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Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
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, 103U/L LIF (Chemicon), 0.1mM beta-mercaptoethanol, and 1×10−4M nonessential amino acids (Invitrogen).

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 10ng/ml bFGF and 10ng/ml activin A (R&D), or 50% MEF feeder supernatant for the MEF-conditioned medium (MEF-CM).

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 4×105 cells per 60mm dish and transduced with the four transcription factors Oct3/4, Sox2, c-Myc and Klf4 by retrovirus-mediated gene transfer (Takahashi et al., 2007a).

Three days after infection, the medium was replaced with ES medium. For feeder cell-induced conditions, the TTFs were digested and transferred to a 60mm dish with mitomycin-C inactivated MEF feeder cells; for non-feeder cell-induced conditions, the TTFs were not passaged on MEF feeder cells until the iPS colonies were picked up.

2.3 Immunofluorescence

Cells were fixed in 4% paraformaldehyde/PBS for 15–20min, permeabilized with 0.2% Triton X-100/PBS for 10min and blocked in 1% BSA for 30min. Cells were incubated with primary antibodies for 1h at room temperature or overnight at 4°C, washed three times with PBS, and incubated with secondary antibody for 1h. SSEA1 antibody was obtained from Millipore (1:50 dilution), and Nanog antibody from Abcam (1:100 dilution).

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 2μg total RNA. PCR reactions were performed with the primer sequences shown in Table 1. For quantitative analysis of Nanog expression, cDNA samples were amplified on ABI PRISM 7000 system by SYBR PrimeScript™ RT-PCR Kit (Takara).


Table 1.

Primer sequences used in RT-PCR.

Gene namePrimer sequencesGene bank

Annealing temperature

Product length

Rex1-FCTAGCCGCCTAGATTTCCANM_00955658406
Rex1-RCCACTTGTCTTTGCCGTTT
Dax1-FGCTGGAGTCTGAACATTGACNM_00743058490
Dax1-RCAGTATGGAGCAGAGGGAT
fbx15-FCTTACTTGGATGCGGTGACNM_01579856319
fbx15-RATTCTGAGGGACTCTTTGG
Dppa5-FAACAACAAGATTCGGGCTAANM_02527458138
Dppa5-RAAACTGGCTTCACTCGATACA
FGF4-FGTGGTGAGCATCTTCGGAGTGGNM_01020262146
FGF4-RGCGTAGGATTCGTAGGCGTTGT
Nac1-FCCACAATGAAGAGGACGAAGNM_02578860225
Nac1-RTTGCCAATCTGGTTGATAAGT
nanog-FATCCCTTCCCTCGCCATCANM_02801658486
nanog-RACCGCTTGCACTTCATCCTTTG
Oct3/4-FTCTGTTCCCGTCACTGCTCTNM_01363356157
Oct3/4-RCCTGTTGTGCTTTTAATCCCTC
Nanog-FTGCTTACAAGGGTCTGCTACTGANM_028016

54 Q-PCR

114
Nanog-RTTGTTTGGGACTGGTAGAAGAATC
GAPDH-FGGCACAGTCAAGGCTGAGAATGNM_00808458143
GAPDH-RATGGTGGTGAAGACGCCAGTA


2.5 Fluorescence-activated cell sorter (FACS) analysis

Cells were trypsinized and resuspended in FACS buffer (PBS+5%FBS); 1×106 cells were stained with 10μl anti-SSEA1 (480) PE (Santa Cruz) for 30min, washed once with PBS, and resuspended in FACS buffer for analysis on a FACS Aria cell sorter (BD).

2.6 Teratoma formation and histological analysis

To evaluate the pluripotency of iPS cells cultured in bFA medium, a suspension containing 1×106 cells was injected subcutaneously into nude mice. Tumors were dissected and immunohistochemistry was performed as previously described (Zhong et al., 2007). Sections were incubated with primary antibodies (Maixin. Bio) for cytokeratin (CK), desmin or neuron-specific enolase (NSE).

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=3). (B, E 5× magnification; A, C, D, F 10× magnification). Scale bars=200μm (B, E) and 100μm (A, C, D, F).


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=20μm) and SSEA-1 (Scale bars=50μm) similarly to F-iPS cells. (B) RT-PCR analysis showed that mouse ES marker genes were expressed in both NOF-iPS and F-iPS cells. (C) Quantitative RT-PCR analysis for Nanog expression in F-iPS and NOF-iPS (n=3). C3, C6 and C8 represent iPS clones 3, 6 and 8.


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 (5×105) were seeded into each well of a 6-well plate and were counted and passaged every 3 days. (B) bFA increased the percentage of SSEA-1+ cells after 10 passages. Each bar represents the mean ± standard deviation (n=3). (C) iPS cell-induced teratomas tissues expressed cytokeratin (CK, epithelial marker), desmin (muscle cell marker), and neuron-specific enolase (NSE) after 10 passages in bFA medium. Scale bars=100μm.


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. Cell Stem Cell 2008:2:151-9
Crossref   Medline   1st Citation  

Eiselleova L, Peterkova, I, Neradil, J, Slaninova, I, Hampl, A, Dvorak, P. Comparative study of mouse and human feeder cells for human embryonic stem cells. Int J Dev Biol 2008:52:353-63
Crossref   Medline   1st Citation  

Hanna J, Wernig, M, Markoulaki, S, Sun, CW, Meissner, A, Cassady, JP. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 2007:318:1920-3
Crossref   Medline   1st Citation  

Huangfu D, Maehr, R, Guo, W, Eijkelenboom, A, Snitow, M, Chen, AE. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol 2008:26:795-7
Crossref   Medline   1st Citation  

Jaenisch R, Young, R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 2008:132:567-82
Crossref   Medline   1st Citation  

Kim JB, Zaehres, H, Wu, G, Gentile, L, Ko, K, Sebastiano, V. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 2008:454:646-50
Crossref   Medline   1st Citation  

Klimanskaya I, Chung, Y, Meisner, L, Johnson, J, West, MD, Lanza, R. Human embryonic stem cells derived without feeder cells. Lancet 2005:365:1636-41
Crossref   Medline   1st Citation  

Lim JW, Bodnar, A. Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics 2002:2:1187-203
Crossref   Medline   1st Citation  

Loh YH, Wu, Q, Chew, JL, Vega, VB, Zhang, W, Chen, X. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 2006:38:431-40
Crossref   Medline   1st Citation  

Mauritz C, Schwanke, K, Reppel, M, Neef, S, Katsirntaki, K, Maier, LS. Generation of functional murine cardiac myocytes from induced pluripotent stem cells. Circulation 2008:118:507-17
Crossref   Medline   1st Citation  

Mikkelsen TS, Hanna, J, Zhang, X, Ku, M, Wernig, M, Schorderet, P. Dissecting direct reprogramming through integrative genomic analysis. Nature 2008:454:49-55
Crossref   Medline   1st Citation  

Ogawa K, Saito, A, Matsui, H, Suzuki, H, Ohtsuka, S, Shimosato, D. Activin-Nodal signaling is involved in propagation of mouse embryonic stem cells. J Cell Sci 2007:120:55-65
Crossref   Medline   1st Citation  

Okita K, Ichisaka, T, Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 2007:448:313-7
Crossref   Medline   1st Citation   2nd   3rd  

Park IH, Zhao, R, West, JA, Yabuuchi, A, Huo, H, Ince, TA. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008:451:141-6
Crossref   Medline   1st Citation   2nd  

Smith AG, Heath, JK, Donaldson, DD, Wong, GG, Moreau, J, Stahl, M. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988:336:688-90
Crossref   Medline   1st Citation  

Soh BS, Song, CM, Vallier, L, Li, P, Choong, C, Yeo, BH. Pleiotrophin enhances clonal growth and long-term expansion of human embryonic stem cells. Stem Cells 2007:25:3029-37
Crossref   Medline   1st Citation  

Takahashi K, Okita, K, Nakagawa, M, Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protocols 2007:2:3081-9
Crossref   1st Citation   2nd  

Takahashi K, Tanabe, K, Ohnuki, M, Narita, M, Ichisaka, T, Tomoda, K. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007:131:861-72
Crossref   Medline   1st Citation   2nd   3rd   4th  

Takahashi K, Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006:126:663-76
Crossref   Medline   1st Citation   2nd   3rd   4th  

Xu C, Inokuma, MS, Denham, J, Golds, K, Kundu, P, Gold, JD. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001:19:971-4
Crossref   Medline   1st Citation  

Yu J, Vodyanik, MA, Smuga-Otto, K, Antosiewicz-Bourget, J, Frane, JL, Tian, S. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007:318:1917-20
Crossref   Medline   1st Citation  

Zhong X, Li, Y, Peng, F, Huang, B, Lin, J, Zhang, W. Identification of tumorigenic retinal stem-like cells in human solid retinoblastomas. Int J Cancer 2007:121:2125-31
Crossref   Medline   1st Citation  


Received 10 January 2009/18 May 2009; accepted 3 June 2009

doi:10.1016/j.cellbi.2009.06.004


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)