1 Maria Infertility Hospital Medical Institute/Maria Biotech, Seoul 130-812, 2 Department of Animal Sciences, Kon-Kuk University, Seoul 143-701 and 3 Maria Infertility Hospital, Seoul 130-812, Korea
4 To whom correspondence should be addressed at Maria Infertility Hospital Medical Institute, 103-11, Sinseol-dong,Dongdaemun-gu, Seoul, 130-812 Korea. e-mail: eykim{at}mariababy.com or sppark{at}mariababy.com
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Abstract |
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Key words: embryonic stem cells/frozenthawed human blastocysts/inner cell mass/in vitro differentiation /STO cell
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Introduction |
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The primary difficulty in establishing hES cells is obtaining an embryo source without inciting ethical concerns. Infertility clinics generally maintain surplus embryos, left over after embryo transfer for pregnancy induction, and frozen for a certain period of time for future implantation. The use of frozen blastocysts that are no longer needed and would otherwise be discarded may not pose an ethical problem. Furthermore, establishment of ES cells from frozen blastocysts would be much easier than using cells in the pronuclear (PN) stage or 2- to 3-day-old frozen donated embryos.
Alternatives to mouse cells have been evaluated, but mouse embryonic fibroblasts (MEFs) are most commonly used as the feeder layer for culture of hES cells to help maintain the pluripotence of stem cells (Thomson et al., 1998; Reubinoff et al., 2000
). Shamblott et al. (1998
) have reported that ready-made STO fibroblast cells can also be used as feeder cells for culture of embryonic germ cells. STO cells are immortal MEFs that produce leukaemia inhibitory factor (LIF) and are more easily maintained than MEFs for the preparation of feeder layers.
In this study, we examined whether hES cell lines can be successfully derived from frozenthawed embryos, that would have been discarded from a routine human IVF-embryo transfer programme, using an STO cell feeder layer. In addition, we differentiated in vitro an hES cell line into specific cells representing three embryonic germ layers.
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Materials and methods |
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Assessment of hES cell growth on the STO cell feeder layer
The growth of hES cells on the STO cell feeder layer was examined from day 1 to day 5 after plating the hES cell clumps. Cell doubling of several selected colonies (n = 10) was evaluated daily by counting along a major axis under the same magnification (x40 or x60) with an inverted microscope (Olympus).
Stem cell marker staining
To assess alkaline phosphatase (AP) activity, hES cell colonies (>15 passages) were fixed in 4% formaldehyde for 15 min and then stained with Fast red TR/naphthol AS-MX for 15 min (Sigma). To detect the human stem cell markers stage-specific embryonic antigen (SSEA)-3 or SSEA-4, ES colonies were fixed with 90% acetone in H2O at room temperature for 15 min. To detect SSEA-1, tumour rejection antigen (TRA) 1-60 or TRA1-81, cells were fixed with 100% ethanol 4°C for 15 min. The monoclonal antibodies against stem cell markers (SSEA-1, MC-480; SSEA-3, MC-631; and SSEA-4, MC-813-70) were supplied by Developmental Studies Hybridoma Bank (Iowa City, IA). Also, monoclonal antibodies against TRA1-60 and TRA1-81 were donated by Peter Andrews, University of Sheffield. Antibody localization was detected using a rabbit anti-mouse antibody conjugated to fluorescein isothiocyanate (FITC; Jackson Immunoresearch Lab, Inc., Baltimore, PA).
Oct-4 expression measured by indirect immunostaining
Oct-4 expression was assessed in undifferentiated hES cells with an H-134 antibody (Santa Cruz, CA). ES colonies were fixed in a 4% paraformaldehyde solution at 4°C for 10 min. Antibody localization was determined with a goat anti-rabbit antibody labelled with FITC
Chromosome analysis
For chromosome analysis, hES cells were cultured in feeder-free Matrigel (Becton Dickinson, 1:20)-coated plates for 46 days. After treatment with 5% colcemid (Gibco), harvested ES cells were stained using a standard G-banding technique. Karyotyping was analysed using a Cytovision program (Applied Imaging Co.).
In vitro differentiation
Embryoid bodies (EBs) were prepared in a bacteriological dish (no. 1007, Becton Dickinson) for 5 days following 0.04% collagenase treatment and mechanical dissection of hES cell colonies. To initiate differentiation, EBs were treated with 1 µM retinoic acid (RA; Sigma) for 1 week and plated onto a 0.1% gelatin-coated dish in differentiation medium (KO-DMEM containing 1 mM glutamine, 0.1 mM -mercaptoethanol, 1% NEAA and 10% FBS) for 3 weeks.
RTPCR analysis
Expression of specific genes in undifferentiated hES cells was analysed by RTPCR analysis with primers for three embryonic germ layer cells in day 5 EBs and in RA-treated plus day 14 differentiated ES cells. Total RNA from samples was extracted using a TRI reagent kit (Sigma) according to the manufacturers instructions. cDNA was synthesized from 1 µg of total RNA using SuperScript II reverse transcriptase (Invitrogen, Grand Island, NY). cDNA samples were subjected to PCR amplification with primers selective for human neurofilament heavy chain (NF-H, 400 bp), keratin (780 bp), enolase (490 bp), cAct (630 bp) and amylase (490 bp) genes. Also, as a control for mRNA quality,
-actin (200 bp) was assayed using the same RTPCR method. The PCR primers are described in Table I. The PCR products were size fractionated by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. Final analysis was obtained in an image analyser (Biorad).
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Results |
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Discussion |
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In the human IVF-embryo transfer programme, surplus embryos left over after embryo transfer to induce pregnancy are maintained frozen for certain periods of time for future implantation. The use of frozen embryos, that are no longer needed and would otherwise be discarded, with patients consent may minimize ethical problems related to the use of human embryos for research. A recently developed culture medium (G1/G2 or P1) has been useful in the development of blastocysts from human zygotes (Gardner et al., 1998; Plachot et al., 2000
). In this study, we used modified CR1aa medium containing 20% hFF to grow human embryos to the blastocyst stage. Our results indicate that frozenthawed blastocysts are more advantageous than frozenthawed PN stage embryos for the establishment of ES cells.
To date, all human ES cell lines have been developed using a layer of so-called feeder cells from mice or human cells that supply components necessary to sustain human stem cells. In this study, we used STO cells as a feeder layer instead of MEF feeders (Thomson et al., 1998; Reubinoff et al., 2000
) or human skin fibroblasts (Hovatta et al., 2003
) for the culture of hES cells to help maintain them as pluripotent stem cells. STO cells are ready-made immortal MEFs that are maintained as easily as other cell lines. Xu et al. (2001
) reported that the frequency of spontaneous differentiation of hES cells is higher when grown under feeder-free conditions using conditioned medium (CM) from STO cells than when grown under feeder-free conditions using CM from MEFs. To circumvent this problem, we prepared an STO cell feeder layer from two to five passages after thawing. Our results indicate that the doubling time of hES cells on the STO cell feeder layer was
36 h, similar to the doubling time of cells grown on MEF feeder cells. Therefore, well maintained STO cell feeders and serum replacement added KO-DMEM medium sufficiently maintain pluripotency and minimize spontaneous differentiation in hES cells cultured for a long duration (>350 doublings). As in other reports (Thomson et al., 1998
; Reubinoff et al., 2000
), our established hES cells grew in tight colonies composed of cells with high nuclear to cytoplasm ratios and prominent nucleoli, as indicated in Figures 1 and 2.
Human ES cells express a series of surface antigens as well as Oc-t , have a normal karyotype and exhibit remarkable long-term proliferative potential. The SSEAs 1, 3 and 4 are globoseries glycolipids. Similar to other reports (Andrews et al., 1996; Thomson and Marshall, 1998
; Thomson et al., 1998
; Reubinoff et al., 2000
), our hES cells express SSEA-3 and SSEA-4 (the epitope recognized by the latter is more readily detected than that in the former) but not SSEA-1. Also, our hES cells express TRA1-60, TRA1-81 (the epitope recognized by the former is more readily detected than by the latter) and AP activity. Cell fate during development depends upon transcription factors that act as molecular switches to activate or repress specific gene expression programmes (Niwa et al., 2000
). Oct-4 (also called Oct-3) is a mammalian POU transcription factor expressed in early embryo cells and germ cells (Botquin et al., 1998
). Oct-4 activity is an essential characteristic of pluripotential founder cell populations in mammalian embryo (Nichols et al., 1998
). Oct-4 has been detected in vitro only in undifferentiated embryonal carcinoma cells, ES cells and embryonic germ cells (Yeom et al., 1996
). Oct-4 expression was confirmed in the undifferentiated hES cells by indirect immunostaining.
However, stem cells can either continue to grow in a pattern of prolonged self-renewal or differentiate. This fate choice is highly regulated by intrinsic signals and the external microenvironment (Odorico et al., 2001). Shuldiner et al. (2000
) reported that none of the growth factors evaluated exclusively directs hES cell differentiation. Also, they demonstrated that pluripotent stem cells express a wide range of receptors for growth factors, and multiple human cell types may be enriched in vitro by specific factors. Among the additive growing factors, RA was identified as a morphogenic and teratogenic compound and as a signalling molecule influencing gene expression in a complex manner via a family of RA receptors (Rohwedel et al., 1999
). In vitro differentiation of ES cells was initially confirmed morphologically and, later, characteristics specific to ES cells were reconfirmed by RTPCR or indirect immunocytochemistry. Early heart development is known to be sensitive to RA concentrations. RA increased the concentrations of
-actin and
-actinin in the cytoplasmic and cytoskeletal fractions of cells at all stages of development (Aranega et al., 1999
). However, in this study, the production of cardiomyocytes was limited, rather than frequent and transient. Differentiation into muscle actin was frequently observed upon addition of RA to culture conditions. After hES cells differentiated in vitro into three embryonic germ layer cells, RTPCR analysis identified expression of brain neurofilament, skin keratin, muscle enolase, heart cardiac actin and pancreatic amylase genes. However, keratin and enolase gene expression was also detected in undifferentiated hES cells. This pattern was similar to that reported by Shuldiner et al. (2000
). In vitro differentiation of hES cells into muscle, neuronal and glial cells was confirmed by indirect immunostaining with monoclonal or polyclonal antibodies. Also, some neurons express the functional neuronal markers GAD and TH, suggesting that these cells may be glutamatergic and dopaminergic neurons, respectively. In addition, when hES cells were injected intramuscularly into 5- to 7-week-old SCID mice (ICR strain), teratoma formation with various differentiated cells (glandular, blood vessel, cartilage, epithelial, etc.) was confirmed histologically (data not shown). Therefore, our hES cells were able to differentiate into cell types of all germ layers both in vitro and in vivo.
hES cell lines will serve as unique models in developmental reseach, toxicology screening and cell therapy (Thomson et al., 1998; Reubinoff et al., 2000
). However, the use of human embryos for development of ES cells is currently a controversial ethical and political problem in many countries. In Korea, the use of frozenthawed embryos destined to be discarded after 5 years in a routine human IVF-embryo transfer programme is legal. We believe we are the first to establish hES cell lines on an STO cell feeder layer. Additional studies underway are focused on the reproducibility of our method and improvement of protocols in order to obtain larger populations of specific differentiated cells.
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Acknowledgement |
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FOOTNOTES |
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References |
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Submitted on August 13, 2003; resubmitted on September 15, 2003; accepted on October 31, 2003.