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Preparation of induced pluripotent stem cells on dishes grafted on oligopeptide under feeder-free conditions
Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 295–301
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Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Preparation of induced pluripotent stem cells on dishes grafted on oligopeptide under feeder-free conditions Akon Higuchi a,b,c,1,*, Feng-ling Lin d,1, Yu-Kai Cheng a, Ta-Chun Kao a, S. Suresh Kumar a,e, Qing-Dong Ling c,f, Chun-Han Hou g, Da-Chung Chen h, Shih-Tien Hsu i, Gwo-Jang Wu j a
Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan Cathay Medical Research Institute, Cathay General Hospital, Taipei 10630, Taiwan d Department of Dermatology, Cathay General Hospital Sijhih Branch, Taipei 22174, Taiwan e Department of Medical Microbiology and Parasitology, Universities Putra Malaysia, Slangor, Malaysia f Institute of Systems Biology and Bioinformatics, National Central University, Taoyuan 32001, Taiwan g Department of Orthopedic Surgery, School of Medicine, National Taiwan University, Taipei 10051, Taiwan h Department of Obsterics and Gynecology, Taiwan Landseed Hospital, Taoyuan 32405, Taiwan i Department of Community Medicine, Taiwan Landseed Hospital, Taoyuan 32405, Taiwan j Graduate Institute of Medical Sciences and Dept. of Obstetrics & Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 January 2013 Received in revised form 7 June 2013 Accepted 23 June 2013 Available online 24 July 2013
Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have potentially therapeutic applications in the treatment of many diseases, due to their unique ability to differentiate into any type of somatic cell. However, the clinical potential of hESCs and hiPSCs is restricted by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer for these cells. We report that hiPSCs can be successfully generated without the use of a feeder layer of MEFs. We generated hiPSCs by transducing human adipose-derived stem cells (hADSCs) with a retrovirus containing pluripotency genes, and the hiPSCs were cultured on synthetic dishes grafted with an oligopeptide derived from vitronectin (VN-dish). On the fourth day after transduction, the hADSCs transduced with pluripotency genes were transferred to a MEF layer for culturing as a control condition or to VN-dishes for culture. The hiPSC colonies in the MEF-cultures were clearly observed at day 14 after transduction, whereas hiPSC colonies were detected on the VN-dishes after the cells were passaged. When 105 hADSCs were seeded on the dishes, the number of colonies generated on the MEFs was 120 28, while the number of colonies generated on VN-dishes was 25 8. Thus, the efficiency of hiPSC generation on the VN-dishes under feederfree conditions was lower than hiPSCs cultured on MEFs. However, the hiPSC colonies from VN-dishes demonstrated alkaline phosphatase activity, and immunohistochemistry suggested that the hiPSCs generated on VN-dishes expressed the pluripotency protein, stage-specific embryonic antigen-4 (SSEA-4), under feeder-free conditions. ß 2013 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Induced pluripotent stem cell Feeder-free culture Adipose-derived stem cell Vitronectin Oligopeptide Biomaterial
1. Introduction Human embryonic stem cells (hESCs) [1] and human induced pluripotent stem cells (hiPSCs) [2,3] have potentially therapeutic applications in the treatment of many diseases, due to their unique ability to differentiate into any type of somatic cell [4]. For example, hESCs and hiPSCs have been differentiated into nerve
* Corresponding author at: Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan. Tel.: +886 3 4227151 34257; fax: +886 3 4252296. E-mail addresses: [email protected], [email protected], [email protected] (A. Higuchi). 1 These authors contributed equally to the work.
cells that secrete dopamine and b cells that secrete insulin, and these cells can be transplanted for the treatment of Parkinson’s disease [5,6] and diabetes [6,7], respectively. The pluripotent nature of these cells could permit the development of a wide range of stem cell-based regenerative therapies and drug discovery platforms [4]. However, the tentative clinical potential of hESCs and hiPSCs is restricted by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer in the culture of these cells. While the addition of leukemia inhibitory factor (LIF) to the culture medium can allow mouse ESCs to proliferate and remain undifferentiated in the absence of a feeder layer of MEFs, this method is not effective for the culture of hESCs or hiPSCs [1,8]. The addition of LIF to the culture medium is insufficient to maintain the pluripotency and
1876-1070/$ – see front matter ß 2013 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jtice.2013.06.022
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self-renewal of hESCs and hiPSCs in a feeder-free culture [4]. The possibility of xenogenic contamination during culture with MEFs restricts the clinical use of transplanted hESCs and hiPSCs [9,10]. Furthermore, the process of culturing hESCs and hiPSCs using feeder layers is elaborate and costly, thereby limiting the largescale culture of these cells. The variability of MEFs between laboratories and across batches also affects the characteristics and the pluripotency of hESCs and hiPSCs [4]. Feeder-free cultures that use synthetic polymers or biomacromolecules as stem cell culture materials offer more reproducible culture conditions and lower the cost of production without introducing xenogenic contaminants. These improvements could increase the potential clinical applications of differentiated hESCs and hiPSCs [4]. Several feeder-free cultures of hESCs and hiPSCs have been reported. Swistowski et al. investigated hESC culture on human albumin- and fibronectin-coated dishes (CellstartTM coating dishes) [11,12]. The pluripotency of the hESCs was maintained for over 25 passages in their study. Rodin et al. reported hESC and hiPSC culture on laminin-511-coated dishes, maintaining pluripotency for 4 months (20 passages) under xeno-free and feederfree conditions [13]. The adhesion of hESCs was found to be dependent on integrin a6b1 [13,14], which binds to laminin-511 [13,15]. Melkoumian et al. developed acrylate dishes grafted with synthetic oligopeptides that were derived from bone sialoprotein (KGGNGEPRGDTYRAY) or vitronectin (KGGPQVTRGDVFTMP), and these surfaces supported the pluripotency of hESCs and hiPSCs for more than 10 passages [16]. Oligopeptides and glycosaminoglycans, such as heparin, play an important role in supporting the pluripotency of hESCs and hiPSCs. Klim reported that hESCs could maintain their pluripotency on surface-immobilized heparinbinding peptides (GKKQRFRHRNRKG) for three months (17 passages) [17]. Fully chemically synthetic materials have been reported as coating materials for cell culture dishes that sustain the pluripotency of long-term hESC cultures [18–20]. Villa-Diaz developed a zwitterionic polymer, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH) and showed that the polymer-coated dishes supported the pluripotency of H9 hESCs for ten passages in a defined serum-free medium [18,19]. However, the PMEDSAH-coated dishes did not support the culture of other hESC cell lines such as BG01 [18]. Although hiPSCs and hESCs can retain their pluripotency under feeder-free conditions, in most cases the reprogramming of somatic cells into hiPSCs has been performed using MEF [2,3] or human somatic cells such as fibroblasts [21] and mesenchymal stem cells [22,23] as feeder layers. Here, we report the reprogramming of human adiposederived stem cells (hADSCs) into hiPSCs without the use of feeder layers (e.g., MEFs) using gene transduction by a retrovirus containing pluripotent genes during cell culture on synthetic dishes grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP). 2. Materials and methods 2.1. Preparation of hADSCs The experiments performed for this research were approved by the ethics committee and institutional review board (IRB) of National Central University, Taiwan Landseed Hospital, and Cathay General Hospital. Adipose tissue from the intestinal omentum of a 53-year-old male was carefully dissected and washed with phosphate-buffered saline (PBS) to remove blood and impurities. The adipose tissue was minced into small pieces (approximately 2 mm3) and digested with 2.5 mg/mL of
collagenase type-IV (GibcoTM, Invitrogen, Grand Island, NY) at 37 8C for 60 min [24,25]. Enzymatic activity was neutralized with Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma–Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS, Biological Industries). The digested solution was centrifuged at 1200 g for 6 min [24,25]. The resulting cells were suspended in erythrocyte lysis buffer (154 mM NH4Cl, 20 mM Tris, pH 7.4) for 2 min to remove red blood cells, followed by neutralization with DMEM containing 10% FBS [24,25]. The cell solution was centrifuged at 1200 g for 6 min. The cells were resuspended in DMEM containing 10% FBS to obtain a suspension of adipose tissue cells (adipose tissue-derived stromal vascular fraction [SVF]). The total number of cells in this suspension was counted by flow cytometry. The adipose-tissue cells were then seeded into polystyrene tissue culture dishes (TCPs) and cultured in DMEM containing 10% FBS for 21 days. The number of hADSCs in the SVF was counted by using flow cytometry with antibodies to CD34 (IM1870U, FITC mouse antihuman CD34, Beckman Coulter, Marseille, France), CD44 (IM1219U, FITC mouse anti-human CD44, Beckman Coulter, Marseille, France), CD73 (550257, PE mouse anti-human CD73, BD Biosciences, San Jose, CA), CD90 (IM1840U, PE mouse antihuman CD90, Beckman Coulter, Marseille, France), and their isotype controls (733179, PE mouse anti-human IgG1 and 41116015, FITC mouse anti-human IgG1, Beckman Coulter, Marseille, France). The total cell number in the primary suspension of adipose tissue cells was also counted by flow cytometry (Coulter EPICSTM XL, Beckman Coulter, Marseille, France) after staining with 7-AAD (A07704, Beckman Coulter, Marseille, France). After culture for 5–7 days, more than 85% of the cells exhibited the mesenchymal stem cell markers CD73 and CD90, which indicated that the cells were primarily hADSCs. These hADSCs were cultured and passaged using conventional culture techniques [26]. The hADSCs purified by the culture method were used to generate hiPSCs by transduction with pluripotent genes using a retrovirus, as described below. 2.2. Establishment of hiPSCs using a retrovirus hiPSCs were generated using the procedures described by Yamanaka and colleagues [3], with some modifications. A brief schematic of the protocol is shown in Fig. 1. First, 293T cells (ATCC) were plated in OPTI-MEM (Invitrogen, Grand Island, NY) containing 10% FBS at 2 106 cells per 100 mm tissue culture polystyrene dish and incubated overnight. Cells were transfected with retrovirus vectors (5 mg of phOct4, phSox2, phcMyc, and phklf4, Addgene) and virus-producing vectors (3.3 mg of pCMV-GagPol and 1.7 mg of pCMV-VSV-G, Cell Biolabs, Inc.) by Lipofectamine 2000 (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. Three days after transfection, the supernatant of the transfectant was collected, filtered through a cellulose acetate filter with 0.45 mm pores (OE67, Whatman, Kent, UK), and centrifuged at 8000 g at 4 8C to obtain the virus pellet. One mL of serum-free DMEM containing penicillin/streptomycin (Invitrogen, Grand Island, NY) was added to the virus pellet, and this retrovirus solution was shaken overnight. The retrovirus solution was stored at 135 8C or used for the transduction of hADSCs as follows. hADSCs were seeded at 1 105 cells per well in tissue culture polystyrene plates (6 wells per plate). The cell medium was replaced with the retrovirus solution containing 4 mg/ml of polybrene (Sigma–Aldrich, St. Louis, MO), and cells were incubated with this solution for two days. Half of the medium was exchanged with fresh DMEM containing 10% FBS after the first 8 h and subsequently every day.
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Fig. 1. Procedure for the preparation of hiPSCs.
2.3. hiPSC culture on MEFs
3. Results and discussion
MEFs were prepared as described previously with some modifications [3]. Briefly, MEFs were isolated from 13.5 days post coitum embryos from ICR mice. The MEFs were cultured on gelatin-coated dishes and were treated with mytomycin C. hADSCs transduced with pluripotent genes were shifted to culture on MEFs on the fourth day after their induction into hiPSCs. Half of the medium (DMEM/F12 [Invitrogen, Grand Island, NY] supplemented with 20% Knockout Serum Replacement [KSR, Invitrogen, Grand Island, NY]) was changed every day.
3.1. hADSC preparation
2.4. hiPSC culture on VN-dishes hADSCs transduced with pluripotent genes were shifted to culture on synthetic dishes grafted with an oligopeptide derived from vitronectin (VN-dishes), which were prepared from polyacrylate-coated dishes grafted with an oligopeptide (KGGPQVTRGDVFTMP) derived from vitronectin (SynthemaxTM, Corning, Tewksbury, MA). The VN-dishes, which were commercially available (SynthemaxTM), were originally developed by Melkoumian et al. [16]. Cells were cultured in serum-free hiPSC culture medium (DMEM/F12 [Invitrogen, Grand Island, NY] supplemented with 20% Knockout Serum Replacement [KSR, Invitrogen, Grand Island, NY]). Half of the medium was changed every day. 2.5. hiPSC characterization The alkaline phosphatase (AP) activity of hiPSCs was measured using an alkaline phosphatase detection kit (SCR004, Millipore, Billerica, MA) according to the manufacturer’s instructions [27]. Immunostaining of stage-specific embryonic antigen-4 (SSEA4) on hiPSCs was performed following the conventional protocol [27]. Cells on dishes were incubated with mouse anti-human SSEA4 antibody (ab16287, Abcam, Cambridge, MA), secondary antibody, and Alexa Fluor 488-anti-mouse IgG (A21202, Invitrogen, Grand Island, NY). The stained cells were analyzed using fluorescence microscopy (Eclipse Ti-U fluorescence inverted microscope, Nikon Instruments, Inc., Tokyo, Japan).
hADSCs were selected for transduction with pluripotent genes and reprogramming to generate hiPSCs. The adipose tissuederived stromal vascular fraction (SVF) was isolated from a solution of digested fat tissue by centrifugation. The cells in the SVF were cultured on conventional TCPs for 21 days to purify and enhance the number of hADSCs in culture. Fig. 2 shows the morphologies of cells in the SVF and cells cultured for 30 h, 4 days, and 21 days. Cells with spherical morphologies were observed after 30 h of culture [Fig. 2(a) and (b)], whereas spindle-shaped cells could be detected on the TCPs after 4 days of culture [Fig. 2(c) and (d)], in agreement with the characteristics of mesenchymal stem cells [6,28–31]. Undifferentiated hADSCs express several surface markers, including CD13, CD29 (b1 integrin), CD44, CD63, CD73, CD90 (Thy-1), CD105 (endoglin), CD166, and CD34 (stem cellassociated marker) [25,32–35]. Therefore, a surface marker analysis of the cells in the SVF and the cells cultured on TCPs for 21 days was performed to evaluate the purity of the hADSCs within each culture. Fig. 3 presents representative flow cytometry results for the expression of CD34, CD44, CD73, and CD90 on the cells in the SVF and the cells cultured on TCPs for 21 days (passage one). Less than 36% of the cells in the SVF expressed CD34, CD44, CD73, and CD90. In contrast, 79.8%, 86.1%, and 94.3% of the cells that had been cultivated on TCPs for 21 days expressed CD44, CD73, and CD90, respectively. Thus, the population of hADSCs was found to increase after cell culture selection on TCPs. The expression of the stem cell-associated marker CD34 has been reported to be at its highest in primary adipose tissue cells, and decreases throughout the culture period [35,36]. Mitchell et al. reported that 60% of SVF cells expressed CD34 at passage 0, but by passage 2 this percentage had dropped to 5.4% [35]. CD34 was expressed on 14.9% of the SVF cells in this study, while only 1.6% of the cells expressed CD34 after being cultured on TCPs for 21 days. This decrease in the cellular expression of CD34 with increasing amounts of time in culture followed the same trend as that reported by Mitchell et al. [35].
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Fig. 2. Morphologies of cells in the SVF (a) and cells cultured for 30 h (b), 4 days (c), and 21 days (d). The scale bar is 40 mm.
Fig. 3. Flow cytometric analysis of cells in the SVF and the first passage of cells cultured from the SVF. (A) Flow cytometry histograms of cells in the SVF stained with anti-CD34, anti-CD44, anti-CD73, and anti-CD90 antibodies, showing the fluorescence intensity and the number of cells. (B) Flow cytometry histograms of the first passage of cells cultured from the SVF and stained with anti-CD34, anti-CD44, anti-CD73, and anti-CD90 antibodies, showing the fluorescence intensity and the number of cells. The dotted line indicates cells labeled with isotype controls.
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Fig. 4. Cell morphology of hADSCs after retroviral transduction with pluripotency genes, shown 0 days [(a) and (d)] and 14 days [(b) and (e)] after transduction and 2 days after the first passage [(c) and (f)]. The hiPSCs were generated on MEFs [(a), (b), and (c)] or on VN-dishes [(d), (e), and (f)].
3.2. Reprogramming of hADSCs into hiPSCs In this study, hiPSCs were generated from hADSCs retrovirally transduced with pluripotency genes. On the fourth day after transduction, hADSCs transduced with pluripotency genes were transferred to culture (1) on MEFs as a control condition, or (2) on VN-dishes, which are synthetic dishes grafted with the cellbinding site of a synthetic oligopeptide of vitronectin [16]. Colony formation is the first criterion used to evaluate hiPSC generation from somatic cells [4]. Therefore, colony formation of hADSCs was evaluated in cells cultured on MEFs and on VN-dishes. hiPSC colonies were observed in cells cultured on MEFs at day 14 after transduction, while hiPSC colonies were not observed in cells cultured on VN-dishes by day 14 after transduction (Fig. 4). However, hiPSC colonies were clearly detected after the first passage (Fig. 4). The number of colonies generated on MEFs was 120 28, while the number of colonies generated on VN-dishes was 25 8 when 105 hADSCs were seeded on the dishes (Fig. 5). The efficiency of hiPSC generation on the VN-dishes under feeder-free conditions was lower than that obtained from culture on MEFs. 3.3. Evaluation of hiPSCs One of the first assays performed to assess the quality of the hiPSC colonies was AP activity staining [27]; in general, hiPSC colonies exhibit positive staining [27]. We evaluated the AP activity of the hiPSCs cultured on VN-dishes, and the results are shown in Fig. 6. AP activity was clearly observed in the hiPSC
colonies cultured on VN-dishes, which indicated that most of the hADSCs cultured on VN-dishes were reprogrammed into hiPSCs. Expression of the pluripotency protein, SSEA-4 was also evaluated on hiPSCs cultured on MEFs or on VN-dishes. Fig. 7 shows immunostaining for SSEA-4 expression in hiPSCs cultured on MEFs or VN-dishes. hiPSCs cultured under either condition showed excellent expression of SSEA-4, indicating that the hADSCs were successively reprogrammed into hiPSCs on MEFs and on VNdishes under feeder-free conditions. 4. Discussion hiPSCs were generated under feeder-free conditions using VNdishes, although the efficiency of generating hiPSC colonies under these conditions decreased to approximately one-fourth of that the efficiency of generating hiPSC colonies using MEF culture. However, the generation of more than 20 colonies in one well of a six-well dish should be sufficient for the selection of hiPSCs with hiPSC-colony morphology from an initial seeding of 105 hADSCs. The hiPSCs generated and cultured on VN-dishes showed high AP activity and expressed the pluripotency protein SSEA-4, suggesting that the hiPSCs generated in this study should maintain a high degree of pluripotency. There have been several reports on the culture of hiPSCs under feeder-free conditions [11–13,16–20]. However, in these reports, hiPSCs were first generated and cultured on MEFs before being cultured under feeder-free conditions. In this study, we reprogrammed hiPSCs from hADSCs on completely synthetic dishes (VN-dishes) using feeder-free culture.
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Fig. 5. Colony formation by hiPSCs on MEFs (a) and on VN-dishes (b). The dish diameter is 3.5 cm.
Fig. 6. Macroscopic (a) and microscopic (b) observation of the AP activity of hiPSCs cultured on VN-dishes. The dish diameter in (a) is 3.5 cm. The scale bar shown in (b) is 50 mm.
Fig. 7. Characterization of hiPSCs cultured on MEFs or on VN-dishes. Cell morphology of hiPSCs on MEFs (a) and VN-dishes (b), analyzed by bright field microscopy. Immunostaining of SSEA-4 expression in hiPSCs cultured on MEFs (c) or on VN-dishes (d). The scale bar is 50 mm.
A. Higuchi et al. / Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 295–301
hADSCs can be easily prepared from a patient’s adipose tissue. Furthermore, it is simple to prepare hiPSCs from hADSCs on VN-dishes. We are now proceeding with the purification of hADSCs under serum-free conditions (i.e., xeno-free conditions), as well as the generation of hiPSCs from hADSCs by transduction with a retrovirus or with nonintegrating episomal lentiviral vectors [37] containing pluripotent genes under completely xeno-free conditions.
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Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Preparation of induced pluripotent stem cells on dishes grafted on oligopeptide under feeder-free conditions Akon Higuchi a,b,c,1,*, Feng-ling Lin d,1, Yu-Kai Cheng a, Ta-Chun Kao a, S. Suresh Kumar a,e, Qing-Dong Ling c,f, Chun-Han Hou g, Da-Chung Chen h, Shih-Tien Hsu i, Gwo-Jang Wu j a
Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan Cathay Medical Research Institute, Cathay General Hospital, Taipei 10630, Taiwan d Department of Dermatology, Cathay General Hospital Sijhih Branch, Taipei 22174, Taiwan e Department of Medical Microbiology and Parasitology, Universities Putra Malaysia, Slangor, Malaysia f Institute of Systems Biology and Bioinformatics, National Central University, Taoyuan 32001, Taiwan g Department of Orthopedic Surgery, School of Medicine, National Taiwan University, Taipei 10051, Taiwan h Department of Obsterics and Gynecology, Taiwan Landseed Hospital, Taoyuan 32405, Taiwan i Department of Community Medicine, Taiwan Landseed Hospital, Taoyuan 32405, Taiwan j Graduate Institute of Medical Sciences and Dept. of Obstetrics & Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 January 2013 Received in revised form 7 June 2013 Accepted 23 June 2013 Available online 24 July 2013
Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have potentially therapeutic applications in the treatment of many diseases, due to their unique ability to differentiate into any type of somatic cell. However, the clinical potential of hESCs and hiPSCs is restricted by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer for these cells. We report that hiPSCs can be successfully generated without the use of a feeder layer of MEFs. We generated hiPSCs by transducing human adipose-derived stem cells (hADSCs) with a retrovirus containing pluripotency genes, and the hiPSCs were cultured on synthetic dishes grafted with an oligopeptide derived from vitronectin (VN-dish). On the fourth day after transduction, the hADSCs transduced with pluripotency genes were transferred to a MEF layer for culturing as a control condition or to VN-dishes for culture. The hiPSC colonies in the MEF-cultures were clearly observed at day 14 after transduction, whereas hiPSC colonies were detected on the VN-dishes after the cells were passaged. When 105 hADSCs were seeded on the dishes, the number of colonies generated on the MEFs was 120 28, while the number of colonies generated on VN-dishes was 25 8. Thus, the efficiency of hiPSC generation on the VN-dishes under feederfree conditions was lower than hiPSCs cultured on MEFs. However, the hiPSC colonies from VN-dishes demonstrated alkaline phosphatase activity, and immunohistochemistry suggested that the hiPSCs generated on VN-dishes expressed the pluripotency protein, stage-specific embryonic antigen-4 (SSEA-4), under feeder-free conditions. ß 2013 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Induced pluripotent stem cell Feeder-free culture Adipose-derived stem cell Vitronectin Oligopeptide Biomaterial
1. Introduction Human embryonic stem cells (hESCs) [1] and human induced pluripotent stem cells (hiPSCs) [2,3] have potentially therapeutic applications in the treatment of many diseases, due to their unique ability to differentiate into any type of somatic cell [4]. For example, hESCs and hiPSCs have been differentiated into nerve
* Corresponding author at: Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan. Tel.: +886 3 4227151 34257; fax: +886 3 4252296. E-mail addresses: [email protected], [email protected], [email protected] (A. Higuchi). 1 These authors contributed equally to the work.
cells that secrete dopamine and b cells that secrete insulin, and these cells can be transplanted for the treatment of Parkinson’s disease [5,6] and diabetes [6,7], respectively. The pluripotent nature of these cells could permit the development of a wide range of stem cell-based regenerative therapies and drug discovery platforms [4]. However, the tentative clinical potential of hESCs and hiPSCs is restricted by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer in the culture of these cells. While the addition of leukemia inhibitory factor (LIF) to the culture medium can allow mouse ESCs to proliferate and remain undifferentiated in the absence of a feeder layer of MEFs, this method is not effective for the culture of hESCs or hiPSCs [1,8]. The addition of LIF to the culture medium is insufficient to maintain the pluripotency and
1876-1070/$ – see front matter ß 2013 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jtice.2013.06.022
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self-renewal of hESCs and hiPSCs in a feeder-free culture [4]. The possibility of xenogenic contamination during culture with MEFs restricts the clinical use of transplanted hESCs and hiPSCs [9,10]. Furthermore, the process of culturing hESCs and hiPSCs using feeder layers is elaborate and costly, thereby limiting the largescale culture of these cells. The variability of MEFs between laboratories and across batches also affects the characteristics and the pluripotency of hESCs and hiPSCs [4]. Feeder-free cultures that use synthetic polymers or biomacromolecules as stem cell culture materials offer more reproducible culture conditions and lower the cost of production without introducing xenogenic contaminants. These improvements could increase the potential clinical applications of differentiated hESCs and hiPSCs [4]. Several feeder-free cultures of hESCs and hiPSCs have been reported. Swistowski et al. investigated hESC culture on human albumin- and fibronectin-coated dishes (CellstartTM coating dishes) [11,12]. The pluripotency of the hESCs was maintained for over 25 passages in their study. Rodin et al. reported hESC and hiPSC culture on laminin-511-coated dishes, maintaining pluripotency for 4 months (20 passages) under xeno-free and feederfree conditions [13]. The adhesion of hESCs was found to be dependent on integrin a6b1 [13,14], which binds to laminin-511 [13,15]. Melkoumian et al. developed acrylate dishes grafted with synthetic oligopeptides that were derived from bone sialoprotein (KGGNGEPRGDTYRAY) or vitronectin (KGGPQVTRGDVFTMP), and these surfaces supported the pluripotency of hESCs and hiPSCs for more than 10 passages [16]. Oligopeptides and glycosaminoglycans, such as heparin, play an important role in supporting the pluripotency of hESCs and hiPSCs. Klim reported that hESCs could maintain their pluripotency on surface-immobilized heparinbinding peptides (GKKQRFRHRNRKG) for three months (17 passages) [17]. Fully chemically synthetic materials have been reported as coating materials for cell culture dishes that sustain the pluripotency of long-term hESC cultures [18–20]. Villa-Diaz developed a zwitterionic polymer, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH) and showed that the polymer-coated dishes supported the pluripotency of H9 hESCs for ten passages in a defined serum-free medium [18,19]. However, the PMEDSAH-coated dishes did not support the culture of other hESC cell lines such as BG01 [18]. Although hiPSCs and hESCs can retain their pluripotency under feeder-free conditions, in most cases the reprogramming of somatic cells into hiPSCs has been performed using MEF [2,3] or human somatic cells such as fibroblasts [21] and mesenchymal stem cells [22,23] as feeder layers. Here, we report the reprogramming of human adiposederived stem cells (hADSCs) into hiPSCs without the use of feeder layers (e.g., MEFs) using gene transduction by a retrovirus containing pluripotent genes during cell culture on synthetic dishes grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP). 2. Materials and methods 2.1. Preparation of hADSCs The experiments performed for this research were approved by the ethics committee and institutional review board (IRB) of National Central University, Taiwan Landseed Hospital, and Cathay General Hospital. Adipose tissue from the intestinal omentum of a 53-year-old male was carefully dissected and washed with phosphate-buffered saline (PBS) to remove blood and impurities. The adipose tissue was minced into small pieces (approximately 2 mm3) and digested with 2.5 mg/mL of
collagenase type-IV (GibcoTM, Invitrogen, Grand Island, NY) at 37 8C for 60 min [24,25]. Enzymatic activity was neutralized with Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma–Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS, Biological Industries). The digested solution was centrifuged at 1200 g for 6 min [24,25]. The resulting cells were suspended in erythrocyte lysis buffer (154 mM NH4Cl, 20 mM Tris, pH 7.4) for 2 min to remove red blood cells, followed by neutralization with DMEM containing 10% FBS [24,25]. The cell solution was centrifuged at 1200 g for 6 min. The cells were resuspended in DMEM containing 10% FBS to obtain a suspension of adipose tissue cells (adipose tissue-derived stromal vascular fraction [SVF]). The total number of cells in this suspension was counted by flow cytometry. The adipose-tissue cells were then seeded into polystyrene tissue culture dishes (TCPs) and cultured in DMEM containing 10% FBS for 21 days. The number of hADSCs in the SVF was counted by using flow cytometry with antibodies to CD34 (IM1870U, FITC mouse antihuman CD34, Beckman Coulter, Marseille, France), CD44 (IM1219U, FITC mouse anti-human CD44, Beckman Coulter, Marseille, France), CD73 (550257, PE mouse anti-human CD73, BD Biosciences, San Jose, CA), CD90 (IM1840U, PE mouse antihuman CD90, Beckman Coulter, Marseille, France), and their isotype controls (733179, PE mouse anti-human IgG1 and 41116015, FITC mouse anti-human IgG1, Beckman Coulter, Marseille, France). The total cell number in the primary suspension of adipose tissue cells was also counted by flow cytometry (Coulter EPICSTM XL, Beckman Coulter, Marseille, France) after staining with 7-AAD (A07704, Beckman Coulter, Marseille, France). After culture for 5–7 days, more than 85% of the cells exhibited the mesenchymal stem cell markers CD73 and CD90, which indicated that the cells were primarily hADSCs. These hADSCs were cultured and passaged using conventional culture techniques [26]. The hADSCs purified by the culture method were used to generate hiPSCs by transduction with pluripotent genes using a retrovirus, as described below. 2.2. Establishment of hiPSCs using a retrovirus hiPSCs were generated using the procedures described by Yamanaka and colleagues [3], with some modifications. A brief schematic of the protocol is shown in Fig. 1. First, 293T cells (ATCC) were plated in OPTI-MEM (Invitrogen, Grand Island, NY) containing 10% FBS at 2 106 cells per 100 mm tissue culture polystyrene dish and incubated overnight. Cells were transfected with retrovirus vectors (5 mg of phOct4, phSox2, phcMyc, and phklf4, Addgene) and virus-producing vectors (3.3 mg of pCMV-GagPol and 1.7 mg of pCMV-VSV-G, Cell Biolabs, Inc.) by Lipofectamine 2000 (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. Three days after transfection, the supernatant of the transfectant was collected, filtered through a cellulose acetate filter with 0.45 mm pores (OE67, Whatman, Kent, UK), and centrifuged at 8000 g at 4 8C to obtain the virus pellet. One mL of serum-free DMEM containing penicillin/streptomycin (Invitrogen, Grand Island, NY) was added to the virus pellet, and this retrovirus solution was shaken overnight. The retrovirus solution was stored at 135 8C or used for the transduction of hADSCs as follows. hADSCs were seeded at 1 105 cells per well in tissue culture polystyrene plates (6 wells per plate). The cell medium was replaced with the retrovirus solution containing 4 mg/ml of polybrene (Sigma–Aldrich, St. Louis, MO), and cells were incubated with this solution for two days. Half of the medium was exchanged with fresh DMEM containing 10% FBS after the first 8 h and subsequently every day.
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Fig. 1. Procedure for the preparation of hiPSCs.
2.3. hiPSC culture on MEFs
3. Results and discussion
MEFs were prepared as described previously with some modifications [3]. Briefly, MEFs were isolated from 13.5 days post coitum embryos from ICR mice. The MEFs were cultured on gelatin-coated dishes and were treated with mytomycin C. hADSCs transduced with pluripotent genes were shifted to culture on MEFs on the fourth day after their induction into hiPSCs. Half of the medium (DMEM/F12 [Invitrogen, Grand Island, NY] supplemented with 20% Knockout Serum Replacement [KSR, Invitrogen, Grand Island, NY]) was changed every day.
3.1. hADSC preparation
2.4. hiPSC culture on VN-dishes hADSCs transduced with pluripotent genes were shifted to culture on synthetic dishes grafted with an oligopeptide derived from vitronectin (VN-dishes), which were prepared from polyacrylate-coated dishes grafted with an oligopeptide (KGGPQVTRGDVFTMP) derived from vitronectin (SynthemaxTM, Corning, Tewksbury, MA). The VN-dishes, which were commercially available (SynthemaxTM), were originally developed by Melkoumian et al. [16]. Cells were cultured in serum-free hiPSC culture medium (DMEM/F12 [Invitrogen, Grand Island, NY] supplemented with 20% Knockout Serum Replacement [KSR, Invitrogen, Grand Island, NY]). Half of the medium was changed every day. 2.5. hiPSC characterization The alkaline phosphatase (AP) activity of hiPSCs was measured using an alkaline phosphatase detection kit (SCR004, Millipore, Billerica, MA) according to the manufacturer’s instructions [27]. Immunostaining of stage-specific embryonic antigen-4 (SSEA4) on hiPSCs was performed following the conventional protocol [27]. Cells on dishes were incubated with mouse anti-human SSEA4 antibody (ab16287, Abcam, Cambridge, MA), secondary antibody, and Alexa Fluor 488-anti-mouse IgG (A21202, Invitrogen, Grand Island, NY). The stained cells were analyzed using fluorescence microscopy (Eclipse Ti-U fluorescence inverted microscope, Nikon Instruments, Inc., Tokyo, Japan).
hADSCs were selected for transduction with pluripotent genes and reprogramming to generate hiPSCs. The adipose tissuederived stromal vascular fraction (SVF) was isolated from a solution of digested fat tissue by centrifugation. The cells in the SVF were cultured on conventional TCPs for 21 days to purify and enhance the number of hADSCs in culture. Fig. 2 shows the morphologies of cells in the SVF and cells cultured for 30 h, 4 days, and 21 days. Cells with spherical morphologies were observed after 30 h of culture [Fig. 2(a) and (b)], whereas spindle-shaped cells could be detected on the TCPs after 4 days of culture [Fig. 2(c) and (d)], in agreement with the characteristics of mesenchymal stem cells [6,28–31]. Undifferentiated hADSCs express several surface markers, including CD13, CD29 (b1 integrin), CD44, CD63, CD73, CD90 (Thy-1), CD105 (endoglin), CD166, and CD34 (stem cellassociated marker) [25,32–35]. Therefore, a surface marker analysis of the cells in the SVF and the cells cultured on TCPs for 21 days was performed to evaluate the purity of the hADSCs within each culture. Fig. 3 presents representative flow cytometry results for the expression of CD34, CD44, CD73, and CD90 on the cells in the SVF and the cells cultured on TCPs for 21 days (passage one). Less than 36% of the cells in the SVF expressed CD34, CD44, CD73, and CD90. In contrast, 79.8%, 86.1%, and 94.3% of the cells that had been cultivated on TCPs for 21 days expressed CD44, CD73, and CD90, respectively. Thus, the population of hADSCs was found to increase after cell culture selection on TCPs. The expression of the stem cell-associated marker CD34 has been reported to be at its highest in primary adipose tissue cells, and decreases throughout the culture period [35,36]. Mitchell et al. reported that 60% of SVF cells expressed CD34 at passage 0, but by passage 2 this percentage had dropped to 5.4% [35]. CD34 was expressed on 14.9% of the SVF cells in this study, while only 1.6% of the cells expressed CD34 after being cultured on TCPs for 21 days. This decrease in the cellular expression of CD34 with increasing amounts of time in culture followed the same trend as that reported by Mitchell et al. [35].
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Fig. 2. Morphologies of cells in the SVF (a) and cells cultured for 30 h (b), 4 days (c), and 21 days (d). The scale bar is 40 mm.
Fig. 3. Flow cytometric analysis of cells in the SVF and the first passage of cells cultured from the SVF. (A) Flow cytometry histograms of cells in the SVF stained with anti-CD34, anti-CD44, anti-CD73, and anti-CD90 antibodies, showing the fluorescence intensity and the number of cells. (B) Flow cytometry histograms of the first passage of cells cultured from the SVF and stained with anti-CD34, anti-CD44, anti-CD73, and anti-CD90 antibodies, showing the fluorescence intensity and the number of cells. The dotted line indicates cells labeled with isotype controls.
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Fig. 4. Cell morphology of hADSCs after retroviral transduction with pluripotency genes, shown 0 days [(a) and (d)] and 14 days [(b) and (e)] after transduction and 2 days after the first passage [(c) and (f)]. The hiPSCs were generated on MEFs [(a), (b), and (c)] or on VN-dishes [(d), (e), and (f)].
3.2. Reprogramming of hADSCs into hiPSCs In this study, hiPSCs were generated from hADSCs retrovirally transduced with pluripotency genes. On the fourth day after transduction, hADSCs transduced with pluripotency genes were transferred to culture (1) on MEFs as a control condition, or (2) on VN-dishes, which are synthetic dishes grafted with the cellbinding site of a synthetic oligopeptide of vitronectin [16]. Colony formation is the first criterion used to evaluate hiPSC generation from somatic cells [4]. Therefore, colony formation of hADSCs was evaluated in cells cultured on MEFs and on VN-dishes. hiPSC colonies were observed in cells cultured on MEFs at day 14 after transduction, while hiPSC colonies were not observed in cells cultured on VN-dishes by day 14 after transduction (Fig. 4). However, hiPSC colonies were clearly detected after the first passage (Fig. 4). The number of colonies generated on MEFs was 120 28, while the number of colonies generated on VN-dishes was 25 8 when 105 hADSCs were seeded on the dishes (Fig. 5). The efficiency of hiPSC generation on the VN-dishes under feeder-free conditions was lower than that obtained from culture on MEFs. 3.3. Evaluation of hiPSCs One of the first assays performed to assess the quality of the hiPSC colonies was AP activity staining [27]; in general, hiPSC colonies exhibit positive staining [27]. We evaluated the AP activity of the hiPSCs cultured on VN-dishes, and the results are shown in Fig. 6. AP activity was clearly observed in the hiPSC
colonies cultured on VN-dishes, which indicated that most of the hADSCs cultured on VN-dishes were reprogrammed into hiPSCs. Expression of the pluripotency protein, SSEA-4 was also evaluated on hiPSCs cultured on MEFs or on VN-dishes. Fig. 7 shows immunostaining for SSEA-4 expression in hiPSCs cultured on MEFs or VN-dishes. hiPSCs cultured under either condition showed excellent expression of SSEA-4, indicating that the hADSCs were successively reprogrammed into hiPSCs on MEFs and on VNdishes under feeder-free conditions. 4. Discussion hiPSCs were generated under feeder-free conditions using VNdishes, although the efficiency of generating hiPSC colonies under these conditions decreased to approximately one-fourth of that the efficiency of generating hiPSC colonies using MEF culture. However, the generation of more than 20 colonies in one well of a six-well dish should be sufficient for the selection of hiPSCs with hiPSC-colony morphology from an initial seeding of 105 hADSCs. The hiPSCs generated and cultured on VN-dishes showed high AP activity and expressed the pluripotency protein SSEA-4, suggesting that the hiPSCs generated in this study should maintain a high degree of pluripotency. There have been several reports on the culture of hiPSCs under feeder-free conditions [11–13,16–20]. However, in these reports, hiPSCs were first generated and cultured on MEFs before being cultured under feeder-free conditions. In this study, we reprogrammed hiPSCs from hADSCs on completely synthetic dishes (VN-dishes) using feeder-free culture.
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Fig. 5. Colony formation by hiPSCs on MEFs (a) and on VN-dishes (b). The dish diameter is 3.5 cm.
Fig. 6. Macroscopic (a) and microscopic (b) observation of the AP activity of hiPSCs cultured on VN-dishes. The dish diameter in (a) is 3.5 cm. The scale bar shown in (b) is 50 mm.
Fig. 7. Characterization of hiPSCs cultured on MEFs or on VN-dishes. Cell morphology of hiPSCs on MEFs (a) and VN-dishes (b), analyzed by bright field microscopy. Immunostaining of SSEA-4 expression in hiPSCs cultured on MEFs (c) or on VN-dishes (d). The scale bar is 50 mm.
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hADSCs can be easily prepared from a patient’s adipose tissue. Furthermore, it is simple to prepare hiPSCs from hADSCs on VN-dishes. We are now proceeding with the purification of hADSCs under serum-free conditions (i.e., xeno-free conditions), as well as the generation of hiPSCs from hADSCs by transduction with a retrovirus or with nonintegrating episomal lentiviral vectors [37] containing pluripotent genes under completely xeno-free conditions.
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