2013), neural progenitor cells (Hori et al., 2005), bone marrow-derived cells (Zhang et al., 2009) and umbilical cord blood cells (Boroujeni and Aleyasin, 2013) may be possible sources from which to

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derive transplantable insulin-producing cells (IPCs). However, the ability of embryonal carcinoma (EC) cells to generate IPCs remains unknown. EC and ES cells are developmentally pluripotent cells which can differentiate into all cell types under the appropriate conditions (Chaerkady et al., 2010). P19 cells are murine EC cells that have been used as a model system for studying early embryonic development and differentiation. Like other EC lines, P19 cells are immortal, proliferate rapidly in culture (Rudnicki and McBurney, 1987) and have a normal karyotype (McBurney and Rogers, 1982). Various treatments cause P19 cultures to differentiate into derivatives of all the three germ layers (MacPherson and McBurney, 1995). In the present study, using this cell line, we examined the effect of neonate mouse pancreas-conditioned medium (PCM) on in vitro differentiation of P19 EC cells into IPCs. During embryogenesis, several growth and transcription factors are involved in pancreas development and β cell differentiation (Ackermann and Gannon, 2007; Jennings et al., 2013). A number of studies have shown that conditioned medium from pancreas culture contains soluble factors that are necessary for pancreatic development. Otonkoski et al. (1996) demonstrated that the culture of human pancreatic epithelial cells in conditioned media from pancreatic fibroblasts stimulated the formation of islet-like cell clusters as well as β cell proliferation. They indicated that hepatocyte growth factor/scatter factor (HGF/SF) secreted by fetal fibroblasts is an important physiological growth factor for human pancreatic β cells (Otonkoski et al., 1996). It was reported previously that pancreatic duct epithelial cells appear to secrete soluble factors that exert proliferative effects on fully mature islet cells (Metrakos et al., 1993). Moreover, application of pancreatic duct-conditioned medium (DCM) to islet cells showed that DCM could promote islet cell survival. This effect appears to be mediated in a paracrine manner by the release of insulinlike growth factor-II (IGF-II) from the duct epithelial cells (Ilieva et al., 1999). In another study, a mouse ES cell line was cultured in low serum concentrations with the addition of conditioned medium from pancreatic buds. Induction by fetal soluble factors of ES cell differentiation into IPCs was observed (Vaca et al., 2006). More recently, Johansson et al. (2009) reported that rat islet endotheliumconditioned culture medium (ECCM) supports pancreatic β cell function; interestingly, both insulin release and total insulin content were increased in the islets cultured in ECCM. Mouse ES cells were cultured using MIN-6 β cell conditioned medium; the data indicated that MIN-6 β cells secreted factors that promoted differentiation of mouse ES cells towards a pancreatic fate (Uroic et al., 2010). Talavera-Adame et al. (2011) found that the differentiation of mouse EBs into pancreatic endocrine progenitors and insulin-producing β-like cells was enhanced by endothelial cell conditioned media; they detected upregulation of PDX-1, Ngn3, Nkx6.1, INS1, INS2, amylin, SUR1, GKS and amylase as well as down-regulation of SST in treated embryoid bodies (EBs). The aim of this study was to investigate whether the factors secreted by neonate pancreatic cells induce IPCs Copyright © 2014 John Wiley & Sons, Ltd.

differentiation during in vitro culture of EC cells. To achieve this, we developed a culture system using an EC cell line, P19, cultured in PCM to monitor the differentiation of these cells into pancreatic β cells. In addition, to provide a clear image of underlying regulatory mechanisms supporting this differentiation, we built gene interaction networks governing the in vitro differentiation of IPCs, based on overexpressed genes and available biological information.

2. Materials and methods 2.1. Culture of the mouse neonate pancreas and preparation of PCM BALB/c pregnant mice were obtained from Azad University of Shahrekord (Iran) and kept under standard housing conditions according to the Ethics Committee of the Medical Sciences School, and 1–2 week-old newborn mice were used. The stomach, pancreas and a small portion of the intestine were dissected together. The whole pancreas was carefully removed from the digestive tracts, minced into 1 mm pieces, immersed in 0.25% trypsin/1 mM EDTA solution and then centrifuged at 200 × g for 2 min. The pancreatic cells were then cultured in RPMI1640 medium (Gibco, cat. no. 306-00303) supplemented with 10% fetal bovine serum (FBS; Gibco, cat. no. 10270-106), penicillin G (50 μg/ml; Sigma, St. Louis, MO, USA, cat. no. P3032) and streptomycin (50 μg/ml; Sigma, cat. no. S1277). Cultures were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The medium was replaced every 48 h. Dithizone (DTZ; diphenylthiocarbozone; Merck, cat. no. DX2370-3) staining was used to confirm the culture of pancreas tissue. Pancreatic cells were passaged at 80% confluence. Conditioned medium was then collected, pooled and stored at –70 ºC. Before use, the conditioned medium was thawed, centrifuged at 5000 × g for 10–15 min at 4 °C and filtered using a 0.2 μm filter (Grainger, cat. no. 12 K960).

2.2. Cell culture and differentiation induction protocol The experiments reported here were carried out with the P19 line of murine EC stem cells. Outline of differentiation protocols is summarized in Figure 1. Undifferentiated P19 cells were grown in minimum essential medium (α-MEM; Gibco-BRL, Carlsbad, CA, USA,, cat. no. 11900073) supplemented with 15% FBS, penicillin 50 μg/ml and streptomycin 50 μg/ml (stage 1). The cells were kept in a humidified cell culture incubator at 37 °C under 5% CO2 with close control of pH. To direct differentiation, cells were grown in suspension for 4 days in non-adherent Petri dishes to induce the formation of embryoid bodies (EBs; stage 2). The EBs were then cultured on 0.1% gelatin-coated Petri dishes containing coverslip in α-MEM supplemented with 3% FBS for an additional 4 days and J Tissue Eng Regen Med (2014) DOI: 10.1002/term

Differentiation of P19 carcinoma stem cells into insulin-producing cells

Figure 1. Schematic representation of the five-stage protocol for differentiation of insulin-producing cells (IPCs) from P19 cells, based on the selection of nestin-positive progenitors. (Right panel) Cell morphology at different stages: EC, embryonal carcinoma; EBs, embryoid bodies; ITSFn, insulin–transferrin–sodium selenite; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; PCM, pancreas-conditioned medium

induced based on the selection of nestin-positive cells (Lumelsky et al., 2001) (stage 3). To select nestin-positive cells, the medium was replaced with serum-free medium including Dulbecco’s modified Eagle’s medium/F12 nutrient mixture (DMEM/F12; Sigma, cat. no. D8900) and 1 mg/ml insulin–transferrin–sodium selenite (ITSFn) media supplement (Sigma, cat. no. I1884). After 4–7 days, the cultures were trypsinized and passed onto 0.1% gelatin-coated six-well plates containing coverslips and grown in N2 medium (Stem Cell Technologies, cat. no. 07152) supplemented with B27 (Invitrogen, cat. no. 17504-044), 5 μg/ml insulin, 10 ng/ml human basic fibroblast growth factor (bFGF; Sigma, cat. no. F0291) and 10 ng/ml human epidermal growth factor (EGF; Sigma, cat. no. E9644) (stage 4) to expand the progenitor cells. After 6–7 days, bFGF and EGF were withdrawn and 10 mM nicotinamide (Sigma, cat. no. N0636) was added to the medium for the next 7–14 days (stage 5). In order to induce the differentiation of cells into IPCs, different concentrations of PCM (25%, 50%, 75% and 100%) were added in stage 5. The medium was changed every 2 days. Cells cultured in medium with no PCM were considered as controls.

2.3. Dithizone staining Dithizone staining was carried out as described previously (Shiroi et al., 2005); 50 mg DTZ in 5 ml dimethylsulphoxide Copyright © 2014 John Wiley & Sons, Ltd.

(DMSO; Sigma, cat. no. D2650) was prepared as stock solution and stored at –20 °C. In vitro DTZ staining was performed by adding 10 μl stock solution to 1 ml culture medium and then incubating at 37 °C for 15–30 min. After rinsing three times the dishes with Hanks’ balanced salt solution [HBSS; sodium chloride 8000 mg/l, potassium chloride 400 mg/l, potassium phosphate monobasic (KH2PO4) 60 mg/l, glucose 1000 mg/l, sodium phosphate dibasic (Na2HPO4, anhydrous) 48 mg/l], crimson-stained clusters were examined using a stereomicroscope. The number of DTZ-positive (DTZ+) cells in the cultures was counted after trypsinization using a haemocytometer under a microscope at × 40 magnification. Different concentrations of PCM (25%, 50%, 75% and 100%) were used for the DTZ assay, which was repeated at least five times.

2.4. Immunofluorescence The following primary antibodies were used in this study: anti-nestin (Sigma, cat. no. N5413), mouse monoclonal proinsulin + insulin, (Abcam, Cambridge, MA, USA, cat. no. ab8304-50), rabbit polyclonal anti-C peptide antibody (Abcam, cat. no. ab14181) and mouse monoclonal insulin receptor-β (Abcam, cat. no. ab8304-100). Cy5.29conjugated anti-rabbit IgG (Abcam, cat. no. ab6564) and FITC-conjugated anti-mouse IgG (Sigma, cat. no. F9137) were used as secondary antibodies. For immunofluorescence, J Tissue Eng Regen Med (2014) DOI: 10.1002/term

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cells were cultured in six-well plates on a cover slip, fixed in 4% paraformaldehyde, rinsed three times with PBS and permeabilized with 0.3% Triton X-100 in PBS for 30 min at 37 °C. Following three washes with PBS, the cells were incubated with 10% normal goat serum (Sigma, cat. no. G9023) in PBS for 30 min to block non-specific binding. Afterwards, the cells were incubated with the relevant primary antibody for 60 min, followed by incubation with the secondary antibody for 30 min. All antibodies were used at 1:1000 dilutions. The coverslips were mounted with 70% glycerol in PBS. Several controls for immunostaining were used, in which the primary antibody was omitted.

2.5. Insulin detection assay by enzyme-linked immunosorbent assay (ELISA) IPCs, undifferentiated P19 EC cells and spontaneously differentiated EBs were grown in six-well plates to estimate the intracellular and secreted insulin levels by ELISA. Before examination, the cells were washed three times with PBS and incubated in fresh serum-free medium with 0.5% bovine serum albumin (BSA) to enable the detection of insulin secretion without interference from the fetal serum. The cells were incubated under these conditions for 3 h at 37 °C and then washed twice with serum-free medium. High-glucose challenge of the cells was achieved by sequential addition of serum-free medium containing low (5.5 mM) or high (25 mM) glucose for 2 h at 37 °C. The conditioned medium was collected and frozen at –70 °C until assayed for insulin content. For measurement of intracellular insulin content, cell pellets were sonicated in acid-ethanol (0.1 N hydrochloric acid in absolute ethanol). The values obtained were normalized relative to the total protein content (Bradford protein assay reagent, Sigma, cat. no. B6916). ELISA was performed on the conditioned media and cellular extract using insulin mouse ultrasensitive ELISA kit (Alpco, cat. no. 80-insmsu-E01).

2.6. RNA extraction and real-time polymerase chain reaction Total cellular RNA was extracted from IPCs, undifferentiated P19 EC cells and spontaneously differentiated EBs using Qiazol lysis reagent (Qiagen, cat. no. 79306). Quantiscript reverse transcriptase (QiagenQuantiTect Reverse Transcription Kit, cat. no. 205311) was used for the synthesis of cDNA with oligo-dT and random primers, according to the manufacturer’s protocol. RT–PCR assays were performed using the Qiagen apparatus (Qiagene, Rotor-Gene, CA, USA). Real time RT–PCRs of undifferentiated stem cell markers, Oct3/4 (POU class 5 homeobox 1), Sox-2 [sex-determining region Y (SRY) box 2] and Nanog (Nanog homeobox) (Khoo et al., 2013), as well as pancreatic β cell genes, pancreatic and duodenal homeobox 1 (PDX-1), insulin 1 (INS1), insulin 2 (INS2), E1A-binding protein p300 (EP300), cAMP-responsive element binding protein 1 (CREB1) and β-2 Copyright © 2014 John Wiley & Sons, Ltd.

microglobulin (β-2 M) cDNAs were performed using specific primers (Table 1) and SYBR Premix Ex Taq (TaKaRa, cat. no. RR081Q). Ep300 and CREB1 were also in our regulatory network of IPCs differentiation (see below). RT samples and negative controls (no template) were run together with test samples, and standard curves were used for each gene tested to analyse the efficiency of the PCR reaction. A melt-curve analysis was performed at the end of each reaction. Expression levels were normalized to individual β-2 M (internal controls). The profile was obtained by plotting relative gene expression levels comparing to undifferentiated P19 EC cells. Symmetrical fold change expression of Oct3/4, Sox-2, Nanog, EP300 and CREB1 were compared between P19 EC and IPCs (see supporting information, Figure S1).

2.7. Statistical analysis All experiments were performed in triplicate, and statistical differences were measured using Student’s t-test when there were two groups to be compared, and one-way analysis of variance (ANOVA) and Duncan’s Test when there were more than two groups. Results are expressed as mean ± standard deviation (SD) and p < 0.05 was considered to be statistically significant.

2.8. Construction of regulatory networks underlying the formation of IPCs Based on the results of real-time PCR, genes overexpressed during the formation of IPCs (INS1, INS2 and PDX-1) were used as input genes against the protein interaction database of the PathwayStudio 9 package (Elsevier, USA). PathwayStudio employs the MedScan text-mining tool to utilize the available information from a huge number of published scientific papers. These relationships with their corresponding reference information are stored in the PathwayStudio database. We enriched Table 1. Real-time PCR primer sequences Gene

Primer sequence

PDX-1

F 5′-TCCACCACCACCTTCCAGCTCA-3′ R 5′-TTCCTCGGGTTCCGCTGTGT-3′ F 5′-ATGGCCCTGTTGGTGCACTTCC-3′ R 5′-AAGAAGCCACGCTCCCCACA-3′ F 5′-AACATGGCCCTGTGGATGCG-3′ R 5′-ACCCAGCTCCAGTTGTGCCA-3 F 5′-TCCATACCGAACAAAGGCCC-3′ R 5′-CCAGATGTGCCCTGTTGTGT-3′ F 5′-AGAAGCAGCACGGAAGAGAG-3′ R 5′-CTTTCTGGTTGTGGCCAAGC-3′ F 5′-CTACCATCTGCCGCTTTG-3′ R 5′-GCCGCAGCTTACACATGTTCT-3′ F 5′-ACAGCAAATGACAGCTGCAAA-3′ R 5′-TCGGCATCGCGGTTTTT-3′ F 5′-CCAAAGGCAAACAACCCACTT-3′ R 5′-CGGGACCTTGTCTTCCTTTTT-3′ F 5′-AGTCGTCAGCATGGCTCGCT-3′ R 5′-TGAGGCGGGTGGAACTGTGT-3′

INS1 INS2 EP300 CREB1 Oct3/4 Sox-2 Nanog β-2 M

J Tissue Eng Regen Med (2014) DOI: 10.1002/term

Differentiation of P19 carcinoma stem cells into insulin-producing cells

the database for the development of pancreas and IPCs by adding information from a large number of papers and updating the database with Pubmed, Medline, etc. Furthermore, to unravel the regulatory network underlying development of IPCs, relations related to different aspects of regulation, including expression, promoter binding and microRNA, were called from the database using a shortest-path algorithm; this selects the shortest paths between a given entity and its group to create shortest-path longevity networks. As a result, the smallest number of links between two nodes is generated using the shortest path. All entities and relations of the network with corresponding references were saved in the company Excel file of the network. As mentioned above, the expression of some predicted transcription factors, including EP300 and CREB1, in differentiated IPCs were confirmed by real-time PCR.

that the differentiating cells expressed nestin, an intermediate filament presented in neural stem cells and possible islet precursors, at stage 3. Figure 3 shows individual nestinpositive cells (C) and a nestin-positive cell cluster with spindle-shaped cells at the periphery (G). These cells were expanded by culture in the presence of bFGF and EGF and then differentiated into IPCs by supplementing the culture medium with nicotinamide and different concentrations of PCM. Using this procedure, cells that displayed expression of pancreatic β cellspecific proteins were obtained. Immunofluorescence evaluation of the differentiated P19 cells showed that these cells were immunoreactive to proinsulin + insulin (Figure 4A), insulin receptor-β (Figure 4B) and C-peptide (Figure 4C) after PCM treatment. Several controls for immunostaining were used, in which the primary antibody was omitted (Figure 4D).

3. Results

3.3. Insulin secretion and content

3.1. Morphological studies

For further determination of the functional status of IPCs developed in our culture system, we analysed glucosedependent insulin release by ELISA. Glucose-inducible insulin secretion was examined in P19 undifferentiated EC cells, spontaneous differentiated EBs and IPCs; the results are shown in Figure 5. The statistical differences between the groups used in the study revealed that, compared to P19 and EB, differentiated IPCs demonstrated an obvious increase in secreted (Figure 5A) and intracellular (Figure 5B) insulin when normalized to the total protein content. Furthermore, IPCs synthesized and secreted insulin in a glucose-regulated manner, as presented in Figure 5. No significant insulin content or secretion was observed in undifferentiated cells.

In this study we used PCM as an inducer to direct IPC differentiation of an EC cell line, P19, into IPCs. Figure 2 shows the morphology of exponentially growing P19 cells at low (A) and high (B) magnifications. When cultured in gelatinized dishes, the cells formed colonies composed of tightly packed polygonal cells of small size and prominent nuclei, as shown in Figure 2. DTZ, a zinc-chelating agent, which stains insulin-containing cells, was used to assess the presence of IPCs after differentiation induction. Using this characteristic of DTZ, IPCs were readily identified as crimson cellular clusters. The results of DTZ staining of P19-derived IPCs and whole pancreas cultures are shown in Figure 2E, F. Distinct DTZ+ cell clusters were observed in PCM-treated P19-derived EB outgrowths, while they could not be seen in the intact undifferentiated P19 cultures (Figure 2C) and were rare in spontaneously differentiated EBs (Figure 2D). To estimate the frequency of the emerged DTZ+ cells in the cultures, the number of crimson cells was counted directly under a microscope after trypsinization following DTZ staining. Table 2 shows the percentage of DTZ+ cells (with respect to total cell number) at different concentrations of PCM. The cell counts showed that in all PCM-treated groups the percentages of DTZ+ cells were significantly higher than those of the untreated group (negative control). There was no significant difference between 50%, 75% and 100% PCM-treated groups (p < 0.05). The highest number of DTZ+ cells was obtained at 75% concentration of PCM (mean 48.58 ± 7.27%).

3.2. Detection of pancreatic β cell-specific proteins In the first step of differentiation induction protocol, P19 cells were cultured in a serum-free medium to select nestin-positive cells. Immunofluorescence analysis showed Copyright © 2014 John Wiley & Sons, Ltd.

3.4. Quantitative analysis of gene expression To determine the effects of PCM on differentiation of P19 EC cells into IPCs, the cells were cultured as EBs in different concentrations of PCM (25%, 50%, 75% and 100%). Untreated EC cells (with no PCM treatment) were also considered as controls. Real-time PCR analysis was used to examine the expression of pancreas-specific genes in differentiated cells. Results from representative experiments are shown in Figure 6. The data showed that, in this culture system, transcriptional factors and ligands essential to pancreatic development, such as PDX-1, INS1 and INS2, were significantly induced (Figure 6A). The expression of PDX-1 was significantly increased in 50% concentration of PCM compared with untreated (P19) and 25%, 75% and 100% PCM-treated cells. PCM also significantly induced INS1 gene expression at a concentration of 75%. Significant differences were observed between 75% PCM-treated cells and all the other groups. Furthermore, the results showed that the highest INS2 gene expression occurred at a concentration of 25%. There was a J Tissue Eng Regen Med (2014) DOI: 10.1002/term

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Figure 2. Morphology of exponentially growing P19 cells at low (A) and high (B) magnifications: undifferentiated cells are tightly packed polygonal cells, with large nucleoli and a high nucleus:cytoplasm ratio. (C–F) Differentiation of P19 EC cells into IPCs and DTZ staining: untreated EC cells are not stained (C); individual cells are DTZ-positive in spontaneously differentiated EBs (D); a cell cluster distinctly stained crimson by DTZ is apparent in P19 EC cell-derived IPCs (E); and culture of whole pancreas (F)

+

Table 2. Percentage of DTZ cells among total cells in different concentrations of PCM PCM (%) 0 25 50 75 100

+

DTZ (%) (mean ± SD) 15.91 ± 3.16c 26.17 ± 4.61b 46.09 ± 4.33a 48.58 ± 7.27a 46.87 ± 6.39a +

Data represent mean ± SD of the means of DTZ cells derived from P19 EC cells in different concentrations of PCM used in the study. PCM induced the differentiation of P19 EC cells at a higher mean than that of the untreated group (negative control). + PCM, pancreas conditioned medium; DTZ , DTZ-positive cells. a,b,c Pairwise comparisons that share a common superscript letter are not significantly different at P < 0.05.

significant difference between 25% PCM-treated cells and all the other groups. To categorize the P19 EC cells, a real-time PCR analysis was initially performed on these cells and IPCs to compare Copyright © 2014 John Wiley & Sons, Ltd.

undifferentiated marker gene expression, including Oct3/4, Sox-2 and Nanog (Figure 6B). The results showed that the expression of the genes was significantly downregulated in differentiated cells. In addition, differentiated cell markers, EP300 and CREB1, were selected based on the predicted regulatory network. These transcription factors were more highly expressed in IPCs than in P19 EC cells (Figure 6B). During differentiation of IPCs from P19 ECs, the expression of EP300 and CREB1 transcription factors was increased by 2.5- and 3.1-fold, respectively. In contrast, sharp decreases in expression of Oct3/4, Sox-2 and Nanog by 4-, 1.5- and 1.5-fold, respectively, were observed.

3.5. Regulatory network underlying the formation of IPCs Figure 6 presents the predicted regulatory network underlying the activation of INS2 and PDX-1 and the formation of IPCs. Table S1 (see supporting information) shows the relation map of this network with the associated references. Cell localization of the network is demonstrated J Tissue Eng Regen Med (2014) DOI: 10.1002/term

Differentiation of P19 carcinoma stem cells into insulin-producing cells

Figure 3. Fluorescence micrographs illustrating the expression of nestin, an intermediate filament presented in neural stem cells and possible islet precursors. (A–D) Individual nestin-positive cells: (A) phase-contrast image of the same field shown in (B–D); (B) nuclei counterstained by Hoechst; (C) expression of nestin in differentiating cells at stage 3; (D) merged image of nestin and Hoechst. (E–H) A nestin-positive cell cluster with spindle-shaped cells at the periphery: (E) phase-contrast image of the same field shown in (F–H); (F) nuclei counterstained by Hoechst; (G) expression of nestin in a cluster of differentiating cells at stage 3; (H) merged image of nestin and Hoechst

Figure 4. Fluorescence micrographs illustrating the expression of pancreatic β cell markers in insulin-producing cells derived from P19 EC cells: staining of IPCs with antibodies against proinsulin + insulin (A); insulin receptor β (B); and (C) C-peptide showing that most of the P19 cells treated with PCM express pancreatic β cell markers. (C) Control for immunostaining; the primary antibody was omitted. Nuclei are counterstained by Hoechst Copyright © 2014 John Wiley & Sons, Ltd.

J Tissue Eng Regen Med (2014) DOI: 10.1002/term

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Figure 5. Determination of secreted (A) and secreted vs intracellular (B) insulin in P19 undifferentiated EC cells (P19), spontaneous differentiated EBs (EB) and insulin-producing cells (IPCs). Significant insulin concentration was observed in PCM–treated IPCs. To normalize the amount of insulin secretion, the total protein of the cells in each well was measured by the Bradford method. The experiment was performed in triplicate; each value represents mean ± SD; small fonts show statistical significance – different letters indicate a statistically significant difference between the compared treatments

Figure 6. Quantitative analysis of genes involved in differentiation of pancreatic cells derived from P19 EC cells. The cells were cultured as EBs in different concentrations of pancreas-conditioned medium (PCM). (A) Relative gene expression of PDX-1, INS1 and INS2 in different concentrations of PCM. (B) Comparison of expression of Oct3/4, Sox-2 and Nanog (undifferentiated stem cell markers) and EP300 and CREB1 (differentiated stem cell transcription factors) between P19 EC (embryonal carcinoma) cells and insulin-producing cells (IPCs). The data are expressed as gene expression relative to β-2 M and are presented as mean ± SD; means with different letters are significantly different at p = 0.05; small fonts show statistical significance – different letters indicate a statistically significant difference between the compared treatments

in Figure S2 (see supporting information). As can be inferred from the regulatory network, INS2 and PDX-1 are the central proteins for induction of IPCs. The network suggests the involvement of KLF1 (Kruppel-like factor 1), EP300, CREB1, ONECUT1 (one cut homeobox 1) and NROB2 (nuclear receptor subfamily 0, group B, member 1) in the induction of IPCs. Figure 7 and Table S1 unravel the positive regulatory effects of several transcription factors, including peroxisome proliferatoractivated receptor-α (PPARα), CREB1, peroxisome proliferator-activated receptor-γ (PPARγ), HNF1 homeobox A (HNF1A), paired box 6 (PAX6), EP300 and CREB1 on crosstalk and activation of PDX-1 and INS2. The network demonstrated that two receptors, including v-kit Copyright © 2014 John Wiley & Sons, Ltd.

Hardy–Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and glucagon-like peptide 1 receptor (GLP1R) are involved in positive activation of INS2 and PDX-1 (Figure 7). As described above, significant overexpression of EP300 and CREB1 in IPCs relative to P19 EC cells was shown by real-time PCR (Figure 6).

4. Discussion In this study, we examined the possibility of differentiation of IPCs from an EC cell line, P19 by using PCM as a natural native inducer. DTZ staining, used to characterize J Tissue Eng Regen Med (2014) DOI: 10.1002/term

Differentiation of P19 carcinoma stem cells into insulin-producing cells

Figure 7. Predicted regulatory network underlying activation of INS2 and PDX-1 and formation of IPCs (insulin-producing cells) from P19 EC (embryonal carcinoma) cells. INS2 and PDX-1are the central proteins for induction of IPCs. In the presented network, blue lines correspond to the expression relation between proteins; red lines show promoter binding interactions between proteins (particularly transcription factor and target protein); grey dotted lines represent interaction regulation. The relationships and their corresponding references are presented in Table S1

the morphological features, showed that P19 cells are able to differentiate into IPCs and form islet-like structures. The expression of specific β cell markers was analysed using immunofluorescence and real-time PCR. The results indicated that P19-derived IPCs were immunoreactive to proinsulin, insulin, C-peptide and insulin receptor-β. Furthermore, expression of genes, including PDX-1, INS1, INS2, EP300 and CREB1, that are related to pancreatic β cell development and function was shown. These cells synthesized and secreted insulin in a glucose-regulated manner, as shown by ELISA. We constructed a regulatory network which demonstrated that INS2 and PDX-1 are the central proteins for induction of IPCs. We provided evidence suggesting that generation of IPCs is the result of the expression ratio between EC-related transcription factors (Oct3/4, Sox-2 and Nanog) and differentiationrelated transcription factors (EP300 and CREB1). To our knowledge, this is the first report on induction of IPCs differentiation of EC cells by neonate mouse PCM. Recently, we introduced a simple accessible non-selective approach to produce IPCs from P19 EC cells by mouse pancreas extract (MPE), as a natural inducer. Altogether, our findings in both studies document that P19 cells can differentiate efficiently to functional IPCs (Ebrahimie et al., 2014). Here, we reproduced and modified the original protocol and adapted a strategy used to generate cells expressing nestin, to derive IPCs from ES cells (Lumelsky et al., 2001). The original protocols for IPCs differentiation of stem cells are based on either selection of nestinpositive cells (Lumelsky et al., 2001) or via a definitive endoderm (DE) route (Wei et al., 2013). By comparing two protocols Wei et al. (2013) confirmed that IPCs can Copyright © 2014 John Wiley & Sons, Ltd.

be induced successfully using either strategy. Mouse ES cells could differentiate more efficient to IPCs by nestinpositive selection (Jafary et al., 2008). However, it is still a matter of controversy whether nestin is an appropriate marker for IPC differentiation. Co-expression of insulin and nestin in early human embryonic pancreas development was determined by Yang et al. (2011); according to their investigation, nestin may not be a specific marker of β cell precursors. In contrast, some studies show that nestin-positive cells were able to differentiate into pancreatic endocrine and exocrine phenotypes (Gao et al., 2003; Zulewski et al., 2001). Recent studies have suggested that human neural progenitor cells, when exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive IPCs (Hori et al., 2005). In pancreas, nestin expression is considered to be a marker for pancreatic stem cells and islet progenitor cells (Kim et al., 2004, 2010). Our results showed that it is possible to generate IPCs from undifferentiated mouse P19 EC cells with the molecular and functional characteristics of pancreatic β cells via a nestin-selection protocol and using PCM as a natural native inducer. These findings are consistent with the earlier demonstrations that nestin-expressing cells can be differentiated into IPCs (Chao et al., 2008; Fujikawa et al., 2005; Kim et al., 2004, 2010; Wei et al., 2013; Zulewski, 2006). ES and EC cells may provide information pertinent to one another. Both of these cell types share the general properties of pluripotent stem cells, in that they exhibit unlimited self-renewal and can give rise to derivatives of all three embryonic germ layers (Chaerkady et al., 2010). They can be considered as complementary tools to study J Tissue Eng Regen Med (2014) DOI: 10.1002/term

A. Mansouri et al.

the developmental mechanisms. The P19 mouse EC line is an excellent and widely used model to analyse regulation of neuronal development and differentiation (Bakhshalizadeh et al., 2011; Esmaeili et al., 2006; MacPherson and McBurney, 1995). P19 cells have a normal male karyotype (McBurney and Rogers, 1982). They are widely available, easily cultured without feeders and do not usually require special growth conditions. In addition, the neurons derived from this cell line have been used successfully in transplantation studies in several mouse models (Astigiano et al., 2005). However, despite these valuable advantages, the ability of EC cells to generate IPCs remains unknown. Here, using P19 cells, a simple and efficient strategy is proposed to serve as a basis for pancreatic linage differentiation studies. Previous studies have shown that both the exocrine and endocrine compartments of the pancreas undergo dramatic expansion after birth and are capable of at least partial regeneration following injury (Keefe et al., 2012). The islets of mouse pancreas undergo further remodelling and maturation for 2–3 weeks after birth (Habener et al., 2005; Wang et al., 2010). Furthermore, in rodents there is clear evidence of pancreatic regeneration after injury (Inada et al., 2008). Damaged pancreas produces various growth factors and hormones, which are required for pancreas development and regeneration (Lee et al., 2008; Shin et al., 2005; Wang et al., 2010). Based on these reports, we hypothesized that primary culture of neonate pancreas fragments can undergo dramatic proliferation and mimic normal regeneration to produce and secrete some soluble factors that are necessary for pancreatic development. Our results support the hypothesis, and also show that PCM alone could efficiently induce P19 differentiation into IPCs. Recently, differentiation of human embryonic stem cells (hESCs) into pancreatic β cells by using a combination of extracellular matrix (ECM) and conditioned medium from an appropriate committed cell line, rat pancreatic β cell line (RIN5F), was reported. The results showed that the ECM alone did not have any effect on gene expression levels. However, conditioned medium alone triggered the expression of Nkx6.1 and cells exposed to both ECM and conditioned medium expressed key β cell markers, including PDX1, insulin and Glut2 (Narayanan et al., 2014). It is interesting to note that in the culture system reported here, PCM alone efficiently induced P19 differentiation into IPCs and the differentiated cells significantly expressed the genes related to pancreatic β cell development and function, such as PDX-1, INS1, INS2, EP300 and CREB1. This discrepancy is probably due to the type of conditioned medium used in the two investigations. The PCM utilized in our study derived from whole pancreas primary culture (see Materials and methods) as a natural native medium. Therefore, PCM provided a tissue-specific micro-environment for pancreatic differentiation. Furthermore, it is possible that the cultured pancreas fragments mimicked the normal regeneration system and produced factors necessary for cell proliferation and differentiation, as well Copyright © 2014 John Wiley & Sons, Ltd.

as tissue repair. Under in vivo conditions, stem cells are recruited to the damaged site for repair, whereby they undergo differentiation into the cell or tissue concerned (Narayanan et al., 2014). Therefore, the main difference between our investigation and that of Narayanan et al. is probably the application of neonate mouse PCM as a natural native inducer to differentiate stem cells into IPCs. Given the important role of ECM in cell differentiation and tissue-specific gene expression, applying a system composed of both pancreatic ECM and PCM would be beneficial. Another difference is the stem cell line used for differentiation induction in these two studies. To our knowledge, there has not been any previous report of IPC differentiation of P19 EC cells using neonate mouse PCM. In many earlier protocols the first stage of neuronal and pancreatic differentiation is similar, based on selection of nestin-expressing cells (Lumelsky et al., 2001). Nestin is an intermediate filament protein transiently expressed during early development in neuronal cells. In pancreas, nestin is considered to be a marker for pancreatic stem cells and islet progenitor cells (Kim et al., 2004, 2010). Since P19 cells are amenable to neuronal differentiation, we reasoned that they are probably appropriate for pancreatic differentiation. Overall, our results demonstrate that it is possible to generate IPCs from undifferentiated mouse P19 EC cells with the molecular and functional characteristics of pancreatic β cells by using neonate mouse PCM as a natural native inducer. We showed that PCM is able to induce pancreatic markers, insulin, proinsulin, C-peptide and insulin receptor-β. Expression of proinsulin is the main characteristic feature of normal pancreatic β cells (Brolén et al., 2005). Immunoreactivity for proinsulin, along with C-peptide, exhibited de novo synthesis and processing of insulin. The insulin receptor is a transmembrane receptor that is activated by insulin, IGF-I and IGF-II (Ward and Lawrence, 2009). These factors stimulate β cell replication and hypertrophy (Rabinovitch et al., 1982; Swenne et al., 1987). Although the components of the PCM were not analysed in this project, a possibility is that soluble factors secreted by pancreatic cells in our culture system are implicated in β cell differentiation via specific receptors expressed in IPCs. It has been reported already that soluble factors are involved in the upregulation of some of the pancreatic endocrine and exocrine markers (Metrakos et al., 1993; Talavera-Adame et al., 2011). We showed here that the PCM from whole pancreas culture exerted an endocrine progenitor differentiation effect on P19 EC cells. Regions of pancreatic endocrine cells could be specifically labelled with DTZ, which efficiently stains β cells in the islet, owing to the presence of zinc in insulin-containing secretory granules (Latif et al., 1988). In our culture system, discrete areas of DTZ staining were identified in differentiated cells derived from EC cells as cellular clusters. Our observations demonstrated that P19 cells could differentiate into functional β cells, capable of producing and J Tissue Eng Regen Med (2014) DOI: 10.1002/term

Differentiation of P19 carcinoma stem cells into insulin-producing cells

secreting insulin in response to glucose. The insulin content was enhanced when the glucose concentration in the medium was increased. These findings are consistent with the earlier observations that both insulin release and total insulin content were increased in islets exposed to ECCM (Johansson et al., 2009). Insulin secretion, especially under glucose induction, is an essential feature of pancreatic β cells. Based on some reports, insulin-positive cells derived from ES cells may not be real IPCs (Rajagopal et al., 2003; Sipione et al., 2004). In some studies, no message for insulin was detectable in culture, which suggested that the cells might be concentrating the hormone from the medium, rather than producing it (Rajagopal et al., 2003; Sipione et al., 2004). Immunoreactivity for proinsulin, insulin and C-peptide in the current study indicated the presence of endogenously produced insulin; this observation is in line with the previous findings of Fujikawa et al. (2005), who claimed that immunoreactivity for proinsulin or C-peptide would indicate that the precursor proinsulin was synthesized by the differentiated cells. Moreover, the intracellular insulin level after exposure to 25 mM glucose was lower in comparison to secreted insulin (Figure 5B). Therefore, it is concluded that the hormone was produced by the differentiated IPCs, rather than being taken up from the culture medium. Also, we found that two mature forms of insulin, INS1 and INS2, were both expressed in the differentiated cells. This is in accord with earlier work (Bai et al., 2005) which revealed INS1 and INS2 mRNA expression in IPCs derived from mouse ES cells, but contrasts with other claims that found no insulin transcripts (Rajagopal et al., 2003) or only INS2 (Sipione et al., 2004). The difference between our results and those of Sipione et al., whose IPCs were glucose-unresponsive and did not express INS1 and rarely glucagon, may be due to the different stem cell lines examined, slight modification of the differentiation protocol and, importantly, using PCM as an inducer in our culture system. Real-time PCR analysis confirmed the expression of PDX-1 by P19 derived-IPCs. PDX-1 is a crucial regulator for pancreatic development (Kunisada et al., 2011). Its expression seems likely to be very important in the in vitro differentiation of ES cells along pancreatic cell lineages (Miyazaki et al., 2004). The previous reports by other investigators suggested that ES cells cultivated with pancreatic rudiments, mesenchyme (Shiraki et al., 2005) or MIN-6 β-cell-conditioned medium could express Hnf1β, PDX-1 and Ptf1 (Uroic et al., 2010). The genes tested here (PDX1, INS-1 and INS-2) did not show a concentrationdependent increase in expression levels (Figure 6A). PDX-1 is the key transcription factor in this context. Transcription factors can be activated with less or more amount of inducer agent. It should be noted that some inconsistency between regulator and target gene may be observed, as their relationship is not quite linear. More investigations seem to be required in future studies. Computational methods of network construction play a pivotal role in the prediction of gene interactions and for Copyright © 2014 John Wiley & Sons, Ltd.

revealing novel genes involved, by the use of available biological data (Hosseinpour et al., 2012). As can be inferred from the regulatory network presented, INS2 and PDX-1 are the central proteins for induction of IPCs. PDX-1 encodes a protein that is a transcriptional activator of several genes, including insulin, somatostatin, glucokinase, islet amyloid polypeptide and glucose transporter type 2. Overexpression of this transcription factor is one of the major components of the observed function of the differentiated cells in this study. Interaction (crosstalk) between PDX-1 and other transcription factors, such as Ngn3, Nkx6.1, MafA and NeuroD1, will extend our understanding of β cell development in future studies. PDX-1 recognizes and binds to the 5′-[CT]TAAT[TG]-3′ regulatory element on the promoter region of downstream genes (An et al., 2010). Activation of INS2 and its interaction with PDX-1 play a vital role in the glucose uptake response of differentiated cells (Barazzoni et al., 2012). The predicted regulatory network (Figure 7; see also supporting information, Table S1) unravelled the positive regulatory effects of several transcription factors, including PPARα, EP300, CREB1, PPARγ, HNF1A and PAX6 on crosstalk and activation of PDX-1 and INS2. It has been documented that PPARα positively regulates the expression of PDX-1 in β cells (Sun et al., 2008; Yessoufou et al., 2009). Positive regulatory effects of PPARγ in β cell differentiation have been confirmed based on in vitro RNA interference studies (Evans-Molina et al., 2009). PPARγ increases the transcription of certain insulin-sensitive genes, thereby improving insulin sensitivity (Nattrass and Bailey, 1999). Network demonstrated that two receptors, KIT and GLP1R, are involved in positive activation of INS2 and PDX-1 (Figure 7). Activation of KIT increases PDX-1 and insulin expression (Li et al., 2007), and similarly, GLP receptor activation enhances β cell proliferation and promotes islet neogenesis via induction of PDX-1 expression (Brubaker and Drucker, 2004). A functionally important loop between locally produced GLP and its cognate receptor has been suggested, based on the fact that GLP antagonist application significantly reduced insulin secretion/production in β-TC-6 insulinoma cells and isolated rat islets (Masur et al., 2005). Positive involvement of PTK2 (positive regulatory effects) and the negative role of mitogen-activated protein kinase 8 (MAPK8; Figure 7; see also supporting information, Table S1) reinforce the central role of phosphorylation in the generation of IPCs. The network suggests the involvement of KLF1, EP300, ONECUT1 and NROB2 in the induction of IPCs. Interestingly, the relationship between KLF1 and NROB2 with insulin production is yet to be reported. These genes are novel candidates for increasing the efficiency of IPC induction. Enrichment of nestin-positive cells, FACS analysis to understand the percentage of cells positive for pancreas β cell markers, and detection of PCM components that regulate the differentiation of P19 cells are the future activities which will extend our understanding of β cell development in future studies. J Tissue Eng Regen Med (2014) DOI: 10.1002/term

A. Mansouri et al.

5. Conclusion In the present study we examined, for the first time, the possibility of differentiation of IPCs from EC cells by using PCM as an inducer. Our results suggest that it is possible to generate IPCs from undifferentiated mouse P19 EC cells with the molecular and functional characteristics of pancreatic β cells. The differentiated cells showed typical islet cell cluster morphology and exhibited a number of markers characteristic of functional pancreatic β cells. Moreover, when stimulated with glucose, these cells synthesized and secreted insulin. The regulatory network showed that INS2 and PDX-1 are the central proteins for induction of IPCs. The designed network and its suggestion of novel regulatory candidates open a new vista for future studies on increasing the efficiency of production of IPCs. This approach led to the prediction of critical members and bottlenecks of the regulatory network that may govern the induction of IPCs. Since P19 EC cells are widely available, easily cultured without feeders and do

not usually require special growth conditions, they would provide a valuable experimental tool for studying pancreatic β cell differentiation and development.

Conflict of interest The authors have declared that there is no conflict of interest.

Acknowledgements The authors would like to express deep thanks to Dr Behnaz Saffar (Head of Research Institute of Biotechnology, Shahrekord University, Iran) for her excellent assistance, Dr Manijeh Mohammadi-Dehcheshmeh (School of Agriculture, Food and Wine, University of Adelaide, Australia) for her assistance in preparation of Figures, and Professor Jeremy Timmis and Dr David A. Ogunniyi (School of Molecular and Biomedical Science, University of Adelaide) for critical review of the manuscript. The work was supported by a grant from Shahrekord University, Iran.

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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web-site.

Copyright © 2014 John Wiley & Sons, Ltd.

J Tissue Eng Regen Med (2014) DOI: 10.1002/term