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a Cellartis AB, Göteborg, Sweden;
b Department of Medical Biochemistry, Göteborg University, Göteborg, Sweden;
c Department of Obstetrics and Gynaecology, Sahlgrenska University Hospital, Göteborg, Sweden;
d Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden
Key Words. Human blastocyst • ICM • Human ES cell • Pluripotency • Subcloning • Differentiation
Henrik Semb, Ph.D., Section of Endocrinology, Lund University, Klinikgatan 26, BMC, B10, SE-22184, Lund, Sweden. Telephone: 46-2223159; Fax: 46-2223600; e-mail: henrik.semb{at}endo.mas.lu.se
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ABSTRACT |
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INTRODUCTION |
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Due to the limited number of available hES cell lines, there is an urgent need for the generation and characterization of more cell lines, as each line may have its own characteristics and advantages for different applications. Furthermore, the availability of more hES cell lines for comparison will aid in defining criteria for bona fide hES cells and the establishment of appropriate and robust methods for maintenance and expansion of hES cells.
Here, we describe the successful establishment of hES cell lines from the ICM by immunosurgery, from spontaneously hatched blastocysts, and from blastocysts after pronase-mediated removal of the zona pellucida. Three of the hES cell lines have been maintained in culture for more than 1 year, during which time high levels of telomerase activity, stable karyotype, and expression of markers characteristic for undifferentiated hES cells were maintained. The cells could be cryopreserved by vitrification without any effect on their ability to re-establish pluripotent hES cell colonies. The pluripotent qualities of these cell lines were demonstrated in several ways. Most importantly, the cells were able to differentiate into cell types originating from each of the three embryonic germ layers (endoderm, mesoderm, and ectoderm) in vitro as well as in vivo. In addition, we subcloned one of our cell lines and showed that it could be propagated in an undifferentiated state while maintaining its pluripotency both in vitro and in vivo, as was previously shown for other hES cell lines [5].
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MATERIALS AND METHODS |
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Blastocysts with intact zona were treated in pronase for 1–3 minutes (Sigma-Aldrich:10 U/ml in ICM-2 [Vitrolife AB] 1–3 minutes in three subsequent drops), washed three times in ICM-2 and plated on MEFs in hES medium supplemented with 125 mg/ml HA and 4 ng/ml hrbFGF. ICM-2 is a blastocyst culture medium containing recombinant human albumin and HA. Pronase-treated zona-free blastocysts selected for immunosurgery were washed twice in antihuman serum antibody (Sigma-Aldrich, 1:5 in ICM-2). After the second wash the blastocysts were placed in a new drop of the antibody and incubated for 30 minutes. The blastocysts were then extensively washed three times in ICM-2 medium followed by three washes in guinea-pig complement serum (Invitrogen, 1:5 in ICM-2). The blastocysts were then incubated for 10 minutes in guinea-pig complement serum and followed by three washes in ICM-2 and placed on MEFs in VitroHES supplemented with 125 µg/ml HA and 4 ng/ml hrbFGF.
ICM outgrowths were passaged to plates with fresh medium and MEF cells by mechanical dissection using Stem Cell ToolTM (Swemed Lab International AB; Billdal, Sweden). Established hES cell lines were routinely passaged every 4–5 days. The hES cell colonies were mechanically cut into pieces, 200 x 200 µm, and removed from the culture dish and transferred to a new culture dish with fresh MEF cells and VitroHES supplemented with 4 ng/ml hrbFGF.
Telomerase Activity
It has been demonstrated that high telomerase activity in ES cells correlates with their ability to divide indefinitely in culture [10]. Degradation of telomeric sequences or end-to-end fusion of chromosomes can lead to genomic instability [11]. To analyze telomerase activity in hES cells a polymerase chain reaction-based enzyme-linked immunosorbent assay was used (Roche Diagnostics GmbH; Mannheim, Germany; http://www.roche.com). The cells were harvested and lysed according to the manufacturer’s instructions.
Karyotyping and FISH
hES cells designated for karyotyping were cultured in hES medium supplemented with 0.1 µg/ml colcemid (Invitrogen) for 1–3 hours. The cells were subsequently trypsinized, fixed, and mounted on glass slides. The chromosomes were visualized by using modified Wright’s staining. For fluorescence in situ hybridization (FISH) analysis, a commercially available kit containing probes for chromosome 13, 18, and 21 and the sex chromosomes (X and Y) was used (MultiVysionTM PB Multicolour Probe Panel; Vysis, Inc.; Downers Grove, IL; http://www.vysis.com) according to the manufacturer’s instructions. For each cell line at least 200 nuclei were analyzed. The slides were analyzed in a fluorescence microscope equipped with appropriate filters and software (CytoVision; Applied Imaging; Santa Clara, CA; http://www.appliedimagingcorp.com).
In Vitro Differentiation
The cells were kept on MEFs without passaging up to 14 days. Medium (hES medium without hrbFGF) was changed every second day. Alternatively, incubating clumps of hES cells in VitroHES without hrbFGF in suspension culture for 4–9 days generated both simple and cystic embryoid bodies (EBs).
Xenografting of hES Cells
Severe combined immunodeficient (SCID) mice, [12] (C.B-17/lcrCrl-scidBR; Charles River Laboratories) were used as animal hosts for the xenografted hES cells. Four- to five-week-old animals were anesthetized with intraperitoneal injections of ketamine hydrochloride (Ketalar; Warner Lambert Nordic AB; Solna, Sweden; http://www.warner-lambert.com; 75 µg/g mouse) and medetomidine hydrochloride (Domitor; Orion Pharma Corporation; Espoo, Finland; http://www.orionpharma.com; 1 µg/g mouse). hES cell colonies were mechanically cut into 200 x 200-µm pieces, washed once in phosphate-buffered saline (PBS) containing 4 mg/ml human serum albumin and penicillin (Cryo-PBS; Vitrolife AB), and 20 cell clusters were injected under the kidney capsule or in the testicular lumen using a 200-µm lumen glass transfer pipette (Swemed Lab International AB). The number of cells transferred was approximately 20,000 to 40,000 per organ. Control animals were either injected with Cryo-PBS or grafted with primary brain cells from a littermate. The mice were resuscitated with intraperitoneal injections of atipamezol (Antisedan; Orion Pharma; 1 µg/g mouse), and kept on a heated pad until consciousness. Palpable tumors started to appear 3 weeks after transplantation. The tumors were allowed to develop for 8 weeks before the animals were sacrificed by cervical dislocation. All animals appeared healthy during the 8-week period, and no animal died due to illness. The tumors were excised and immediately fixed in 4% paraformaldehyde, incubated for 24 hours, washed or stored in 70% ethanol, and processed in a Tissue-Tek paraffin infiltrator (Sakura Fine Technical; Tokyo, Japan; http://www.sakuraus.com), and paraffin-embedded. The tumors were subsequently sectioned in 6–8 µm sections with a Microm HM 360 (Microm GmbH; Walldorf, Germany). Samples were stained for morphology using hematoxylin and eosin with a Sakura DRS-601 stainer. To determine the origin (human, mouse) and tissue type within teratomas, we combined histopathological and marker expression analysis. To evaluate whether the tissues were of human origin we used several human-specific antibodies, e.g., anti-E-cadherin and anti-human nuclei (see below). To confirm the presence of tissues derived from all three germ layers, we focused on tissues that can be easily distinguished by histopathological methods, e.g., neuroectoderm, cartilage, kidney tubuli, and gut-like epithelium. To strengthen these conclusions, we also used antibodies against markers characteristic for derivatives of the germ layers, e.g., 
-smooth muscle actin, desmin, nestin, ß-III-tubulin, -fetoprotein, and HNF3ß.
Subcloning
The cells were washed inside the wells twice with cloning medium, 150 µl, 500 µl, and 1,000 µl for the 96-, 48-, 24-well plates, respectively. The inner part of the colonies was cut with a 300-µm Stem Cell ToolTM and subsequently incubated with 0.5 mM EDTA for 20 minutes at 37°C. The cells were triturated carefully with a pipette and diluted either in knockout (KO)-DMEM medium (GIBCO) supplemented with 15% concentrated conditioned medium, 3.5 mM glucose, 1 mM Glutamax (invitrogen-sigma-albumin), 1% NEAA (GIBCO), and 4 ng/ml bFGF, KO-DMEM medium supplemented with 15% FCS, 3.5 mM glucose, 1 mM Glutamax, 1% NEAA, and 4 ng/ml bFGF, or KO-DMEM medium supplemented with 20% serum replacement (GIBCO) SR, 3.5 mM glucose, 1 mM Glutamax, 1% NEAA, and 4 ng/ml bFGF. Single cells were picked and put into individual wells with MEF-coated plates. To confirm the colony-forming ability of the cells, positive controls were performed (e.g., 10, 100, 1,000 cells/ml or smaller clusters) as well as negative controls (wells without dissociated hES cells). Subclones were preferentially obtained in 15% concentrated conditioned medium of hES cells grown in presence of FCS.
Histochemical Staining for Alkaline Phosphatase
Histological staining for alkaline phosphatase was carried out using a commercially available kit (Sigma-Aldrich) following the manufacturer’s instructions.
Immunocytochemistry
The cells were fixed in 4 % paraformaldehyde for 15 minutes at room temperature, washed in PBS, and exposed to the primary antibodies overnight at 4°C. As secondary antibodies, we used fluorescein isothiocyanate (FITC)- and Cy-3-conjugated antibodies (1:50, Southern Biotech; Birmingham, AL; http://www.southernbiotech.com). The monoclonal antibodies (mAb) directed against SSEA-1, SSEA-3, and SSEA-4 (Developmental Studies Hybridoma Bank, The University of Iowa; Iowa City, IA) were used at 1:200, whereas the TRA-1-60 and TRA-1-81 mAbs (Santa Cruz Biotechnology; Santa Cruz, CA; http://www.southernbiotech.com) were used at 1:500. Neuroectodermal precursor cells and neurons were detected by a nestin mAb (BD Biosciences; Stockholm, Sweden; 1:100), and ß-tubulin-III mAb (Sigma-Aldrich; 1:100), respectively. Endodermal cells were recognized by a mAb against Cdx2 (gut endoderm, visceral endoderm) (BioGenex, Nordic BioSite; Täby, Sweden; 1:200; http://www.innogenex.com), and polyclonal antibodies against -1-fetoprotein ([AFP] Sigma-Aldrich; 1:2000) and HNF3ß (Santa Cruz Biotechnology; 1:500). Mesodermal cells were detected by a desmin antibody (Chemicon; Temecula, CA; http://www.chemicon.com; 1:200). hES cell-derived cells were detected by mAbs against human nuclei (Chemicon; 1:100) and human E-cadherin (Zymed Laboratories; South San Francisco, CA; http://www.zymed.com; 1:500). Some cultures were double stained with DAPI (4'-6'Diamidino-2-phenylindole, final concentration 0.1 µg/ml, Sigma-Aldrich) for 5 minutes.
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RESULTS |
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). Due to the limited number of embryos used in this study, we can, however, not conclude anything about the relative efficiencies of the different methods. Whereas cell line SA002 was derived from a spontaneously hatched blastocyst, cell lines FC018, AS034, and AS038 were established from pronase-treated blastocysts (Table 1). Immunosurgery was used for establishing cell lines SA121 and SA181. One to two weeks after plating, the expanded ICM was transferred to a fresh MEF-coated IVF-cell culture dish by mechanical dissection. Successful propagation of the ICM was associated with the appearance of ES-like cells in the outgrowth, whereas differentiated cells, presumably representing primitive endoderm and trophectoderm, either died or disappeared upon repeated passaging (Fig. 1).
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In the presence of mouse feeders and human recombinant bFGF, all six hES cell lines gave rise to large compact multicellular colonies of cells with the characteristic hES cell morphology, i.e., a high ratio of nucleus to cytoplasm and prominent nucleoli. Some of these lines have been passaged more than 120 times. Initially, the different cell lines could not be discriminated morphologically from each other except for cell line AS038, which never developed a clear distinguishable border towards the mouse feeders (data not shown). However, with time the morphology of AS038 became indistinguishable from the other cell lines.
Karyotype analyses carried out at different passages (from passage three to passage 76) indicated a normal stable karyotype in four of the cell lines (Fig. 2A, Table 1
). In two of the cell lines chromosomal aberrations were apparent, trisomy 13 in cell line SA002 (Fig. 2B), and triploid karyotype in FC018 (Table 1).
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, Table 1). The overall percentage of visible AP-positive cells within a colony varied from 60% to 90% in all cell lines. The hES cells lines were further characterized by expression analysis of five cell surface markers: SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, and the intermediate filament protein nestin [1, 14–17]. Figure 3 depicts examples of the expression of these markers in undifferentiated colonies from cell line SA002. The results of the expression analysis are summarized in Table 1. Whereas all cell lines expressed SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, they were, with a few exceptions (see below), negative for nestin and SSEA-1.
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, Table 1), whereas Oct-4 was downregulated concomitant with differentiation (data not shown).
Nestin is expressed in a variety of stem/precursor cell populations of neuroectodermal and mesodermal origin, and we found it useful for detecting differentiated hES cells that could not be recognized by morphological criteria. Generally, nestin-positive colonies often appeared during suboptimal growth conditions and seemed to result in irreversible commitment. Interestingly, whereas TRA-1-60 and TRA-1-81 were initially expressed normally in cell lines FC018 (passage 25) and AS038 (passage 41) (Fig. 4C and 4D, Table 1), a patchy expression pattern of SSEA-3 and SSEA-4 was observed (Fig. 4F, Table 1). Moreover, additional signs of cell differentiation were the appearance of SSEA-1- and nestin-expressing cells (Fig. 4B and 4E, Table 1). However, with time both line FC018 (passage 118) and AS038 (passage 72) expressed SSEA-4 and Oct-4 uniformly within the colonies (Fig. 4G and 4I), whereas nestin was no longer expressed (Fig. 4H).
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). In summary, three of the six characterized hES cell lines, AS034, SA121, and SA181, exhibited the morphology, genotype, telomerase activity, and marker expression characteristics for previously reported pluripotent stem cell lines with a normal karyotype [1, 2, 13].
Analysis of In Vitro Pluripotency of hES Cell Lines
Similar to mouse ES cells, hES cells spontaneously form three-dimensional aggregates of differentiated cells known as EBs when grown in suspension. Upon continued in vitro culture of EBs, a variety of ectodermal, endodermal, and mesodermal germ layer derivatives, such as hematopoietic, endothelial, cardiac, skeletal muscle, and neuronal cell lineages appear [21]. We could show that all hES cell lines are capable of generating both simple and cystic EBs. Marker expression analysis and morphological examination of plated EBs revealed derivatives of all three germ layers, including areas of beating heart muscle-like cells (data not shown). However, EB formation is not an exclusive pathway for initiating hES cell differentiation. An alternative efficient and timesaving method to induce spontaneous differentiation of hES cells is simply by keeping the colonies on mouse feeders for more than 7 days without passaging. Similar to EB formation, this method gives rise to a variety of cell types derived from all three germ layers. The vast majority of cells within the differentiated colonies expressed neuroectodermal cell markers, such as nestin and ß-III-tubulin (Fig. 5A and 5B). These markers were preferentially expressed within typical rosette-like structures during early stages of differentiation (data not shown). Derivatives of mesoderm were confirmed by desmin stainings, and the appearance of synchronously beating cardiomyocyte-like cells (Fig. 5C, data not shown). Endodermal derivatives appeared later during differentiation in the periphery of the colonies and were identified by the expression of AFP, Cdx2, and HNF3ß (Fig. 5D–5F). In summary, based on in vitro differentiation, all cell lines displayed the potential to form derivatives of all three embryonic germ layers. Importantly, these characteristics remained the same after repeated freezing-thawing cycles (data not shown).
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). Importantly, SSEA-1 and nestin were not detected in undifferentiated colonies (Fig. 6B, data not shown). Furthermore, the subclone was capable of differentiating into ectodermal, mesodermal, and endodermal cell types both in vitro and in vivo (Table 1).
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, Table 1, data not shown). However, two of the cell lines (FC018, AS038) consistently formed fluid-filled cyst-like structures composed of hES cell-derived connective tissue and epithelial cells (Fig. 7B, Table 1). Infrequently, these cysts contained small solid teratomas consisting of cell types derived from several germ layers (data not shown). Thus, in summary, whereas all cell lines, including lines with an abnormal karyotype, exhibited pluripotent differentiation qualities in vitro, pluripotency in vivo was only consistently observed in lines SA002, AS034, AS034.1, SA121, and SA181
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DISCUSSION |
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In addition to the previously reported immunosurgery protocol [1, 23], we report the successful derivation of hES cell lines from a spontaneously hatched blastocyst, and from blastocysts after enzymatic removal of the zona pellucida by pronase. A possible drawback of only removing the zona is overgrowth by the trophectoderm and the possible generation of trophectoderm stem cell lines. However, this should also be taken into account when using immunosurgery since this method does not guarantee complete removal of trophectoderm cells. Nevertheless, due to the morphology of trophoectoderm cells, i.e., flattened and polarized as they mature [24], they can be discriminated from the ICM once plated on MEFs. The fact that all of our cell lines expressed Oct-4, which normally is downregulated during differentiation and in trophectoderm stem cells [18, 19], substantiates our conclusion that the derived pluripotent stem cells were derived from the ICM.
Although four of our cell lines appeared phenotypically similar to previously derived hES cell lines [1, 2, 13], two of the cell lines (FC018 and AS038) were phenotypically different. Instead of generating solid teratomas upon xenografting, they developed into cyst-like structures. In contrast, when differentiated in vitro these lines generated cell types from all three germ layers. Except for their morphology in culture, which was similar to the "normal" hES cell lines, FC018 and AS038 also exhibited aberrant expression patterns of some of the markers characteristic for undifferentiated and differentiated hES cells, respectively. However, with time, the expression of these markers became reminiscent of normal hES cells. Whether the temporal shift in marker expression pattern in these cell lines can be explained by an initial mixed population of cells which went through selection, or whether cells were more prone to spontaneously differentiate initially, is presently unclear.
Our results indicate that adding early differentiation markers, such as nestin, to the list of markers whose expression patterns are analyzed in undifferentiated hES cells improves the detection of early hES cell differentiation. We show that the expression of nestin, a marker for stem/precursor cells of neuroectodermal and mesodermal origin, precede visible morphological signs of differentiation. We also found that the frequent spontaneous commitment to the neuroectodermal pathway of cell lines FC018 and AS038 correlate with loss of developmental potential to form derivatives of the three germ layers in vivo.
By definition, clonal expansion of hES cells is a prerequisite for the strict definition of pluripotent cell lines. Currently, the culture conditions for clonal expansion of hES cells are suboptimal. Unlike mouse ES cells, hES cells die at a high rate when they are dissociated into single cells [5]. Depending on which cell line was used, only 0.1%-1% of plated single cells was able to generate colonies that could be propagated. Among the few clones that survived, the majority were lost due to irreversible differentiation. We found that concentrated conditioned medium from hES cells grown in the presence of FCS promoted cell survival and maintenance of an undifferentiated fate. In general, our experience is that culture conditions that may be rate limiting for maintaining undifferentiated growth of hES cells include MEF quality and density, changes in the osmolarity, pH, and temperature of the medium, as well as the presence of supplements, such as ß-mercaptoethanol.
The stable maintenance of diploid chromosome number in the majority of our cell lines indicates that our cell lines maintained a stable karyotype in vitro after extensive passaging and repeated freezing/thawing cycles. The fact that two of the stem cell lines were chromosomally abnormal is not surprising taking into account that Hardarson et al. [25] recently found that only 42% of surplus IVF embryos were chromosomally normal at the blastocyst stage.
Consistent with previous reports, xenografting of most of our cell lines (SA002, AS034, AS034.1, SA118, and SA121) generated solid teratomas consisting of endodermal, mesodermal, and ectodermal cell type derivatives [1, 2, 5]. However, some of the cell lines (FC018, AS038) preferentially generated fluid-filled cyst-like structures of human origin. We have yet to determine the cellular identity and origin of these cysts. Potentially, these cells may be of trophoectoderm origin, since it was recently shown that hES cells are capable of differentiating into extraembryonic derivatives, such as trophoblast cells [26]. Alternatively, FC018 and AS038 may represent primitive endoderm lineages. However, the facts that undifferentiated colonies of these cell lines expressed significant levels of Oct-4, and that they lacked expression of primitive endoderm markers, such as Gata4 and AFP [27, 28], suggest that it is unlikely that they are of extraembryonic or primitive endoderm origin (data not shown).
Needless to say, more knowledge is needed about basic hES cell biology, such as the regulatory pathways that govern self-renewal and differentiation, before it will be possible to appreciate the potential applications of hES cells in basic science and cell-replacement therapy.
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ACKNOWLEDGMENT |
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FOOTNOTES |
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* These authors contributed equally. 
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REFERENCES |
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