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a Centre for Stem Cell Biology and Developmental Genetics, University of Newcastle, Newcastle upon Tyne, UK;
b School of Biological and Biomedical Sciences, University of Durham, Durham, UK;
c Newcastle Fertility Centre at Life, Newcastle Health Service, Newcastle upon Tyne, UK
Key Words. Human embryonic stem cells • Pluripotency • Differentiation • Autogeneic feeder
Correspondence: M. Stojkovic, Ph.D. Centre for Stem Cell Biology and Developmental Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK. Telephone: 44-191-241-8638; Fax: 44-191-219-4747; e-mail: mio-drag.stojkovic{at}ncl.ac.uk
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ABSTRACT |
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We report here the culture of hESCs on a novel feeder cell system, comprising fibroblast-like cells derived from the spontaneous differentiation of hESCs. Isogenicity of the hESCs and hESC-derived fibroblasts was confirmed by micro satellite analysis. The nature of the hESC-derived fibroblasts was identified by the expression of specific markers. This feeder system permits continuous growth of undifferentiated and pluripotent hESCs, as demonstrated by the expression of specific hESC markers, by the formation of teratomas after injection of hESCs into severely combined immunodeficient mice, and by in vitro differentiation of hESCs into differentiated cells of ectodermal, endodermal, and mesodermal origin. Feeder cells derived from hESCs offers a potentially more secure autogeneic and genotypically homogenous system for the growth of undifferentiated hESCs.
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INTRODUCTION |
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The use of feeder cells for the prolonged culture of undifferentiated hESCs, however, does limit their medical application: xenogeneic and allogeneic feeders bear the risk of transmitting pathogens and other unidentified risk factors [7, 8, 13], not all human feeders and cell-free matrices support the culture of undifferentiated hES cells equally well [7, 13], and the availability of human cells from aborted fetuses or Fallopian tubes is relatively low.
We previously described [11] the derivation of a new and fully characterized hESC line (hES-NCL1). After culture in a feeder-free system, the hES-NCL1 and commercially available hESC (H1 line) were found to spontaneously differentiate into cells with fibroblast-like morphology. We show in this article that the latter cells can be used as an autogeneic feeder system that efficiently supports the growth and maintenance of pluripotency of both autogeneic and allogeneic undifferentiated hESCs.
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MATERIALS AND METHODS |
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)—were transferred into T-75 flasks (Iwaki). The cells were cultured for a further 3 days to produce a confluent primary monolayer of hES-dFs (for hESC-derived fibroblasts). Fibroblast-like cells derived from hES-NCL1 and H1 cells were passaged weekly and cultured for an additional 12 weeks in DMEM supplemented with FCS. These cells were either cryopreserved or passaged. After mitotic inactivation of hES-dFs, passages 2–12 were used for growth of hES-NCL1 or H1 cells.
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To identify the nature of feeder cells, hES-dFs (passage 5) derived from hES-NCL1 (passage 19) were compared with human foreskin fibroblasts (HFFs, passage 4; American Type Culture Collection [ATCC], Teddington, UK; http://www.lgcpromochem.com/atcc) using flow cytometry analysis. Briefly, hES-dFs were harvested using 0.05% trypsin/0.53 M EDTA (Invitrogen, Paisley, Scotland; www.invitrogen.com) and suspended in staining buffer (phosphate-buffered solution [PBS] +5% FCS) at a concentration of 106 cells/ml. One hundred µ1 of the cell suspension was stained with 0.2 µg of CD31 (PECAM-1), CD71 (transferrin receptor), CD90 (Thy-1), and CD106 (VCAM-1) antibodies (all available from BD Biosciences, Oxford, UK; http://www.bd.com) at 4°C for 20 minutes. Three washes in staining buffer were carried out before staining with secondary antibody, goat anti-mouse immunoglobulin G and M fluorescein isothiocyanate (IgG/M–FITC, 6 µg/ml final concentration; Sigma-Aldrich Co., Dorset, UK; www.sigmaaldrich.com) used at 4°C for 20 minutes. Cells were washed again three times and resuspended in staining buffer before being analyzed with fluorescence-activated cell sorter (FACS Calibur, BD Biosciences) using the CellQuest software. At least 10,000 events were acquired for each sample, and propidium iodide staining (1 µg/ml) was used to distinguish live from dead cells.
Growth of hESCs on hES-dFs
To test whether the stem cell lines were able to keep their pluripotency when grown on hES-dFs, both hES-NCL1 and H1 cells were cultured on 
-irradiated (75,000 cells/cm2) autogeneic or allogeneic hES-dFs. The culture medium was embryonic stem cell medium containing knockout DMEM (Invitrogen), 100 mM ß-mercaptoethanol (Sigma), 1 mM L-glutamine (Invitrogen), 100 µM nonessential amino acids, 20% serum replacement (SR; Invitrogen), 1% penicillin-streptomycin (Invitrogen), and 4 ng/ml basic fibroblast growth factor (bFGF; Invitrogen). ES medium was changed daily. hESCs were passaged every 4–5 days by incubation in 1 mg/ml collagenase IV (Invitrogen) for 5–8 minutes at 37°C, or they were mechanically dissociated and then removed to plates with freshly prepared hES-dFs.
Recovery of hES-dF–Conditioned Medium
Mitotically inactivated hES-dFs recovered from hES-NCL1 or H1 cells were cultured in T-25 flasks (Iwaki) with addition of ES medium for 10 days. hES-dF–conditioned media were collected every day, centrifuged at 200 g for 3 minutes, filtered with 0.2 µm syringe filter (Millipore, Watford, England; www.millipore.com), then frozen at –80°C. After thawing, the medium was equilibrated for 2 hours at 5% CO2 and 37°C and then used for the feeder-free culture of hESCs.
Growth of hESCs in Feeder-Free System Using hES-dF–Conditioned Media
The hES-NCL1 and H1 cells were passaged and then removed to plates precoated with Matrigel (BD Biosciences, Bedford, MA; http://www.bdbioscience.com), as previously described [16]. ES media conditioned by feeders derived from hES-NCL1 or H1 cells were changed every 48 hours.
Cryopreservation of hESCs and hES-dFs
To see whether frozen-thawed hES-dFs still support undifferentiated growth of cryopreserved hESCs, hES-dFs (passages 3 and 8) were frozen at –80°C using FCS supplemented with 10%(v/v) dimethyl sulfoxide (Sigma). Clumps of hESCs were vitrified using protocol as previously described [19].
Characterization of hESCs Cultured on hES-dFs
To investigate whether hES-NCL1 or H1 lines grown on autogeneic or allogeneic hES-dFs maintain their undifferentiated and pluripotent state, we performed immunocytochemical live staining of hESC cell-surface markers as follows: primary antibodies TRA-1-60 (1:10), SSEA-4 (1:5) (Developmental Studies Hybridoma Bank, DSHB, Iowa City, http://www.uiowa.edu/~dshbwww), and GTCM-2 (1:2; a kind gift from Dr. M. Pera, Monash University, Clayton, Victoria) were added to hESCs for 20 minutes at 37°C. The samples were gently washed three times with ES medium before being incubated with the secondary antibodies (Sigma) conjugated to FITC at 37°C for 20 minutes. All samples were again washed three times with ES medium and subjected to fluorescence microscopy. The bright field and fluorescent images were obtained using a Zeiss microscope and the AxioVision software (Carl Zeiss, Jena, Germany; http://www.zeiss.com).
For the flow cytometry analysis the hES colonies were collected using collagenase IV treatment (1 mg/ml for 5 minutes), followed by brief trypsin incubation (1 minute at 0.025% trypsin/0.25 M EDTA). The hESCs were suspended in staining buffer (PBS +5% FCS) at a concentration of 106 cells/ml. A total of 100 µ l of the cell suspension was stained with Tra-1-60(10 µg/ml final concentration; Chemicon International, Temecula, California; http://www.chemicon.com). Three washes in staining buffer were carried out before staining with secondary antibody, goat anti-mouse IgM–FITC (6 µg/ml final concentration; Sigma) Cells were washed again three times and resuspended in staining buffer before being analyzed with FACS Calibur (BD Biosciences) using the CellQuest software. At least 10,000 events were acquired for each sample, and propidium iodide staining (1 µg/ml) was used to distinguish live from dead cells.
Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis
The reverse transcription was carried out to investigate the presence of specific hESC markers in both hESC lines and hES-dFs derived from hES-NCL1 or H1 cells. RT was done using the cells with a cDNA II kit (Ambion, Huntingdon, UK; http://www.ambion.com), according to manufacturer’s instructions. In brief, hESCs collected after collagenase IV treatment (1 mg/ml for 5 minutes) were submerged in 1003l of ice-cold cell lysis buffer and lysed by incubation at 75°C for 10 minutes. Genomic DNA was degraded by incubation with DNAse I for 15 minutes at 37°C. RNA was reverse transcribed using M-MLV reverse transcriptase and random hexamers, following manufacturer’s instructions. PCR reactions were carried out using the following primers:
OCT-4F: 5'-GAA GGT ATT CAG CCA AAC-3'
OCT-4R: 5'-CTT AAT CCA AAA ACC CTG G-3'
REX1F: 5'-GCGTACGCAAATTAAAGTCCAGA-3'
REX1R: 5'-CAGCATCCTAAACAGCTCGCAGAAT-3'
NANOGF: 5'-GATCGGGCCCGCCACCATGAGTGTG-GATCCAGCTTG-3'
NANOGR: 5'-GATCGAGCTCCATCTTCACAC-GTCTTCAGGTTG-3'
TERTF: 5'-CGGAAGAGTGTCTGGAGCAAGT-3'
TERTR: 5'-GAACAGTGCCTTCACCCTCGA-3'
FOXD3F: 5'-GGA GGG AGG GGG CAA TGC AC-3'
FOXD3R: 5'-CCC CGA GCT CGC CTA CT-3'
GAPDHF: 5'-GTCAGTGGTGGACCTGACCT-3'
GAPDHR: 5'-CACCACCCTGTTGCTGTAGC-3'
PCR products were run on 2% agarose gels and stained with ethidium bromide. Results were assessed on the presence or absence of the appropriate size PCR products. Reverse transcriptase negative controls were included to monitor genomic contamination.
Karyotype Analysis of hESCs
To investigate the stability of hES-NCL1 and H1 cells grown on autogeneic hES-dFs, the karyotype of hESCs was determined by standard G-banding procedure.
Tumor Formation in Severe Combined Immunodeficient (SCID) Mice
The pluripotential nature of hESC-NCL1 grown on hES-dFs to build teratomas and form all three germ layers under in vivo conditions was estimated using SCID mice. All procedures involving mice were carried out in accordance with institution guidelines and institution permission. Approximately 3,000 hESCs (passage 23) were injected in testis and kidney of adult SCID mice. After 21–90 days, mice were sacrificed, and the tissues were dissected, fixed in Bouins overnight, processed, and sectioned according to standard procedures, then counterstained with either hematoxylin and eosin (H&E) or Weigerts stain. Sections were examined using bright field light microscopy, then photographed as appropriate.
In Vitro Differentiation of hESCs
Colonies of hES-NCL1 (passages 21 and 38) and H1 (passage 11) were removed from their autogeneic hES-dFs and cultured under feeder-free conditions in ES medium. After 5–14 days spontaneous differentiation was observed, and differentiated cells were passaged and cultured under the same conditions. Cells were fixed in 4% paraformaldehyde in PBS (Sigma) for 30 minutes, and then permeabilized for an additional 10 minutes with 0.1% Triton X (Sigma). The blocking step was 30 minutes with 2% FCS in PBS. Differentiated cells with beating cardiomyocytes were incubated with antibody against nestin (1:200; Chemicon), or 
-actinin (sarcomeric) (1:800; Sigma), or -fetoprotein (1:500; Sigma) for an additional 2 hours. Each antibody was detected by using the corresponding secondary antibodies conjugated to FITC. The nuclei of cells were stained using propidium iodide or Hoechst 33342 for 5 minutes. The bright field and fluorescent images were obtained using a Zeiss microscope and the AxioVision LE Rel. 4.2 software (Carl Zeiss).
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RESULTS |
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) which appeared as long, flat cells with an elongated, condensed nucleus (Fig. 1C) were further used for establishment of hES-dFs. Micro satellite analysis confirmed that the hES-NCL1 cells (Fig. 2A) and their autogeneic hES-dFs (Fig. 2B) have a common genetic origin. Flow cytometry (Fig. 3) revealed that very few cells showed expression of mesenchymal cell-specific markers CD106 (V-CAM1) and CD71 (transferrin receptor), and none expressed the endothelial-specific cell marker CD31 (PECAM-1). On the contrary, 94% and 82% of the hES-dF cells were stained with the CD44 and CD90 (THY-1) antibodies, respectively. Both antibodies were also presented in human foreskin fibroblasts (HFFs; Fig. 3).
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) or on Matrigel (Fig. 4G) were dense and compact, and they exhibited the typical morphology of hESCs (Fig. 4B). The hES-NCL1 cells cultured on autogeneic hES-dFs or on Matrigel with the addition of autogeneic hES-dF–conditioned medium expressed markers typical of hESCs, such as the cell surface markers TRA-1-60 (Fig. 4D), SSEA-4 (Fig. 4F), and GTCM-2 (Fig. 4H). H1 cells cultured on autogeneic hES-dFs or on Matrigel with the addition of autogeneic hES-dF–conditioned medium expressed TRA-1-60 (Fig. 5D, H) and SSEA-4 (Fig. 5F).
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). In addition, hES-NCL1 cells cultured on Matrigel with allogeneic (H1) hES-dF–conditioned medium expressed cell surface marker TRA-1-60 (Fig. 6H). Flow cytometry analysis revealed that hES-NCL1 grown on either autogeneic or allogeneic feeders expressed TRA-1-60 antigen (passage 42, 66.3%; passage 20, 58.9%). When grown on autogeneic or allogeneic feeders, 70.8% and 68.6% of H1 cells expressed TRA-1-60 (passage 15 and 14, respectively).
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) when grown on their autogeneic feeders. The fibroblast-like cells derived from both hESC lines also expressed the telomerase reverse transcriptase (TERT) and REX1 in early passages, but none of the other markers characteristic of ESCs (see Fig. 7A, B).
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The hESC cells grafted into SCID mice consistently developed into teratomas, demonstrating the pluripotency of hES-NCL1 cells grown on autogeneic hES-dFs. Teratomas were primarily restricted to the site of injection, and their histological examination revealed advanced differentiation of structures representative of all three embryonic germ layers, including cartilage, skin, muscle, primitive neuroectoderm, neural ganglia, secretory epithelia, and connective tissues (Fig. 8).
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). This demonstrates that both hESC lines grown on autogeneic feeders have the ability to differentiate into cells of all three germ layers also under in vitro conditions.
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DISCUSSION |
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The use of feeder cells or conditioned media of xenogeneic or allogeneic origin addresses the concerns about the inter-and intra-species transfer of viruses [23]. Human feeders recovered from the same or different donors have different abilities to support hESC growth [13], and ethical concerns have been raised regarding the derivation of feeders from aborted human fetuses. In addition, MEF cells can perform optimally only between the fourth and sixth passages [12, 13], and new MEF batches need to be continuously isolated from mouse fetuses [7, 13].
In this study we describe spontaneous differentiation of hES-NCL1 cells into fibroblast-like cells. Moreover, under the conditions described here, the commercially available H1 line [5] spontaneously differentiates into fibroblast-like cells. The nature of these cells was supported by similar expression in hES-dFs and HFFs for certain characteristic markers, in particular, CD44 and CD90, which are both known to be present in mesenchymal and fibroblast cells [24, 25]. The fibroblast-like cells derived from both hESC lines also expressed TERT and REX1 in early passages, but after injection of these cells into SCID mice, no formation of teratoma was observed (data not shown).
We used such differentiated cells derived from two hESC lines (hES-NCL1 and H1) as feeders, and we have shown that both lines cultured on autogeneic (isogenically related) or allogeneic feeder cell systems maintain their pluripotency. The expression of specific cell surface markers determined in our study by immunostaining and flow cytometry corroborate with the results described previously by Henderson and colleagues [26], which used the same technique to evaluate the pattern of cell surface antigen expression by the H7 and H14 lines grown on MEF. In addition, we found that hESCs differentiate into fibroblast-like cells also under serum-free conditions, and our results show that media conditioned by hES-dFs efficiently supports growth of hESCs in feeder-free culture systems.
Taken together, there are several potential advantages for using hES-dFs as feeder cells: (a) feeder cells derived from hESCs offers more secure autogeneic or genotypically homogenous systems for prolonged growth of undifferentiated hESCs; (b) feeders differentiated from the first clinical-grade hESC line could be used worldwide as initial monolayers for growth of isolated ICMs to eliminate transfer of pathogens; (c) the long proliferation time of already derived hESC lines allows screening for viral contamination; (d) medium conditioned by hES-dFs can be used for feeder-free growth of hESCs, thus avoiding potential viral transfer from the MEF-conditioned media used to date; (e) due to the low bioburden, embryonic tissues perform better support in vitro than adult tissues [13]; (f) derivation and culture of hES-dFs is fully controlled and not time consuming; (g) in vitro studies on cell-to-cell contacts and identification of isolated soluble factors could significantly improve cell culture, cell transplantation, and tissue engineering, avoiding at the same time expensive tissue biopsy and unnecessary sacrifice of animals.
In conclusion, we have developed a specific and unique feeder cell system from spontaneously differentiated hESCs. This feeder cell system derived from hESCs successfully prolongs growth of undifferentiated and cytogenetically stable hESCs and eliminates risk factors and concerns about using xenogeneic or unknown allogeneic feeders.
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ACKNOWLEDGMENTS |
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REFERENCES |
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