Nanotopography Promotes Pancreatic Differentiation of Human

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Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells Jong Hyun Kim, Hyung Woo Kim, Kyoung Je Cha, Jiyou Han, Yu Jin Jang, Dong Sung Kim, and Jong-Hoon Kim ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b06985 • Publication Date (Web): 22 Feb 2016 Downloaded from http://pubs.acs.org on February 24, 2016

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Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells Jong Hyun Kim†,# , Hyung Woo Kim‡,#, Kyoung Je Cha‡, §, Jiyou Han†, Yu Jin Jang†, Dong Sung Kim*,‡, and Jong-Hoon Kim*,† †

Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of

Life Sciences and Biotechnology, Science Campus, Korea University, 145 Anam-ro, Seongbukgu, Seoul, 02841, Korea ‡

Department of Mechanical Engineering, Pohang University of Science and Technology

(POSTECH), 77 Cheongam-Ro, Nam-gu, Pohang, Gyeongbuk, 37673, Korea * Address

§

correspondence to [email protected], [email protected].

Present address: Ultimate Manufacturing Technology Group, Korea Institute of Industrial

Technology (KITECH), Techno Sunwan-ro Yuga-myeon Dalseong-gun, Deagu 711-880, Korea #

JH Kim and HW Kim contributed equally to this work as first authors

*

DS Kim and JH Kim contributed equally to this work as corresponding authors.

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ABSTRACT

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nano-patterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200-nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1+ pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200-nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

Keywords: polystyrene nanopore surfaces, nano-injection molding, human embryonic stem cells, induced pluripotent stem cells, pancreatic differentiation


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Human pluripotent stem cells, including human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are capable of differentiating into a wide spectrum of cell types. Thus, these cells are considered a useful source of various somatic cells that may reconstitute lost or injured tissues to treat a variety of human diseases. Our and other studies demonstrate that human ES and iPS cells can be efficiently differentiated into specific cell types, including neurons, liver cells, and pancreatic cells that function in animal models of different diseases.1-4 However, most differentiation methods developed so far mainly rely on biochemical soluble factors including cytokines, growth factors, and/or small molecules that inhibit or stimulate particular intracellular signaling networks in defined culture conditions.5-7 Recent encouraging studies, including our own, demonstrate that sequential treatments of cytokines or growth factors under three-dimensional (3D) culture conditions may further increase the differentiation yield or function of stem cell-derived somatic cells.8,9 Nevertheless, to derive clinically relevant somatic cells from human pluripotent stem cells, current in vitro differentiation systems should be further refined and optimized to more closely mimic the microenvironment in which desired target tissues develop during normal embryonic development. More recently, increasing evidence suggests that not only biochemical soluble factors but also biophysical and mechanical forces from surrounding extracellular microenvironments strongly influence various cellular behaviors by actively regulating gene expression.10 Some of these findings suggest that surface properties mimicking physiologically relevant extracellular matrix (ECM) characteristics, such as physical nanotopographies and matrix stiffness, play a crucial role in controlling not only the self-renewal but also the differentiation of stem cells.11,12 Many notable studies have been performed not only to obtain insights into stem cell differentiation in response to nanotopographies but also to provide a new solution for directing


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differentiation of stem cell by combining biochemical and biophysical properties. For example, polymethylmethacrylate and polycarbonate 120-nm nanopits were found to promote osteogenesis and support long-term maintenance of human adult mesenchymal stem cells.13,14 In addition, efficient and rapid neural differentiation was achieved from human ES cells when cultured on polyurethane acrylate 350-nm ridge/groove patterns without the use of growth factors or cytokines.15 We also showed that polystyrene (PS) nanopillar and nanopore surfaces regulate osteogenic and adipogenic differentiation of human adipose-derived stem cells,16 respectively. Thus, these studies strongly suggest that proliferation and differentiation of stem cells could be controlled by the topographical surface texture created by nanoscale patterns. In particular, cells physically interact with nanotopographies on surfaces through attachment points (e.g., integrins, cadherins, gap junctions) linking cytoskeletal elements to extracellular binding sites, and these interactions strongly affect in vitro cell adhesion, cell morphology, and consequently gene expression.17-18 Nanotopographies that increase cell adhesion can effectively regulate cell functions by mechanotransduction through the cell membrane19-21, whereas nanotopographies that induce low cell adhesion allow cells to form spheroids of specific cell types by promoting cell-cell interaction.22,23 However, few studies have attempted to investigate the effects of biophysical cues or the combined effects of biochemical and biophysical cues on the differentiation of ES or iPS cells compared with adult stem cells. Pancreatic cells in the islets of Langerhans play a vital role in the maintenance of blood glucose homeostasis by secreting insulin. Thus, degeneration of β cells results in diabetes mellitus, and exogenous insulin injection is the only major therapeutic option for controlling the disease. Although transplantation of the whole pancreas or isolated islets may have positive therapeutic outcomes, the shortage of donor islets is still problematic. We and others show that


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insulin-producing cells can be derived from human pluripotent stem cells under 2D and 3D conditions.3,24-26 However, the differentiation yield and/or function of stem cell-derived cells is still relatively low compared with normal pancreatic cells.27 The developing pancreas forms complex structures, playing a bifunctional role in exocrine digestion and endocrine hormonal regulation. Particularly, ECMs surrounding the developing pancreas play crucial roles in organogenesis by generating signals that control complex developmental processes, including branching morphogenesis.28 These findings strongly suggest that cell fate determination and differentiation of β cells can be controlled by surrounding physical cues. However, no attempt has yet been made to address this issue. In the present study, we developed PS nanopore-patterned (NPo) surfaces with different nanopore diameters (130, 170, 200, or 230 nm) to investigate the effect of nanotopographical cues in different stages of pancreatic differentiation of human ES and iPS cells. We showed that 200-nm NPo surfaces strongly promote differentiation into pancreatic progenitors that can give rise to pancreatic endocrine cells.


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RESULTS Nanofabrication of PS NPo plates by thermal nanoimprinting and nano-injection molding A three-step nanofabrication process for PS NPo surfaces consisting of aluminum anodizing, nickel nano-electroforming, and PS nano-molding (i.e., thermal nanoimprinting or nano-injection molding) is schematically shown in Fig. 1. The fabrication processes of NPo surfaces are similar to those previously reported.16,29 In this study, anodic aluminum oxide (AAO) nano-templates with NPo surfaces were fabricated through a two-step aluminum anodization process in which the diameter of nanopores was easily controlled by varying a widening time (Fig. 1A). The nickel nano-mold inserts for PS nano-molding were then cost-effectively manufactured by a nano-electroforming process on the AAO nano-templates (Fig. 1B-C). To determine the optimal diameter of nanopores for pancreatic differentiation of human ES and iPS cells, PS NPo surfaces with various nanopore diameters (130, 170, 200, or 230 nm) with a depth of ~500 nm and interpore distance of ~500 nm were replicated by thermal nanoimprinting (Fig. 1D-1). Representative scanning electron microscope (SEM) images of replicated PS NPo patterns are shown in Supplementary Fig. 1. After determining the optimal diameter of nanopores for pancreatic progenitor differentiation, PS cell culture plates with the optimized NPo surface29 were replicated through nano-injection molding (Fig. 1D-2). An SEM image of the PS NPo surface on the cell culture plate replicated through nano-injection molding is shown in Fig. 1G. PS cell culture plates with flat (FL) surfaces were also produced by injection molding, with a mold insert having a mirror-like surface used as a control group. The FL and NPo surfaces were treated by oxygen plasma to enhance both surface hydrophilicity and cell attachment to the surfaces. The water contact angles of oxygen plasma-treated PS surfaces were stabilized around 60° (Fig. 1E and Supplementary Fig. 1), which is a suitable contact angle range for adequate cell attachment


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to the surface (Fig. 1F). The protein adsorption levels were similar with each other for all prepared surfaces (NPo and FL surfaces) when treated with 0.2% FBS after fibronectin coating (The detailed results are described in supplementary information, Supplementary Fig.2). From these results, we could suppose that the chemistry of all surfaces is identical, suggesting the changes in topography are crucial in modulating the pancreatic differentiation of human pluripotent stem cells in this study.

Determination of optimal nanopore diameter for pancreatic differentiation of human pluripotent stem cells The human ES and iPS cells used in the present study expressed pluripotent stem cell markers, including OCT4, NANOG, and SSEA4 (Supplementary Fig. 3). We first determined the optimal nanopore diameter for effectively directing the differentiation of human ES and iPS cells into pancreatic cells or the endoderm, a founder tissue of the pancreas. Different diameters of nanopores (130, 170, 200, or 230 nm) with a depth of ~500 nm and an inter-pore distance of ~500 nm were tested for cell attachment and initial differentiation of human ES cells. Human ES cells grown on mouse embryonic fibroblast (MEF) feeder cell layers were harvested and passaged two times under feeder-free conditions (mTeSR medium supplemented with bFGF) to remove a residual population of MEF feeder cells. The expended human ES cells were dissociated and grown further on NPo or FL surfaces under the feeder-free condition. The number of human ES cells attached on NPo plates did not significantly differ depending on nanopore diameter and was comparable to that on control FL plates (Fig. 2A). Interestingly, however, when human ES cells were differentiated for 6 days, a trend of increased expression of


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PDX1, a marker of pancreatic progenitors,28 was observed as the pore diameter increased in the NPo plates (Fig. 2B). The highest PDX1 expression was detected when cells were differentiated on 200-nm nanopores. A lower but similar pattern of expression was observed for FOXA2, a key endodermal gene that controls PDX1 expression in the developing pancreas (Fig. 2B).30 The expression of NKX6.1, which regulates β cell maturation,31 did not markedly change across the 6 days of differentiation, but a moderate increase was also detected on 200-nm nanopores (Fig. 2B). Based on this result, 200-nm NPo culture plates were used for the following experiments. Previous studies report variations in cell behaviors, including proliferation and differentiation, between different human ES and iPS cell lines.32 Thus, we examined the adherence and proliferation capabilities of multiple human ES and iPS cell lines on 200-nm NPo surfaces before inducing pancreatic differentiation. We found that all human ES and iPS cell lines tested attached and proliferated on 200-nm NPo plates without a significant difference from control FL plates (Supplementary Fig. 4). Interestingly, however, SEM imaging showed clear morphological changes of cells on NPo plates compared with FL plates. The leading edge of human ES cells on NPo plates stretched out several filopodia, whereas lamellipodia without noticeable filopodia projections were observed on FL plates (Fig. 2C). Immunofluorescence analyses revealed that levels of F-actin-binding phalloidin and cell-cell/cell-matrix adhesionassociated vinculin and E-cadherin were upregulated at the margin and center of human ES cell clusters grown on NPo plates compared with FL plates (Fig. 2D-F). Despite clear morphological differences, the expression levels of pluripotency markers (OCT4, NANOG, and alkaline phosphatase) in ES and iPS cells were not significantly different between NPo and FL plates (Fig. 2E, G-I; Supplementary Fig. 5). The expression of endodermal (SOX17 and FOXA2), mesodermal (T, brachyury transcription factor), and


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ectodermal (SOX1) markers were not detected or were detected at very low levels in ES and iPS cells on both NPo and FL plates (Fig. 2G-I). These data suggest that 200-nm NPo plates provide a favorable environment for pancreatic commitment upon differentiation of human ES and iPS cells and induce no significant change in pluripotent properties of these cells.

Endodermal differentiation of human pluripotent cells on NPo plates We next investigated whether the 200-nm NPo plates direct human ES and iPS cells toward pancreatic cell fates during different steps of differentiation by analyzing the expression of stagespecific markers (Supplementary Fig. 6). To test the effect of only physical cues of NPo plates on endodermal differentiation, human ES and iPS cells were first differentiated on NPo or FL surfaces in the absence of growth factors or cytokines for 3 days and stained with an antibody against SOX17, an endoderm marker. Immunostaining analysis showed that the number of SOX17+ cells was significantly increased when ES and iPS cells were differentiated on NPo surfaces compared with conventional FL plates (Fig. 3A; FL, 9.30 ± 2.74% vs. NPo, 20.17 ± 2.19%). In line with this observation, we found significant increases in the expression of SOX17 and an additional endodermal marker CXCR4 on NPo surfaces at the mRNA level as determined by quantitative polymerase chain reaction (qPCR) (Fig. 3B). NPo surfaces did not significantly change the expression of neuroectodermal (SOX1), mesodermal (T), and primitive endodermal (SOX7) genes from the baseline levels observed on control FL plates. Next, we tested whether stimulation with physical nanotopography in combination with biochemical soluble factors may further increase endodermal differentiation. To address this hypothesis, human ES cells were differentiated on NPo surfaces in the presence of wnt3a and activin A, both of which promote


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endodermal induction.33,34 Immunofluorescence staining and fluorescence-activated cell sorting (FACS) analyses for SOX17 and CXCR4 showed that although combined physical and biochemical stimulation profoundly increased the differentiation efficiency of ES cells into endoderm, a similar level of endodermal induction was also achieved by the same growth factor treatment on FL plates (Fig. 3C and D).

Differentiation into pancreatic progenitors on NPo plates The pancreas develops from the posterior part of the foregut endoderm.35 Therefore, the regionspecific patterning of differentiating endodermal cells is essential for efficiently deriving pancreatic cells in vitro. To investigate the effect of nanopore topography on differentiation into pancreatic progenitors, endodermal cells were first derived from human ES cells in the presence of wnt3a and activin A and were then further differentiated on NPo or FL plates for an additional 4 days without cytokines or growth factors. qPCR analyses revealed that NPo surfaces significantly upregulated PDX1 as observed in Fig. 2 and also increased the expression of ALB (albumin), which is a protein produced in the liver (Fig. 4A). On the other hand, other regionspecific genes of the gut tube, including SOX2/BARX1 (stomach) and CDX2/CES2 (intestine), as well as SOX7, a marker gene of the extra-embryonic endoderm, were downregulated when cells were differentiated on NPo surfaces (Fig. 4A). We next attempted to enhance NPo-induced pancreatic differentiation by treatments with developmentally relevant soluble factors. Immunostaining analyses showed that the percentage of PDX1+ cells on control FL plates was 24.9%, even after 4 days of combined treatment with retinoic acid (RA) and noggin, which favor pancreatic specification of human ES cell-derived


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endoderm36 (Fig. 4B, C). Interestingly, the number of PDX1+ cells was increased by up to 72.3% on NPo surfaces by the same treatment, whereas no significant difference in the number of EdU+ proliferating cells was found between control FL and NPo surfaces (Fig. 4B, C). These observations were further validated in two human iPS cell lines: ANFXF-1 derived from adipocytes and UNFXF-1 derived from urine (Supplementary Fig. 7A). A recent study demonstrated that the homeodomain transcription factor NKX6.1 is both necessary and sufficient to specify pancreatic endocrine progenitors into insulin-producing cells in cooperation with PDX1.37 Although PDX1+ cells were efficiently derived from human ES and iPS cells on NPo plates, very few NKX6.1+ cells were observed among the PDX1+ cell population. Thus, we treated differentiating cells with pancreatic development-associated factors bFGF, activin βB, and vascular endothelial growth factor (VEGF) on FL or NPo surfaces for an additional 4 days (Supplementary Fig. 6). Strikingly, the expression of NKX6.1 was increased on NPo surfaces, so that the number of NKX6.1+ cells among the PDX1+ cell population was significantly higher on NPo plates (64.6% of PDX1+ cells; 78.5% of total cells were PDX1+) than on control FL plates (24.9% of PDX1+ cells; 28.53% of total cells were PDX1+) (Fig. 4D and Supplementary Fig. 7B for iPS cells). Instead of PDX1+ cells, a preferential differentiation into cells expressing CK19, a marker of the pancreatic duct, were observed on FL plates (Fig. 4E). qPCR data supported the effect of a NPo surface, showing strong upregulation of PDX1, NKX6.1, and NKX2.2 together with NEUROG3 and NEUROD1, which are additional pancreatic endocrine progenitor genes. Concomitantly, SOX17 expression was decreased at this stage of differentiation (Fig. 4F). These results indicate that a matrix surface with 200-nm nanopores and developmentally relevant factors efficiently induced the posterior foregut patterning of endoderm and subsequent differentiation into pancreatic progenitors.


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Differentiation of NPo-induced pancreatic progenitors into endocrine cells Several transcription factors, including NEUROG3, NEUROD1, ISL1, and PAX6, play important roles in the differentiation of pancreatic endocrine progenitors into β cells.38-41 To test whether pancreatic progenitors produced on NPo surfaces have the potential to give rise to β cells, cells were further differentiated for 4 days in the presence of exendin-4, which promotes the maturation of endocrine progenitors in human fetal pancreatic islets (Supplementary Fig. 6).42 The numbers of NEUROD1+ and ISL1+ cells consistently increased when cells were differentiated on NPo plates compared with control FL plates (Fig. 5A). Additionally, FACS analysis revealed that a greater percentage of cells expressing CD200, a surface marker of pancreatic endocrine cells, was obtained on NPo plates (Fig. 5B). Expression profile data obtained from the human ES cell PCR array also confirmed the clear upregulation of genes associated with pancreatic lineages, including INS (insulin), GCG (glucagon), PDX1, NEUROD1, and PAX6, by NPo surfaces (Fig. 5C). We next tested whether adding additional factors promote the differentiation of pancreatic endocrine cells on NPo surfaces. Immunostaining showed that the treatment with exendin-4, together with insulin-like growth factor (IGF) and hepatocyte growth factor (HGF) for the last 4 days (Supplementary Fig. 6) profoundly increased the number of ISL1+ cells (Fig. 5D). Furthermore, immunostaining and FACS analyses using antibodies tested on human pancreatic tissue (Fig. 5E) demonstrated that 30-37% of cells differentiated on NPo surfaces were positive for insulin, and the insulin+ cells also co-expressed c-peptide (Fig. 5F and G). A few cells positive for glucagon or somatostatin were also found on NPo surfaces (Fig. 5H). As expected, a


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much lower level of pancreatic differentiation was achieved on control FL plates (Supplementary Fig. 8). Importantly, expression of MafA, a pancreatic transcriptional factor that induces insulin mRNA expression, was increased on NPo plates compared with control FL plates (Fig. 5I). In line with this observation, ELISA assay detected a significantly higher level of glucose-induced insulin secretion from cells on NPo plates compared with cells on FL plates (Fig. 5J).

TAZ downregulation involves in NPo-induced induction of pancreatic differentiation To investigate a possible mechanism underlying NPo-mediated pancreatic differentiation, we focused on the stage of conversion of human ES and iPS cell-derived endoderm into pancreatic progenitors, as NPo plates showed the most powerful effect at this transition (Fig. 4). As expected, Western blot analysis confirmed that NPo surfaces induced a marked increase in PDX1 protein together with a reduction in CK19 protein in differentiating ES cells (Fig. 6A). Interestingly, differentiation on NPo plates led to reduced expression of TAZ, a transcriptional regulator that mediates TGFβ-dependent Smad nucleocytoplasmic shuttling (Fig. 6A and B).43 In addition, the level of pSmad2/3 was also decreased in cells differentiating on NPo plates, whereas a noticeable change in pSmad 7 level was not observed (Fig. 6A). The reduction of both TAZ and pSmad2/3 coincided with a marked upregulation of neurogenin 3 (Fig. 6A), a key transcription factor in the specification of pancreatic endocrine cells.44 Immunostaining analysis of cells differentiating on NPo plates showed strong TAZ signals in occasional CK19+ cell clusters, but a low level of punctate TAZ distribution was detected in predominant PDX1+ cells (Fig. 6C). As expected, relatively low numbers of PDX1+ cells were found in FL plates, but a


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similar staining pattern of TAZ was observed in both PDX1+ and CK19+ cells (Supplementary Fig. 9A and B). Furthermore, gene knockdown experiments using short interfering RNA (siRNA) revealed that silencing of TAZ gene increased the expression of PDX1 in pancreatic progenitor cells on FL plates, as determined by Western blot analysis (Supplementary Fig. 9C). Disruption of TGFβ-activated Smad signaling has been shown to promote differentiation into pancreatic endocrine cells not only from the developing pancreas but also from pluripotent stem cells.45,46 These results thus suggest that NPo-induced downregulation of TAZ may disturb TGFβ/Smad signaling and contribute to enhanced pancreatic differentiation on NPo surfaces.


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DISCUSSION In this study, we first investigated the effect of PS NPo surfaces, which were replicated by a nano-molding process with nickel nano-molds, on pancreatic differentiation of human ES and iPS cells. Along with various stem cell-nanotopography interaction studies, many different types of nanofabrication techniques have been developed for realizing nanotopographical surfaces.18,47,48 Generally, however, a large number of culture plates are needed to fully understand the effects of distinct nanotopographies on cell functions, from simple comparative experiments to combinatorial high-throughput screening.49 It is important to develop nanomanufacturing systems that can make high-quality polymeric NPo substrates in large numbers at a relatively low cost. Moreover, it is valuable to impart the nanotopography on a PS substrate to compare cell-nanotopography interactions with validated and grounded cell studies using PS culture platforms such as culture dishes, flasks, and chambers.50 However, micro/nanofabrication of thermoplastic materials such as PS still poses significant challenges associated with the nanomanufacturing of metallic stamps and nano-molding of thermoplastic polymeric materials.51 To overcome these challenges, we developed a mass nanofabrication method for PS cell culture platforms with nanopore arrays by not only developing a nano-electroforming process to achieve a metallic nano-stamp but also optimizing injection molding conditions for successful replication of nanopore arrays.29 In this study, we fabricated four different types of nickel nano-mold inserts having diameters of nanopillar cavities ~ 130, 170, 200, or 230 nm, and multiple PS NPo surfaces were replicated through thermal nanoimprinting at the screening stage where the effect of nanopore diameters on pancreatic differentiation of pluripotent stem cells was investigated. The nickel nano-mold insert withstanding rapid high pressure and temperature changes during the thermal nanoimprinting enables to replicate many identical PS NPo surfaces. Once the


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optimal diameter of nanopores was determined (200-nm nanopores for the pancreatic differentiation in this case), a large number of PS NPo surfaces were replicated through the nanoinjection molding with the nickel nano-mold insert for the extensive cellular studies. The nanoinjection molding process is a one-step replication of nano-topographical platforms utilizing a nano-mold insert (also called a nano-stamp) in addition to a conventional injection molding machine. In this regard, the nano-injection molding process with PS material would be industrially feasible for the fast and cost-effective mass production of cell culture platforms with nanotopography. During early organogenesis, biomechanical forces generated from surrounding tissues regulate crucial cellular processes, including proliferation, spatial rearrangement, and differentiation.1,52 Using multiple human ES and iPS cell lines, we tested four diameters of nanopores and demonstrated that 200-nm nanopores did not affect the pluripotency of human ES and iPS cells but promoted pancreatic differentiation. Similar attempts have recently been made using nanoscale patterns with different chemistry and topography with the purpose of maintaining stem cells and generating neural cells.14,15 Other studies show that human ES cells change their behaviors depending on nanoscale roughness, possibly through feedback regulation of mechanosensory integrin-mediated adhesion, myosin II, and E-cadherin.12 A more recent study using gradient nanopattern array demonstrates that ES cells form more compact colonies and express higher levels of pluripotency markers on nanopillars (120-170 nm in diameter) under feeder-free conditions.53 In our study, before inducing pancreatic differentiation, human ES and iPS cells showed no significant differences in the expression of undifferentiated markers on 200nm nanopores. However, we found clear differences in the expression of proteins associated with cell-matrix interactions as well as the shape of filopodia and lamellipodia in cells grown on


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nanopores, similar to the previous study using nanopillars.53 Thus, a long-term study with continuous passages would be required if nanopores affect the pluripotency of human pluripotent stem cells. Importantly, we found that PDX1 expression was gradually upregulated as the pore diameter increased, reaching a plateau at 200 nm. Therefore, the present and previous findings indicate that undifferentiated and differentiating human ES and iPS cells are able to sense and respond to nanotopographical patterns. In addition, these results suggest that mechanical or physical cues as well as biochemical soluble factors are critical factors determining cell fate during the differentiation of stem cells. Despite the encouraging results of previous studies, few studies have investigated the influence of nanotopographies on the differentiation of human pluripotent stem cells.12,54 Indeed, primitive cells in developing organs are continuously exposed to complex chemical and microor nanotopographical cues that are not sufficiently provided from conventional in vitro culture systems.55,56 Thus, this issue should be carefully considered for the purpose of directing the differentiation of stem cells, particularly ES cells derived from early developing embryos and iPS cells that have similar properties to ES cells. In the present study, we provide the first evidence that nanopore patterns successfully promoted the pancreatic differentiation of human ES and iPS cells. We found that 200-nm NPo surfaces upregulated expression of PDX1 (pancreas) with ALB (liver) and downregulated expression of SOX2/BARX1 (stomach) and CDX2/CES2 (intestine) in the absence of biochemical soluble factors. Both the pancreas and liver originate from the posterior foregut and are located between the stomach and duodenum, which is the first section of the small intestine.57 Thus, we speculate that 200-nm NPo surfaces contributed to the region-specific patterning of human pluripotent stem cell-derived endoderm, although the exact mechanism remains to be elucidated.


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More importantly, our results emphasize the cooperative effect of biochemical and mechanical cues on the pancreatic commitment of differentiating endoderm. By adding the soluble factors RA and noggin, which attenuate liver induction,58 we obtained an approximately 3-fold increase in the percentage of PDX1+ pancreatic progenitors on NPo surfaces compared with control FL surfaces. Likewise, subsequent differentiation in the presence of bFGF, activin βB, and VEGF profoundly increased the subpopulation of NKX6.1+ pancreatic endocrine cells among PDX1+ pancreatic progenitors on NPo surfaces. Therefore, these findings indicate that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells. Although we obtained a high purity of PDX1+ pancreatic progenitor cells (72%) using NPo surfaces and cytokines, the final purity of β cells was approximately 37% of total differentiated cells. As pancreatic progenitors give rise to other pancreatic cell types, including α cells, γ cells, and δ cells, further fine-tuning of the microenvironment during the final step of differentiation may increase the yield of β cells. Understanding how NPo directs the commitment of human ES and iPS cell-derived endoderm to pancreatic progenitor cells is important for optimizing the yield of β cells. A growing body of evidence suggests that mechanical stimulation can be transduced into biochemical signals,59,60 but the molecular mechanism underlying this mechanotransduction is largely unknown. Recent studies showed that self-renewal and differentiation of mesenchymal stem cells can be modulated, when cultured on nanotopographical surfaces.61,62 Particularly, an optimized nanopit topography (NSQ50) promoted colocalization of integrins and bone morphogenetic protein 2 receptors, enhancing osteogenic activity of mesenchymal stem cells.62 Thus these studies strongly imply that mechanical stimulations may be able to regulate stem cell


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fates by activating adhesions and cytoskeleton. Our results demonstrate that NPo-induced upregulation of PDX1 in human ES cell-derived endoderm is associated with downregulation of TAZ, suggesting that NPo-mediated mechanotransduction is associated with TAZ signaling. TAZ (WWTR1) conveys mechanical signals produced by the ECM into the nucleus, regulating gene expression in different cell types.63 A previous study demonstrates that in response to TGFβ, TAZ binds Smad molecules and promotes nuclear accumulation of Smads (Fig. 6D), which is required for the maintenance of human ES cell self-renewal.43 In our study, how mechanical input produced by NPo decreases TAZ expression remains to be determined. However, we found that the reduction of TAZ was associated with a decrease of activated Smads (pSmad2/3), and these events occurred concomitantly with an upregulation of NGN3 and PDX1. Previous studies show that inhibition of TGFβ-dependent Smad2/3 activation promotes the differentiation of human ES cells into pancreatic endocrine cells in the developing pancreas by increasing NGN3 expression.44 In addition, focal adhesion kinase signaling, which mediates mechanotransduction, affects activation of the TGFβ/Smad2/3 pathway and influences the commitment of human ES cells into pancreatic endocrine cells.45 Taking the present and previous findings into account, we speculate that mechanical inputs produced by NPo surfaces reduce TAZ and disturb Smad2/3 signaling, eventually promoting the differentiation of human ES and iPS cells into pancreatic endocrine cells (Fig. 6D). The direct correlation between TAZ and PDX1 has not yet been studied. However, a recent study showed that nanotopographical cues increase the expression of HNF1α, a transcription factor that binds to the distal enhancer element of the human PDX1 promoter.64,65 Thus, it will be interesting to explore possible roles of TAZ in the regulation of PDX1 expression in normal pancreatic organogenesis as well as the in vitro differentiation of pluripotent stem cells.


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CONCLUSIONS In summary, we provide the first direct evidence that differentiating human ES and iPS cells sense nanotopographical cues and efficiently direct their differentiation into pancreatic cell fates. We found that NPo surfaces efficiently promoted differentiation of human pluripotent stem cells into endoderm with a posterior foregut identity. Our results also showed that NPo surfaces dramatically increased the efficiency of commitment of human ES and iPS cell-derived endoderm into pancreatic progenitors, which were capable of giving rise to pancreatic endocrine cells in vitro. Our findings also suggest that TAZ is a key player in the NPo-induced mechanotransduction that facilitates the pancreatic differentiation of pluripotent stem cells. Recent rapid advances in nanofabrication techniques enable us to mimic microenvironments through different features of nanoscale surfaces. We developed a mass nanofabrication process based on thermal nanoimprinting and nano-injection molding for imparting nanotopographies on PS substrates in large numbers, with high quality, and at a relatively low cost. Thus, our study serves as an important example for future investigations exploring the optimal nanotopographical cues for differentiating stem cells into other cell types for cell therapies.


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MATERIALS AND METHODS Nanofabrication of PS NPo plates AAO nano-templates with different sizes of nanopores, which function as master templates during nano-electroforming, were fabricated through a two-step aluminum anodization process (Fig. 1A). A nanopore surface on the AAO nano-template was formed into a desired dimension in a phosphoric acid bath by controlling several processing parameters, including an anodization time of 3 h, applied voltage of 195 V, and temperature of -5°C, resulting in the same nanopore depth of ~500 nm and interpore distance of ~500 nm, whereas different widening times of 2, 3, 4, or 5 h resulted in nanopore diameters of 130, 170, 200, or 230 nm, respectively. Nickel nanomold inserts (also called stamps) with nanopillars in a reverse shape of the nanopores in AAO surfaces were manufactured by a nickel nano-electroforming process (Fig. 1B). A 20 nm-thick nickel seed layer was deposited on the AAO nano-templates using an electron beam evaporator for uniform conductivity. The nano-electroforming process was then carefully carried out in a nickel sulfamate bath by controlling electric current densities up to the desired thickness to reduce internal stress of the electrodeposited nickel layer. The electroformed part was then machined and polished to make a nano-mold insert with a constant thickness of 1 mm over an area of 2×2 cm2 (Fig. 1C). To determine the optimal nanopore size for the pancreatic differentiation of human pluripotent stem cells, a small number of PS (GP125EI, Kumho Chemical, Seoul, Korea) NPo surfaces with different nanopore diameters (130, 170, 200, and 230 nm) were replicated through the thermal nanoimprinting process (Fig. 1D-1). An imprinting temperature of 95°C and pressure of 5 MPa were applied for the nano-replication of PS NPo surfaces. After determining the


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optimal nanopore diameter, a large number of culture plates containing 200-nm NPo surfaces were produced by a nano-injection molding process (SE50D, Sumitomo, Tokyo, Japan) with the nano-mold insert (Fig. 1D-2). Based on our previous work,29 an optimal processing condition was applied to the nano-injection molding: a melting temperature of 220°C, mold temperature of 90°C, injection speed of 24 mm s-1, packing pressure of 120 MPa, and packing time of 1 s.

Surface treatment of PS cell culture plates All substrates were treated by oxygen plasma (VITA1; Femto Science, Gyeonggi-do, Korea) with a plasma power of 50 W for 10 s before cell culture to enhance surface hydrophilicity and cell attachment. The hydrophobic bare PS FL and NPo surfaces had contact angle greater than 90° (SmartDrop, Femtofab, Kyeongsangbuk-do, Korea), whereas the plasma-treated surfaces had contact angle less than 60°, which was appeared to be a suitable contact angle range for adequate cell attachment to the surface. Though the hydrophobic recovery of the plasma treated PS surfaces was observed; in other words, the contact angle increased with time, the contact angle were stabilized around 60° (Fig. 1E and Supplementary Fig. 1), which is known to as sufficient for cell attachment (Fig. 1F).

Maintenance of human ES and iPS cells Human ES cell lines (H1 and BGO1; WiCell Research Institute, Madison, WI) and iPS cell lines (ANFXF-1, UNFXF-1, originated from human urine, kindly provided Dr. Hwang D.Y., CHA


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University, Gyeonggi-do, Korea) were maintained on a feeder layer of mouse embryonic fibroblasts (MEFs). MEFs were harvested from 13.5-day-old CF1 mouse embryos (Orient Bio, Seoul, Korea). Human ES cells were cultured in Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 mixture (DMEM/F12, 1:1, Gibco, Grand Island, NY) supplemented with 20% KnockOut serum replacement (Gibco), 1 mM nonessential amino acids (Invitrogen, Grand Island, NY), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St Louis, MO), 100 U/ml penicillin G (Gibco), 100 μg/ml streptomycin (Gibco), and 8 ng/ml basic fibroblast growth factor (bFGF, Peprorech, Rocky Hill, NJ). Human ES cells were passaged at weekly intervals by dissociating with 1 mg/ml collagenase type IV (Invitrogen) and cultured in a standard gas atmosphere containing humidified 5% CO2 at 37°C.

Feeder-free culture and spontaneous differentiation on FL and 200-nm NPo surfaces. Human ES and iPS cell lines were routinely maintained on a layer of mitotically inactive mouse embryonic fibroblast (MEF) feeder cells. Expanded human ES and iPS cells were harvested from feeder cell layers and grown in mTeSR medium for feeder-free cultures before use in differentiation experiments. Briefly, cells were cultured in matrigel (BD Biosciences, San Jose, CA)-coated culture dishes containing mTeSR1 media (Stemcell Technologies, Vancouver, BC, Canada) without MEF feeder cells and passaged two times under feeder-free conditions. To initiate spontaneous differentiation, cells were dispersed with TrypLE select (Invitrogen) for 3 min. The resulting single-cell suspension was washed twice with DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin. Dissociated single cells were plated on conventional FL or NPo culture dishes containing mTeSR1 medium


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supplemented with 10 μM Rho-associated protein kinase inhibitor Y-27632 (ROCK inhibitor, Calbiochem, Merck, Rockland, MA). The dishes were pretreated with 5 μg/ml fibronectin (Roche, Mannheim, Germany) for 2 h before use. To induce spontaneous differentiation, cells were cultured on FL or NPo surfaces in the absence of growth factors in Roswell Park Memorial Institute Medium 1640 (RPMI-1640, Gibco) containing 2% FBS for 4-6 days. To direct the differentiation into endoderm, cells were treated with wnt3a and activin A on FL or NPo plates for 4 days in DMEM containing B27 supplement (Gibco).

Directed differentiation into pancreatic cells The experimental scheme for the pancreatic differentiation process is described in Supplementary Fig. 5. To induce endodermal differentiation, single-cell dissociated human ES or iPS cells were plated at 2×106 cells on FL or NPo surfaces in mTeSR1 media. Cells were then treated with 100 ng/ml activin A (R&D Systems, Minneapolis, MN) and 30 ng/ml wnt3a (R&D Systems) in RPMI-1640 medium on day 1 and further differentiated in the presence of activin A (100 ng/ml) alone for an additional 2 days in the same culture medium containing 0.2% FBS. To differentiate into pancreatic progenitors, cells were grown in DMEM containing 2 μM RA (Sigma-Aldrich) and 50 ng/ml noggin (R&D Systems) for 4 days and cultured in the presence of 1 ng/ml bFGF (Peprorech), 50 ng/ml activin βB (R&D Systems), 30 ng/ml VEGF (R&D Systems), and 100 μM ascorbic acid (Sigma-Aldrich) for an additional 4 days. To derive pancreatic endocrine cells, cells were cultured in CMRL-1066 medium (Invitrogen) containing B27 supplement, 50 ng/ml exendin-4 (Sigma-Aldrich), 30 ng/ml HGF (ProSpec, East Brunswick, NJ, USA), and 30 ng/ml IGF-1 (R&D Systems) for a final 8 days.


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Reverse transcription PCR (RT-PCR) and qPCR analyses Total RNA was isolated from cells grown on FL or NPo surfaces using TRIzol reagent (Invitrogen), and cDNA was synthesized from 2 μg total RNA using the SuperScript III FirstStrand Synthesis Kit (Invitrogen). PCR was subsequently carried out using AccuPower PCRPremix (Bioneer, Daejeon, Korea). The primer sequences and reaction conditions used in this study are listed in Supplementary Table 1. Relative band intensities were determined using a spectrum imaging system (UVP, Upland, CA). The levels of target mRNAs were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). qRT-PCR was performed using iQTM SYBR Green Supermix (Bio-Rad, Hercules, CA). Primer sequences used for qPCR are listed in Supplementary Table 2. Each qPCR mixture consisted of 10 μl 2× iQTM SYBR Green Supermix, 2 μl cDNA template (50 ng), 2 μl of a set of primers, and 6 μl nuclease-free water. The cycle threshold (CT) values of target genes were analyzed using the CFX-96 real-time PCR system (Bio-Rad). The comparative CT method (2-ΔΔCt) was used to determine the relative quantification of target genes by normalizing PCR products to levels of GAPDH.

PCR Array The expression of a panel of pluripotency- and differentiation-associated genes (84 genes) in cells differentiated on FL or NPo surfaces was analyzed using the Human Embryonic Stem Cell RT2 Profiler PCR Array (SABiosciences, Qiagen, Valencia, CA). Data analysis was performed using online SA Biosciences and Cluster software (RT2 profiler PCR array data analysis version


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3.5, SA Biosciences). The primers used are provided in Supplementary Table 3. mRNA levels were normalized to that of GAPDH.

Western blotting analysis Cells were harvested in RIPA lysis buffer (Upstate, Charlottesville, VA) supplemented with a cocktail tablet of protease inhibitors (Roche). A total of 50 μg of proteins were separated by NuPage 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (Invitrogen). Membranes were incubated with primary antibodies overnight at 4°C. Primary antibodies included cytokeratin 19 (1:200, M0888, Dako, Glostrup, Denmark), TAZ (1:500, 560235, BD Biosciences), pSmad 7 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA), pSmad 2/3 (1:400, 3191, Cell Signaling Technology, Beverly, MA), neurogenin 3 (1:100, sc-23832, Santa Cruz Biotechnology), PDX1 (1:200, AF2419, R&D Systems), β-actin (1:200, sc-1615, Santa Cruz Biotechnology) and GAPDH (1:400, sc-365062, Santa Cruz Biotechnology). The membranes were then rinsed with TBS-T buffer and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:10000, sc-2020, sc-2005, sc-2054, Santa Cruz Biotechnology) for 1.5 h at room temperature. Immunoreactive proteins were detected using ECL Plus reagents (GE Healthcare, Pittsburg, PA).

Immunofluorescence staining Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) in phosphate buffered saline (PBS). Immunostaining was carried out using standard protocols as previously described66, and alkaline


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phosphatase activity staining was performed using Alkaline Phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Primary antibodies included SSEA4 (1:200, MAB4304, Millipore Billerica), anti-Oct4 (1:400 sc-5279, Santa Cruz Biotechnology), Nanog (1:200, AF1997, R&D Systems), Foxa2 (1:400, AF2400, R&D Systems), Sox17 (1:400, AF1924, R&D Systems), Alexa 488-conjugated Phalloidin (1:400, A12379, Invitrogen), β-catenin (1:400, sc-7199, Santa Cruz Biotechnology), PDX1 (1:200, AF2419, R&D Systems), CK19 (1:200, M0888, Dako), TAZ (1:200, 560235, BD Biosciences), NKX6.1 (1:50, F55A12, Developmental Studies Hybridoma Bank, Iowa City, IA), Islet (1:200, AF1837, R&D Systems), NeuroD-1 (1:300, sc-1084, Santa Cruz Biotechnology), Insulin (1:500, 4011-0F, Linco, St Charles, MO), C-peptiden (1:200, 4011-01F, Linco), Glucagon (1:500, G2654, Sigma-Aldrich), and Somatostatin (1:400, A0566, DAKO). An Apotome-Axiovert 200 M fluorescence microscope (Carl Zeiss, Thornwood, NY) and confocal laser scanning microscope (Carl Zeiss) were used to visualize cells after counterstaining with 4’,6-diamidino-2phenylindole (DAPI, 1:1000, 100-43-6, Sigma-Aldrich).

siRNA treatment Inhibition of the TAZ gene expression was induced by short interfering RNA (si-TAZ) oligonucleotide, siGENOME human WWTR1 siRNA-SMART pool (GE Healthcare Dharmacon Inc., Lafayette, CO, USA). For knockdown experiment, human ES cell-derived endodermal cells were treated with si-C (negative control RNA, Qiagen) or si-TAZ (40 nmol/L) using lipofectamine® 3000 reagent (invitrogen) for 12 h according to the manufacturer’s instruction and further differentiated into pancreatic progenitors for an additional 3.5 days.


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EdU staining For in vitro proliferation assay, cells were incubated with 5-ethynyl-2’-deoxyuridine (EdU) for 1 h, and EdU incorporation was detected using a Click-iT EdU imaging Kit (Invitrogen) according to the manufacturer’s instructions. A confocal laser scanning microscope (Carl Zeiss) was used to visualize cells after counterstaining with DAPI (1:1000, Sigma-Aldrich).

Flow cytometric analysis To quantitatively analyze the derivation of definitive endoderm and pancreatic cells, cells were harvested at the indicated stage of differentiation and subjected to FACS. For intracellular marker staining, cells were fixed, permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences), and stained with FITC-conjugated antibody against Sox17 (1:20, 552205, BD Biosciences) or PDX1 (1:200, AF2419, R&D Systems). For surface marker staining, cells were washed with PBS supplemented with 1% FBS and incubated for 30 min at 4°C with antibodies against CXCR4 (1:20, 555974, BD Biosciences) or CD200 (1:25, 552475, BD Biosciences). Data were acquired for each sample using a FACS Caliber cytometer equipped with a FL1, FL2 laser and analyzed using FlowJo software (version 7.2, BD Biosciences).

Insulin content and secretion assays Cells differentiated on FL or NPo surfaces were washed four times with PBS and cultured in 3.3 mM glucose-containing RPMI-1640 medium supplemented with 10 mM HEPES buffer (Gibco)


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and 10% FBS for 1 h at 37°C. Insulin secretion was then triggered by culturing cells in the presence of 25 mM glucose in the same culture medium for an additional 2 h. The supernatant was harvested, and the level of secreted insulin was measured using an ELISA kit (DAKO) according to the manufacturer’s instructions. To normalize the amount of insulin secretion, the total protein concentration of cells in each condition was measured by the Bradford method.

Statistical analysis Data are shown as mean values of at least three independent experiments performed in triplicate and expressed as mean±standard deviation (SD). Pairwise comparisons were performed using Student’s t-tests. For multiple comparisons, one-way ANOVA was performed followed by Tukey’s post-hoc tests. Statistical significance was set at p

Nanotopography Promotes Pancreatic Differentiation of Human

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