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Cell Biology International (2009) 33, 1268–1273 (Printed in Great Britain)
Role of MEF feeder cells in direct reprogramming of mousetail-tip fibroblasts
Mengfei Chenab, Xuerong Suna, Ruzhang Jianga, Wenjuan Shenc, Xiufeng Zhonga, Bingqian Liua, Ying Qia, Bing Huanga, Andy Peng Xiangb and Jian Gea*
aState Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xian Lie Nan Road, Guangzhou 510060, China
bCenter for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, Guangzhou 510080, China cDepartment of Pathophysiology, Medical College of Jinan University, Guangzhou 510632, China Abstract Pluripotent stem cells can be induced from somatic cells by the transcription factors Oct3/4, Sox2, c-Myc and Klf4 when co-cultured with mouse embryonic fibroblast (MEF) feeder cells. To date, the role of the feeder cells in the reprogramming process remains unclear. In this study, using a comparative analysis, we demonstrated that MEF feeder cells did not accelerate reprogramming or increase the frequency of induced pluripotent stem (iPS) cell colonies. However, feeder conditions did improve the growth of primary iPS colonies and were necessary for passaging the primary colonies after reprogramming was achieved. We further developed a feeder-free culture system for supporting iPS growth and sustaining pluripotency by adding bFGF and activin A (bFA) to the medium. These data will facilitate the generation of human iPS cells without animal feeders for regenerative medicine. Keywords: Induced pluripotent stem cells, Embryonic stem cells, Reprogramming, fibroblasts, MEF feeder cells, Feeder-free. *Corresponding author. Tel.: +86 20 87331374; fax: +86 20 87333271. 1 Introduction Somatic cells can be reprogrammed into a pluripotent stem cell state through transduction of the transcription factors Oct3/4, Sox2, c-Myc and Klf4 (Okita et al., 2007; Takahashi and Yamanaka, 2006). These four factors can also reprogram human somatic cells into a pluripotent state (Park et al., 2008; Takahashi et al., 2007b; Yu et al., 2007), which could allow the creation of patient-specific stem cells and have enormous therapeutic potential for human diseases (Hanna et al., 2007; Mauritz et al., 2008). In previous studies, mouse embryonic fibroblast (MEF) feeder cells were adopted to provide a reprogramming microenvironment for generating iPS cells after gene transduction (Okita et al., 2007; Park et al., 2008; Takahashi et al., 2007a,b; Takahashi and Yamanaka, 2006). MEF feeder cells produce multiple proteins and soluble factors, including activin A, TGFβ, bFGF, Wnts and BMP4 (Eiselleova et al., 2008; Lim and Bodnar, 2002; Soh et al., 2007), which are important for maintaining embryonic stem (ES) cell proliferation and pluripotency. However, it is not clear whether the induced reprogramming process is actually improved by factors secreted by the MEF feeder cells. The feeder cells could also be mixed with the iPS colonies when they are picked up by morphological criteria screening. Therefore, the purpose of this study was to analyze the role of MEF feeder cells in reprogramming mouse tail-tip fibroblasts (TTFs) by a comparative analysis. Our findings confirmed that the MEF cells were not necessary for the four factors to initiate reprogramming, but they were important for maintaining the proliferation of iPS cells after reprogramming was achieved. 2 Materials and methods 2.1 Cell cultures To culture primary TTFs, the skin was peeled from tail tips of 1 month BALB/c mice and incubated in DMEM (Gibco) containing 10% FBS (Hyclone) for 5–7 days. Cells that migrated out of the graft pieces were passaged to new plates. ES and iPS cells were cultivated on mitomycin C-inactivated MEFs in ES-DMEM containing 15% FBS, 103 For feeder-free culture of iPS cells, the plate was coated with polylysine and the medium used was ES-DMEM (with 15% knockout serum replacement), containing 10 2.2 Virus production and retroviral infection pMXs-based retroviral vectors for Oct3/4, Sox2, Klf4 or c-Myc (Addgene) were introduced into EcoPack2-293 cells (Clonetech) using the Fugene 6 transfection reagent (Roche). TTFs were seeded at 4 Three days after infection, the medium was replaced with ES medium. For feeder cell-induced conditions, the TTFs were digested and transferred to a 60 2.3 Immunofluorescence Cells were fixed in 4% paraformaldehyde/PBS for 15–20 2.4 RT-PCR for marker genes All cells were depleted of feeder cells after two passages, after which total RNA was extracted using TRI Reagent (Ambion). A RevertAid™ First Strand cDNA Synthesis Kit (Fermentas) was used to synthesize complementary DNA from 2 Table 1. Primer sequences used in RT-PCR.
2.5 Fluorescence-activated cell sorter (FACS) analysis Cells were trypsinized and resuspended in FACS buffer (PBS 2.6 Teratoma formation and histological analysis To evaluate the pluripotency of iPS cells cultured in bFA medium, a suspension containing 1 3 Results 3.1 Generation of iPS colonies with or without MEF feeder cells Eight days after retroviral infection, many small compact ES-like colonies grew out from the donor TTFs in dishes with or without MEF feeder cells (Fig. 1A,D). The primary iPS colonies co-cultured with feeder cells (Fig. 1B) grew more quickly than those without feeders (Fig. 1E). 
Fig. 1 MEF feeder cells improve the proliferation of primary iPS colonies. (A, D) iPS colonies outgrowth with (A) or without (D) feeder cell conditions 8 days after infection. (B, E) F-iPS colonies (B) grew more rapidly than NOF-iPS colonies (E) 12 days after infection. The background cells in D and E contain only donor TTFs. (C, F) Morphology of F-iPS (C) and NOF-iPS cells (F) when co-cultured with MEF feeder. (G) MEF feeder did not obviously improve iPS colony numbers at any time point. Each bar represents the mean ± standard deviation (n The numbers of ES-like colonies were counted on days 8, 10 and 12 after infection (Fig. 1G); the data showed that MEF feeder cell conditions did not obviously increase the number of iPS colonies at any time point. Reprogramming efficiency was similar with or without feeder conditions (&007E;0.02% on day 12). Thus the time needed to generate iPS colonies and the efficiency of induced reprogramming were not improved by the MEF feeder cells. Several homogeneous iPS cell lines were obtained by passaging the primary iPS colonies on MEF feeder cells. There was no significant difference in morphology between F-iPS (MEF feeder cell condition-derived iPS cells, Fig. 1C) and NOF-iPS cells (non-MEF feeder cell condition-derived iPS cells, Fig. 1F). However, when these two kinds of primary iPS colonies were passaged in the absence of feeder cells, the iPS cells proliferated very slowly and displayed a non-colony state, indicating that factors produced by the MEF feeder cells were important for the propagation of iPS cells. 3.2 ES marker expressed in F-iPS and NOF-iPS cells To determine whether NOF-iPS cells expressed similar mouse ES markers to F-iPS, the cells were stained with SSEA-1 and Nanog antibody. The results showed that all the F-iPS and NOF-iPS cell lines stained positively for Naong and SSEA-1 (Fig. 2A). 
Fig. 2 ES markers expressed in F-iPS and NOF-iPS cells. (A) Immunofluorescence confirmed that NOF-iPS expressed ES markers such as Nanog (Scale bars RT-PCR analysis was performed to detect whether ES marker genes including Nanog, Oct3/4, Rex1, Dax1, Fgf4, Nac1, Fbx15, and Dppa5 were expressed differently in NOF-iPS and F-iPS cells. The results showed upregulated expression of ES marker genes both in F-iPS and NOF-iPS cells when compared with uninfected TTFs (Fig. 2B). The expression levels were similar in the NOF-iPS and F-iPS cells, but slightly lower than in the ES cells' control. We further quantitatively analyzed the expression pattern of pluripotent gene Nanog by RT-PCR. This demonstrates that the expression levels of Nanog were different in NOF-iPS and F-iPS, but both were comparable to ES (Fig. 2C). Previous studies have also shown lower expression of ES marker genes in iPS cells than in ES, implying that iPS are similar but not identical to ES cells (Mikkelsen et al., 2008; Okita et al., 2007; Takahashi et al., 2007b; Takahashi and Yamanaka, 2006). 3.3 Culture of iPS cells in feeder-free conditions The above results demonstrate that MEF feeder cells are not necessary for the four factors to initiate reprogramming, but some unidentified factors produced by MEF feeder cells may have contributed to the proliferation of iPS cells after reprogramming was achieved. To develop a feeder-free culture system for optimizing the propagation of mouse iPS cells, we compared the abilities of bFGF plus activin A (bFA) and MEF-CM to maintain the proliferation of iPS cells. iPS cells grown in ES-DMEM differentiated spontaneously and failed to sustain growth after six passages when cultured in feeder-free conditions. In contrast, iPS cells cultured in medium containing bFA sustained ES-like colonies and proliferation (Fig. 3A). By FACS analysis, the percentages of SSEA-1 positive iPS cells cultured in bFA medium was comparable to that of MEF-CM conditions after 10 passages; in contrast, the SSEA+ cells were obviously decreased when cultured in ES-DMEM (Fig. 3B). In another study, the percentage of SSEA+ cells was 15–28%, which is lower than for ES (Brambrink et al., 2008). Furthermore, immunohistochemical analysis confirmed that iPS cells maintained pluripotency and differentiated into various tissues in teratomas when cultured in bFA medium (Fig. 3C). 
Fig. 3 Culture of iPS cells in the absence of feeder cells. (A) Growth curve of iPS cells cultured in feeder-free conditions. Cells (5 4 Discussion Mouse and human ES cells are generally co-cultured with mitomycin-C-inactivated or γ-irradiated MEF cells, as this system has been shown to be efficient for long-term maintenance of the pluripotency. However, preparing feeder cell layers is time-consuming and requires the destruction of embryo. Furthermore, co-culture with animal cells will expose human ES cells to animal pathogens. Therefore, many efforts have been made to develop feeder-free culture systems for mouse (Smith et al., 1988) and human (Klimanskaya et al., 2005; Xu et al., 2001) ES cells. iPS cells can be induced with fewer transcription factors (Kim et al., 2008), and higher efficiency of reprogramming has been achieved (Huangfu et al., 2008), but little is known about the function of MEF feeders in reprogramming. We have confirmed that co-culture with MEF feeder cells did not accelerate reprogramming or enhance the frequency of iPS colonies (Fig. 1G). Furthermore, NOF-iPS displayed ES-like properties of proliferation and pluripotency similar to F-iPS. Therefore, we concluded that MEF cells were not necessary for the four factors to initiate reprogramming in mouse TTFs. We also observed that MEF feeder cells improved proliferation in the primary iPS colonies, and the selected primary iPS colonies could not be passaged in non-feeder cell conditions. Previous studies have also reported that the iPS cells could not remain undifferentiated when cultured in the absence of feeder cells (Takahashi et al., 2007b; Takahashi and Yamanaka, 2006). These findings indicate that factors produced by MEF feeders are important for accelerating iPS cells' proliferation and maintaining pluripotency after the reprogramming process is complete. The transcription factors Oct4, Sox2 and Nanog occupy actively transcribed genes such as Lefty2, STAT3 and FGF2, etc. (Jaenisch and Young, 2008; Loh et al., 2006). In addition, a serum-free culture supplemented with activin A significantly enhanced mouse ES cell propagation without affecting pluripotency (Ogawa et al., 2007). We hypothesized that the lower expressed genes in iPS (Fig. 2B) could be redeemed by downstream factors, which were also secreted by MEF feeders. In view of this, we examined the ability of bFGF plus activin A (bFA) to support iPS growth in feeder-free conditions. The results confirmed that bFGF and activin A supplements promote iPS cells proliferation and sustain pluripotency in the absence of feeder cells. Direct reprogramming of adult cells provides a new strategy for generating patient-tailored pluripotent stem cells that will be invaluable for disease research and cell replacement therapies. Since the reprogramming process initiated by the four factors does not depend on MEF feeder cell conditions, our findings will facilitate the production of applicable human iPS cells without animal feeder in the future. We presume that human iPS cells can be induced without MEF feeder cells, and the donor cells or additional factors may be used to expand human iPS cells in xeno-free conditions for regenerative medicine. Acknowledgments We would like to thank Drs Duan Shan and Gao Qianying for critical suggestions, and Dr Jing Zhuang for critical reading of this manuscript. This study was supported by the National Basic Research Program of China (973 program) NO: 2007CB512207 and the National Natural Science Foundation of China NO: 30672275, NO: 30500555 and NO: 30600695. References Brambrink T, Foreman, R, Welstead, GG, Lengner, CJ, Wernig, M, Suh, H. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. 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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355 Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB) |
Abstract