Hum. Reprod. Advance Access originally published online on January 24, 2007
Human Reproduction 2007 22(5):1231-1238; doi:10.1093/humrep/del523
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Testing of nine different xeno-free culture media for human embryonic stem cell cultures
1 REGEA, Institute for Regenerative Medicine, University of Tampere, Tampere University Hospital, Tampere, Finland 2 The Finnish Defense Forces, Helsinki, Finland 3 Karolinska Institute, CLINTEC, Karolinska University Hospital Huddinge, Stockholm, Sweden
4 To whom correspondece should be addressed at: Kristiina Rajala, REGEA, Institute for Regenerative Medicine, University of Tampere, Tampere University Hospital, 33520 Tampere, Finland. E-mail: kristiina.m.rajala{at}regea.fi
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Abstract |
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BACKGROUND: Human embryonic stem cells (hESC) are excellent candidates for cell replacement therapies. However, currently used culture conditions contain animal-derived components that bear a risk of transmitting animal pathogens and incorporation of non-human immunogenic molecules to hESC.
METHODS: Nine xeno-free culture media were compared with the conventional serum replacement (ko-SR) containing media in the culture of hESC on human feeder cells. Cultured hESC were characterized immunocytochemically and by fluorescence-activated cell sorter analysis. The differentiation potential of hESC cultured with xeno-free media was determined with the RT–PCR analysis.
RESULTS: The hESC cultured in xeno-free media differentiated or the proliferation decreased substantially. Under some test conditions, the morphology of the feeder cells was altered considerably. The hESC cultured with human serum underwent excessive differentiation in the beginning of culture, but a fraction of hESC was able to adapt to culture conditions containing 20% of human serum.
CONCLUSIONS: None of the studied xeno-free media was able to maintain the undifferentiated growth of hESC. The medium containing 20% human serum was found to sustain undifferentiated hESC proliferation to some extent, yet was inferior to the conventional ko-SR-containing medium.
Key words: human embryonic stem cell/human serum/xeno-free culture conditions
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The first human embryonic stem cell (hESC) lines were derived and cultured on mitotically inactivated mouse embryonic fibroblast (MEF) cell layer using a medium containing fetal bovine serum (FBS) (Thomson et al., 1998
). Under the FBS-containing conditions, many cells died and the hESC colonies underwent excessive differentiation (Amit et al., 2000; Reubinoff et al., 2000; Amit and Itskovitz-Eldor, 2002). Amit et al. (2000) described serum-free culture conditions for hESC, using a commercial serum replacement (KnockOut-Serum Replacement, ko-SR, Invitrogen) instead of FBS in the hESC culture medium. It was shown that ko-SR supplemented with basic fibroblast growth factor (bFGF) was able to support a prolonged growth of hESC in an undifferentiated state, and a higher cloning efficiency was obtained than in the FBS-containing medium (Amit et al., 2000). We have systematically compared different ko-SR concentrations for culturing hESC and found 20% to be the optimal concentration for the undifferentiated propagation of hESC derived and propagated on human feeder cells (Koivisto et al., 2004). The use of ko-SR in the hESC culture medium provides more standardized culture conditions compared with FBS, which is highly variable from lot to lot. Currently, ko-SR has mostly replaced the use of FBS in hESC cultures in most laboratories. Unfortunately, ko-SR still contains animal proteins such as bovine serum albumin (BSA) and hence is not completely free of xeno-derived components (Price et al., 1998). Human serum has also been used to replace FBS in hESC culture with some success. Richards et al. (2002) and Ellerström et al. (2006) derived hESC lines using culture medium containing human serum. Ellerstöm et al. managed to propagate hESC line in the human-serum-containing culture medium in an undifferentiated state for over 20 passages.
The exposure of hESC to xeno-products such as animal sera or proteins risks the contamination of hESC with undefined retroviruses and other animal pathogens (Amit et al., 2003). In addition, certain animal molecules such as the sialic acid Neu5Gc are incorporated and expressed on hESC cultured in the presence of animal-derived products. Such non-human antigens result in an immune response in humans (Martin et al., 2005). The use of MEF feeder cells in hESC culture is another major concern when aiming at developing xeno-free culture conditions for hESC. Various types of human feeder cells have been successfully used in maintaining hESC cultures (Richards et al., 2002). Richards et al. (2003) showed human adult skin fibroblasts to be the best feeder cell type in a comparative evaluation of 11 different human adult, fetal and neonatal fibroblast feeder types. Our group has derived and cultured hESC lines using commercial human foreskin fibroblasts as feeder cells since 2002 (Hovatta et al., 2003; Inzunza et al., 2005).
Various feeder-free culture conditions have also been reported for the culture of hESC (Xu et al., 2001; Amit et al., 2004; Beattie et al., 2005; Klimanskaya et al., 2005; Stojkovic et al., 2005). Some of these methods have been xeno-free by containing recombinant or human-derived extracellular matrixes and xeno-free media. A few hESC culture studies have been reported with X-Vivo 10 medium that contains only human-sourced recombinant proteins supplemented with recombinant human bFGF, stem cell factor, recombinant human flt3 ligand and leukaemia inhibitory factor (LIF) (Li et al., 2005) or a high concentration of bFGF (Genbacev et al., 2005). Recently, Ludwig et al. (2006) described a feeder-free derivation and culture of hESC using defined medium (TeSR1) including protein components solely from recombinant sources or purified from human material. However, feeder-free culture methods may induce chromosomal abnormalities in hESC due to the adaptation to more demanding growth conditions and enzymatic passaging methods often utilized in the feeder-free cultivation of hESC (Draper et al., 2004; Mitalipova et al., 2005). In feeder-free conditions, the importance of high concentrations of exogenously added growth factors and other factors increases. The heterogeneity of culture conditions and the variety and high concentrations of growth factors tested in sustaining undifferentiated growth of hESC reflect the fact that knowledge about the maintenance of self-renewal and pluripotency of hESC is still inadequate.
The use of xeno-derived components in the culture of hESC essentially limits the future clinical use of hESC-based therapies, and finding alternatives to replace these xeno-derived components has been one of the major focuses of hESC research during the past few years. Some commercial xeno-free serum replacements and media have recently become available. In order to find optimal culture conditions with low concentrations of bFGF using post-natal foreskin fibroblasts as feeder cells, we systematically tested eight commercially available or published xeno-free media and human serum in the culture of more than one hESC line.
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Culture media
Two commercial culture media, X-Vivo 10 and X-Vivo 20 (both from Cambrex Bio Science, Walkersville, MD, USA), published TeSR1 medium (Ludwig et al., 2006
; Thomson and Ludwig, 2006) with modifications, five commercially available serum replacements—LipuminTM 10x, SerEx 10x (both from PAA Laboratories GmbH, Pasching, Austria), SR3 (Sigma, St Louis, MO, USA), serum substitute supplement (SSS) (Irvine Scientific, Santa Ana, CA, USA) and Plasmanate (Bayer Healthcare, West Haven, CT, USA)—and human serum (Sigma) were tested in the culture of hESC, as shown in Table I.
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The control hESC medium contained 80% (vol/vol) KnockOut Dulbecco's modified Eagle's medium (DMEM) (Gibco Invitrogen, Carlsbad, CA, USA) and 20% (vol/vol) ko-SR (Invitrogen) supplemented with 2 mM Glutamax (Invitrogen), 0.1 mM
-mercaptoethanol (Invitrogen), 0.1 mM MEM non-essential amino acids (Cambrex Bio Science), 50 U penicillin/ml–50 µg streptomycin/ml (Cambrex Bio Science) and recombinant human bFGF (R&D Systems, Minneapolis, MN, USA) at 8 ng/ml. For feeder-free culture experiments, the hESC medium was supplemented with 80 ng/ml of human bFGF.
Xeno-free serum replacements LipuminTM 10x, SerEx 10x, SR3 and SSS were tested at 10 and 20% concentrations and Plasmanate at 20 and 40% concentrations in KnockOut DMEM supplemented as the control hESC medium without ko-SR. X-Vivo 10 medium contained X-Vivo 10 basal medium and 0.12 ng/ml transforming growth factor 1 (TGF1, Sigma) and was supplemented as the control hESC medium without ko-SR. X-Vivo 20 medium contained X-Vivo 20 basal medium and was supplemented as the control hESC medium without ko-SR. The human-serum-containing culture medium contained KnockOut DMEM, 10 or 20% (vol/vol) of heat-inactivated, sterile filtered human serum (H1388, Sigma) and 50 mg/l L-ascorbic acid 2-phosphate and was supplemented as the control hESC medium without ko-SR. For feeder-free culture experiments, 20% human-serum-containing culture medium was supplemented with 80 ng/ml of human bFGF.
The modified TeSR1 medium contained DMEM/F12 basal medium (Invitrogen) supplemented with 16.5 mg/ml human serum albumin (Sigma), 196 µg/ml insulin (Invitrogen), 108 µg/ml human holo-transferrin (Sigma), 1:500 chemically defined lipid concentrate (Invitrogen), 2 mg/l reduced glutathione (Sigma), 1:1000 trace elements B and C solution (Cellgro, Herndon, VA, USA), 6 mg/l thiamine hydrochloride (Sigma), 0.02 mg/l sodium selenite (Sigma), 41.5 mg/l lithium chloride (Sigma), 0.1 mg/ml 
-aminobutyric acid (Sigma), 0.127 µg/ml pipecolic acid (Sigma), 0.6 ng/ml TGF1 (Sigma) and 50 mg/l L-ascorbic acid 2-phosphate (Sigma) supplemented as the control hESC medium without ko-SR. The modification made to the published TeSR1 medium (Ludwig et al., 2006; Thomson and Ludwig, 2006) was the use of 8 ng/ml of bFGF in the experiments with human foreskin fibroblast feeder cells. Our previous testing has showed that there is no improved effect of higher concentration of bFGF for the growth of undifferentiated hESC in the presence of human foreskin fibroblasts (unpublished results); hence low concentration of bFGF was used for the experiments performed with human foreskin fibroblast feeder cells. For feeder-free culture experiments, the TeSR1 medium was supplemented with 100 ng/ml of human bFGF.
hESC cultures using human foreskin fibroblast feeder layer
Human ESC lines HS181 (passages 60 and 62), HS237 (passages 59, 61 and 74), HS293 (passages 42 and 49) and HS306 (passage 50) derived at the Karolinska Institute, Stockholm, Sweden, were used for the culture experiments. The Karolinska Institute has an approval of the Ethics Committee of the Karolinska Institute for derivation, characterization and differentiation of hESC lines. REGEA, Institute for Regenerative Medicine, University of Tampere, Finland, has the approval of the Ethical Committee of Pirkanmaa Hospital District to culture hESC lines derived at the Karolinska Institute. These cell lines have been derived and cultured on human foreskin fibroblasts as feeder cells, and the lines have been characterized earlier (Hovatta et al., 2003; Inzunza et al., 2005). Commercially available human foreskin fibroblast cells (CRL-2429, ATCC, Manassas, VA, USA) were used as feeder cells for the culture of hESC. Before plating the hESC, the feeder cells were mitotically inactivated by irradiating with 40 Gy. The hESC were adapted to the test culture conditions by gradually increasing the concentration of the test medium and decreasing the concentration of the control hESC culture medium every second day during the adaptation phase. The hESC were cultured in a humidified + 37°C, 5% CO2 incubator. The growth of hESC was monitored microscopically and culture media were changed daily. The hESC cultures were passaged mechanically every 7–10 days to new feeder cells. Every test media experiment was performed with at least two different hESC lines. The experiments failing to maintain hESC undifferentiated were repeated for second time in order to verify the results of the first experiments.
Feeder-free culture of hESC
Human ESC line HS237 (passage 78) was used for the feeder-free culture experiments. The hESC were adapted to the TeSR1 and 20% human-serum-containing media on human feeder cells by gradually increasing the concentration of the test medium and decreasing the concentration of the control hESC culture medium every second day during the adaptation phase. After adaptation phase, hESC were plated onto 12-well plates (CellBIND Surface, Corning, Inc., Corning, NY, USA) containing 10 µg/cm2 human collagen IV (Sigma), 0.2 µg/cm2 human vitronectin (Sigma), 5 µg/cm2 human fibronectin (Sigma) and 5 µg/cm2 human laminin (Sigma) coating mixture. The hESC were cultured in a humidified + 37°C, 5% CO2 incubator. The growth of hESC was monitored microscopically and culture media were changed daily. The feeder-free hESC cultures were passaged mechanically every 7–10 days and plated onto a new 12-well plate containing the coating mixture. The experiments which failed to maintain hESC undifferentiated were repeated for a second time in order to verify the results of the first experiments.
Immunofluoresence for Nanog and stage-specific embryonic antigen-1
The hESC colonies were fixed in culture dishes with 4% paraformaldehyde in phosphate-buffered saline (PBS) (0.01 M, pH 7.4) for 20 min at room temperature (RT), followed by washing with PBS (2 x 5 min). The cells were permeabilized and blocked with 0.1% Triton X-100, 1% BSA (Sigma) and 10% normal donkey serum (Sigma) in PBS for 45 min at RT and then washed once with 0.1% Triton X-100, 1% BSA and 1% normal donkey serum in PBS. Primary antibodies, polyclonal goat anti-human Nanog at a dilution of 1:200 and monoclonal mouse anti-human stage-specific embryonic antigen-1 (SSEA-1) at 1:200 (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used. Primary antibodies were incubated overnight at 4°C. The cells were washed (3 x 5 min) with 1% BSA in PBS and probed with secondary antibodies: rhodamine-red-conjugated donkey anti-mouse immunoglobulin (Ig) M at 1:400 (Jackson ImmunoResearch Europe Ltd, Cambridgeshire, UK) and Alexa Fluor 488 donkey anti-goat IgG at 1:800 (Invitrogen) for 1 h in the dark at RT. Human ESC labelled only with secondary antibodies were used as negative controls. After incubation, the cells were washed with PBS (3 x 5 min) and mounted in Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA, USA). Human ESC line HS237 cultured with the hESC medium was used as a control in immunofluoresence analysis. The labelled cells were viewed and photographed with a Nikon Eclipse TE2000-S phase contrast microscope with fluorescence optics and a Nikon COOLPIX 5400 camera.
Fluorescence-activated cell sorter analysis
The hESC (HS237) cultured in medium containing 20% human serum were analysed using a fluorescence-activated cell sorter (FACS). The cells were dissociated from the culture dish with TrypleTM Select (Invitrogen) for 15 min at 37°C and resuspended in 1 ml FACS buffer I (2% FBS, 0.01% sodium azide in PBS) and counted with a haemacytometer. A total of 0.2 x 106 cells were recovered, half of which were probed for 15 min at 4°C with a 1:500 dilution of monoclonal mouse anti-human SSEA-4 (Santa Cruz Biotechnology, Inc.) and the other half with monoclonal mouse anti-human SSEA-1 in FACS buffer I. The cells were then washed with FACS buffer I and probed with FACS buffer I containing a 1:500 dilution of r-phycoerythrin-conjugated goat anti-mouse IgG or r-phycoerythrin-conjugated goat anti-mouse IgM (both from Invitrogen) for 15 min in the dark at 4°C. The cells were then washed once with FACS buffer I, once with FACS buffer II (0.01% sodium azide in PBS) and fixed with 1% formaldehyde in PBS. HS237 cells cultured in a hESC medium were used as a control and treated similarly. The samples were analysed using BD FACSAriaTM equipment (BD Biosciences, Franklin Lakes, NJ, USA). Acquisition was set for 10 000 events per sample. The data were analysed using FACSDiva Software version 4.1.2.
In vitro differentiation and RT–PCR analysis
The pluripotency of the hESC line HS237 cultured with the modified TeSR1 medium was analysed with the RT–PCR analysis. The embryoid bodies (EB) were formed by mechanically dissecting upward-growing hESC colonies at passage 7 and transferring the resulting pieces onto a culture dish without feeder cells. The EBs were cultured in a modified TeSR1 medium without bFGF for 23 days before the isolation of RNA. The medium was changed every 2–3 days. The hESC line HS181 cultured in a hESC medium was used as a control, and samples were prepared similarly. Total RNA was isolated from EBs (TeSR1, n = 25; control hESC medium, n = 5) using RNeasy mini kit (Qiagen, Valencia, CA, USA). The RNA extraction was performed according to the manufacturer's instructions. The concentration and quality of isolated RNA were determined using a ND-1000 Spectrophotometer (NanoDrop Technologies, USA). Complementary DNA (cDNA) was synthesized from 50 ng of total RNA using Sensiscript Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions. The expression of markers characteristic of ectoderm (neurofilament 68 kDa, sense 5'-GAG TGA AAT GGC ACG ATA CCT A-3'; antisense 5'-TTT CCT CTC CTT CTT CAC CTT C-3'), endoderm (
-fetoprotein, sense 5'-GCT GGA TTG TCT GCA GGA TGG GGA A-3'; antisense 5'-TCC CCT GAA GAA AAT TGG TTA AAA T-3') and mesoderm (-cardiac actin, sense 5'-GGA GTT ATG GTG GGT ATG GGT C-3'; antisense 5'-AGT GGT GAC AAA GGA GTA GCC A-3') development in EBs were determined using RT–PCR primers (Proligo, Sigma). Glyceraldehyde 3-phosphate dehydrogenase (sense 5'-AGC CAC ATC GCT CAG ACA CC-3'; antisense 3'-GTA CTC AGC GGC CAG CAT CG-5') was used as a housekeeping control. One microlitre of cDNA was used as template in the PCR reactions. The negative control contained sterilized water instead of cDNA template. The PCR reactions were carried out in the Eppendorf Mastercycler as follows: denaturation at 95°C for 3 min and 40 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 30 s and extension at 72°C for 1 min, followed by final extension at 72°C for 5 min. The PCR products were analysed with electrophoresis on 1.5% agarose gel containing 0.4 µg/ml ethidium bromide (Sigma) and DNA standard (MassRulerTM DNA Ladder Mix, Fermentas).
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Human ESC were gradually adapted to different test media, using an increasing proportion of test media (with ratios of test media to control hESC media at 20:80, 50:50 and 80:20) up to 100% during the first week of culture. Different concentrations of commercially available serum replacements and media were used to evaluate the growth and maintenance of undifferentiated hESC. None of the eight xeno-free culture media or serum replacements tested were able to maintain the undifferentiated growth of hESC on human feeder cells. The differentiation of hESC already began during the adaptation process with all test media, as indicated by the change in colony morphology. The colonies became thinner and lost their angular shape and sharp edges. The number of undifferentiated colonies diminished significantly after the third adaptation phase (80:20) at all concentrations (10 and 20%), tested with LipuminTM-, SerEx-, SSS-, SR3-, X-Vivo-10- (100%), X-Vivo-20- (100%) and Plasmanate- (20 and 40%) containing media.
The results were consistent in all hESC lines examined and in repeated experiments. The differentiation was first judged by morphology and then confirmed by immunofluoresence analysis. The hESC colonies grown with the test culture media in all tested concentrations showed an increased expression of a marker common to the differentiated hESC (SSEA-1) and were negative for a marker common to the undifferentiated hESC (Nanog) (Figure 1).
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The morphology of the feeder cells used was found to change under some test conditions. Feeder cells shortened, became spherical and started to detach in X-Vivo-10-, X-Vivo-20-, SR3- and LipuminTM-containing media (Figure 1). In some test conditions, the growth of the colonies was also reduced significantly (X-Vivo 10, SSS, SerEx, modified TeSR1). Human ESC were all differentiated (except in modified TeSR1 media) when the adaptation process was complete, and it was impossible to passage the colonies further. The modified TeSR1 media were able to maintain the undifferentiated growth of hESC on feeder cells for seven passages. The hESC colonies in modified TeSR1 media began to grow upwards after seven passages, and the experiment was aborted.
The upward-growing hESC colonies cultured in the modified TeSR1 medium using feeder cells were differentiated in vitro into EBs, which were analysed with RT–PCR. Ectoderm (neurofilament 68 kDa) and mesoderm (-cardiac actin) specific markers were detected in the RT–PCR analysis. However, an endoderm (-fetoprotein) specific marker was not detected (Figure 2). These results show a defective pluripotency of hESC cultured with the modified TeSR1 medium. Our results clearly show that the various xeno-free test media were not able to maintain the undifferentiated growth and the pluripotency of hESC on human feeder cells.
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Human ESC were gradually adapted to human-serum-containing media using an increasing proportion of test media. The human-serum-containing medium was tested with concentrations of 10 and 20% of heat-inactivated, sterile filtered human serum. The hESC colonies underwent excessive differentiation during the first passages, as the concentration of human serum was gradually increased and the concentration of ko-SR in the culture medium was decreased. The differentiation was indicated by the change of hESC colony morphology. The colonies got thinner and some lost their angular shape and defined borders. The medium containing 10% of human serum was able to maintain the undifferentiated hESC growth for nine passages on human feeder cells. At the end of passage 10, all colonies had differentiated completely and the differentiation was confirmed by immunofluoresence staining with Nanog and SSEA-1 (Figure 3). As the colonies in the culture medium containing 20% of human serum were passaged further, they began to regain their thicker, undifferentiated morphology at passage level 8. At passage level 11, the colonies showed undifferentiated morphology (Figure 3), although they were notably thinner than the hESC cultured in the presence of a control hESC culture medium (Figure 3). At passage level 11, the hESC cultured in the 20% human serum medium were stained with a panel of immunocytochemical antibodies specific to hESC markers: Nanog, OCT-3/4, SSEA-4 and SSEA-1 (Figure 3). The expression of the markers was not complete, and parts of the colonies were differentiated, although there was no expression of SSEA-1 (Figure 3). As a control, hESC cultured in a control hESC medium were stained with Nanog and SSEA-1 and showed a strong expression of Nanog and no expression of SSEA-1 (Figure 3).
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Human ESC cultured with 20% human-serum-containing medium on human feeder cells were further analysed with an FACS. In total, 0.1 x 106 cells were labelled with SSEA-4 and 0.1 x 106 with SSEA-1. According to the FACS analysis, 35% of the hESC were positive for SSEA-4 (Figure 4). Of the control hESC, 80% were positive for SSEA-4. The 20% human serum culture medium was found to sustain an undifferentiated hESC proliferation to some extent, however, being inferior to the currently used culture medium containing ko-SR.
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The control hESC medium, TeSR1 medium and a medium containing 20% human serum were used to evaluate the growth and maintenance of undifferentiated hESC on extracellular matrix coating mixture without human foreskin fibroblast feeder cells. The attachment of hESC was poor; from the hESC colony pieces which were plated with each medium, only 30% attached in the TeSR1 medium, 55% in 20% human-serum-containing medium and 68% in the control hESC medium. From the colony pieces attached, only 30% formed colonies in the TeSR1 medium, whereas 75% formed colonies in the 20% human-serum-containing medium and hESC medium (Figure 5). After second passaging, only minor colony formation was observed in the hESC control medium and in 20% human-serum-containing medium, whereas no colony formation was observed in the TeSR1 medium and the experiment was terminated.
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Discussion |
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In this study, nine commercially available or published xeno-free media and serum replacements were compared with the conventionally used serum replacement (ko-SR) containing medium in the culture of hESC. These xeno-free reagents were not able to maintain the undifferentiated growth of hESC. The cell proliferation decreased during the adaptation to the test media, and after the complete adaptation, the hESC quickly differentiated. The use of commercially available X-Vivo 10 medium has been previously described by two research groups (Genbacev et al., 2005
; Li et al., 2005) in feeder-free culture conditions of hESC. Li et al. supplemented the X-Vivo 10 medium with recombinant human bFGF, stem cell factor, recombinant human flt3 ligand and LIF, whereas Genbacev et al. used a high concentration of bFGF. Recently, Ludwig et al. (2006) reported feeder-free derivation and culture of hESC using defined medium (TeSR1) including protein components solely from recombinant sources or purified from human material. In addition, they used a combination of collagen IV, fibronectin, laminin and vitronectin coating from human sources instead of a feeder cell layer. They managed to derive two hESC lines in these animal-product-free conditions, although neither one maintained stable karyotype. The TeSR1 medium contains various ingredients, of which many are used in high concentrations, making the medium very expensive. Also, the purified human matrix components used in the coating are expensive. Unfortunately, neither X-Vivo 10 medium nor TeSR1 with low concentration of bFGF were able to sustain the undifferentiated growth of our hESC lines on human foreskin feeder layer. The proliferation of hESC cultured in the modified TeSR1 medium decreased significantly after the adaptation process. Human ESC colonies began to grow upwards, making the passaging impossible. The modified TeSR1 media were able to maintain the undifferentiated growth of hESC for seven passages on human feeder cells. However, the pluripotency of hESC was incomplete, as an endoderm-specific marker was not detected in the RT–PCR analysis of the hESC cultured in the modified TeSR1 medium. We also tested feeder-free cultivation of hESC in the TeSR1 medium. However, the attachment and colony formation in the TeSR1 medium was poor compared with the control hESC medium, and we were able to maintain hESC in the TeSR1 medium only for two passages.
The morphology of the feeder cells used was found to change under some test conditions. Feeder cells shortened, became spherical and started to detach in X-Vivo-10-, X-Vivo-20-, SR3- and LipuminTM-containing media. Because feeder cells are an important part of our culture system, this might have a critical effect on the differentiation of hESC. It is reported that ascorbic acid deficiency in a fibroblast culture causes, among other things, an easy disaggregation of the cells from the intracellular matrix by protease action (Schafer et al., 1967). The role of ascorbic acid in a cell culture is to function as an antioxidant for the cells. Ascorbic acid is not available in a standard basal culture medium and needs to be added to the medium as a stable phosphate (Geesin et al., 1993). The ko-SR (Gibco Invitrogen) contains ascorbic acid (Price et al., 1998), but it is not known whether the commercially available serum replacements and media tested contain L-ascorbic acid. The absence or presence of other components in the tested culture media may also have influenced the decreased proliferation and differentiation of hESC. It is certain that the ingredients of the control and the test culture media differ because once the control media is entirely replaced, the differentiation of hESC is excessive. Our results clearly show that the eight different culture media tested were not able to support the undifferentiated growth of hESC. These results suggest that unknown components either present in or absent from the tested media compared with ko-SR induce the differentiation of hESC.
Human serum has previously been used in hESC culture with some success. Richards et al. (2002) derived an hESC line using 20% human-serum-containing culture medium and were able to propagate hESC in an undifferentiated state for 10 passages. However, it was later observed that a prolonged use of human serum beyond the 10th passage led to the increased differentiation of hESC (Richards et al., 2003). Recently, Ellerström et al. were able to derive and propagate an hESC line in the human-serum-containing medium for over 20 passages, without problems of excessive differentiation. In our study, human serum medium (containing L-ascorbic acid and 20% of human serum) was found to maintain undifferentiated hESC growth on human feeder cells to some extent (11 passages), but the hESC underwent an excessive differentiation in human serum culture media in the beginning of the experiments. As Richards et al. (2003) stated, human serum may contain some unknown factors that promote the differentiation of hESC, whereas ko-SR contains factors that enable an undifferentiated hESC culture. Since a fraction of the cells could be passaged despite the excessive differentiation in the beginning of the experiment and since the colony morphology improved after several passages in 20% human serum medium, the population of cells was able to adapt to culture conditions with human serum. The hESC cultured in media containing 10% of human serum were not able to adapt to these culture conditions. This is most likely due to a lack of sufficient serum proteins in the culture media. In particular, the amount of albumin, which is the major blood protein, seems to be essential for hESC survival in vitro. This notion is supported by the fact that BSA is the major constituent of ko-SR (Price et al., 1998). Although human serum can provide nutritive supplementation for hESC, it is a complex mixture containing compounds both beneficial and detrimental to hESC, which means that each lot should be carefully tested prior to use.
The addition of L-ascorbic acid to the culture medium could play a role in the ability of a subpopulation of hESC to survive in the human-serum-containing culture medium. The hESC cultured in 20% human serum medium maintained their undifferentiated morphology with smooth, angular colony shape for at least 11 passages, even though the colonies were thinner when compared with those cultured in the control hESC medium. We also tested feeder-free culture of hESC in 20% human-serum-containing medium. The attachment and colony formation in the human-serum-containing medium was considerably better than that in the TeSR1 medium. We also observed that the morphology of the cells in the colonies was considerably different than that in the control hESC medium and TeSR1 medium.
The use of human serum in the hESC culture medium is a xeno-free alternative to ko-SR, but similar to FBS, human serum is not of defined composition and is thus suboptimal. The commercial human sera available are pooled from a heterogeneous group of donors. Pooling sera from selected donors might provide more homogenous sera for hESC culture. Yet another concern with the use of human serum is the potential transmission of extremely serious pathogens such as human immunodeficiency virus, which has a long latent preclinical period and could go undetected in routine donor screenings (Mallon et al., 2006). The optimal solution for the xeno-problem of ko-SR would be the use of a similarly defined serum replacement containing purified human or human recombinant components.
The various culture conditions reported are difficult to compare because each group has used a different base media, matrix or feeders, cell lines and cell passage numbers. Although hESC have been shown to grow in each of these conditions, it is unclear which, if any, of these culture conditions are optimal. The use of a single substrate for hESC growth is desirable, but the substrates used are still undefined components and may have a lot-to-lot variability (Hoffman and Carpenter, 2005). It is also unclear whether the hESC maintained in these different substrates are equivalent. In fact, the gene expression signature of hESC is reported to be different when cultured with the ko-SR-containing medium and FBS (Skottman et al., 2006).
Even though several improvements in hESC culture conditions have been achieved during the past few years, a completely xeno-free, defined culture and derivation methods for hESC, which could be reproduced in various laboratories culturing hESC lines worldwide, have not been established. Cells used for human transplantation are regulated by the European Union (EU). According to new EU directives (2003/94/EC and 2004/24/EC), hESC for transplantation must be cultured according to good manufacturing practice (GMP) requirements. In order to derive clinical-grade hESC lines, the used derivation methods and all constituents of culture must be of GMP grade. Continuous research for identifying the mechanisms of self-renewal of hESC and improving the current culture conditions is essential in order to establish completely xeno-free culture systems that meet the requirements of GMP and enable the large-scale production of hESC.
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Acknowledgements |
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We thank the personnel of Regea, Institute for Regenerative Medicine, for their support in the stem cell research. This work was supported by the Academy of Finland, TEKES, the Finnish Funding Agency for Technology and Innovation, the Competitive Research Funding of the Pirkanmaa Hospital District, the Finnish Defence Forces and the Swedish Research Council.
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Submitted on October 6, 2006; resubmitted on December 13, 2006; accepted on December 27, 2006.
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