Small Molecules Differentiate Definitive Endoderm ... - Springer Link

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May 27, 2014 - endoderm (DE) will identify a key step in hepatocytes and beta cell development ... endoderm 1 (IDE1) is known to play an important role in the ...

Appl Biochem Biotechnol (2014) 173:1727–1736 DOI 10.1007/s12010-014-0960-9

Small Molecules Differentiate Definitive Endoderm from Human Induced Pluripotent Stem Cells on PCL Scaffold Elham Hoveizi & Sirus Khodadadi & Shima Tavakol & Oveis Karima & Mohammad Ali Nasiri-Khalili

Received: 20 December 2013 / Accepted: 13 May 2014 / Published online: 27 May 2014 # Springer Science+Business Media New York 2014

Abstract Human induced pluripotent stem cells (hiPSCs) are attractive sources of cells for disease modeling in vitro, and they may eventually provide access to cells/tissues for the treatment of many degenerative diseases. Stepwise differentiation from hiPSCs to definitive endoderm (DE) will identify a key step in hepatocytes and beta cell development and may prove useful for transplantation therapy for liver diseases and diabetes. Inducer of definitive endoderm 1 (IDE1) is known to play an important role in the regional specification of DE. Here, we have investigated the effect of stimulation with IDE1 on the development of hiPSCs into DE cells in three-dimensional (3D) cultures. The differentiation was determined by immunofluorescence staining with Sox17, FoxA2, and goosecoid (Gsc) and also by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. In this study, we showed that hiPSCs with 6-day IDE1 treatment (as chemical tool) on poly(ε-caprolactone) (PCL) nanofibrous scaffold were able to differentiate into DE cells. Keywords IDE1 . Definitive endoderm . hiPSCs . PCL

E. Hoveizi (*) Department of Biology, Faculty of Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran e-mail: [email protected] S. Khodadadi : M. A. Nasiri-Khalili Department of biosciences and biotechnology, Malek Ashtar University of Technology, Tehran, Iran S. Tavakol Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran O. Karima Department of clinical biochemistry, Faculty of medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran


Appl Biochem Biotechnol (2014) 173:1727–1736

Introduction Human induced pluripotent stem cells (hiPSCs) can be maintained in vitro for extended periods without immune rejection and ethical challenges, and hiPSCs are potential sources for generating a variety of specialized human cells needed for clinical applications. hiPSCs hold the promise of serving as a source of hepatocytes and beta cells [1, 2]. Recently, there has been superior advancement in directed differentiation of hiPSCs into beta cells [3] and hepatocytes [4], two cell types of excellent medical value. During vertebrate gastrulation, epiblast cells are specified to the three principal germ layers. Definitive endoderm (DE) gives rise to the intestinal epithelium and various cell types including the thyroid, lung, liver, and pancreas. To obtain pancreatic or hepatic progenitors, the first vital stage is to induce stem cells to differentiate into DE cells [5–8]. Considering the endoderm, a number of signaling pathways have been identified that control its differentiation in vertebrates, including high levels of nodal/activin A, BMPs, Wnt, and fibrolast growth factor (FGF). Also, during in vivo embryonic development, components of the extracellular matrix (ECM) including biochemical soluble signals and macromolecules play an important role in the control of cellular interactions, cell proliferation, migration, and differentiation [9–11]. Tissue engineering provides an excellent model system in which cells may adhere and aid in shaping and defining in vivo cell growth. Recsmports described that three-dimensional (3D) cultures can be a mimicked structure and biological function of native ECM. Engineering a 3D nanoenvironment can provide mechanical support and regulate cell activities [12, 13]. Nanometer diameter fibers as nanoenvironment are most usually generated by electrospinning, a technique that can be used for fabricating microfibers and nanofibers. Natural and synthetic materials can be used for fabrication of nanofibrous scaffolds from numerous biodegradable polymers, such as poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and the copolymer poly(lactide-co-glycolide) (PLGA). PCL is Food and Drug Administration (FDA)-approved and has a long history of safe use in humans. The diameter of PCL fibers (which is obtained by electrospinning method) is about nanometer that is one of the main advantages of PCL scaffold. Such small-size fibers could physically mimic the structural dimension of the ECM. Consequently, because of low thickness of PCL fibers, it will provide more surface for cellular adherence, migration, proliferation, and function. PCL scaffold provides highly porous microstructure with interconnected pores which is conductive to cell proliferation and differentiation [14, 15]. Today, more effective and selective methods are needed to direct the differentiation of different stem cells to produce of particular cell lineages. One approach to potentially improve current differentiation protocols is the use of 3D scaffolds, which has been shown to enhance cellular function and differentiation potential [15]. The aim of this study was to define the interplay of IDE1 stimulation during DE induction of hiPSCs in 3D culture. In this study, differentiation was investigated by investigating the expression of DE-specific markers, ultrastructure, and function of differentiated cells.

Materials and Methods Scaffold Preparation In order to obtain appropriate polymer solution, 10 %w/v of PCL (Mw 80,000, Sigma) was dissolved in chloroform (CHCl3). The polymer solution was delivered to a metal needle connected to a high-voltage power supply. Upon applying a high voltage, a fluid jet was ejected from the needle. As the jet accelerated toward a grounded collector, the solvent evaporated, and a charged polymer fiber was deposited on the collector in the form of a nanofibrous mat. The typical

Appl Biochem Biotechnol (2014) 173:1727–1736


parameters for electrospinning were as follows: 12 kV (voltage), 10 cm (distance between tip and receptor), 0.5 ml/h, 55–60 % (humidity), and 20–24 °C (temperature). After electrospinning, all the mats were placed in a vacuum drying oven for a couple of days. ImageJ software was used to determine the fiber diameter from scanning electron microscopy (SEM) analysis. Typically, 100 diameters were measured. Finally, the histogram of fiber diameter distribution was plotted. SEM Observation For analysis of the morphology of the electrospun fibers, the samples were sputter-coated with gold and examined using SEM (XL-30, Philips, Netherland). Cell-containing scaffold specimens were fixed with 2.5 % glutaraldehyde, followed by washing with phosphate-buffered saline (PBS) and dehydrated in a series of sequentially increasing concentration of ethanol solutions. Cell Seeding on PCL Scaffolds At the first stage of cell seeding, scaffold was cut to the required dimensions for 24-well tissue culture plates and anchored with rings. Then, nanofibrous scaffolds were exposed to ultraviolet irradiation for 24 h and washed two times with PBS, then soaked in culture medium supplement with 2X pen/strep (Gibco) and 5X amphotericin B (Gibco) overnight at 37 °C. In the end, hiPSCs were seeded at a density of 5×104 cells/well on nanofibrous PCL scaffolds and incubated under standard conditions. hiPSC Cell Culture and Induction of Differentiation In Vitro In the present study, we used the hiPS cell line (gift [16]) which was generated from the human cells by overexpression of Oct4, Sox2, KLF4, and c-Myc. These cells exhibited a round morphology with small gray drop like typical tight colonies. hiPSCs were routinely cultured on mitomycin C (Sigma)treated mouse embryonic fibroblast (MEF) feeders, and they were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (Gibco) supplemented with 10 % KnockOut Serum Replacement (Gibco), 5 % fetal bovine serum (Invitrogen), 2 mM L-Glu (Gibco), 1 mM 2-mercaptoethanol (Gibco), 1 mM nonessential amino acids (Gibco), 1 % P/S (Gibco), and 10 ng/ml bFGF (Invitrogen). hiPSCs were incubated at 95 % humidity, with 5 % CO2 at 37 °C. The medium was changed daily, and cells were subcultured approximately once every 8 days by using collagenase type IV (1 mg/ml). For differentiation, 4-day-old embryoid bodies (EBs) were trypsinized, and 2.5×104 cells/cm2 were mixed in DMEM/F12 supplemented with 2 μM IDE1 and cultured on PCL scaffolds for 6 days. As a control, a group of hiPSC-derived EBs were cultured on PCL scaffolds in the absence of differentiation factors for 6 days. Immunofluorescence Staining The cells were fixed by incubation in 4 % paraformaldehyde/PBS (PFA, Sigma-Aldrich) for 20 min, permeabilized with 0.5 % Triton X-100 in PBS for 10 min, and blocked for 30 min at room temperature with 5 % BSA. The cells were then reacted with primary antibodies (human Sox17 (1:20, polyclonal goat IgG, R&D, AF1924), human goosecoid (Gsc) (1:300, polyclonal goat IgG, Santa Cruz, (N-12), sc-22234), and human FoxA2 (1:500, polyclonal rabbit IgG, Millipore, AB4125)) at 4 °C for 24 h; then, the cells were incubated with secondary antibodies including Alexa Fluor 488 donkey anti-goat (1:200, Gibco, A-11058) or Alexa Fluor 594 donkey anti-rabbit (1:200, Gibco, A-21207) at room temperature for 1 h. As a control, fixed hiPSCs were stained with the secondary antibodies only. To determine the percentage of


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positively differentiated cells, the cells were imaged with a fluorescent microscope, and positive cells were quantified in 20 randomly selected fields. Image tool software was used for cell counting from fluorescence microscopy images. RNA Extraction and Quantitative RT-PCR The messenger RNA (mRNA) expression patterns of different genes involved in DE development were studied using quantitative reverse transcriptase-polymerase chain reaction (qRTPCR). Total RNA was extracted by QIAzol lysis reagent and treated with DNase I (Ambion). Then, RNA (2–5 μg) was reverse-transcribed using the TaqMan Reverse Transcription Kit (Applied Biosystems). qRT-PCR reactions were carried out in the 48-well optical reaction plates on StepOne™ Real-Time PCR machine using primers listed in Table 1. The threshold cycle (Ct) of each target gene obtained from StepOne software (Applied Biosystems) was normalized by GAPDH as internal standard gene. The comparative 2−ΔΔCt method was applied to calculate the relative quantity of target gene expression in each sample to control. PCR reactions were performed on a mastercycler gradient machine (Eppendorf, Germany) (initial denaturation at 95 °C for 30 s, annealing at 57–62 °C for 30 s, extension for 35 s at 72 °C, followed by 46 cycles). Statistical analyses were performed by REST software. MTT Assay for Cell Viability A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, USA) assay was used to measure relative cell viability. After 1-, 3-, 5-, and 7-day incubation, cell viability was evaluated. Table 1 Primers and the reaction conditions for qRT-PCR of differentiation definitive endoderm cells from hiPSCs Gene


Annealing Lenght temperature (°C) (bp)

Homo sapiens forkhead box A2 (FOXA2)




Homo sapiens goosecoid homeobox (Gsc)




Homo sapiens SRY (sex determining F 5' - CATGGTGTGGGCTAAGGACG-3' region Y)-box 17 (Sox17) R 5' - AGCGCCTTCCACGACTTG-3'



Homo sapiens Nanog homeobox (NANOG)





Homo sapiens POU class 5 homeobox 1 (POU5F1)




Homo sapiens SRY (sex determining F 5' - TCATGGTTTGGGCCAAGGAC-3' region Y)-box 7 (SOX 7) R 5' - GCCTTCCACGACTTTCCCAG-3'


Homo sapiens SRY (sex determining F 5' - atctccaactcgcagggcta-3' region Y)-box 1 (SOX1) R 5' - ggctccgacttcaccagaga-3' Homo sapiens T, brachyury homolog F 5' - ACAGGTACCCAACCCTGAGG-3' (mouse) (T) R 5' - TGGGGTACTGACTGGAGCTG-3' GAPDH F 5' - TCGCCAGCCGAGCCA-3' R 5' - CCTTGACGGTGCCATGGAAT-3'

100 4108





Appl Biochem Biotechnol (2014) 173:1727–1736


Statistical Analysis All values in the figures of present study indicate means ± standard deviation (SD), and all determinations were repeated three times. Statistical analysis were performed by one-way ANOVA followed by unpaired Student’s t test, and P values less than 0.05 were considered significant.

Results Morphology of Electrospun Nanofiber and Cell Seeding Figure 1 shows the morphology of the randomly oriented electrospun PCL scaffold that successfully fabricates. The fibers’ diameters were analyzed by ImageJ and ranged from 100 to 400 nm. The average diameter was 200 nm (Fig. 1). The cell morphologies on the PCL scaffold are shown in Fig. 1. After culturing for a week, the cells adhere and cover all the surface of the scaffolds (Fig. 1). These observations showed that PCL scaffold (with uniform and continuous mats) could be sufficient to provide a suitable substrate for cell adhesion and spreading. Immunostaining Analysis We used IDE1 to induce differentiation of hiPSCs into DE cells. Here, we determined whether IDE1 could induce DE formation from hiPSCs in 3D culture. Immunostaining demonstrated that IDE1 caused a significant increase in the number of cells expressing Sox17, FoxA2, and Gsc as DE markers. Control staining of the differentiated hiPSCs after IDE1 induction, omitting the primary antibody, was negative (Fig. 2). Five images per well were captured by a high-content imaging system, and an algorithm (that determines the percentage of DE-positive cells vs total DAPI-positive nuclei) was used to

Fig. 1 Scanning electron micrographs showing the morphology of plated/differentiated hiPSCs on PCL scaffold 7 days after seeding. a The fibers of PCL scaffold were randomly entangled to form a strong, flexible, and porous 3D matrix (scale bar 10 μm). b The fibers of PCL scaffold with higher magnification (scale bar 2 μm). c Plated hiPSCs on PCL scaffold that grew and differentiated on PCL scaffold 7 days after seeding (scale bar 20 μm). d hiPSCs on PCL scaffold with higher magnification (scale bar 10 μm). e Diameters of the nanofibers


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evaluate the activity of the IDE1. IDE1 caused to increase the percentage of Sox17-positive cells to 45 % of total cells, FoxA2-positive cells to 45 %, and Gsc-positive cells to 45 % of the total cells after 6 days of differentiation (Fig. 2). These results confirmed that treatment with IDE1 could significantly induce differentiation of hiPSCs into DE cells in 3D culture. Definitive Endoderm-Specific Gene Expression The mRNA expression of DE-specific genes was observed during hiPSC differentiation by qRT-PCR at day 6. qRT-PCR analyses revealed that IDE1 treatment at 2 μM resulted in a significant increased expression of DE genes such as FoxA2, Sox17, and Gsc (Fig. 3). We also observed a significant decrease in expression of brachyury, a mesendoderm marker; Sox1, an ectoderm marker; and Sox7, a visceral endoderm marker; Nanog and Oct4, pluripotent markers compared to a control experiment without IDE1 induction (Fig. 3). In this study, the control group did not express mRNAs of the DE genes (Fig. 3). Cell Viability Cell viability and proliferation are dependent on the interaction between the cells and the biomaterial scaffold. According to our results, within the nanoenvironment provided by PCL scaffold, hiPSCs tended to survive more than monolayer culture. MTT results indicated that

Fig. 2 Immunofluorescence performed for Sox17, FoxA2, and Gsc as DE-specific proteins by differentiated hiPSCs on nanofibrous PCL scaffold by IDE1 after 6 days. As a control, fixed hiPSCs were stained with the secondary antibodies only. Staining of nucleus was performed by DAPI (×40). *P

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