Induced pluripotent stem cells (iPSCs) have attracted the attention of many surgeons and scientists for cell replacement therapies. Nanofibrous biocompatible scaffolds have been shown to promote better cell adhesion and improve stem cell differentiation. In the present study, after fabrication using electrospinning technique and surface modifications, the characteristics of Polyethersulfone (PES) nanofibers were determined by scanning electron microscopy (SEM), ATR-FTIR and MTT assay. Then, the hepatogenic potential capacity of iPSCs was evaluated using Real-Time RT-PCR and immunocytochemistry (ICC) after cultured on collagen coated polyethersulfone (PES/COL) scaffolds. After scaffolds characterization, analysies of two important definitive endoderm specific markers (such as Sox17 and Foxa2) using Real-Time RT-PCR and ICC indicated increase that thesein their mRNA and protein levels were increased after 5 days of hepatogenic induction. In addition, to determine hepatic differentiation of iPSCs cultured on PES/COL, the expression of albumin and α-FP was evaluated by ICC after 20 days. Real-Time RT-PCR analysis showed increased expression of albumin, TAT, cytokeratin19 and Cyp7A1 genes over during the course of the differentiation program. In conclusion, our results demonstrated that PES/COL nanofibrous scaffolds could be a proper substrate to significantly increase for the hepatogenic differentiation potential of iPSCs to significantly increase in their potential and it could also be introduced as a promising candidate for liver tissue engineering applications.
Keywords: Stem Cell, Nanobiotechnology, Liver/hepatocytes, Cell Differentiation
List of abbreviations: iPSCs, Induced pluripotent stem cells; PES, Polyethersulfone; PES/COL, collagen coated polyethersulfone; αFP, α-Fetoprotein; ATR-FTIR, Attenuated total reflection Fourier transform infrared spectroscopy; CK19, Cytokeratin19; ICC, immunocytochemistry;
Liver failure is an important clinical challenge since itand is one of the leading causes of death in the worldwide. Currently, whole-organ liver transplantation is the only curative treatment. Donor shortage and possible immune rejection limit the efficiency of transplantation and subsequently increase the number of people living with end stage of liver disease . As an alternative to liver transplantation, tissue engineering has focused on developing cell-based therapies to overcome the scarcity of liver donors, so that hepatocytes generated by differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in culture can provide an unlimited supply of such cells for transplantation . iPSCs, which have been generated by Yamanaka and colleagues, are very similar to ESCs in morphology, differentiation potential, telomerase activity, surface markers and gene expression profiles . These cells are pluripotent and have differentiation potential into three germ layers lineages, including endodermal hepatocyte-like cells , . Importantly, several studies have shown that the hepatocyte-like cells generated from iPSCs express many transcription factors and markers of hepatocytes ,,. Therefore, with due to their unique properties, iPSCs have been proposed as a very valuable and unlimited transplantable source of hepatic cells source for patients with end-stage liver diseases . While Since the hepatocytes differentiated from patient-specific iPSCs are genetically identical cells, immune rejection as well as ethical issues associated with human ES cells would be prevented and an in vitro model for liver disease would be provided . Tissue engineering has been used to develop compatible scaffolds that possesseswith similar structure and properties to match with native extracellular matrix (ECM), which comprises a complex network of nanoscale fibers forming highly structured local microenvironments . Electrospinning is a valuable and readily controllable method for preparation of these scaffolds, which has been widely studied for fabrication of nanofibers with a high surface area and porosity . Nanofibers improve the nutrient transfer as well as exchange of oxygen and other metabolites. Several nanofibrous scaffolds such as collagen coated poly(L-lactic acid) and PCL/collagen/polyethersulfone (PES) composite have been reported as suitable substrates for hepatocytes [13-15]. As a biocompatible and non-biodegradable polymer, PES had has been mainly used in membrane materials with applications such as filtration and hemodialysis . Recently, several studies showed that PES nanofibrous scaffolds fabricated by electrospinning could be a suitable substrate to support proliferation and differentiation of different cells and stem cells. For instance, these scaffolds have the potential for feeder-free culture of pluripotent stem cells because of its 3-dimensional3D structure and bioactivity which that enhances proliferation, differentiation and infiltration of embryonic stem cells . Kinasiewicz et al observed that PES membranes support the growth of hepatic cells growth in 3D and even after transplantation to SCID/NOD mice . In addition, human hepatoblastoma cell line (HepG2) grow on PES membrane and maintain their crucial functions . For liver regeneration, a 3D spatial architecture could enhance liver-specific gene expressions and function. Furthermore, topographical cues and porosity are essential for nutrient transfer of the scaffold and growth factors are essential for the cell attachment, proliferation, migration and differentiation to hepatocytes cells mainly because hepatocytes consume about 10 folds times more higher oxygen compared to other cells . In addition, data shows that hepatocytes exhibit better viability and attachment on chitosan nanofibers than film . Despite several studies a volume of research abouton differentiation of iPSCs into hepatocyte-like cells in two-dimensional space, the robust protocols for iPSCs derived hepatocytes that sustain a high level of biological function on PES nanofibers remain elusive. It seems that a combination of iPSCs and nanofibrous scaffolds has the potential for treatment for of liver disease. In addition, as the most abundant ECM protein in liver cells regulating hepatocyte behavior and gene function, collagen is used as a coating for nanofibers to improve cell attachment . The goal of this study was to evaluate the hepatic differentiation capacity of iPSCs on fabricated and modified PES nanofibers with collagen coating (PES/Col) in vitro.
2. Materials and methods
Nanofibers were produced by electrospinning methods according to the protocol previously reported by Shabani et al. . Briefly, Polyethersulfone powder (Ultrason E6020P, MW: 58,000 Da, USA) was dissolved in N,N-Dimethylformamide (Merck, USA) at 25%wt. Then, the solution was fed into a 10-ml glass syringe and driven by a syringe pump. A voltage of 20 KV was applied by a High DC power between the tip of the needle and the collector at a distance of 15 cm.
2.2. Surface modification
After scaffold fabrication, plasma treatment was performed under the optimized conditions of 40 kHz frequency with a cylindrical quartz reactor (Diener Electronics, Germany) by introducing pure oxygen into the reaction chamber at 0.4 (mbar) pressure and then the glow discharge was ignited for 5 min. For collagen grafting, plasma-treated sheets were punched and immersed in 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS, country) solution (5mg/mL) for 12 h. Then, the scaffolds were treated with 1mg/ml collagen I (Advanced Biomatrix, PureCol) solution overnight. Nanofibrous scaffolds (PES/COL) were soaked overnight in culture medium supplemented with 2X pen/strep (Sigma) and 5X amphotericin B (Sigma) at 37°C.
2.3. Attenuated total reflection Fourier transform infrared spectroscopy
After plasma treatment and collagen grafting, surface chemical modifications were investigated by attenuated total reflection Fourier transform infrared (ATR-FTIR). The spectra were recorded using an Equinox 55 spectrometer (Bruker Optics) equipped with a deuterated triglycine sulfate (DTGS) detector and a diamond ATR crystal.
2.4. Characterization of the nanofibers
The morphology of nanofibers was examined by scanning electron microscopy (SEM; Hitachi S-4500, Japan) with and without stem cells. The cell-loaded scaffolds were rinsed with phosphate buffered saline (PBS, sigma, USA) before scanning and were fixed in 2.5% glutaraldehyde. After dehydration through a graded series of ethanol, the scaffolds were vacuum dried and sputter-coated with gold.
2.5. iPSCs culture and hepatogenic differentiation
The iPSCs were kindly donated by Stem Cells Technology Research Center cell bank that and were routinely maintained on mitotically inactivated mouse embryonic fibroblast (MEF) as feeders . Culture media medium consisted of DMEM/F12 (Gibco) supplemented with 15% Knock Out Serum Replacement (SR), 2 mM L-Glu, 1 mM 2-mercaptoethanol, 1 mM nonessential amino acids, 50 U/ml Penicillin/Streptomycin and 10 ng/mL bFGF, all from Invitrogen. The medium was changed daily, and approximately once every 5 days, iPSCs colonies were detached using collagenase type IV (1 mg/mL) and sub-cultured. To generate hepatocytes, monolayers of iPSCs harvested using collagenase then were plated on PES/COL nanofiber at a density of 2.5×105 cells per well in MEF-conditioned media for one week. iPSCs were differentiated into hepatocytes as previously described . In the first step, the culture medium was replaced with 100ng/ml Activin A (R&D Systems) in RPMI/B27 medium (Invitrogen) for 5 days followed with 20ng/ml BMP4 (Peprotech) and 10ng/ml FGF-2 (Invitrogen) in RPMI/B27. After 5 days, the medium was changed replaced with RPMI/B27 supplemented bywith 20ng/ml HGF (Peprotech) and finally for 5 days with 20ng/ml Oncostatin-M (R&D Systems) in Hepatocyte Culture Media (Lonza) supplemented with SingleQuots (without EGF).
2.6. MTT assay
The attachment and proliferation ability of iPSCs on the PES/COL nanofibers were was evaluated via MTT assay during 4 days. Briefly, sterilized nanofibers were placed in cell culture plate, seeded at a cell density of 4 × 103 cells per cm2 and incubated at 37 °C with 5% CO2. At 24, 48 and 72 h after cell seeding, MTT solution (3-[4,5-dimethyl-thiazolyl-2]-2,5-diphenoltetrazolium bromide) with a final concentration of 5 mg/ml in DMEM was added to each well. After 3 h incubation, the medium was removed and the formazan crystals were solubilized with dimethylsulfoxide (DMSO). Absorbance of the converted dye was read at a wavelength of 570 nm in a micro-plate reader. Data were obtained as the mean values of three repeats.
2.7. Immunofluorescence Staining
In order to evaluate the hepatic differentiation, the cultured differentiated iPSc were fixed with 5% paraformaldehyde (PFA; Sigma-Aldrich) for 30 min in definitive endoderm and hepatocyte steps. Then, they were permeabilized with 0.2% triton X-100 for 10 min and blocked in 10% goat serum in PBS for 1 h at 37°C. For definitive endoderm step, primary antibodies against Human Sox17 (1:20; Polyclonal Goat IgG, R&D, AF1924), human Foxa2 (1:500, Polyclonal rabbit IgG; Millipore, AB4125) and for hepatogenic differentiation, albumin (1:200, Santa Cruz, Sc-46293),
α-FP (1:200, Santa Cruz, Sc-8108) and antibodies were diluted in 1% BSA in PBS overnight. Secondary antibodies included Alexa fluor 488 donkey anti-goat (1:200; Gibco, A-11058) or Alexa Fluor 594 donkey anti-rabbit (1:200; Gibco, A-21207). The nuclei were counterstained with DAPI (0.1 μg/ml, Sigma-Aldrich, D8417) for 5 min.
2.8. RNA extraction and Real-Time RT-PCR
Total RNA was isolated using Roche High pure RNA isolation kit (Ref: 11828665001). Then, reverse transcription of 1 μg of total RNA to cDNA was performed using the M-MuLV Reverse Transcriptase kit (Fermentas) and a random hexamers primer in a total reaction volume of 15 μl.
The polymerase chain reaction (PCR) parameters included denaturation at 95°C for 3 min and then 40 cycles at 95 °C for 20 s, annealing at 60 °C for 30 s and elongation at 72 °C for 30 s. For Real-Time RT-PCR, the reactions were set up in duplicate with the SYBR premix ExTaq (Takara) in the 96-well optical reaction plates on Step One Plus TM Real-Time PCR machine. The reaction was carried out at 95 °C for 2 min, followed by 40 amplification cycles (each 5 s, at 95 °C, 30 s at 60 °C) with fluorescence detection and a final step of melting curve analysis. Primer sequences are illustrated in Table 1.
3.1. Scaffold characterization
PES and PES/COL scaffolds were morphologically characterized by SEM, which showed non-woven, randomly oriented nanofibers with porous structures (Fig 1). The plasma treatment and collagen coating had no significant effect on the structure of scaffolds (data not shown). Optical micrographs of iPSCs colonies before starting differentiation are shown in Fig 2A and after culture and differentiation to hepatocyte on nanofibrous PES during 20 days in Fig 2B and C. The cells penetrated and adhered well onto the nanofibrous hybrid scaffolds and then expanded on the surface of nanofibers by proliferation during the period of study, which confirmed biocompatibility of these mats. ATR-FTIR spectroscopy confirmed grafting of collagen onto the surface of PES nanofibers with three peaks at 1649, 2930 and 3325 cm-1 that were related to amide A and amid II bands respectively existing in collagen backbone (Fig3).
MTT assay was used to for biocompatibility evaluation of scaffolds in addition to the SEM analysis on iPSCs cultured scaffolds. Results of MTT assay were demonstrated that the proliferation rate of iPSCs was significantly enhanced when cultured on PES/COL in comparisonrelative to the TCPS during days 3 and 4 after cell seeding (Fig4).
3.3. Differentiation of iPSCs to Definitive Endoderm
We have used a four-step protocol by modifying a previous method , so that iPSc were routinely cultured without MEF and treated with 100 ng/ml Activin A for 5 days. During this period, we evaluated the endoderm identity of these differentiated cells on nanofibers by ICC and Real Time RT-PCR. Our ICC results showed the expression of major transcription factors of the definitive endoderm such as Foxa2, Sox17, and which were significantly higher in differentiated cells (Fig5). The cells gradually exhibited morphological change and had alsoa high proliferation toopotential. Moreover, endoderm identity of these differentiated cells was further confirmed by Real-Time RT-PCR analysis and the results also showed significantly increase in the expression of Sox17, and Foxa2 but the increase in GATA4 was not significant during differentiation (Fig6).
3.4. Differentiation of iPSCs to Hepatocyte cells
To determine in vitro hepatogenic differentiation of iPSCs on PES/COL nanofibers, the expressions of albumin and αFP were was evaluated by ICC analysis after 20 days. As shown in Fig. 7, the differentiated cells were intensely stained for albumin and αFP (Fig7). These markers were not expressed in iPSCs used as the control.
3.5. Gene Expression Pattern during hepatogenic Differentiation of iPSCs
The expression of human liver cell markers, namely albumin, CK19, TAT and Cyp7A1 was also evaluated by Real Time RT-PCR in the differentiated cells on the surface of nanofibrous scaffolds. Real Time RT-PCR revealed that the expression levels of albumin, CK19, TAT, Cyp7A1 were was significantly increased in differentiated cells on scaffolds comparison to the undifferentiated iPSCs (Fig8).
The importance of iPSCs in tissue engineering is promising; however, bioartificial liver engineering still seems to be out of reach. Several studies report the hepatogenic potential of iPSCs by using directed developmental signaling, but up to recent years, the hepatocyte-directed differentiation of iPSCs has mainly been done in 2D culture, and so far the fully functional hepatocytes which maintaining its their characteristics in 3D is restrictedhave not been developed . In addition, scaffolds provide a suitable place for cell attachment through increasing surface area as well as supporting stem cell proliferation and differentiation , . So that the combination Combination of iPSCs with scaffolds have has many several beneficial effects such as slow diffusion of small molecules, metabolites and fabrication of hepatic constructs for possible liver tissue engineering purposes. In the present study, our results were demonstrated that iPSCs differentiate into hepatocyte in the presence of PES nanofibers coated with collagens as well as growth factors, which is probably useful as a delivery vehicle for hepatocyte transplantation. Feng et al. reported that collagen coated electrospun poly(L-lactic acid) nanofibers could be a suitable microenvironment for hepatocytes adhesion and proliferation . In addition, Kazemnejad et al. had used PCL/Col/PES composite scaffolds for differentiation of hMSCs into hepatocyte like cells and their results showed a significant increase in hepatocyte-specific markers in comparison to the controls . A recent publication reported that nanofibers enhance the differentiation of ES cells into neural lineages, as well as human MSCs into hepatoblasts , . Our SEM result of the collagen-grafted PES nanofibers showed a highly porous and non-woven architecture that mimics ECM structure. Recently, Shabani et al showed that PES/COL scaffolds enhance infiltration and biomineralization of stem cells . In fact, the high proliferative capacity of iPSCs demands better filtration and nutrient supply, which would be provided by the special structure of PES scaffold. In addition, as functional units of liver, hepatocytes consume 5 to 10 folds more oxygen compared to other cells. Moreover, this scaffold has capacity for expansion of various types of stem cells and as well as differentiation enhancement of stem cell into osteoblasts both in vivo and in vitro  , . Our experiments clearly confirmed the beneficial effects of PES/COL scaffold not only by providing a suitable support for iPSCs proliferation but also promoting their hepatogenic differentiation during 4 weeks, and maintaining hepatocyte stability after differentiation. Dunn et al. cultured hepatocytes in a sandwich configuration between two layers of collagen I gel, and haswhich prolonged the time of cultures displaying hepatocyte-specific functions up to several weeks . It seems that collagen, as the most abundant ECM protein in liver, plays a major role in the hepatogenic differentiation of iPSCs and improves cells-scaffold interactions . Our results showed that iPSc differentiate to definitive endoderm on PES/COL scaffolds by using Activin A for 5 days with a typical expression of definitive endoderm associated markers such as Sox17, FaxA2 and GATA4 in protein and mRNA level. Several reports suggest that Fox a family and GATA factors are pre-initiation players of the endodermal gene transcription ,.
Based on the our knowledge of endoderm development, Nodal is necessary for endoderm induction from pluripotent stem cells, but in this study, Activin A was used as a surrogate for Nodal is used because of its high price. The differentiation protocol used in this study completely recapitulates the development of liver tissue in four define definite steps. During embryogenesis, hepatic endoderm is affected by secreted factors from the mesodermal cells. BMP4 and bFGF growth factors are secreted from the cardiac mesoderm at the time of hepatic induction . After 20 days of differentiation, the cells were harvested and assessed for expression of α-FP and albumin, which confirmed differentiation toward hepatoblasts cells. Apart from ICC, we applied Real Time RT-PCR to detect the expression of liver cell markers such as albumin, CK19, Cyp7A1 and TAT over 20 days of induction on PES/COL scaffold, that which positively confirmed the ICC results, too. In future, in vivo studies would show the exact mechanism of PES/COL nanofibrous scaffold for in improvement of hepatogenic differentiation as well as maturation of hepatocytes cells.
According to aforementioned results, collagen I coated nanofibers demonstrated an efficient contribution to hepatogenic differentiation of iPSCs and could be considered as a new potential for liver tissue engineering and regenerative medicine.
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Fig. 1 Morphology of Polyethersulfone nanofibers at two magnifications (×1000, ×5000). Collagen coated polyethersulfone at two magnifications (×1000, ×5000).
Fig. 2 A: Optical micrographs of induced pluripotent stem cell (iPSC) colonies before starting differentiation. B, C: SEM analysis of iPSCs culture on PES/COL nanofibers after 20 days of differentiation.
Fig. 3 Attenuated total reflection Fourier transform infrared spectra of PES nanofibers (PES) and collagen grafted PES nanofibers (PES-COL).
Fig. 4 MTT cell proliferation assay of iPSCs on collagen coated polyethersulfone scaffold and tissue culture polystyrene (TCPS) during a 1, 2, 3 and 4 days of culture period (asterisks show significant difference between the groups at P<0.05)
Fig. 5 The immunochemical analysis of differentiated iPSc on day 5 for definitive endoderm markers Foxa2 and Sox17 with nuclear counterstaining (DAPI).
Fig. 6 Real Time RT-PCR analyses of the expressions level of Definitive Endoderm genes at day 5 after differentiation of iPSc on collagen-grafted PES nanofibers. The expression levels were normalized to control group.
Fig. 7 The immunocytochemical analysis of differentiated iPSCs on day 20 for α-FP (green) and Albumin (Red) with nuclear counterstaining (DAPI).
Fig. 8 Quantitative RT-PCR analyses of the expressions level of hepatic cell genes at day 20 after hepatogenic differentiation of iPSc on collagen-grafted PES nanofibers. The expression levels were normalized to control group.
Table 1. Human primers used in Real Time RT-PCR studies.
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