Derivation, Characterization, and Differentiation of Human Embryonic Stem Cells
http://www.100md.com
《干细胞学杂志》
a Cellartis AB, G?teborg, Sweden;
b Department of Medical Biochemistry, G?teborg University, G?teborg, Sweden;
c Department of Obstetrics and Gynaecology, Sahlgrenska University Hospital, G?teborg, Sweden;
d Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, G?teborg, 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@endo.mas.lu.se
ABSTRACT
The inner cell mass (ICM) of the preimplantation blastocyst contains a core of cells, termed the epiblast, that have the potential to generate somatic and germ cells of the embryo. It has previously been demonstrated that human embryonic stem (hES) cell lines, exhibiting a stable developmental potential to form derivatives of the three germ layers after prolonged culture in vitro, can be generated by the isolation and culturing of the human ICM . So far, the available knowledge of conditions for deriving, characterizing, and culturing undifferentiated hES cells is largely based on a relatively few successfully isolated cell lines. Furthermore, the underlying mechanisms that control the developmental decisions of hES cells in culture remain essentially unknown. It has been suggested that the poor success rates in developing ES cells in species other than mice is either due to fundamental biological differences between the species or simply due to technical factors associated with the derivation and culture conditions . For example, unless spontaneous differentiation of hES cells is prevented, the cells become gradually restricted and lose the characteristics of ES cells. Frequent passaging and optimization of the quality of the culture conditions and feeder cells may overcome this problem. However, many problems remain with regard to the optimal maintenance and expansion of hES cells.
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 .
MATERIALS AND METHODS
Methods for Deriving hES Cell Lines
The most commonly used method for deriving hES cell lines is by immunosurgical isolation of the ICM from the human blastocyst . Here, we show that hES cell lines can be established from pronase-treated and hatched blastocysts as well (Fig. 1). 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).
Figure 1. Human ES cell derivation from pronase-treated and hatched blastocysts. A) Blastocyst before pronase treatment. B) Outgrowth of pronase-treated blastocyst shown in (A) 6 days after pronase treatment. C) hES colony (passage 6) derived from the ICM in (B). D) Spontaneously hatched blastocyst. E) Outgrowth of the blastocyst shown in (D) 5 days after plating. F) hES colony (passage 3) derived from the ICM in (E).
Table 1. Summary of hES cell characterization
Characterization of hES Cell Lines
To analyze the long-term pluripotency and replicative immortality of the six newly established hES cell lines SA002, FC018, AS034, AS038, SA121, SA181, and subclone AS034.1 (see below), we used previously defined criteria for in vitro and in vivo characterization of hES cells by examining the morphology, marker expression, telomerase activity, karyotype, and pluripotency in vitro and in vivo.
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).
Figure 2. Karyotype of hES lines (see also Table 1). A) Normal 46XY karyotype from cell line SA181. B) XX karyotype from line SA002 with a trisomy 13. The arrow in (B) points to the trisomic chromosome 13.
Similar to undifferentiated pluripotent cultures of human germ cells and previously established hES cell lines, all our cell lines possessed high levels of alkaline phosphatase (AP) activity (Fig. 3B, 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 . 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.
Figure 3. Immunohistochemical marker expression analysis of cell line SA002 (passage 21). The colonies were analyzed 5 days after passaging. A) Morphology, (B) Alkaline Phosphatase (AP), (C) SSEA-1, (D) SSEA-3, (E) SSEA-4, (F) TRA-1-81, (G) TRA-1-60, and (H) Oct-4. Scale bar: 25 μm.
Oct-4 is a POU-domain transcription factor that is essential for establishment of ES cells from the ICM . Similar to previous reports , we found that undifferentiated hES cells expressed Oct-4 (Fig. 3H, 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).
Figure 4. Immunohistochemical marker expression analysis of cell line FC018. The colonies were analyzed 5 days after passaging. A-F: passage 25, G-I: passage 118. (A) Morphology, (B) SSEA-1, (C) TRA-1-60, (D) TRA-1-81, (E, H) nestin, (F, G) SSEA-4, and (I) Oct-4. Scale bar: 25 μm.
Finally, all six hES cell lines expressed high levels of telomerase activity that were maintained even after more than 50 passages (Table 1).
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 .
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 . 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 ?-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 HNF3? (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).
Figure 5. In vitro differentiation of cell line SA002. The colonies were analyzed after 12 days after passaging. A) Nestin-positive neuronal precursors, B) ?-III-tubulin-positive postmitotic neurons, C) desmin-positive mesodermal cells, D, E, F) -fetoprotein-positive (D), HNF3?-positive (E), and Cdx2-positive (F) endodermal cells. Scale bar: 25 μm.
Since none of the described hES cell lines were clonally derived, it cannot be excluded that multiple precursor or stem cells committed to different lineages may coexist within a population of homogeneously appearing cells. Theoretically, this would imply that, until proven, it cannot be stated that a single hES cell is capable of forming derivatives of all three embryonic germ layers. In our initial efforts to subclone cell line AS034, we obtained one clone, AS034.1. To promote cell survival, we used concentrated conditioned medium from hES cells grown in presence of FCS as cloning medium (Materials and Methods). However, the overall yield was low; on average from approximately 103 dissociated single cells one colony resulted. Characterization of subclone AS034.1 revealed that it behaved comparably to the other cell lines in terms of the expression of SSEA-4, SSEA-3, TRA-1-60, and TRA-1-81 (Fig. 6). 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).
Figure 6. Immunohistochemical marker expression analysis of subclone AS34.1. The colonies were analyzed 3 days after passaging. A-F: passage 12, G: passage 34. A) Morpholgy; B) SSEA-1; C) SSEA-3; D) SSEA-4; E) TRA-1-60; F) TRA-1-81, and G) Oct-4. Scale bar: 25 μm.
Analysis of In Vivo Pluripotency of hES Cell Lines
When clusters of hES colonies are xenotransplanted to SCID mice, they form teratomas consisting of cell types derived from ectoderm, mesoderm, and endoderm. To analyze the in vivo pluripotency of the derived hES cell lines, we transplanted clusters of hES cells under the kidney capsule of SCID mice. Eight weeks after the transplantation the mice were sacrificed and teratomas were analyzed. To distinguish human cells from mouse cells, we used antibodies specific for human-specific nuclear antigen and human E-cadherin. Xenografting of hES cluster resulted in two morphologically distinct structures in the kidneys. From a majority of the cell lines (SA002, AS034, AS034.1, SA121, and SA181) solid teratomas, consisting of highly differentiated cells and tissues derived from all three germ layers, such as gut epithelium; glandular epithelium (endoderm); cartilage, bone, smooth muscle, striated muscle, and kidney glomeruli-like structures (mesoderm); and pigment epithelial cells, neural epithelium, hair follicles, and stratified squamous epithelium (ectoderm) formed (Fig. 7, 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
Figure 7. Teratoma analysis. A, B) Low-power view of a solid tumor from cell line SA121 (A) and a fluid-filled cyst generated from cell line FC018 (B). C, D) Derivatives of the ectoderm. Pigmented epithelium (arrowhead) and neuroepithelium (arrow) are shown in C and hair follicles (arrow) in D. E, F) Derivatives of the mesoderm. Cartilage (arrowhead) and bone (arrow) are shown in E, and skeletal muscle in F. G, H) Derivatives of the endoderm. Gut-like epithelium with mucous-containing cells (arrowhead) and glandular epithelium are shown in G and H, respectively. Scale bars: 100 μm.
DISCUSSION
We acknowledge Katarina Andersson, Karin Axelsson, and Angelica Niklasson for assistance with derivation, characterization, and propagation of hES cells; Anita Sj?gren and Thorir Hardarson for culture of human embryos; Gunilla Caisander for karyotyping and FISH analysis; Inger Bryman and staff at the IVF clinic at the Sahlgrenska University Hospital; Monalill Lundqvist at IVF clinic at the Uppsala University Hospital; and Matts Wikland at Fertility Center Scandinavia for supplying embryos. We thank Marie Rehnstr?m and Ulrika Karlsson for assistance in subcloning; Gabriella Brolén for support in stainings and cell culture; and Peter Sartipy for general assistance. The work was supported by grants from the Cell Therapeutics Scandinavia AB, Swedish Research Council (H.S.), Juvenile Diabetes Research Foundation (H.S.), and Inga Britt och Arne Lundbergs Forskningsstiftelse (H.S.).
FOOTNOTES
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b Department of Medical Biochemistry, G?teborg University, G?teborg, Sweden;
c Department of Obstetrics and Gynaecology, Sahlgrenska University Hospital, G?teborg, Sweden;
d Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, G?teborg, 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@endo.mas.lu.se
ABSTRACT
The inner cell mass (ICM) of the preimplantation blastocyst contains a core of cells, termed the epiblast, that have the potential to generate somatic and germ cells of the embryo. It has previously been demonstrated that human embryonic stem (hES) cell lines, exhibiting a stable developmental potential to form derivatives of the three germ layers after prolonged culture in vitro, can be generated by the isolation and culturing of the human ICM . So far, the available knowledge of conditions for deriving, characterizing, and culturing undifferentiated hES cells is largely based on a relatively few successfully isolated cell lines. Furthermore, the underlying mechanisms that control the developmental decisions of hES cells in culture remain essentially unknown. It has been suggested that the poor success rates in developing ES cells in species other than mice is either due to fundamental biological differences between the species or simply due to technical factors associated with the derivation and culture conditions . For example, unless spontaneous differentiation of hES cells is prevented, the cells become gradually restricted and lose the characteristics of ES cells. Frequent passaging and optimization of the quality of the culture conditions and feeder cells may overcome this problem. However, many problems remain with regard to the optimal maintenance and expansion of hES cells.
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 .
MATERIALS AND METHODS
Methods for Deriving hES Cell Lines
The most commonly used method for deriving hES cell lines is by immunosurgical isolation of the ICM from the human blastocyst . Here, we show that hES cell lines can be established from pronase-treated and hatched blastocysts as well (Fig. 1). 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).
Figure 1. Human ES cell derivation from pronase-treated and hatched blastocysts. A) Blastocyst before pronase treatment. B) Outgrowth of pronase-treated blastocyst shown in (A) 6 days after pronase treatment. C) hES colony (passage 6) derived from the ICM in (B). D) Spontaneously hatched blastocyst. E) Outgrowth of the blastocyst shown in (D) 5 days after plating. F) hES colony (passage 3) derived from the ICM in (E).
Table 1. Summary of hES cell characterization
Characterization of hES Cell Lines
To analyze the long-term pluripotency and replicative immortality of the six newly established hES cell lines SA002, FC018, AS034, AS038, SA121, SA181, and subclone AS034.1 (see below), we used previously defined criteria for in vitro and in vivo characterization of hES cells by examining the morphology, marker expression, telomerase activity, karyotype, and pluripotency in vitro and in vivo.
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).
Figure 2. Karyotype of hES lines (see also Table 1). A) Normal 46XY karyotype from cell line SA181. B) XX karyotype from line SA002 with a trisomy 13. The arrow in (B) points to the trisomic chromosome 13.
Similar to undifferentiated pluripotent cultures of human germ cells and previously established hES cell lines, all our cell lines possessed high levels of alkaline phosphatase (AP) activity (Fig. 3B, 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 . 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.
Figure 3. Immunohistochemical marker expression analysis of cell line SA002 (passage 21). The colonies were analyzed 5 days after passaging. A) Morphology, (B) Alkaline Phosphatase (AP), (C) SSEA-1, (D) SSEA-3, (E) SSEA-4, (F) TRA-1-81, (G) TRA-1-60, and (H) Oct-4. Scale bar: 25 μm.
Oct-4 is a POU-domain transcription factor that is essential for establishment of ES cells from the ICM . Similar to previous reports , we found that undifferentiated hES cells expressed Oct-4 (Fig. 3H, 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).
Figure 4. Immunohistochemical marker expression analysis of cell line FC018. The colonies were analyzed 5 days after passaging. A-F: passage 25, G-I: passage 118. (A) Morphology, (B) SSEA-1, (C) TRA-1-60, (D) TRA-1-81, (E, H) nestin, (F, G) SSEA-4, and (I) Oct-4. Scale bar: 25 μm.
Finally, all six hES cell lines expressed high levels of telomerase activity that were maintained even after more than 50 passages (Table 1).
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 .
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 . 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 ?-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 HNF3? (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).
Figure 5. In vitro differentiation of cell line SA002. The colonies were analyzed after 12 days after passaging. A) Nestin-positive neuronal precursors, B) ?-III-tubulin-positive postmitotic neurons, C) desmin-positive mesodermal cells, D, E, F) -fetoprotein-positive (D), HNF3?-positive (E), and Cdx2-positive (F) endodermal cells. Scale bar: 25 μm.
Since none of the described hES cell lines were clonally derived, it cannot be excluded that multiple precursor or stem cells committed to different lineages may coexist within a population of homogeneously appearing cells. Theoretically, this would imply that, until proven, it cannot be stated that a single hES cell is capable of forming derivatives of all three embryonic germ layers. In our initial efforts to subclone cell line AS034, we obtained one clone, AS034.1. To promote cell survival, we used concentrated conditioned medium from hES cells grown in presence of FCS as cloning medium (Materials and Methods). However, the overall yield was low; on average from approximately 103 dissociated single cells one colony resulted. Characterization of subclone AS034.1 revealed that it behaved comparably to the other cell lines in terms of the expression of SSEA-4, SSEA-3, TRA-1-60, and TRA-1-81 (Fig. 6). 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).
Figure 6. Immunohistochemical marker expression analysis of subclone AS34.1. The colonies were analyzed 3 days after passaging. A-F: passage 12, G: passage 34. A) Morpholgy; B) SSEA-1; C) SSEA-3; D) SSEA-4; E) TRA-1-60; F) TRA-1-81, and G) Oct-4. Scale bar: 25 μm.
Analysis of In Vivo Pluripotency of hES Cell Lines
When clusters of hES colonies are xenotransplanted to SCID mice, they form teratomas consisting of cell types derived from ectoderm, mesoderm, and endoderm. To analyze the in vivo pluripotency of the derived hES cell lines, we transplanted clusters of hES cells under the kidney capsule of SCID mice. Eight weeks after the transplantation the mice were sacrificed and teratomas were analyzed. To distinguish human cells from mouse cells, we used antibodies specific for human-specific nuclear antigen and human E-cadherin. Xenografting of hES cluster resulted in two morphologically distinct structures in the kidneys. From a majority of the cell lines (SA002, AS034, AS034.1, SA121, and SA181) solid teratomas, consisting of highly differentiated cells and tissues derived from all three germ layers, such as gut epithelium; glandular epithelium (endoderm); cartilage, bone, smooth muscle, striated muscle, and kidney glomeruli-like structures (mesoderm); and pigment epithelial cells, neural epithelium, hair follicles, and stratified squamous epithelium (ectoderm) formed (Fig. 7, 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
Figure 7. Teratoma analysis. A, B) Low-power view of a solid tumor from cell line SA121 (A) and a fluid-filled cyst generated from cell line FC018 (B). C, D) Derivatives of the ectoderm. Pigmented epithelium (arrowhead) and neuroepithelium (arrow) are shown in C and hair follicles (arrow) in D. E, F) Derivatives of the mesoderm. Cartilage (arrowhead) and bone (arrow) are shown in E, and skeletal muscle in F. G, H) Derivatives of the endoderm. Gut-like epithelium with mucous-containing cells (arrowhead) and glandular epithelium are shown in G and H, respectively. Scale bars: 100 μm.
DISCUSSION
We acknowledge Katarina Andersson, Karin Axelsson, and Angelica Niklasson for assistance with derivation, characterization, and propagation of hES cells; Anita Sj?gren and Thorir Hardarson for culture of human embryos; Gunilla Caisander for karyotyping and FISH analysis; Inger Bryman and staff at the IVF clinic at the Sahlgrenska University Hospital; Monalill Lundqvist at IVF clinic at the Uppsala University Hospital; and Matts Wikland at Fertility Center Scandinavia for supplying embryos. We thank Marie Rehnstr?m and Ulrika Karlsson for assistance in subcloning; Gabriella Brolén for support in stainings and cell culture; and Peter Sartipy for general assistance. The work was supported by grants from the Cell Therapeutics Scandinavia AB, Swedish Research Council (H.S.), Juvenile Diabetes Research Foundation (H.S.), and Inga Britt och Arne Lundbergs Forskningsstiftelse (H.S.).
FOOTNOTES
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