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Cell Biology International (2012) 36, 537–543 (Printed in Great Britain)
Replacement of mouse embryonic fibroblasts with bone marrow stromal cells for use in establishing and maintaining embryonic stem cells in mice
Chae Hyun Lee*, Jun Hong Park*, Jae Hee Lee†, Ji Yeon Ahn†, Jong Heum Park*, Bo‑Ram Lee‡, Dae Yong Kim‡ and Jeong Mook Lim*†1
*Department of Agricultural Biotechnology, Seoul National University, Seoul 151921, Korea, †Laboratory of Stem Cell and Bioevaluation, WCU Biomodulation Program, Seoul National University, Seoul 151742, Korea, and ‡Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Seoul 151742, Korea


We have investigated the use of BMSC (bone marrow stromal cell) as a feeder cell for improving culture efficiency of ESC (embryonic stem cell). B6CBAF1 blastocysts or ESC stored after their establishment were seeded on to a feeder layer of either SCA-1+/CD45/CD11b BMSC or MEF (mouse embryonic fibroblast). Feeder cell activity in promoting ESC establishment from the blastocysts and in supporting ESC maintenance did not differ significantly between BMSC and MEF feeders. However, the highest efficiency of colony formation after culturing of inner cell mass cells of blastocysts was observed with the BMSC line that secreted the largest amount of LIF (leukaemia inhibitory factor). Exogenous LIF was essential for the ESC establishment on BMSC feeder, but not for ESC maintenance. Neither change in stem cell-specific gene expression nor increase in stem cell aneuploidy was detected after the use of BMSC feeder. We conclude that BMSC can be utilized as the feeder of ESC, which improves culture efficiency.


Key words: bone marrow stromal cell (BMSC), embryonic stem cell (ESC), feeder cell

Abbreviations: BMSC, bone marrow stromal cell, DMEM, Dulbecco's modified Eagle's medium, EB, embryoid body, ESC, embryonic stem cell, FBS, fetal bovine serum, ICM, inner cell mass, JAK, Janus kinase, LIF, leukaemia inhibitory factor, MEF, mouse embryonic fibroblast, NOD, non-obese diabetic, PI, propidium iodide, SCID, severe combined immunodeficiency, STAT, signal transducer and activator of transcription

1To whom correspondence should be addressed (email limjm@snu.ac.kr).


1. Introduction

Since the first establishment of mouse ESC (embryonic stem cell) by Evans and Kaufman (1981), much effort has been expended to culture stem cells efficiently. MEF (mouse embryonic fibroblast) have often been used for both the establishment and maintenance of ESC, and LIF (leukaemia inhibitory factor) has routinely been used to maintain stem cell characteristics during in vitro culture (Wobus et al., 1984; A, 2007; Kern and Zevnik, 2009). The MEF–LIF system overcomes the disadvantages of other co-culture systems, which can induce karyotypic abnormalities and alteration of differentiation capacity (Suemori and Nakatsuji, 1987). Nevertheless, MEF have been only utilized for several sub-passage (Karatza and Shall, 1984; Conner, 2001), and their preparation is complicated and time-consuming, typically requiring using large numbers of animals.

To improve the efficiency of current stem cell co-culture system, we adapted it to include BMSC (bone marrow stromal cell). BMSC lines can be maintained for much longer than MEF (>10 times), and can easily be stored following rapid freezing. Furthermore, they contain various promoting factors for cell vitality that may maintain stem cell characteristics or facilitate stem cell transformation of progenitor cells. The potential use of BMSC as feeder cells for maintenance of ESC (in place of MEF) was initially addressed in different species (Dravid et al., 2006; Cui et al., 2009). However, there are no data on ESC establishment. Considering the current trend in stem cell engineering that emphasizes the importance of stem cell niche, the use of BMSC as feeder cells may provide information on the control of stem cell maintenance and differentiation. Based on this rationale, we have used BMSC in establishing ESC lines as well as in maintaining established ESC in mice. Whether LIF enrichment supports BMSC feeder for ESC self-renewal was also examined.

2. Materials and methods

2.1 Animals

The Institutional Review Board and Institutional Animal Care and Use Committee of Seoul National University approved the use of animals and the relevant experimental procedures (approval nos. SNU-050331-2 and SNU-070423-5). Animal management, breeding and surgery were performed according to the standard protocols of Seoul National University.

2.2. Preparation of MEF and BMSC

For the preparation of MEF from pregnant ICR mouse, the visceral organs, head, and extremities of foetuses were removed, and the remaining tissue cut into small pieces, which were incubated in trypsin–EDTA solution (Invitrogen). The fibroblasts retrieved after enzymatic treatment were incubated in DMEM (Dulbecco's modified Eagle's medium; Invitrogen) supplemented with 10% (v/v) FBS (fetal bovine serum; HyClone) and 1% (v/v) penicillin–streptomycin. The bone marrow cells were collected from tibias and femurs of adult ICR mouse, suspended in ice-cold PBS, dispersed using a 23-gauge needle and treated with red blood cell lysis buffer (eBioscience) for 10 min at room temperature. The cells were filtered through a 70-mm nylon mesh and plated on a 100-mm tissue culture dish containing DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin solution. Three days after plating, non-adherent cells were discarded with the supernatant. Cells that were sub-passaged more than eight times were used in ESC culture. To characterize immunophenotypically BMSC, the cells were suspended in PBS and incubated with SCA1, CD45 and CD11b antibodies. They were washed with PBS and analysed by FACSCalibur™ (BD Biosciences). The data have been analysed using BD Cell/Quest Pro Software (BD Biosciences).

2.3. Preparation of feeder cells and detection of LIF in the culture system

To prepare the feeder cells, BMSC and MEF were treated with 16 μg/ml mitomycin C for 3 h. To quantify secreted LIF levels, mitomycin C-treated cells (1×106/dish) were plated on to 60-mm culture dishes. The growth medium was replaced every 24 h after seeding. Culture supernatants were collected, centrifuged twice to remove cell debris, and analysed using Quantikine Immunoassay kit (R&D Systems).

2.4. ESC establishment and characterization

Blastocysts obtained from B6CBAF1 mouse were seeded on to MEF or BMSC feeder layers in 4-well culture dishes containing KDMEM (knockout DMEM; Invitrogen) supplemented with a 3:1 mixture of FBS and knockout serum replacement and 1% (v/v) penicillin–streptomycin, with or without 5000 or 20000 pg/ml LIF. After ICM (inner cell mass) colony formation was confirmed, the colony-forming cells were trypsinized and replated. To determine whether the colony-forming cells had stem cell-like activity, the expression of the stem cell marker proteins and the stem cell-specific genes, as well as cell karyotypes and differentiation in vitro and in vivo, were monitored as previously described (Gong et al., 2008). To assess the capacity to differentiate in vitro, EB (embryoid body) formation was induced by the suspension culture of the established ESC in LIF-free medium for as few as 4 days. EB formation was identified by the expression of the 3 germ layer-specific markers, S100, α-fetoprotein and smooth muscle actin. Subcutaneous transplantation of ESC into NOD (non-obese diabetic)–SCID (severe combined immunodeficiency) mice was also attempted to confirm their differentiation activity in vivo to form teratomas.

2.5. Chromosome analysis

Chromosome numbers in ESC established on BMSC and MEF feeders were counted at the 10th passage. ESC were trypsinized, fixed with 70% ethanol and resuspended in PBS containing 0.1% (v/v) Triton X-100 (Sigma–Aldrich), 50 μg/ml PI (propidium iodide; Sigma–Aldrich) and 2 mg of RNaseA (Roche). They were immediately analysed by flow cytometry (FACSCalibur™; BD Biosciences). For further karyotypic analysis, ESC were suspended in 0.075 M KCl for 15 min at 37°C, placed in hypotonic solution, and fixed in a 3:1 mixture of methanol and acetic acid. Chromosome spreads, produced by dropping suspensions of fixed cells on to glass slides were stained with Giemsa solution (Invitrogen).

2.6. Analysis of relative mRNA levels by real-time PCR

Cells were stored at −75°C in RNA later (Ambion) and subsequently used for isolation of total RNA, which was used as the template for cDNA synthesis with a Superscript III first-strand synthesis system (Invitrogen). Expression of stem cell-specific genes was quantified by real-time PCR (Bio-Rad). β-Actin was used as an internal control for normalization, and relative mRNA levels were calculated using the 2−ΔΔCT method. Sequence-specific primers (listed in Supplementary Table S1 available at http://www.cellbiolint.org/cbi/036/cbi0360537add.htm) were designed using Primer3 software (Whitehead Institute/MIT, Center for Genome Research).

2.7. Statistical analysis

All experiments were performed thrice, and a generalized linear model (PROC-GLM or ANOVA) was employed, using SAS software (SAS), to evaluate inter-group differences. When ANOVA produced a statistically significant result, the effects of different treatments were compared using the least-squares method. A P<0.05 was considered significant statistically.

3. Results

3.1 Characterization of BMSC lines

All three BMSC lines were positive for SCA-1, but negative for CD45 and CD11b (Figure 1a), and there were significant differences (P<0.0079) in LIF secretion between the three SCA-1+/CD45/CD11b BMSC lines (Figure 1b). Compared with control MEF cells, all three BMSC lines exhibited similar or higher LIF gene expression and LIF protein secretion. LIF protein levels in the conditioned media of BMSC ranged from 42.4 to 74.7 pg/ml. Two of the BMSC lines (BMSC-1 and BMSC-3) showed lower LIF gene expression and LIF secretion than the other (BMSC-2).

3.2. Influence of BMSC line on ESC establishment

Regardless of the feeder cell line used, LIF significantly increased ESC colony formation (Table 1). With BMSC as the feeder, increase in ESC establishment when LIF supplementation was from 5000 to 20000 pg/ml, that is, significantly greater than that observed when LIF supplementation was increased from 0 to 5000 pg/ml. Moreover, ESC formation in 20000 pg/ml LIF was greater with a BMSC feeder layer than with MEF, although most blastocyst ICM cells (98–100%; P = 0.8179) formed cell clumps, regardless of feeder cell type and LIF concentration.


Table 1 Colony formation by blastocyst-derived ICM cells cultured on feeder layers of either MEF or BMSC in medium supplemented with different concentrations of LIF

Model effect of treatment on the number of ICM cells formed cell clump and ESC-like colonies were 0.8179 and 0.0002 respectively.

Feeder cells No. (%)† of ICM cells formed
Kinds Subtypes Conc. of LIF in medium (pg/ml) No. of blastocysts seeded* Cell clump ESC-like colonies
MEF 20000 45 45 (100) 9 (20)a
BMSC BMSC1-low LIF 20000 30 30 (100) 7 (23)ab
BMSC2-high LIF 20000 32 32 (100) 12 (37)b
BMSC3-low LIF 20000 35 35 (100) 12 (34)ab
BMSC2-high LIF 5000 9 9 (100) 1(11)abc
BMSC3-low LIF 5000 9 9 (100) 1(11)abc
BMSC1-low LIF 0 54 53 (98) 0 (0)c


Blastocysts were retrieved on day 4 of natural mating.


Percentage of the number of blastocysts seeded.


abcDifferent superscripts within the same column indicate significant differences, P<0.05.



The ESC colonies established on BMSC feeders were passaged >10 times without evident morphological changes. Several randomly selected lines expressed the stem cell-specific genes, Oct4, Nanog, Rex1, Cripto, Sox2 and Gdf3 (Figure 2). They were also positive for the stem cell marker proteins OCT4 and SSEA1. EB formation was subsequently identified by the expression of the three germ layer-specific markers, S100, α-fetoprotein and smooth muscle actin. Subcutaneous transplantation of ESC into NOD–SCID mice induced teratomas consisting of cells derived from the 3 germ layers (Figure 2). There was no significant difference in incidence of chromosomal aneuploidy in the ESC co-cultured in the MEF or BMSC layer (Figure 3). Karyotyping of ESC cultured in different batches of BMSC showed that on average 80% of ESC maintained on BMSC feeder layers contained a normal number of chromosomes (20 pairs).

3.3. Influence of BMSC line on ESC maintenance

Continuous culture of ESC was used to measure the effects of BMSC on ESC maintenance. Regardless of passage number and culture condition, ESC seeded individually on to BMSC feeder layers reformed colonies after passage. Neither LIF secretion activity nor BMSC passage number influenced this process. No difference in ESC activity was detected in randomly selected ESC lines, and no significant differences were found in stem cell-specific gene expression (P>0.1768) among ESC lines maintained under the different conditions (Figure 4).

4. Discussion

BMSC can replace MEF as the feeder cells in ESC culture systems. Although BMSC were used in this capacity, the addition of LIF to the culture medium was essential for the formation of ESC colonies from ICM cells obtained from blastocysts. However, exogenous LIF was not required for the re-cloning and maintenance of individually established ESC. The expression of stem cell-specific genes did not vary significantly between ESC maintained under different growth conditions (LIF-free versus LIF-enriched medium, BMSC versus MEF feeders). The secretion of LIF by BMSC lines may explain why ESC maintenance does not depend on exogenous LIF. Use of BMSC sub-passaged >20 times and minimizing of LIF enrichment contribute to improving the efficiency of stem cell co-culture system.

LIF is a crucial factor for the culture of ESC that expresses heteromeric receptors, comprising gp130 and the LIF receptor. The binding of LIF to these receptors activates JAK (Janus kinase)–STAT (signal transducer and activator of transcription) pathways responsible for stem cell establishment and maintenance (Heinrich et al., 2003; Kristensen et al., 2005; Okita and Yamanaka, 2006). As we have shown, the different BMSC lines secreted greater amounts of LIF (42 to 75 pg/ml), compared with MEF. However, in a previous study using different MEF lines (Lee et al., 2009), concentrations of secreted LIF ranged from 146 to 175 pg/ml. Thus, we cannot conclude that MEF secrete lower amounts of LIF than BMSC. From a different viewpoint, exogenous concentrations of LIF as low as 40 pg/ml are sufficient to support ESC maintenance in the presence of feeder cells. The transformation of blastocyst-derived ICM cells into ESC requires more LIF, >10 times, compared with exogenous LIF requirement for ESC maintenance.

The BMSC lines used secreted different amount of LIF, but there was no difference in the supportive action of BMSC on stem cell maintenance between high and low LIF-secreting BMSC lines. In the presence of BMSC, better colony formation was detected with the largest amount of LIF than at any other dose. In contrast, exogenous LIF does not appear necessary for ESC self-renewal in the presence of BMSC feeder. There may be subtle difference of exogenous LIF derived from feeder cells or supplementation in terms of biological activity, chemical structure and/or receptor compatibility, which modifies LIF requirement in the BMSC co-culture system.

We have confirmed that BMSC provide a suitable environment for ESC maintenance without the need for exogenous factors. BMSC releases soluble factors that stimulate self-renewal and growth of stem cells. Furthermore, BMSC are more easily maintained in vitro for longer than MEF (Meirelles Lda and Nardi, 2003), a great advantage for feeder cells in stem cell culture that directly contributes to abiding by the 3Rs of an experimental animal study.

In our previous experiments with a 3D culture (hydrogel) system, changes in cell niche could induce consequent changes in the major signals for stem cell maintenance, including those transduced by the JAK–STAT, Akt1 and Smad1/4/5 pathways (Lee et al., 2010). Our results also demonstrate that scaffold-dependent non-cellular niche and cellular niche created by BMSC may also modulate stem cell self-renewal signalling. Change or modification of stem cell self-renew signalling by BMSC does not seem to alter profoundly stem cell activity, as confirmed by detecting similar gene expression in ESC cultured under the presence of BMSC and MEF. However, it will be necessary to examine the relationship between exogenous factors secreted from BMSC and cell niche in regulating the establishment, maintenance and differentiation of stem cells.

Author contribution

Chae Hyun Lee did the experimentation and manuscript writing. Jun Hong Park and Jae Hee Lee did the data analysis and interpretation. Ji Yeon Ahn did data analysis and interpretation. and reviewed the paper. Jong Heum Park did data analysis and interpretation. Bo-Ram Lee did data collection and/or assembly. Dae Yong Kim did data analysis and interpretation. Jeong Mook Lim was involved in conception and design, data analysis and interpretation, obtaining financial support and writing the paper.

Acknowledgements

The English in the original of this document has been checked by at least two professional editors, both native speakers of English. For a certificate, see: http://www.textcheck.com/certificate/index/k2BSNQ.

Funding

This work was supported by the Stem Cell Research Center of the 21st Century Frontier Research and Program funded by the MEST (Ministry of Education, Science and Technology), Republic of Korea [grant number SC-5160]. This study was also supported by educational grants from Brain Korea 21 and WCU programs [grant number R31-10056] through the National Research Foundation of Korea funded by MEST.

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Received 22 July 2011/28 November 2011; accepted 6 February 2012

Published as Cell Biology International Immediate Publication 6 February 2012, doi:10.1042/CBI20110395


© The Author(s) Journal compilation © 2012 International Federation for Cell Biology


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