An Autogeneic Feeder Cell System That Efficiently Supports Growth of Undifferentiated Human Embryonic Stem Cells

Authors


Abstract

Human embryonic stem cells (hESCs) have great potential as a source of cells for therapeutic uses, but their culture requires the support of mouse or human cells, either directly as a feeder cell layer or indirectly as a source of conditioned medium in feeder-free culture systems. Unfortunately, the risks of cross-transfer of pathogens from xenogeneic or allogeneic feeders or cell by-products limit their medical applications. In addition, not all human feeders support the growth of hESCs equally well, and ethical concerns have been raised regarding the derivation of feeder cells from aborted human fetuses.

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.

Introduction

Human embryonic stem cells (hESCs) have the ability to differentiate into almost all adult cell types and hold great promise for regenerative medicine [14]. Pluripotent hESC lines have now been derived from the inner cell mass (ICM) of surplus day 5 to day 8 human blastocysts [512], and continuous culture of isolated ICM cells and hESCs in an undifferentiated state requires the presence of feeder layers such as mouse embryonic fibroblast (MEF) cells [5, 6], STO (SIM mouse embryo-derived thioquanine and ouabain resistant) cells [12], or a variety of human fetal, neonatal, and adult cells [7, 8, 1315]. Alternatively, hESCs may be cultured on dishes coated with animal-based ingredients with the addition of MEF cell-conditioned medium [16, 17] or in the presence of the specific pharmacological inhibitor of glycogen synthase kinase-3 [18].

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.

Materials and Methods

Recovery of hESC-Derived Fibroblasts

hES-NCL1 (passages 14 and 31) and H1 (passage 49) cells were transferred into feeder-free and noncoated T-25 flasks (Iwaki, Asahi, Japan; http://www.bibby-sterilin.com), using Delbucco's modified Eagle's medium (DMEM), supplemented with 10% HyClone defined fetal calf serum (FCS; HyClone, Logan, UT; http://www.hyclone.com) at 37°C in a 5% CO2 atmosphere. After 36 hours, the number of attached colonies was evaluated; after one week, cells with fibroblast-like morphology—that is, flat cells with elongated nucleus and branching pseudopodia (Fig. 1)—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.

Figure Figure 1..

Morphology and characterization of hES-dFs (human embryonic stem cell–derived fibroblasts). Note peripheric spontaneous differentiation of (A) hES-NCL1 and (B) H1 cells into fibroblast-like cells in feeder-free conditions. (C): The hES-dF cells show typical fibroblast morphology—that is, flat cells with elongated nucleus and branching pseudopodia (filopodia). Bars: (A) 200 μm; (B) 100 μm; (C) 50 μm.

Identification of hESC-Derived Fibroblasts

To confirm that hES-dF cells are from the same origin as hES-NCL1 cells, we compared DNA genotype of undifferentiated hES-NCL1 cells (passage 19) and hES-dFs (passage 5). Total genomic DNA was extracted from both samples and amplified with 11 micro satellite markers—D3S1358, vWA, D16S539, D2S1338, Amelogenin, D8S1179, D21S11, D18S51, D19S433, TH01, and FGA—then analyzed on an ABI 377 sequence detector using Genotype software (Applied Biosystems, Foster City, CA; http://www.appliedbiosystems.com).

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 γ-irradiated (75,000 cells/cm2) autogeneic or allogeneic hES-dFs. The culture medium was embryonic stem cell medium containing knockout DMEM (Invitrogen), 100 mM β-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 α-actinin (sarcomeric) (1:800; Sigma), or α-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).

Results

When colonies of both hESC lines were transferred and cultured in the absence of mouse embryonic fibroblasts, 86.8% (H1) and 90.0% (hES-NCL1) of the colonies attached showing the first signs of spontaneous differentiation. Of these, only colonies with fibroblast-like cells (Fig. 1A, B) 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).

Figure Figure 2..

Micro satellite comparison of (A) hES-NCL1 cells (passage 19) and (B) feeders (passage 5) derived from hES-NCL1 reveals identical profiles, indicative of a common genetic origin.

Figure Figure 3..

Flow cytometry analysis of fibroblast-like cells (passage 4) derived from hES-NCL1 (left panel) and human foreskin fibroblasts (HFFs) (passage 4; right panel) for the presence of CD31, CD44, CD71, CD90, and CD106. The red line represents the staining with the isotype control, and the green line shows staining with specific antibodies.

Thus far, the hES-NCL1 line has been cultured on autogeneic hES-dFs for over 44 passages and on feeder derived from H1 cells for over 22 passages. H1 cells were grown on autogeneic feeder for over 18 passages and on allogeneic feeder (derived from hES-NCL1) for 17 passages. Both the hES-NCL1 and H1 lines were grown on Matrigel in the presence of autogeneic hES-dF–conditioned medium for 14 and 12 passages, respectively. We found that both fresh and cryopreserved hESC colonies grown on fresh or cryopreserved hES-dFs (Fig. 4A, C, E) 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).

Figure Figure 4..

Morphology and characterization of hES-NCL1 cells grown on (A–F) autogeneic hES-dF monolayers or (G, H) under feeder-free conditions with addition of autogeneic hES-dF–conditioned medium. (A): Five-day-old vitrified human embryonic stem cells (hESCs) cultured on frozen-thawed hES-dFs (passage 8). (B): Higher magnification of the same hESC colony. Note the typical morphology of human embryonic stem cells (hESCs): small cells with prominent nucleoli. (C, E): hESCs grown on hES-dFs stained with antibody recognizing the (D) TRA-1-60 and (F) SSEA-4 epitopes. (G): hESCs grown on Matrigel stained with antibody recognizing (H) the GTCM2 epitope . Bars: (A, E–H) 200 μm; (C, D) 100 μm; (B) 50 μm.

Figure Figure 5..

Morphology and characterization of H1 cells grown on (A–F) autogeneic hES-dF monolayer or (G, H) under feeder-free conditions using hES-dF-conditioned medium. (A): Four-day-old human embryonic stem cell (hESC) colony (passage 11) cultured on hES-dF (passage 4). (B): Higher magnification of the same hESC colony shows typical morphology of hESCs. (C, E): H1 cells (passage 13) grown on autogeneic hES-dFs (passage 6) stained with antibody recognizing the TRA-1-60 (D) and SSEA-4 (F) epitopes. H1 cells (passage 10) grown on (G) Matrigel with addition of autogeneic hES-dF–conditioned medium stained with antibody recognizing (H) the TRA-1-60 epitope. Bars: (A, C, D) 200 μm; (E–H) 100 μm; (B) 50 μm.

To test whether allogeneic hES-dFs support undifferentiated growth of hESCs, we cultured hES-NCL1 on hES-dFs derived from H1 and H1 cells on hES-dFs derived from hES-NCL1 cells. Indeed, the allogeneic feeder also supported undifferentiated growth of hESCs, as demonstrated by the presence of specific cell surface markers (Fig. 6A–F). 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).

Figure Figure 6..

Morphology (A, C, E) and characterization (B, D, F) of hES-NCL1 and H1 cells grown on autogeneic or allogeneic hES-dFs. (A, C): hES-NCL1 cells (passage 18) grown on allogeneic (derived from H1 cells) feeder were stained with antibody recognizing the (B) TRA-1-60 and (D) SSEA-4 epitopes. (E): H1 cells (passage 11) grown on allogeneic (derived from hES-NCL1 cells) feeder stained with antibody recognizing (F) the TRA-1-60 epitope. H1 cells (passage 10) grown on (G) Matrigel with addition of allogeneic (hES-NCL1) hES-dF–conditioned medium stained with antibody recognizing (H) the TRA-1-60 epitope. Bars: (A, B, G, H) 200 μm; (C–F) 100 μm.

RT-PCR analysis of both stem cell lines showed positive expression of OCT-4, NANOG, FOXD3, TERT, and REX-1 (Fig. 7A, B) 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).

Figure Figure 7..

Characterization (A, B) and karyotyping (C, D) of hES-NCL1 and H1 cells grown on autogeneic hES-dF monolayer. Reverse transcription polymerase chain reaction (RT-PCR) analysis of (A) undifferentiated hES-NCL1 (passage 29) and (B) H1 (passage 14) cells grown on inactivated autogeneic hES-dF cells. The PCR products were obtained using primers specific for (lane 1) OCT-4, (lane 2) NANOG, (lane 3) FOXD3, (lane 4) TERT, (lane 5) REX1, and (lane 6) GAPDH. PCR was processed with (+) or without (−) reverse transcriptase. (C): hES-NCL1 (passage 31) grown on hES-dF (passage 10) show a normal female karyotype (46, XX). (D): H1 (passage 16) grown on hES-dF (passage 3) show a normal male karyotype (46, XY). Abbreviations: NC, negative control.

Karyotyping of the hESCs showed that both hES-NCL1 and H1 lines grown on autogeneic feeders have normal female or male karyotypes (Fig. 7C, D).

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).

Figure Figure 8..

Histological analysis of teratomas formed from grafted colonies of hES-NCL1 (passage 23) grown on inactivated autogeneic hES-dF in testis (A–C) and kidney (D–F) of severe combined immunodeficient (SCID) mice. (A): Neural epithelium (ne). (B): Aggregation of glandular cells with characteristic appearance of secretory acini (sa). (C): Cartilage (cart). (D): Wall of respiratory passage showing epithelium (ep), submucosa (sm), and submucosal glands (sg). The epithelium contains occasional ciliated cells and numerous goblet cells secreting mucin (m). (E): Two types of epithelia: respiratory (top), keratinised skin (bottom). Submucosal glands (sg) located beneath pseudostratified ciliated (in parts) epithelium (ep). Structures of the skin include epidermis (ed), dermis (dm), and cornified layer (c). Note that the stratum granulosum (arrow) is characterized by intracellular granules, which contribute to the process of keratinization. Occasional mitotic indices (m) are seen in the basal layer. (F): High magnification image of skin, showing greater detail of dermis (dm), epidermis (ed), and cornified layer (c). Again, the stratum granulosum is visible (arrow). Scale bars: (A, B, C) 100 μm; (D, E) 25 μm; (F) 17.5 μm.

When hES-NCL1 and H1 cells grown on hES-dFs were cultured in the absence of hES-dFs, spontaneous differentiation into neuronal precursor, beating cardiomyocytes, and endodermal cells was observed (Fig. 9). 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.

Figure Figure 9..

Spontaneous differentiation of hES-NCL1 (A–C) and H1 (D–E) cells grown on autogeneic feeders and then in feeder-free culture conditions. Human embryonic stem cells (hESCs) from both lines differentiate into (A, D) neuronal precursor cells, (B,E) beating cardiomyocytes, and (C,F) endodermal cells. Specific staining the green color—represents cells stained with (A, D) nestin, (B, E) α-actinin (sarcomeric), and (C, F) α-fetoprotein antibodies. Red and blue colors represent cell-nuclei stained with (A, D) propidium iodide or (F) Hoechst 33342, respectively. In (B, E, C), the bright field and fluorescent images were recorded at the same time. Scale bars: (A–C) 100 μm; (F) 50 μm.

Discussion

The specific role of feeder cells in initial and prolonged culture of hESCs is not clear. It is known that feeder cells provide an ideal environment, including detoxification of culture medium and secretion of many unique proteins that participate in cell growth, as well as extracellular matrix formation and remodeling [20]. To date, numerous different feeder cells systems have been reported, with differing potentials for supporting growth of undifferentiated hESCs, including MEF; STO; fetal muscle, skin, and foreskin cells; adult Fallopian tube epithelial cells; and adult marrow cells [7, 8, 1215]. hESCs can also be grown on coated dishes with MEF cell–conditioned medium [16, 17, 21] or serum replacement that contains animal ingredients [22].

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.

Acknowledgements

We thank M. Choudary and A. Elliott for the technical support; Complement Genomics, Sunderland for sample analysis; and Dr. M. Pera for the donation of specific antibody. This work was supported by Newcastle University Hospitals Special Trustees, One Northeast Regional Development Agency, Newcastle Health Charity, the Department of Health and MRC (UK) grant no. G0301182.

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