First published online July 12, 2007
Stem Cells
Vol. 25 No.
10
October 2007, pp.
2601
-2609
doi:10.1634/stemcells.2006-0814; www.StemCells.com
© 2007 AlphaMed Press
A Novel Culture Technique for Human Embryonic Stem Cells Using Porous Membranes
Sinae Kima,
Seong Eun Ahna,
Jae Ho Leea,
Do-Seon Limb,
Kwang-Soo Kima,c,
Hyung-Min Chunga,
Soo-Hong Leea
aCHA Stem Cell Institute, Pochon CHA University, Seoul, Korea;
bDepartment of Dental Hygiene, College of Health Sciences, Eulji University, Seongnam, Gyeonggi-do, Korea;
cMolecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, USA
Key Words. Human embryonic stem cells • Cell culture technique • Porous membrane • No enzyme treatment Effective isolation of cultured cells
Correspondence: Soo-Hong Lee, Ph.D., CHA Stem Cell Institute, Pochon CHA University, P.O. Box 135-081, 606-16 Yoeksam 1-dong, Gangnam-gu, Seoul, Korea 135-081. Telephone: +82-2-3468-3688; Fax: +82-2-3468-3373; e-mail: lee.soohong{at}gmail.com
Received on December 18, 2006;
accepted for publication on June 27, 2007.
First published online in STEM CELLS EXPRESS July 12, 2007.
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ABSTRACT
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We have developed a novel culture technique for human embryonic stem cells (hESCs) using a porous membrane with feeder cells. The feeder cells were seeded and attached to the bottom of a porous membrane and, subsequently, hESCs were cultured on the top of the membrane. This porous membrane technique (PMT) allowed hESCs to be successfully cultured and to be effectively and efficiently separated from the feeder cell layer without enzyme treatment. hESCs being cultured by PMT were observed to interact with feeder cells through pores of membrane, where the interaction was dependent on the pore size of the membrane used. It was also revealed that the number of attached hESC colonies depended on the concentration of feeder cells on the bottom of the membrane. On the other hand, hESC colonies did not attach to porous membrane, as feeder cells were in the presence of culture dish, not the porous membrane. The hESCs cultured on porous membranes not only exhibited expression of several undifferentiated markers and a normal karyotype, but they also formed teratomas consisting of three germ layers in in vivo study. Compared with the mechanical isolation technique conventionally used, PMT significantly decreased mouse vimentin gene expression in cultured hESCs. Thus, a PMT for hESC culture would be a useful tool to exclude enzyme treatment and to reduce contamination from feeder cells simultaneously.
Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION
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Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of human blastocysts and can be differentiated toward each of the three germ layers that are potentially able to organize the entire human body [1, 2]. Studies of hESCs not only provide important clues of early human development but also play a critical role as a cell therapy source for regenerative disease treatment.
Recently, cell therapy using hESCs has Received greater attention following the initial hESC culture techniques reported in 1998 [1]. However, the application of hESCs for clinical cell therapy is limited because current culture techniques have been unable to retain highly pure cells that can be used as a clinical source. In other words, the hESCs cultured by currently developed techniques are unstable due to problems that arise from enzyme treatment procedures as well as contamination produced from animal feeder cells. Thus, it is critical to develop novel culture techniques for hESCs with high purity for clinical applications.
A conventional hESC culture method developed by Thomson et al. was to coculture with mitotically inactivated fetal mouse fibroblast feeder (mouse embryonic fibroblast [MEF]) cells [1]. They reported that mouse embryonic stem (ES) cells could maintain their stemness only in the presence of leukemia inhibitory factor in medium; however, due to unique characteristics and unlike mouse ES cells, hESCs could not maintain stemness. Furthermore, hESCs are able to rapidly differentiate without feeder cells, implying that feeder cells are essentially required to maintain hESC stemness [2].
As hESCs are transferred to new culture dishes to effectively isolate and expand them, conventional hESC culture techniques use an enzyme such as collagenase, trypsin, or dispase [3–5]. The enzyme treatment is of great advantage for large-scale bulk expansion of hESCs for a short period without laborious and time-consuming steps. However, continuous exposure to enzymes can cause cytogenic aberrations of hESCs [6, 7]. Therefore, Heins et al. developed a mechanical isolation technique (MIT) that physically separates hESCs from feeder cells by scratching the boundary between the cell layers with a finely drawn Pasteur pipette [8]. This technique has been reported to allow hESCs to maintain normal karyotype even after more than 120 passages. Even though this mechanical technique is able to avoid cytogenic aberrations caused by enzyme treatment, it too has a high possibility of contamination by feeder cells during the hESC isolation process. In addition, this MIT is too laborious and time-consuming to secure large enough amounts of hESCs to apply for cell therapy.
Oh et al. expanded hESCs by combining a MIT and collagenase treatment after coculturing them on feeder STO cells (mouse embryonic fibroblast cell line) [9]. Using this combined technique, they were able to shorten the processing time, but this process is still laborious and time-consuming and does not solve the problematic contamination by feeder cells and the damage incurred by enzyme treatment. A recent study attempted to overcome the labor- and time-intensive steps in mechanical isolation by using an automatic isolation machine [10]. However, the automatic equipment used in the study is expensive and might generate more serious contamination caused by machine inaccuracies.
A number of studies have reported that animal sources of nutrients and feeder cells during the hESC culture limit their usage in clinical applications [11, 12]. In order to reduce or remove contamination and infection from animal sources, human feeder cells, including human fetal fibroblasts and human bone marrow cells, were developed for culturing human ES cells [4, 13–15]. However, human feeder cells make it difficult to prolong passage numbers and to produce sufficient hESCs for clinical therapy because human feeder cells are not able to maintain continuous hESC stemness as much as animal feeder cells such as STO and MEF [4, 13]. Furthermore, in spite of human cells, there is still a challenge of efficient isolation of hESCs from feeder layer to perfectly prevent unknown problems developing in the future. Hence, it is critical to develop novel culture techniques to selectively isolate hESCs without cell contamination or damage for use in clinical applications. Recently, several groups showed that hESCs can be successfully maintained in a feeder cell-free condition [3, 16, 17]. However, these approaches are still not optimal because they cannot retain hESC culture as much as conventional culture techniques using feeder cells. In addition, these procedures are expensive due to the necessity to use high concentrations of basic fibroblast growth factor (bFGF) and laminin.
In the present study, we hypothesized that a porous membrane between hESCs and feeder cells would be able to significantly reduce hESC contamination produced by the current culture techniques and thus provide an efficient and effective procedure to easily isolate hESCs from feeder cells without enzyme treatment. Herein, we developed a novel hESC culture technique by using porous membrane to reduce contamination from feeder cells and to exclude enzyme treatment.
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MATERIALS AND METHODS
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Culture of Human Embryonic Stem Cells on Porous Membrane
The undifferentiated hESC line CHA-hES3 (passage number 85) was cultured as previously described [18]. The hESCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco-BRL, Gaithersburg, MD, http://www.invitrogen.com) supplemented with 20% serum replacement (Gibco-BRL), 1 mM L-glutamine (Gibco-BRL), 1% penicillin-streptomycin (Gibco-BRL), 1% nonessential amino acids (Gibco-BRL), 0.1 mM mercaptoethanol (Gibco-BRL), and 4 ng/ml bFGF (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). STO feeder cells (American Type Culture Collection, Manassas, VA, http://www.atcc.org) were grown in DMEM (Gibco-BRL) containing 10% fetal bovine serum (Gibco-BRL), 1% penicillin-streptomycin, 1% nonessential amino acids, and 0.1 mM-mercaptoethanol. Mitomycin C-treated STO feeder cells (1.5 x 105, 2.5 x 105, 3.5 x 105, or 4.5 x 105 cells per insert) were seeded and cultured on an inverted transwell cell culture insert for six-well plates (1-µm pore poly(ethylene terephthalate) membrane; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) for 1 day (Scheme 1A). The following day, hESC clumps (30 clumps per insert) were seeded inside of the insert. After the first 2 days, the medium was refreshed every day. After 5 days of postseeding of hESCs, the number of colonies and the number of cells in each colony were counted. The fully grown hESCs that cultured for 5–6 days were mechanically isolated and transferred into a prepared insert with fresh feeder cells. To confirm whether this technique is applicable to other cell lines, another human embryonic stem (hES) cell line, H9 (passage number 54; WiCell Research Institute, Madison, WI, http://www.wicell.org), was also used with the same protocol as described above except utilizing MEF as feeder cells instead of STO. This porous membrane technique (PMT) was compared with MIT by seeding hESCs onto the feeder layer directly. In addition, feeder cells were seeded in the culture well to provide conditioned medium and, subsequently, hESCs were cultured on porous membrane as described above (Scheme 1B). All characteristic and analytical data were acquired from CHA3 and H9 hES cells passaged 10 times by PMT and MIT.
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Scheme 1. Overall strategies to culture human embryonic stem cells using porous membrane techniques. Feeder cells are located at the bottom of porous membrane (A) or they are on the culture flask (B). Abbreviations: hESC, human embryonic stem cell; PMT, porous membrane technique.
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Pore Size of Porous Membrane to Prevent Feeder Cell Migration
The mitomycin C-treated feeder cells (STO and MEF) were seeded on the bottom of a porous membrane with 1-, 3-, and 8-µm pore sizes, respectively (600 cells per mm2 for STO and 840 cells per mm2 for MEF). The next day, the prepared samples were transferred into six-well dishes and then incubated for 5 days. The cells were removed from the top and bottom of the membrane by trypsinization and counted. The migration rate was calculated by dividing the number of cells that had migrated upward the porous membrane by the total number of cells as follows: migrated feeder cells (%) = (number of cells on the top of porous membrane/total cell number) x 100.
RNA Extraction and Reverse Transcription-Polymerase Chain Reaction
RNA extraction was performed using TRIzol (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) following the instructions of the manufacturer. After DNase (Invitrogen) treatment to samples, 1 µg of total RNA was used for cDNA synthesis (Superscript First-Strand Synthesis System; Invitrogen) following the manufacturer's protocol. Briefly, the reverse transcription reaction was carried out in 20 µl of mixtures (1 x reverse transcription [RT] buffer, 1.25 mM MgCl2, 5 mM dithiothreitol, 2.5 g random hexamer, 0.5 mM each dATP, dCTP, dGTP, and dTTP, and 50 units of Superscript II enzyme) at 42°C. After the reverse transcript reaction, RNA was degraded by 2 units of Escherichia coli RNase H. Polymerase chain reaction (PCR) was performed in 50 µl of reaction buffer containing 2 U of Takara Taq (Takara, Otsu, Japan, http://www.takara.co.jp), 1 x PCR buffer, 0.8 mM dNTP mixture, and 100 pmol of specific primers. Standard PCR conditions were as follows: 10 minutes at 94°C followed by cycles of 40-second denaturation at 94°C, 30-second annealing at 59°C, and 30-minute extension at 72°C. The primer sequences and expected sizes of amplified products are shown in Table 1. In addition, vimentin expression was measured by real-time RT-PCR in an ExiCycler (Bioneer, Daejeon, Korea, http://www.bioneer.com). One microliter of each RT reaction was amplified in a 20-µl PCR assay volume containing 2.0 mM MgCl2, 0.5 µM each primer (Table 1), and 1x Greenstar PCR Master Mix (Bioneer). Samples were incubated in the ExiCycler for an initial denaturation at 94°C for 10 minutes followed by 40 PCR cycles. Each cycle proceeded at 94°C for 40 seconds, 59°C for 30 seconds, and 72°C for 30 seconds. Relative quantification was calculated using the 2-(
CT) method [19]. To confirm amplification of specific transcripts, melting curve profiles (cooling the sample to 40°C and heating slowly to 95°C with continuous measurement of fluorescence) were produced at the end of each PCR. PCR products were also analyzed on ethidium bromide-stained agarose gels to ensure that a single band of the expected size of DNA was obtained.
Immunocytochemistry and Histology
To observe undifferentiated markers of hESCs, colonies and derivatives cultured on transwell inserts were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 minutes. After treatment with 1% normal goat serum for 30 minutes at room temperature, the cells were incubated with human-specific antibodies for Oct-4 (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), stage-specific embryonic antigen (SSEA)-1 (1:100; Chemicon, Temecula, CA, http://www.chemicon.com), SSEA-4 (1:100; Chemicon), and Tra-1–81 (1:100; Chemicon) for 24 hours at 4°C. After washing, bound primary antibodies were detected by incubation with rhodamine-conjugated goat anti-mouse IgG (1:800; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) for 1 hour. The stained samples were washed and mounted with Vectashield mounting solution with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). For localization of alkaline phosphatase (AP), cells were fixed in 4% paraformaldehyde for 20 minutes. After washing, AP was detected with ES Cell Characterization Kit (Chemicon) according to the manufacturer's instructions. All fluorescence images were acquired and analyzed using Zeiss ApoTome microscopy (Carl Zeiss, Jena, Germany, http://www.zeiss.com).
Karyotype Analysis
Chromosome analysis was performed with a slightly modified standard method [20]. After 3 days of replating, hESCs were incubated with 100 µl of colcemid (Gibco-BRL) for 3 hours at 37°C in a CO2 incubator and then trypsinized. After hypotonic solution treatment (1% citrate buffer), lysed cells were fixed in methanol/glacial acetic acid (3:1). G banding was performed for identification of chromosomes.
Scanning Electron Microscope
The morphology of the hESCs cultured by porous membrane technique was examined using a scanning electron microscope (SEM; S-4700; Hitachi, Tokyo, http://www.hitachi.com). First, samples were fixed in 2.5% glutaraldehyde for 1 hour and washed with 0.1 M phosphate buffer and then fixed again in 1% osmium tetraoxide solution. Next, the samples were dehydrated in ascending grades of ethanol, dried, and mounted on an aluminum stub using a double-sided carbon tape. The specimens were coated with platinum using an Ion Sputter Coater (E-1030; Hitachi) and examined at an acceleration voltage of 10 kV.
Teratoma Formation
Approximately 30,000 ESCs cultured by PMT were injected into the subcutaneous dorsum of 6-week-old nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org). The resulting tumors were removed after 12 weeks of hESC transplantation. The acquired teratoma were frozen and serially sectioned (8 µm) by a cryostat (Leica, Heerbrugg, Switzerland, http://www.leica.com). Sectioned slides were histologically examined by special stains including hematoxylin-eosin stain, Masson's trichrome, Alcian blue, and periodicity/aryl hydrocarbon receptor nuclear translocator/simple-minded stain. Images were analyzed using an inverted microscopy system (ECLIPSE TE2000; Nikon, Kanagawa, Japan, http://www.nikon.com).
Statistical Analysis
The results were evaluated in mean ± standard deviations. Data were analyzed by Statistical Package for Social Sciences 10.0 software (SPSS Inc., Chicago, http://www.spss.com), and a value of p < .05 was considered significantly different.
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RESULTS
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Culture of Human Embryonic Stem Cells on Porous Membrane
In order to separate hESCs from feeder cell layers and to eliminate the enzyme treatment step, we attempted to use transwell inserts with porous membranes (Scheme 1). To this end, feeder cells were attached on the bottoms of membranes by turning the transwell insert upside down. After the transwell membrane was inverted again and placed into an individual well of a six-well plate, two hESC lines (CHA-3 and H9) were seeded and cultured on the top of the porous membrane with feeder cells attached to the bottom (Scheme 1A).
First, STO and MEF feeder cell migration was observed in transwell inserts with different pore sizes (1, 3, and 8 µm). As shown in Figure 1A, both STO and MEF feeder cells on the bottoms of porous membranes migrated upward through the pores, and their migration rates increased as membrane pore size increased. For example, STO feeder cells at 3-µm and 8-µm pore sizes resulted in 1.6% and 9.3% cell migration rates, and MEF also showed a similar cell migration rate depending on pore size of the membrane (Fig. 1A). This demonstrates that larger pore sizes allowed cells to easily migrate through the membranes with fewer disturbances. On the other hand, a 1-µm pore size did not allow STO and MEF feeder cells to migrate. Thus, the 1-µm pore size was sufficient to reduce direct interaction between hES cells and feeder cell migration. After 30 clumps of hESCs (CHA3 and H9) were seeded in each transwell insert with different concentrations of STO or MEF feeder cells (360, 600, 840, or 1,080 cells per mm2), optimal colony formation was observed at a feeder cell concentration of 600 cells per mm2 and 840 cells per mm2 for CHA3 and H9, respectively (Fig. 1B). This result suggests that hESC attachment would also be optimized by an appropriate feeder cell concentration depending on the type of hES cell line (Fig. 1B).
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Figure 1. CHA3 and H9 human embryonic stem cells cultured using a porous membrane. (A): Migration rate of feeder STO cells for CHA3 and mouse embryonic fibroblasts for H9 through porous membrane with different pore sizes. The migration rate was calculated by counting the cell numbers on the tops and bottoms of transwells inserts after 6 days post-seeding of feeder cells (* and **; p < .001). (B): The number of attached hESC colonies was counted after 30 colonies were seeded on the porous membranes and cultured for 2 days (*, **, and ***; p < .001). Abbreviations: hES, human embryonic stem; hESC, human embryonic stem cell.
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Figure 2 shows the CHA3 and H9 hESC morphologies observed after 2 and 5 days of conventional culture (Fig. 2A), porous membrane culture (Fig. 2A, 2B), and conditioned medium culture (Fig. 2C). In the conventional culture technique, the hESCs seeded on the feeder cells begin to push away the feeder cells and then attach and grow on the culture dish. As shown in Figure 2A, the porous membrane technique using transwell inserts also allowed hESCs to attach and grow successfully. Both CHA3 and H9 hES cell lines stably attached on porous membrane with the same efficiency, strongly suggesting that PMT could be generally applied to different hES cell lines. In addition, we explored whether hESCs could be similarly maintained on the porous membrane without feeder cells attached to it (Scheme 1B). In this condition, both CHA3 and H9 hESCs failed to attach and grow on the top of the membrane, and they detached completely after washing with PBS (conditioned medium in Fig. 2C). This result demonstrates that hESC attachment and maintenance are greatly affected not only by feeder cells very closely located at the opposite side of hESCs on porous membrane but also by direct interaction between hES cells and feeder cells through pores. On CHA3 hES cell line (Fig. 3A), PMT exhibited a slightly lower cell number per colony after 5 days but not significantly different compared with conventional culture technique (p > .05). Similarly, a comparable cell number per colony was observed for H9 hESCs using PMT.
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Figure 2. Comparison of hESC morphology cultured by conventional culture technique, PMT, and conditioned medium. (A): The differential interference contrast images of CHA3 and H9 human embryonic stem cells during culture by a conventional culture technique and PMT. (B): Morphologies of attached CHA3 and H9 human embryonic stem (hES) cell line on porous membrane at 3 days post-seeding. (C): On conditioned medium, CHA3 and H9 hES cell lines failed to attach on the porous membrane so that they were completely removed by washing, irrespective of pore sizes. Abbreviation: PMT, porous membrane technique.
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Figure 3. The cell number per colonies of (A) CHA3 and (B) H9 cultured for 5 days (#, p > .05). Abbreviations: hESCs, human embryonic stem cells; PMT, porous membrane technique.
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Characterization of hESCs Cultured by Porous Membrane Technique
In order to characterize hESC differentiation, we analyzed the expression of several undifferentiated stem cell markers such as Oct-4, SSEA-1, SSEA-4, TRA-1–81, and AP by immunocytochemistry and histology (Fig. 4A–4E). Differential interference contrast images of samples show that hESCs cultured by PMT attached and grew successfully on the membrane. In immunocytochemistry results, the blue and red colors represent nuclei stained by DAPI and undifferentiation markers, respectively. Cultured cells expressed Oct-4, SSEA-4, TRA-1–81, and AP but not SSEA-1, confirming that hESCs maintained their stemness during PMT culture. In a tilted vertical image of Figure 4A (Fig. 4F), hESCs stained in red were closely located onto feeder STO cells (stained in blue) through the porous membrane barrier without pushing away the feeder cells as they did in conventional culture (Fig. 2A). This observation suggests that hESCs may grow in a more uniform condition without losing interactions with feeder cells by PMT, compared with conventional culture methods. Finally, we performed karyotype analysis and found that hESCs cultured by PMT exhibit normal human chromosome 46, XY (Fig. 4G).
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Figure 4. Characterization of human embryonic stem cells (hESCs) cultured on porous membranes with feeder STO cells attached to the opposite side. (A–D): Differential interference contrast and fluorescence images of hESCs cultured on the porous membrane. Fluorescence images were acquired after staining hESCs with several stemness markers (red represents each stemness marker and blue represents nuclei by 4,6-diamidino-2-phenylindole staining). (A): Oct4; (B): SSEA-4; (C): TRA-1–81; (D): SSEA-1; (E): An inverted image after AP staining; (F): Vertical (yz plane) image of panel (A). This illustrates that hESCs directly interact with feeder STO cells at the interface of the porous membrane. (G): Karyotype of the hESC by G banding. Karyotype (46, XY) demonstrates that hESCs cultured on porous membrane maintain normal chromosomes. Abbreviations: AP, alkaline phosphatase; SSEA, stage-specific embryonic antigen.
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Comparison of the PMT and the Mechanical Isolation Technique by RT-PCR and Real-Time PCR
Compared with the MIT, in which hESCs are directly cocultured with mouse feeder cells, our PMT using 1-, 3-, and 8-µm pore sizes was remarkably effective in reducing contamination of mouse feeder cells (Fig. 5A, 5B). Vimentin of mouse cells was used as a barometer of feeder cell contamination in hESCs. The hESCs cultured by MIT showed significant vimentin expression, suggesting the presence of mouse feeder cells coexisting with hESC colonies. In contrast, the hES cells cultured by PMT detected no vimentin (1- or 3-µm pore size) or marginal level (8-µm pore size). Consistent with this observation, our real-time PCR analysis confirmed that expression levels of vimentin from mouse cells were significantly lower when both CHA3 and H9 hESCs were cultured by PMT than when by MIT, in particular when 1- or 3-µm pore size was used.
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Figure 5. Comparison of mouse vimentin gene expression of the hESCs (CHA3 and H9) cultured by porous membrane technique (1-, 3-, and 8-µm pore size) and conventional technique. After 10 times passage, human embryonic stem cell lines were used for analysis. (A): Reverse transcription-polymerase chain reaction analysis (CHA3 hESCs). (B): Quantification of vimentin (mouse fibroblast marker). The porous membrane technique significantly decreased the expression of vimentin, a mouse protein marker, compared with the conventional culture technique (*, p < .001). Abbreviations: hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast; MIT, mechanical isolation technique; PMT, porous membrane technique.
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The Morphologies of Human ES Cells Cultured by PMT
An important issue in PMT is whether there is a direct interaction between hESCs and feeder cells. To address this, we performed SEM analysis of hESC culture on PMT at the cross-section part. As shown in Figure 6A and 6B, direct interaction between hES cell and feeder cells through 3-µm pores was distinctly observed. In particular, feeder cells on the bottom of the membrane progressively migrate upward through the 3-µm pores to interact with hES cells (Fig. 6C). This result suggested that direction interaction between hES cells and feeder cells is considerably important to culture and maintain hES cell lines. Despite the 1-µm-small pore, cellular bodies such as lamellipodia and filopodia were found in pores of the membrane (Fig. 6D, arrows). Thus, the 1-µm pore would also be able to allow hES cells to interact with feeder cells, although the interaction would be much less than those at bigger pore sizes (3 and 8 µm).
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Figure 6. Scanning electron microscope images of CHA3 hES cells that interact with feeder cells through 3-µm pores (A–C) and (D) 1-µm pore, respectively. Abbreviation: hES, human embryonic stem.
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Teratoma Formation of Human ES Cells Grown on Porous Membrane
When hESCs was transplanted into the subcutaneous dorsum of NOD/SCID mice, teratomas were observed at the site of hESC transplantation after 12 weeks. According to histological examination, the teratomas developed from hESCs contained all three embryonic germ layers, including cartilage (mesoderm), neuronal rosettes (ectoderm), secretory epithelium (endoderm), and muscle fibers (mesoderm) (Fig. 7).
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Figure 7. In vivo differentiation in teratomas of CHA3 human embryonic stem cells (hESCs) cultured by porous membrane technique. Histology and transgene expression in teratomas formed in the subcutaneous dorsum of nonobese diabetic/severe combined immunodeficient mice after 12 weeks of hESC transplantation. (A): Alcian blue stain (cartilage; mesoderm); (B): Hematoxylin-eosin stain (neuronal rosettes; ectoderm); (C): Periodicity/aryl hydrocarbon receptor nuclear translocator/simple-minded stain (secretory epithelial sheath; endoderm); (D): Masson's trichrome stain (muscle fibers; mesoderm) (scale bar = 100 µm).
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DISCUSSION
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The present study is to develop a new method for expansion of human embryonic stem cells by culturing them on the porous membranes of transwell inserts that have STO feeder cells attached to the other side of the membrane (Scheme 1A). Many studies have attempted to develop culture methods to expand stable hESCs without altering their karyotype [1, 4, 8]. However, many challenges remain toward developing clinical application because hESC stemness and expansion usually require contact with feeder cells and enzyme treatment, which might induce alterations of karyotype. Heins et al. reported a mechanical isolation method, which separates hESCs from feeder cells using a glass pipet without enzyme treatment [8]; however, this technique is a laborious and time-consuming method of isolating hESCs. Furthermore, feeder cells may not be completely removed from the system, resulting in contamination and the inability to use the hESC population for clinical cell therapy.
In this study, we used transwell inserts with porous membranes with pore sizes of 1, 3, and 8 µm. As 3- and 8-µm porous membranes were used, hESC attachment increased as compared with 1 µm (data not shown). However, porous membranes with greater than 3-µm pore sizes allowed feeder cells on the bottom of the membrane to migrate upward, whereas 1-µm pores rarely allowed migration (Fig. 1A). Thus, despite the lower hESC attachment rate, the 1-µm pore size was expected to be an optimal pore size to reduce the possibility of hESCs being contaminated by feeder cell migration. Unfortunately, the 1-µm pore size failed to prolong hESC (both CHA3 and H9) passage number past 15 times, whereas the 3-µm pore size used as an alternative allowed hESCs to be cultured until at least passage number 25, and they are still under observation. This result might be due to increased interaction between hES cells and feeder cells through bigger pores than 1 µm. The concentration of feeder cells also greatly affected the hESC attachment rate (Fig. 1B), validating the argument that feeder cell concentration is important to hESC culture even in alternative systems to conventional hESC culture [18].
Interestingly, when feeder cells were located in the bottom of the culture well, not on the bottom of the porous membrane, hESCs failed to attach to the membrane (Scheme 1B; Fig. 1A). This result possibly suggested that direct interaction between hES cells and feeder cells would be required for hESC culture at the environmental conditions used in this study. Another suggestion would be that local concentrations of cytokines secreted by feeder cells were more efficiently delivered into hESCs because hESCs and feeder cells were located very closely at opposite side of porous membrane. On PMT for hESC culture, pore size density in porous membrane would be another factor that might significantly affect hESC attachment and growth. The pore density used in this study is 1.6 x 106 pores per cm2. An increase in pore density may cause a higher hESC attachment rate because it increases interaction with feeder cells as well as the efficiency of cytokine delivery.
It is highly possible that MIT increases hESC contaminations depending on practical transfer skill. Furthermore, since there is no distinct boundary between hESCs and feeder cells, MIT might induce more serious contamination problems even with careful isolation of hESCs from feeder cells during transfer. However, hESCs cultured by PMT can be isolated efficiently during transfer with rare contamination from feeder cells because feeder cells only attached on the opposite side of the membrane. Thus, the PMT will provide a potentially useful method to generate hESCs for clinical applications by reducing contamination from feeder cells and by excluding the need for enzyme treatment. Furthermore, by controlling feeder cell concentration and/or direct contact between hESCs and feeder cells with small pore size of membrane, it would be unnecessary to mitotically inactivate the feeders, thus avoiding the requirement for using toxic substances (e.g., mitomycin) or radiation.
In the conventional hESC culture technique using feeder cells, hESCs on feeder cells are usually passaged every 5–7 days. Initially, hESCs seeded on feeder cells directly interact with feeder cells so that they can preserve their stemness without differentiation. However, inner cells of hESCs cannot interact with feeder cells continuously as hESCs are gradually growing over culture time, wherein bulk culture for hESC expansion is not allowed. As shown in Figure 6, feeder cells on bottom of membrane progressively migrate upward through pores followed by interaction with hESCs on whole area where hESCs attached. This suggests that PMT also would give a powerful advantage to expand hESCs on large scale for a short culture period, in particular by appropriately combining low concentration enzyme treatment.
In conclusion, we have developed a novel culture method for hESCs using a porous membrane as a physical barrier between feeder cells and hESCs. This PMT method allows hESCs to be successfully cultured and transferred without enzyme treatment, resulting in a significantly reduced contamination from feeder cells. Furthermore, we can expand this porous membrane technique further for expansion of the hES cell line on a large scale. Thus, this novel technique will provide a useful tool to culture hESCs for clinical application.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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This study was supported by a Grant (SC2190) of the Stem Cell Research Center funded by Korea Ministry of Science and Technology, Republic of Korea, and Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-311-D00440). H.-M.C. and S.-H.L. contributed equally to this work.
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REFERENCES
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- Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.[Abstract/Free Full Text]
- Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.[CrossRef][Medline]
- Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.[CrossRef][Medline]
- Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.[CrossRef][Medline]
- Hovatta O, Mikkola M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409.[Abstract/Free Full Text]
- Buzzard JJ, Gough NM, Crook JM et al. Karyotype of human ES cells during extended culture. Nat Biotechnol 2004;22:381–382.[CrossRef][Medline]
- Mitalipova MM, Rao RR, Hoyer DM et al. Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol 2005;23:19–20.[CrossRef][Medline]
- Heins N, Englund MC, Sjoblom C et al. Derivation, characterization, and differentiation of human embryonic stem cells. STEM CELLS 2004;22:367–376.[Abstract/Free Full Text]
- Oh SK, Kim HS, Park YB et al. Methods for expansion of human embryonic stem cells. STEM CELLS 2005;23:605–609.[Abstract/Free Full Text]
- Joannides A, Fiore-Heriche C, Westmore K et al. Automated mechanical passaging: a novel and efficient method for human embryonic stem cell expansion. STEM CELLS 2006;24:230–235.[Abstract/Free Full Text]
- Stojkovic M, Lako M, Strachan T et al. Derivation, growth and applications of human embryonic stem cells. Reproduction 2004;128:259–267.[Abstract/Free Full Text]
- Amit M, Itskovitz-Eldor J. Maintenance of human embryonic stem cells in animal serum- and feeder layer-free culture conditions. Methods Mol Biol 2006;331:105–113.[Medline]
- Cheng L, Hammond H, Ye Z et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.[Abstract/Free Full Text]
- Richards M, Tan S, Fong CY et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. STEM CELLS 2003;21:546–556.[Abstract/Free Full Text]
- Amit M, Margulets V, Segev H et al. Human feeder layers for human embryonic stem cells. Biol Reprod 2003;68:2150–2156.[Abstract/Free Full Text]
- Klimanskaya I, Chung Y, Meisner L et al. Human embryonic stem cells derived without feeder cells. Lancet 2005;365:1636–1641.[CrossRef][Medline]
- Xu C, Rosler E, Jiang J et al. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. STEM CELLS 2005;23:315–323.[Abstract/Free Full Text]
- Ahn SE, Kim S, Park KH et al. Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells. Biochem Biophys Res Commun 2006;340:403–408.[CrossRef][Medline]
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–408.[CrossRef][Medline]
- Dutrillaux B, Viegas-Pequignot E. High resolution R- and G-banding on the same preparation. Hum Genet 1981;57:93–95.[CrossRef][Medline]
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Abstract
Introduction
