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Derivation and characterization of four new human embryonic stem cell lines: the Danish experience
RBMOnline - Vol 12. No 1. 2006 119-126 Reproductive BioMedicine Online; www.rbmonline.com/Article/1969 on web 31 October 2005
Article Derivation and characterization of four new human embryonic stem cell lines: the Danish experience Helle Lysdahl pursued her graduate work at Aarhus University, Denmark, where she was awarded her MSc in 1998 from the Faculty of Science and her PhD in 2002 from the Faculty of Health Sciences. Since 2003 she has been working as assistant professor at the Laboratory for Stem Cell Research, Aalborg University, Denmark. Her main research areas are establishing human embryonic stem cell lines and investigating different differentiation patterns of human embryonic stem cells.
Dr Helle Lysdahl Helle Lysdahl1,4, Anette Gabrielsen2, Stephen L Minger3, Minal J Patel3, Trine Fink1, Karsten Petersen2, Peter Ebbesen1, Vladimir Zachar1 1 Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg Oest; 2Ciconia, Aarhus Private Hospital, Saralyst Allé 50, 8270 Hoejbjerg, Denmark; 3Stem Cell Biology Laboratory, Wolfson Centre for AgeRelated Diseases, King’s College London, London SE1 1UL, UK 4 Correspondence: Tel: +45 9635 7548; Fax: +45 9635 7816; e-mail: [email protected]
Abstract In September 2003, legislation approved in Denmark legalized work on surplus human embryos from IVF for clinical purposes to establish human embryonic stem (ES) cell cultures. The aim of this study was to establish such stem cell lines. Fresh surplus embryos were donated after informed consent from the donors. Embryos were cultured into blastocysts and using the immunosurgery procedure, inner cell masses were isolated and cultured on irradiated human foreskin fibroblasts in KnockOut D-MEM supplemented with KnockOut Serum Replacement, bFGF, and LIF. Within a period of 12 months, 198 embryos were donated. Four isolated inner cell masses developed into putative ES cell lines, CLS1, CLS2, CLS3, CLS4, which have now been continuously cultured for eight months, corresponding to 30 passages. These cells expressed markers for undifferentiated human ES cells: stage-specific embryonic antigen-4, tumour-related antigen (TRA)-1–60, TRA-1–81, OCT4, NANOG, SOX2, and FGF4. The cells expressed high levels of telomerase activity, had a normal karyotype, and have been successfully cryopreserved and thawed. Finally, the cells displayed the potential to differentiate in vitro into cell types originating from all three germ layers. It is thought that the cell lines described in this study are the first human ES cells established in Denmark. Keywords: human embryonic stem cells, human feeders, LIF
Introduction Human embryonic stem (ES) cells are pluripotent stem cells, which can be established from the inner cell mass (ICM) of human blastocysts (Thomson et al., 1998; Reubinoff et al., 2000; Hovatta et al., 2003; Pickering et al., 2003). ES cells can be propagated indefinitely in vitro while retaining a normal karyotype and potential for differentiating into derivatives originating from the three embryonic germ layers. Human ES cells are promising candidates for cell-based treatment of disorders that involve loss of specific cell types, such as osteoarthritis, type I diabetes, Parkinson’s disease, and other degenerative diseases. In spite of the high potential
value in regenerative medicine, many basic questions remain unanswered, and more research is needed in order to make protocols using ES cell-based therapies a routine part of medical practice. In September 2003, legislation approved in Denmark legalized work on surplus human embryos from IVF for clinical purposes to establish human ES cell lines. With permission from the Regional Ethical Committee in Aarhus, the Laboratory for Stem Cell Research, Aalborg University and the Fertilization Clinic at Ciconia, Aarhus Private Hospital aimed at establishing such stem cell lines. The first established human ES cells were cultured on mitotically inactivated mouse embryonic fibroblasts as feeder
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Article - First human ES cells established in Denmark - H Lysdahl et al. cells (Thomson et al., 1998; Reubinoff et al., 2000). Recently, it was shown that human ES cells incorporate Neu5Gc, a nonhuman sialic acid, under conditions using mouse feeder layers and serum replacements (Martin et al., 2005). Hence, the use of non-human materials is not suitable for generating cells to be used for therapeutic purposes in humans. Improving the culture conditions for ES cells by avoiding animal products has been described in a number of publications. Instead of mouse feeder cells, human fetal muscle, fetal skin cells, adult skin fibroblasts, adult Fallopian tubal epithelial cells, adult marrow cells, placental fibroblasts, and foreskin fibroblasts have been successfully used (Richards et al., 2002, 2003; Cheng et al., 2003; Hovatta et al., 2003; Simón et al., 2005). Culturing human ES cells without feeder cells has been improved by coating the culture dishes with extracellular matrix, Matrigel, or laminin and using either conditioned media from the mouse feeders as culture media (Xu et al., 2001) or by coating with Matrigel and using a culture media containing noggin and high basic fibroblast growth factor (bFGF) concentration (Xu et al. 2005). This paper describes the successful establishment of four new human ES cell lines, using human feeder cells and Knockout Serum Replacement.
Materials and methods Establishing and culturing human ES cells Fresh surplus human embryos from IVF for clinical purposes were donated after informed consent and approval from the Regional Ethical Committee in Aarhus (journal number 20010164). The embryos were donated on either day 2 or day 3 after fertilization and cultured to the blastocyst stage in either ISM2 media (Medicult, Copenhagen, Denmark) or blastocyst media (Cook Denmark, Bjaeverskov, Denmark). The inner cell mass (ICM) was isolated by first removing the zona pellucida with a 10 mg/ml protease pronase E solution (Sigma-Aldrich, Copenhagen, Denmark) and then by removing the trophectoderm by immunosurgery using 20% rabbit antihuman whole serum and 20% guinea pig complement (SigmaAldrich) (Solter and Knowles, 1975). The lysed trophectoderm was removed using a blastomere biopsy pipette (Swemed Lab International AB, Billdal, Sweden) with an inner diameter of 40–55 µm.
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The isolated ICM were cultured on irradiated (35 Gy) human foreskin fibroblasts (American Type Culture Collection; LGC Promochem, Boras, Sweden) in 35-mm culture dishes (Corning Biotechline, Copenhagen, Denmark) in Knockout Dulbecco’s modified Eagle’s medium supplemented with 2 mmol/l Lglutamine (InVitrogen, Copenhagen, Denmark), 0.1 mmol/l β-mercaptoethanol, 1000 IU/ml leukaemia inhibitor factor (LIF) (Sigma-Aldrich), 4 ng/ml basic fibroblast growth factor (bFGF) (BioSource Nordic, Denmark), and 20% Knockout Serum Replacement (InVitrogen). The medium used in culture of the feeder cells before inactivation was Iscove’s Modified Dulbecco’s Medium (IMDM; (InVitrogen) supplemented with 10% fetal calf serum (FCS) (Biotech line, Copenhagen, Denmark). After 10–15 days compact, individually cell colonies appeared from the ICM derived outgrowths. These colonies were mechanically dissociated into small clumps using
a scalpel (Aesculap, B. Braun Medical, Copenhagen, Denmark) and replated on irradiated feeder cells in fresh medium. Every 7th to 9th day, individual colonies containing a uniform undifferentiated morphology were mechanically dissociated into clumps and replated.
Freezing ES cells Vitrification in VitriPlugs (Astro Med.Tec/Mobil-Tec Elektronik GmbH, NordicCell, Copenhagen, Denmark) closed with straws using 20% ethylene glycol, 20% dimethyl sulphoxide (DMSO), and 0.5 mol/l sucrose (Sigma-Aldrich) as cryoprotectants was performed as previously described (Reubinoff et al., 2001).
Characterization of ES cells Cell surface markers characteristic of undifferentiated human ES cells were detected using immunocytochemistry. The cells were incubated with Hoechst (Molecular Probes, Invitrogen) before fixation with 4% formaldehyde and incubation with antibodies against stage-specific embryonic antigen (SSEA)-4, SSEA-1, tumour-related antigen (TRA)-1–60, and TRA-1–81 (Santa Cruz; AH Diagnostics, Aarhus, Denmark). The SSEA were diluted 1:100 and the concentration of the TRA was 30 μg/ml. Using Cy5-conjugated goat anti-mouse immunoglobulin (IgG and IgM) secondary antibody (Chemicon; AH Diagnostics), the presence of surface markers was determined using an Axiovert microscope equipped with a AxioCam camera and the image processing was done with the aid of AxioVision software package (Zeiss, Brock and Michelsen A/S, Copenhagen, Denmark). The expression of genes characteristic for pluripotent human ES cells was determined using real-time reverse transcriptase– polymerase chain reaction (RT-PCR). The mRNA from the ES cells was isolated using the AURUM total RNA isolation kit (Biorad, Copenhagen, Denmark) according to the procedure of the manufacturer and synthesized to cDNA using the iScript cDNA synthesis kit (Biorad) according to the procedure of the manufacturer. Levels of the pluripotent gene markers were measured in iCycler (Bio Rad, Stockholm, Sweden) with octamer binding transcription factor-4 (OCT-4) primers 5′-TTCGCAAGCCCTCATTTCACC-3′ (upper) and 5′CCCCCTGGCCCATCACCTC-3′ (lower), NANOG primers 5′CAACTGGCCGAAGAATAGCAATGGTGT-3′ (upper) and 5′AGGAAGAGTAGAGGCTGGGGTAGGTAGGTG-3′ (lower), SOX2 primers 5′-GCAGGATCGGCCAGAGGAGGAGGGAA GC-3′ (upper) and 5′-GAGGGGAGGCGGGCGGGGGTGTC3′ (lower), and fibroblast growth factor 4 (FGF4) primers 5′AGAGCGGCGCCGGCGACTACCTG-3′ (upper) and 5′-AA GATGCTCACCACGCCCCGCTCCAC-3′ (lower). 18S rRNA was used as an internal endogenous standard. All primers were purchased from DNA-Technology, Aarhus, Denmark. The telomerase activity in the human ES cells was analysed using the Telo TAGGG telomerase PCR ELISA kit (Roche, Copenhagen, Denmark) according to the procedure of the manufacturer. The karyotypes of the human ES cells were determined using published protocols (Gosden et al., 1992). Briefly, the human ES cell colonies were incubated with 10 µg/ml colcemid solution (Gibco, Grand Island, New York, USA) for 2 h at 37°C in 5% CO2. The cells were washed with PBS, trypsinized, and neutralized
Article - First human ES cells established in Denmark - H Lysdahl et al. with human ES cell culture medium. Pellets were resuspended and incubated with 0.075 mol/l KCl for 17 min at 37°C. The cells were then fixed with 3:1 methanol:glacial acetic acid three times and dropped onto precleaned chilled slides. Chromosome spreads were Giemsa banded and photographed. At least 20 metaphase spreads and five banded karyotypes were evaluated for chromosomal karyotype and the presence of rearrangements. The in vitro differentiation potential of each of the four ES cell lines was determined by immunocytochemistry (Minger et al., 1996). Spontaneously differentiated cells were cultured in fourwell chamber slides (LabTek NUNC, Biotechline, Copenhagen, Denmark) coated with 0.1% gelatin (Sigma-Aldrich) for one week in feeder medium before being incubated with Hoechst (Molecular Probes, Invitrogen), fixed with 4% formaldehyde, and incubated with the primary antibodies. The ectodermal cells were determined by neurogenin 1 (diluted 1:200), nestin (diluted 1:100), and neuronal nuclei (diluted 1:100). The mesodermal cells were determined by osteopontin (diluted 1:60), smooth muscle actin (diluted 1:4000), and sarcomeric actin (diluted 1:2000). The endodermal cells were recognized by albumin (diluted 1:500) and alpha-fetoprotein (diluted 1:200). Cells originating from either the endodermal layer or the mesodermal layer were also detected by GATA-4 (diluted 1:700) and Nkx 2.5 (diluted 1:900). Using Cy5conjugated goat anti-mouse IgG and IgM, goat anti-rabbit IgG, and mouse anti-goat IgG (Chemicon; AH Diagnostics, Aarhus, Denmark) secondary antibodies, the presence of differentiation markers was determined using an Axiovert microscope equipped with a AxioCam camera and the image processing was done with the aid of AxioVision software package (Zeiss, Brock and Michelsen A/S, Copenhagen, Denmark).
Results Establishing human ES cell lines Within one year, 198 surplus day 2 or day 3 embryos were donated to stem cell research (Table 1). These were further cultured and 24 (12%) developed to the blastocyst stage. In this study only fresh surplus embryos were used. The kind of embryo that typically developed into the blastocyst stage was donated from couples who did not wish to have their surplus embryos cryopreserved. The blastocysts were not classified, as all were used irrespective of their quality. Using the immunosurgery procedure, 23 (∼12%) ICM were isolated. In the four (2%) cases in which ES cell lines were established, the isolated ICM seemed very compacted and had a size of 40–55 µm corresponding to the inner diameter of the blastomere biopsy pipette (Figure 1B). In one case, only lysed trophectoderm was isolated following immunosurgery. The isolated ICM were transferred to dishes containing 2.5 × 105 cell/ml inactivated human feeders and after 10–15 days colonies of cells with stem cell-like morphology developed (Figure 1C). In this experiment, it seemed important that the growth medium was supplemented with LIF. Without the addition of LIF, although the ICM attached to the feeders and in some cases outgrowth appeared within 10–14 days, these colonies stopped growing after the first mechanical passage. However, because of the low number of isolated ICM in this study, this needs to be examined further, before conclusions about the addition of LIF can be made.
Characterization of human ES cell lines The four human ES cell lines CLS1, CLS2, CLS3, and CLS4 were characterized in vitro at various times of culture, using previously defined criteria, by examining the morphology, the expression of cell surface markers (passages 9–14 and 20–27), the gene expression pattern (passages 9–14 and 18–20), the levels of telomerase activity (passages 9–14 and 18–24), the karyotype (passages 6–7 and 15–20), the recovery after freezing, and in vitro differentiation potential (passages 24–27). CLS1, CLS2, and CLS4 have been continuously cultured for 8 months (corresponding to 30 passages). CLS3 was not growing stably within the 1st month, but it has now proliferated continuously for 7 months, corresponding to 22 passages. The cells were mechanically dissected in clumps every seventh to 10th day and transferred to dishes containing new inactivated feeder cells. The clumps attached to the feeders within a day and ES cells started growing out from the clump. Within 7 or 10 days high density ES cell colonies developed containing both undifferentiated and spontaneously differentiated cells (Figure 1D). The spontaneously differentiated cells constituted 10–50% of the colonies. The cells within these multicellular colonies showed high ratios of nucleus to cytoplasm, characteristic of ES cells. Phenotypically undifferentiated colonies of ES cells were chosen for molecular and biochemical characterization. All four human ES cell lines were shown to express the cell-surface markers SSEA-4, TRA-1–60, and TRA-1–81, which are characteristic of undifferentiated human ES cells (Figure 2), the ES cellassociated mRNAs for OCT4, NANOG, SOX2, and FGF4 (Figure 3), and to display high levels of telomerase activity (Figure 4). In contrast, none of the human ES cells expressed the cell-surface marker SSEA-1, which is characteristic of differentiated ES cells (Figure 2). Karyotype analysis carried out before and after cryopreservation indicated a normal and stable karyotype in all cell lines: CLS1 46XY, CLS2 46XY, CLS3 46XX, and CLS4 46XY (Figure 5). All cell lines have been cryopreserved. After thawing, the cells kept dividing, showing that they survived freezing and thawing. Furthermore, all cell lines maintained a normal karyotype after thawing. The pluripotency of the human ES cell lines was examined in vitro. Spontaneously differentiated human ES cells were induced by culturing the cells for more than 10 days without passaging. The differentiated cells were mechanically dissociated and transferred to gelatinized slides. This method gave rise to a variety of cell types originating from all three germ layers. Derivatives of ectoderm were confirmed by positive immunoreactivity for neurogenin 1, nestin, and neuronal nuclei, mesodermal derivatives were confirmed by osteopontin, smooth muscle actin, sarcomeric actin, GATA-4, and Nkx 2.5 immunoreactivity and derivatives of endoderm were confirmed by albumin, alpha-fetoprotein, GATA-4, and Nkx 2.5 immunostaining (Figure 6).
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Table 1. Development of surplus embryos. No. of embryos donated
No. reaching the blastocyst stage (%)
No. of isolated ICM (%)
No. from which stem cell-like cells were isolated (%)
198
24 (12)
23 (~12)
4 (2)
Figure 1. Derivation of human embryonic stem cells. (A) Day 6 blastocyst. (B) Isolated inner cell mass by immunosurgery right after plating on the feeder layer. (C) Ten days after plating the inner cell mass on feeders. Colony of cells with stem cell-like morphology has developed. (D) Human embryonic stem cell culture day 7 after passaging. Cells growing in the centre are differentiated. Original magnifications: A: ×200, B: ×200, C: ×100, D: ×40.
Figure 2. Immunocytochemical staining of CLS4 determining the surface markers. (A) ( Stage-specific antigen (SSEA) 1. (B) ( SSEA-4. (C)) Tumuor-related antigen (TRA)-1–60. (D) ( TRA-1–81. (E)) Negative control for mouse immunoglobulin G. Original magnifications: ×100.
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Figure 3. Expression of gene markers for the human embryonic stem cell lines: CLS1 and CLS2 determined by real-time reverse transcription-polymerase chain reaction. Vertical axis: relative expression levels of the genes: expression level of the gene in relation to expression level of 18S. In parallel with each cell line, the gene level of 18S was determined. Horizontal axis: gene marker. Bars represent the mean of two experiments.
Figure 4. The telomerase activity of four human embryonic stem cell lines, determined by a polymerase chain reaction-based enzyme-linked immunosorbent assay. Vertical axis: absorbance value. Horizontal axis: cell type. Bars represent the difference between the absorbance of the sample and the absorbance of the negative control (sample treated with RNase). Positive control cell extract (immortalized telomerase expressing human kidney cells) was included in the kit.
Figure 5. The karyotype 46 XY of CLS1.
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Figure 6. Immunocytochemical staining of the human embryonic stem cell lines determining the in vitro differentiation potential. Derivatives of the ectoderm were confirmed by (A) neurogenin 1, (B) nestin, and (C) neuronal nuclei, derivatives of the mesoderm were confirmed by (D) osteopontin, (E) smooth muscle actin, (F) sarcomeric actin, (I) GATA-4, and (J) Nkx 2.5, and derivatives of the endoderm were confirmed by (G) albumin, (H) alpha-fetoprotein, (I) GATA-4, and (J) Nkx 2.5. (K) Mouse immunoglobulin IgG was negative control for nestin, neuronal nuclei, osteopontin, smoothe muscle actin, albumin, and sarcomeric actin. (L) Rabbit IgG was negative control for neurogenin 1 and GATA4. (M) Goat IgG was negative control for Nkx 2.5 and alpha-fetoprotein. Original magnifications: ×100.
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Discussion This study reports the derivation and characterization of four new human embryonic stem (ES) cell lines. On the basis of the characteristics described in this study, these four lines are believed to be the first reported human ES cell lines established in Denmark. Only fresh surplus human embryos from IVF for clinical purposes were used. Such spare embryos did not very often develop into blastocysts, as the best quality embryos were either implanted or cryopreserved. Only in cases where the couples did not want their good quality surplus embryos cryopreserved for further treatment and donated the remaining embryos to stem cell research, were good quality blastocysts obtained and stem cell lines established. The four human ES cell lines established in this study were characterized in vitro by criteria previously defined in different laboratories deriving human ES cells (Thomson et al., 1998; Reubinoff et al., 2000; Henderson et al., 2002; Hovatta et al., 2003; Pickering et al., 2003; Strelchenko et al., 2004; Verlinsky et al., 2005). Using immunocytochemistry, it was demonstrated that undifferentiated ES cells expressed the human ES cellspecific cell surface markers SSEA-4, TRA-1–60, and TRA-1– 81. Furthermore, it was demonstrated that the undifferentiated ES cells lack the expression of SSEA-1, which is only expressed on differentiated derivatives of human ES cells. In addition to cell-surface markers, real-time RT-PCR confirmed that the human ES cells expressed genes characteristic for human ES cells. These genes included OCT-4, the POU transcription factor, which is a marker for the pluripotency of the mouse ES cells (Nichols et al., 1998), NANOG, a homeobox-containing transcription factor, which maintains pluripotency in mouse ES cells (Chambers et al., 2003), SOX2, the HMG domain-containing transcription factor, essential for pluripotent development and maintenance in mice (Avilion et al., 2003), and FGF4, which is a target gene for SOX2 (Yuan et al., 1995). In addition, the ES cells expressed high levels of telomerase activity, had a normal chromosomal complementation, and generated undifferentiated human ES cell colonies subsequent to cryopreservation and thawing. The pluripotency of the ES cell lines were demonstrated in vitro by immunocytochemistry. Spontaneously differentiated ES cells expressed markers of different cells originating from the three germ layers, ectoderm, mesoderm, and endoderm. Although one method of demonstrating pluripotency of human ES cells is to implant undifferentiated colonies into immunocompromised mice, the data obtained from subsequent teratoma formation merely confirms the in vitro data (Thomson et al., 1998; Reubinoff et al., 2000; Heins et al., 2004; Inzunza, 2005). On the basis of the in vitro differentiation data demonstrating a number of different cell types derived from all three germ layers, as well as the cell gene expression and cell surface antigen expression patterns, the human ES cell lines described in this study fulfil all standard characteristics of human ES cell lines.
This study confirms that human ES cell lines can be established using commercially available human foreskin fibroblasts as feeder cells and KnockOut Serum Replacement, as described previously (Hovatta et al., 2003; Inzunza et al., 2005). However, in this culture system animal components are still present in terms of the feeder’s culture medium and the Serum Replacement. This is not suitable for cells aiming at transplantation. Furthermore, to obtain human ES cells for transplantation, good manufacturing practice (GMP) conditions are necessary. At the Laboratory for Stem Cell Research, Aalborg University, such conditions are available. It is intended to establish human ES cell lines free of animal compounds and using the GMP facilities for different kinds of transplantation purposes. To summarise, using immunosurgery, ICM were isolated from day 6 blastocysts and cultured on irradiated human foreskin fibroblasts as feeder cells. Well-defined colonies of ES celllike cells appeared after 10–15 days of isolation. These were mechanically dissociated and transferred to new irradiated feeder cells, and four human ES cell lines have been maintained in culture for more than 8 months. The cells express markers characteristic for pluripotent and undifferentiated ES cells, display high levels of telomerase activity, and maintain a stable karyotype over extended passaging. The ES cells were re-established after cryopreservation and their pluripotency was demonstrated by in vitro differentiation studies showing their potential to differentiate into cell types originating from ectodermal, mesodermal, and endodermal layers.
Acknowledgements We thank Mette Boegh Ringgaard and Ole Jensen for excellent technical assistance. None of the authors have financial or commercial interests.
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Article Derivation and characterization of four new human embryonic stem cell lines: the Danish experience Helle Lysdahl pursued her graduate work at Aarhus University, Denmark, where she was awarded her MSc in 1998 from the Faculty of Science and her PhD in 2002 from the Faculty of Health Sciences. Since 2003 she has been working as assistant professor at the Laboratory for Stem Cell Research, Aalborg University, Denmark. Her main research areas are establishing human embryonic stem cell lines and investigating different differentiation patterns of human embryonic stem cells.
Dr Helle Lysdahl Helle Lysdahl1,4, Anette Gabrielsen2, Stephen L Minger3, Minal J Patel3, Trine Fink1, Karsten Petersen2, Peter Ebbesen1, Vladimir Zachar1 1 Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg Oest; 2Ciconia, Aarhus Private Hospital, Saralyst Allé 50, 8270 Hoejbjerg, Denmark; 3Stem Cell Biology Laboratory, Wolfson Centre for AgeRelated Diseases, King’s College London, London SE1 1UL, UK 4 Correspondence: Tel: +45 9635 7548; Fax: +45 9635 7816; e-mail: [email protected]
Abstract In September 2003, legislation approved in Denmark legalized work on surplus human embryos from IVF for clinical purposes to establish human embryonic stem (ES) cell cultures. The aim of this study was to establish such stem cell lines. Fresh surplus embryos were donated after informed consent from the donors. Embryos were cultured into blastocysts and using the immunosurgery procedure, inner cell masses were isolated and cultured on irradiated human foreskin fibroblasts in KnockOut D-MEM supplemented with KnockOut Serum Replacement, bFGF, and LIF. Within a period of 12 months, 198 embryos were donated. Four isolated inner cell masses developed into putative ES cell lines, CLS1, CLS2, CLS3, CLS4, which have now been continuously cultured for eight months, corresponding to 30 passages. These cells expressed markers for undifferentiated human ES cells: stage-specific embryonic antigen-4, tumour-related antigen (TRA)-1–60, TRA-1–81, OCT4, NANOG, SOX2, and FGF4. The cells expressed high levels of telomerase activity, had a normal karyotype, and have been successfully cryopreserved and thawed. Finally, the cells displayed the potential to differentiate in vitro into cell types originating from all three germ layers. It is thought that the cell lines described in this study are the first human ES cells established in Denmark. Keywords: human embryonic stem cells, human feeders, LIF
Introduction Human embryonic stem (ES) cells are pluripotent stem cells, which can be established from the inner cell mass (ICM) of human blastocysts (Thomson et al., 1998; Reubinoff et al., 2000; Hovatta et al., 2003; Pickering et al., 2003). ES cells can be propagated indefinitely in vitro while retaining a normal karyotype and potential for differentiating into derivatives originating from the three embryonic germ layers. Human ES cells are promising candidates for cell-based treatment of disorders that involve loss of specific cell types, such as osteoarthritis, type I diabetes, Parkinson’s disease, and other degenerative diseases. In spite of the high potential
value in regenerative medicine, many basic questions remain unanswered, and more research is needed in order to make protocols using ES cell-based therapies a routine part of medical practice. In September 2003, legislation approved in Denmark legalized work on surplus human embryos from IVF for clinical purposes to establish human ES cell lines. With permission from the Regional Ethical Committee in Aarhus, the Laboratory for Stem Cell Research, Aalborg University and the Fertilization Clinic at Ciconia, Aarhus Private Hospital aimed at establishing such stem cell lines. The first established human ES cells were cultured on mitotically inactivated mouse embryonic fibroblasts as feeder
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Article - First human ES cells established in Denmark - H Lysdahl et al. cells (Thomson et al., 1998; Reubinoff et al., 2000). Recently, it was shown that human ES cells incorporate Neu5Gc, a nonhuman sialic acid, under conditions using mouse feeder layers and serum replacements (Martin et al., 2005). Hence, the use of non-human materials is not suitable for generating cells to be used for therapeutic purposes in humans. Improving the culture conditions for ES cells by avoiding animal products has been described in a number of publications. Instead of mouse feeder cells, human fetal muscle, fetal skin cells, adult skin fibroblasts, adult Fallopian tubal epithelial cells, adult marrow cells, placental fibroblasts, and foreskin fibroblasts have been successfully used (Richards et al., 2002, 2003; Cheng et al., 2003; Hovatta et al., 2003; Simón et al., 2005). Culturing human ES cells without feeder cells has been improved by coating the culture dishes with extracellular matrix, Matrigel, or laminin and using either conditioned media from the mouse feeders as culture media (Xu et al., 2001) or by coating with Matrigel and using a culture media containing noggin and high basic fibroblast growth factor (bFGF) concentration (Xu et al. 2005). This paper describes the successful establishment of four new human ES cell lines, using human feeder cells and Knockout Serum Replacement.
Materials and methods Establishing and culturing human ES cells Fresh surplus human embryos from IVF for clinical purposes were donated after informed consent and approval from the Regional Ethical Committee in Aarhus (journal number 20010164). The embryos were donated on either day 2 or day 3 after fertilization and cultured to the blastocyst stage in either ISM2 media (Medicult, Copenhagen, Denmark) or blastocyst media (Cook Denmark, Bjaeverskov, Denmark). The inner cell mass (ICM) was isolated by first removing the zona pellucida with a 10 mg/ml protease pronase E solution (Sigma-Aldrich, Copenhagen, Denmark) and then by removing the trophectoderm by immunosurgery using 20% rabbit antihuman whole serum and 20% guinea pig complement (SigmaAldrich) (Solter and Knowles, 1975). The lysed trophectoderm was removed using a blastomere biopsy pipette (Swemed Lab International AB, Billdal, Sweden) with an inner diameter of 40–55 µm.
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The isolated ICM were cultured on irradiated (35 Gy) human foreskin fibroblasts (American Type Culture Collection; LGC Promochem, Boras, Sweden) in 35-mm culture dishes (Corning Biotechline, Copenhagen, Denmark) in Knockout Dulbecco’s modified Eagle’s medium supplemented with 2 mmol/l Lglutamine (InVitrogen, Copenhagen, Denmark), 0.1 mmol/l β-mercaptoethanol, 1000 IU/ml leukaemia inhibitor factor (LIF) (Sigma-Aldrich), 4 ng/ml basic fibroblast growth factor (bFGF) (BioSource Nordic, Denmark), and 20% Knockout Serum Replacement (InVitrogen). The medium used in culture of the feeder cells before inactivation was Iscove’s Modified Dulbecco’s Medium (IMDM; (InVitrogen) supplemented with 10% fetal calf serum (FCS) (Biotech line, Copenhagen, Denmark). After 10–15 days compact, individually cell colonies appeared from the ICM derived outgrowths. These colonies were mechanically dissociated into small clumps using
a scalpel (Aesculap, B. Braun Medical, Copenhagen, Denmark) and replated on irradiated feeder cells in fresh medium. Every 7th to 9th day, individual colonies containing a uniform undifferentiated morphology were mechanically dissociated into clumps and replated.
Freezing ES cells Vitrification in VitriPlugs (Astro Med.Tec/Mobil-Tec Elektronik GmbH, NordicCell, Copenhagen, Denmark) closed with straws using 20% ethylene glycol, 20% dimethyl sulphoxide (DMSO), and 0.5 mol/l sucrose (Sigma-Aldrich) as cryoprotectants was performed as previously described (Reubinoff et al., 2001).
Characterization of ES cells Cell surface markers characteristic of undifferentiated human ES cells were detected using immunocytochemistry. The cells were incubated with Hoechst (Molecular Probes, Invitrogen) before fixation with 4% formaldehyde and incubation with antibodies against stage-specific embryonic antigen (SSEA)-4, SSEA-1, tumour-related antigen (TRA)-1–60, and TRA-1–81 (Santa Cruz; AH Diagnostics, Aarhus, Denmark). The SSEA were diluted 1:100 and the concentration of the TRA was 30 μg/ml. Using Cy5-conjugated goat anti-mouse immunoglobulin (IgG and IgM) secondary antibody (Chemicon; AH Diagnostics), the presence of surface markers was determined using an Axiovert microscope equipped with a AxioCam camera and the image processing was done with the aid of AxioVision software package (Zeiss, Brock and Michelsen A/S, Copenhagen, Denmark). The expression of genes characteristic for pluripotent human ES cells was determined using real-time reverse transcriptase– polymerase chain reaction (RT-PCR). The mRNA from the ES cells was isolated using the AURUM total RNA isolation kit (Biorad, Copenhagen, Denmark) according to the procedure of the manufacturer and synthesized to cDNA using the iScript cDNA synthesis kit (Biorad) according to the procedure of the manufacturer. Levels of the pluripotent gene markers were measured in iCycler (Bio Rad, Stockholm, Sweden) with octamer binding transcription factor-4 (OCT-4) primers 5′-TTCGCAAGCCCTCATTTCACC-3′ (upper) and 5′CCCCCTGGCCCATCACCTC-3′ (lower), NANOG primers 5′CAACTGGCCGAAGAATAGCAATGGTGT-3′ (upper) and 5′AGGAAGAGTAGAGGCTGGGGTAGGTAGGTG-3′ (lower), SOX2 primers 5′-GCAGGATCGGCCAGAGGAGGAGGGAA GC-3′ (upper) and 5′-GAGGGGAGGCGGGCGGGGGTGTC3′ (lower), and fibroblast growth factor 4 (FGF4) primers 5′AGAGCGGCGCCGGCGACTACCTG-3′ (upper) and 5′-AA GATGCTCACCACGCCCCGCTCCAC-3′ (lower). 18S rRNA was used as an internal endogenous standard. All primers were purchased from DNA-Technology, Aarhus, Denmark. The telomerase activity in the human ES cells was analysed using the Telo TAGGG telomerase PCR ELISA kit (Roche, Copenhagen, Denmark) according to the procedure of the manufacturer. The karyotypes of the human ES cells were determined using published protocols (Gosden et al., 1992). Briefly, the human ES cell colonies were incubated with 10 µg/ml colcemid solution (Gibco, Grand Island, New York, USA) for 2 h at 37°C in 5% CO2. The cells were washed with PBS, trypsinized, and neutralized
Article - First human ES cells established in Denmark - H Lysdahl et al. with human ES cell culture medium. Pellets were resuspended and incubated with 0.075 mol/l KCl for 17 min at 37°C. The cells were then fixed with 3:1 methanol:glacial acetic acid three times and dropped onto precleaned chilled slides. Chromosome spreads were Giemsa banded and photographed. At least 20 metaphase spreads and five banded karyotypes were evaluated for chromosomal karyotype and the presence of rearrangements. The in vitro differentiation potential of each of the four ES cell lines was determined by immunocytochemistry (Minger et al., 1996). Spontaneously differentiated cells were cultured in fourwell chamber slides (LabTek NUNC, Biotechline, Copenhagen, Denmark) coated with 0.1% gelatin (Sigma-Aldrich) for one week in feeder medium before being incubated with Hoechst (Molecular Probes, Invitrogen), fixed with 4% formaldehyde, and incubated with the primary antibodies. The ectodermal cells were determined by neurogenin 1 (diluted 1:200), nestin (diluted 1:100), and neuronal nuclei (diluted 1:100). The mesodermal cells were determined by osteopontin (diluted 1:60), smooth muscle actin (diluted 1:4000), and sarcomeric actin (diluted 1:2000). The endodermal cells were recognized by albumin (diluted 1:500) and alpha-fetoprotein (diluted 1:200). Cells originating from either the endodermal layer or the mesodermal layer were also detected by GATA-4 (diluted 1:700) and Nkx 2.5 (diluted 1:900). Using Cy5conjugated goat anti-mouse IgG and IgM, goat anti-rabbit IgG, and mouse anti-goat IgG (Chemicon; AH Diagnostics, Aarhus, Denmark) secondary antibodies, the presence of differentiation markers was determined using an Axiovert microscope equipped with a AxioCam camera and the image processing was done with the aid of AxioVision software package (Zeiss, Brock and Michelsen A/S, Copenhagen, Denmark).
Results Establishing human ES cell lines Within one year, 198 surplus day 2 or day 3 embryos were donated to stem cell research (Table 1). These were further cultured and 24 (12%) developed to the blastocyst stage. In this study only fresh surplus embryos were used. The kind of embryo that typically developed into the blastocyst stage was donated from couples who did not wish to have their surplus embryos cryopreserved. The blastocysts were not classified, as all were used irrespective of their quality. Using the immunosurgery procedure, 23 (∼12%) ICM were isolated. In the four (2%) cases in which ES cell lines were established, the isolated ICM seemed very compacted and had a size of 40–55 µm corresponding to the inner diameter of the blastomere biopsy pipette (Figure 1B). In one case, only lysed trophectoderm was isolated following immunosurgery. The isolated ICM were transferred to dishes containing 2.5 × 105 cell/ml inactivated human feeders and after 10–15 days colonies of cells with stem cell-like morphology developed (Figure 1C). In this experiment, it seemed important that the growth medium was supplemented with LIF. Without the addition of LIF, although the ICM attached to the feeders and in some cases outgrowth appeared within 10–14 days, these colonies stopped growing after the first mechanical passage. However, because of the low number of isolated ICM in this study, this needs to be examined further, before conclusions about the addition of LIF can be made.
Characterization of human ES cell lines The four human ES cell lines CLS1, CLS2, CLS3, and CLS4 were characterized in vitro at various times of culture, using previously defined criteria, by examining the morphology, the expression of cell surface markers (passages 9–14 and 20–27), the gene expression pattern (passages 9–14 and 18–20), the levels of telomerase activity (passages 9–14 and 18–24), the karyotype (passages 6–7 and 15–20), the recovery after freezing, and in vitro differentiation potential (passages 24–27). CLS1, CLS2, and CLS4 have been continuously cultured for 8 months (corresponding to 30 passages). CLS3 was not growing stably within the 1st month, but it has now proliferated continuously for 7 months, corresponding to 22 passages. The cells were mechanically dissected in clumps every seventh to 10th day and transferred to dishes containing new inactivated feeder cells. The clumps attached to the feeders within a day and ES cells started growing out from the clump. Within 7 or 10 days high density ES cell colonies developed containing both undifferentiated and spontaneously differentiated cells (Figure 1D). The spontaneously differentiated cells constituted 10–50% of the colonies. The cells within these multicellular colonies showed high ratios of nucleus to cytoplasm, characteristic of ES cells. Phenotypically undifferentiated colonies of ES cells were chosen for molecular and biochemical characterization. All four human ES cell lines were shown to express the cell-surface markers SSEA-4, TRA-1–60, and TRA-1–81, which are characteristic of undifferentiated human ES cells (Figure 2), the ES cellassociated mRNAs for OCT4, NANOG, SOX2, and FGF4 (Figure 3), and to display high levels of telomerase activity (Figure 4). In contrast, none of the human ES cells expressed the cell-surface marker SSEA-1, which is characteristic of differentiated ES cells (Figure 2). Karyotype analysis carried out before and after cryopreservation indicated a normal and stable karyotype in all cell lines: CLS1 46XY, CLS2 46XY, CLS3 46XX, and CLS4 46XY (Figure 5). All cell lines have been cryopreserved. After thawing, the cells kept dividing, showing that they survived freezing and thawing. Furthermore, all cell lines maintained a normal karyotype after thawing. The pluripotency of the human ES cell lines was examined in vitro. Spontaneously differentiated human ES cells were induced by culturing the cells for more than 10 days without passaging. The differentiated cells were mechanically dissociated and transferred to gelatinized slides. This method gave rise to a variety of cell types originating from all three germ layers. Derivatives of ectoderm were confirmed by positive immunoreactivity for neurogenin 1, nestin, and neuronal nuclei, mesodermal derivatives were confirmed by osteopontin, smooth muscle actin, sarcomeric actin, GATA-4, and Nkx 2.5 immunoreactivity and derivatives of endoderm were confirmed by albumin, alpha-fetoprotein, GATA-4, and Nkx 2.5 immunostaining (Figure 6).
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Table 1. Development of surplus embryos. No. of embryos donated
No. reaching the blastocyst stage (%)
No. of isolated ICM (%)
No. from which stem cell-like cells were isolated (%)
198
24 (12)
23 (~12)
4 (2)
Figure 1. Derivation of human embryonic stem cells. (A) Day 6 blastocyst. (B) Isolated inner cell mass by immunosurgery right after plating on the feeder layer. (C) Ten days after plating the inner cell mass on feeders. Colony of cells with stem cell-like morphology has developed. (D) Human embryonic stem cell culture day 7 after passaging. Cells growing in the centre are differentiated. Original magnifications: A: ×200, B: ×200, C: ×100, D: ×40.
Figure 2. Immunocytochemical staining of CLS4 determining the surface markers. (A) ( Stage-specific antigen (SSEA) 1. (B) ( SSEA-4. (C)) Tumuor-related antigen (TRA)-1–60. (D) ( TRA-1–81. (E)) Negative control for mouse immunoglobulin G. Original magnifications: ×100.
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Figure 3. Expression of gene markers for the human embryonic stem cell lines: CLS1 and CLS2 determined by real-time reverse transcription-polymerase chain reaction. Vertical axis: relative expression levels of the genes: expression level of the gene in relation to expression level of 18S. In parallel with each cell line, the gene level of 18S was determined. Horizontal axis: gene marker. Bars represent the mean of two experiments.
Figure 4. The telomerase activity of four human embryonic stem cell lines, determined by a polymerase chain reaction-based enzyme-linked immunosorbent assay. Vertical axis: absorbance value. Horizontal axis: cell type. Bars represent the difference between the absorbance of the sample and the absorbance of the negative control (sample treated with RNase). Positive control cell extract (immortalized telomerase expressing human kidney cells) was included in the kit.
Figure 5. The karyotype 46 XY of CLS1.
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Figure 6. Immunocytochemical staining of the human embryonic stem cell lines determining the in vitro differentiation potential. Derivatives of the ectoderm were confirmed by (A) neurogenin 1, (B) nestin, and (C) neuronal nuclei, derivatives of the mesoderm were confirmed by (D) osteopontin, (E) smooth muscle actin, (F) sarcomeric actin, (I) GATA-4, and (J) Nkx 2.5, and derivatives of the endoderm were confirmed by (G) albumin, (H) alpha-fetoprotein, (I) GATA-4, and (J) Nkx 2.5. (K) Mouse immunoglobulin IgG was negative control for nestin, neuronal nuclei, osteopontin, smoothe muscle actin, albumin, and sarcomeric actin. (L) Rabbit IgG was negative control for neurogenin 1 and GATA4. (M) Goat IgG was negative control for Nkx 2.5 and alpha-fetoprotein. Original magnifications: ×100.
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Discussion This study reports the derivation and characterization of four new human embryonic stem (ES) cell lines. On the basis of the characteristics described in this study, these four lines are believed to be the first reported human ES cell lines established in Denmark. Only fresh surplus human embryos from IVF for clinical purposes were used. Such spare embryos did not very often develop into blastocysts, as the best quality embryos were either implanted or cryopreserved. Only in cases where the couples did not want their good quality surplus embryos cryopreserved for further treatment and donated the remaining embryos to stem cell research, were good quality blastocysts obtained and stem cell lines established. The four human ES cell lines established in this study were characterized in vitro by criteria previously defined in different laboratories deriving human ES cells (Thomson et al., 1998; Reubinoff et al., 2000; Henderson et al., 2002; Hovatta et al., 2003; Pickering et al., 2003; Strelchenko et al., 2004; Verlinsky et al., 2005). Using immunocytochemistry, it was demonstrated that undifferentiated ES cells expressed the human ES cellspecific cell surface markers SSEA-4, TRA-1–60, and TRA-1– 81. Furthermore, it was demonstrated that the undifferentiated ES cells lack the expression of SSEA-1, which is only expressed on differentiated derivatives of human ES cells. In addition to cell-surface markers, real-time RT-PCR confirmed that the human ES cells expressed genes characteristic for human ES cells. These genes included OCT-4, the POU transcription factor, which is a marker for the pluripotency of the mouse ES cells (Nichols et al., 1998), NANOG, a homeobox-containing transcription factor, which maintains pluripotency in mouse ES cells (Chambers et al., 2003), SOX2, the HMG domain-containing transcription factor, essential for pluripotent development and maintenance in mice (Avilion et al., 2003), and FGF4, which is a target gene for SOX2 (Yuan et al., 1995). In addition, the ES cells expressed high levels of telomerase activity, had a normal chromosomal complementation, and generated undifferentiated human ES cell colonies subsequent to cryopreservation and thawing. The pluripotency of the ES cell lines were demonstrated in vitro by immunocytochemistry. Spontaneously differentiated ES cells expressed markers of different cells originating from the three germ layers, ectoderm, mesoderm, and endoderm. Although one method of demonstrating pluripotency of human ES cells is to implant undifferentiated colonies into immunocompromised mice, the data obtained from subsequent teratoma formation merely confirms the in vitro data (Thomson et al., 1998; Reubinoff et al., 2000; Heins et al., 2004; Inzunza, 2005). On the basis of the in vitro differentiation data demonstrating a number of different cell types derived from all three germ layers, as well as the cell gene expression and cell surface antigen expression patterns, the human ES cell lines described in this study fulfil all standard characteristics of human ES cell lines.
This study confirms that human ES cell lines can be established using commercially available human foreskin fibroblasts as feeder cells and KnockOut Serum Replacement, as described previously (Hovatta et al., 2003; Inzunza et al., 2005). However, in this culture system animal components are still present in terms of the feeder’s culture medium and the Serum Replacement. This is not suitable for cells aiming at transplantation. Furthermore, to obtain human ES cells for transplantation, good manufacturing practice (GMP) conditions are necessary. At the Laboratory for Stem Cell Research, Aalborg University, such conditions are available. It is intended to establish human ES cell lines free of animal compounds and using the GMP facilities for different kinds of transplantation purposes. To summarise, using immunosurgery, ICM were isolated from day 6 blastocysts and cultured on irradiated human foreskin fibroblasts as feeder cells. Well-defined colonies of ES celllike cells appeared after 10–15 days of isolation. These were mechanically dissociated and transferred to new irradiated feeder cells, and four human ES cell lines have been maintained in culture for more than 8 months. The cells express markers characteristic for pluripotent and undifferentiated ES cells, display high levels of telomerase activity, and maintain a stable karyotype over extended passaging. The ES cells were re-established after cryopreservation and their pluripotency was demonstrated by in vitro differentiation studies showing their potential to differentiate into cell types originating from ectodermal, mesodermal, and endodermal layers.
Acknowledgements We thank Mette Boegh Ringgaard and Ole Jensen for excellent technical assistance. None of the authors have financial or commercial interests.
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