Derivation of Human Embryonic Stem Cell Lines in Serum Replacement Medium Using Postnatal Human Fibroblasts as Feeder Cells
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《干细胞学杂志》
a Department of Clinical Sciences, Division of Obstetrics and Gynecology,
b Department of Medical Nutrition at Novum,
c Department of Laboratory Medicine, and
d Department of Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
Key Words. Human embryonic stem cells ? Serum replacement medium ? Foreskin fibroblasts ? Characterization ? Pluripotency
Correspondence: Outi Hovatta, M.D., Ph.D, Department of Clinical Sciences, Division of Obstetrics and Gynecology, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden; Telephone: 46-8-58580000; Fax: 46-8-58587575; e-mail: Outi.Hovatta@klinvet.ki.se
ABSTRACT
Derivation and culture of human embryonic stem cells (hESCs) from the inner cell mass (ICM) of blastocysts fertilized in vitro and establishment of permanent lines were originally carried out using media that contained fetal calf serum (FCS) and fetal mouse fibroblasts as feeder cells . The potential of transplanted hESCs in cases of severe degenerative diseases has been clearly recognized .
Animal-derived components, nonhuman sera, and feeder cells in the cultures bear a risk of transmitting animal pathogens to hESCs. Such materials are therefore not desirable in cell lines in regard to cell transplantation in humans.
Existing hESC lines, originally derived using fetal mouse fibroblasts and FCS, have been successfully cultured as nondifferentiated ESCs without feeder cells on Matrigel, using conditioned media from cultures of mouse feeder cells . These cultures were a step forward, although they were not entirely free of animal material. Human fetal fibroblasts and cells from fallopian tubes of women, obtained at surgery, have also enabled nondifferentiated growth of already existing hESCs, and the use of human serum in cultures has also been reported, with one line also derived in such conditions . Recently, derivation of an hESC line on fetal mouse feeder cells but using serum replacement (SR) was reported . We have derived and propagated new hESC lines using human foreskin fibroblasts as feeder cells .
Amit et al. described serum-free culture conditions for a line that had originally been derived using the conventional FCS-mouse fibroblast system. They used SR medium and postnatal human fibroblasts as feeder cells. The same group also described a system involving culture of hESCs on fibronectin and adding transforming growth factor ?1, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor to the culture medium . However, there are many differentiated cells at the margins of their colonies, as shown by their pictures. In these experiments, they used hESC lines that had been originally derived on mouse fibroblasts and in medium containing FCS.
We have also used SR medium for the culture of hESC lines that we originally derived in FCS-containing medium but with human foreskin fibroblasts as feeder cells . In a systematic survey, we found that adding bFGF at 8 ng/ml to culture medium containing 20% SR medium gave the best support for nondifferentiated growth of these hESCs .
We now describe fully characterized hESC lines (HS293 and HS306) that had been derived from the beginning using SR medium in the culture and human foreskin fibroblasts as feeder cells.
MATERIALS AND METHODS
The isolated ICM of the donated blastocyst received in October 2003 became attached to the feeder cells and grew to form a colony of typical hESCs (Fig. 2A). After 12 days, the first splitting onto new feeder cells was carried out, and thereafter the cells were passaged at 5- to 8-day intervals. This line, HS293, has been in continuous culture for 56 passages, with a doubling time of 24–36 hours. It was mechanically split and transferred to new dishes when the aggregate reached 2.5 to 6.4 x 103 cells. The size of the transferred aggregates has been 1 to 2 x 103 cells. Spontaneous differentiation was observed in 10%–20% of the cells in 1 out of 20 colonies during the 5- to 8-day culture period. The line has been frozen and thawed, and it grows well after thawing.
Figure 2. Colonies of lines (A) HS293 (passage level 12) and (B) HS306 (passage level 15) growing on the feeder layer. Magnification x40.
The other ESC line (HS306) from a blastocyst was received in February 2004. It has been similarly processed and is now at passage level 42 (Fig. 2B). It has similar characteristics to the line HS293 (Fig. 3). Its pluripotency in vitro has been analyzed, and the same markers were expressed as in line HS293.
Figure 3. Immunofluorescence staining for markers characterizing undifferentiated human embryonic stem cells and for markers of the three embryonic germ cell layers. Undifferentiated HS293 (passage 49) and HS306 (passage 40) expressed (A, F) Oct-4, (B, G) TRA-1-60, (C, H) TRA-1-81, and (D, I) SSEA-4 and did not express (E, J) SSEA-1. Embryoid bodies of HS293 (passages 49–51) and HS306 (passages 33–37) contained (K, N) ectoderm shown by Nestin expression, (L, O) mesoderm shown by bone morphogenetic protein-4 (BMP-4) expression, and (M, P) endoderm shown by alpha fetoprotein (AFP) expression. (A–E, K–M): HS293; (F–J, N–P): HS306. Specific staining is green (Alexa 488), and nuclear background staining is blue (DAPI). Original magnification (C–E, J, M–O) x10 and (A, B, F–I, K, L, P) x40.
The growing cells of both lines expressed markers characteristic of hESCs. Colonies of different sizes stained positively for Oct-4 (Figs. 3A, 3F), TRA-1-60 (Figs. 3B, 3G), TRA-1-81 (Figs. 3C, 3H), and SSEA-4 (Fig. 3D, 3I). In addition, undifferentiated colonies showed positive staining for GCTM-2 and alkaline phosphatase activity (data not shown). Staining for SSEA-1 was negative (Fig. 3E, 3J). Pluripotency in vivo of the line HS293 was shown by teratoma formation in beige/SCID mice. Tissue components could be identified representing ectodermal (Fig. 4A), mesodermal (Figs. 4B, 4C), and endodermal (Fig. 4D) tissue.
Figure 4. Teratomas formed after injection of the human embryonic stem cells to beige/severe combined immunodeficiency mice with tissue components of all three embryonic germ cell layers. Ectoderm , mesoderm , and endoderm . Original magnification x100.
The embryoid bodies formed from lines HS293 and HS306 expressed SOX-1, ND-1, alpha-cardiac actin, and AFP as constituents of the three germ cell layers (Fig. 5), as revealed by RT-PCR. The three germ layers were also confirmed using immunofluorescent staining against Nestin, BMP-4, and AFP, as seen in Figures 3K–3M, which correspond to HS293, and Figures 3N–3P, which correspond to HS306.
Figure 5. Embryoid bodies from lines HS293 and HS306 expressed markers for the three embryonic germ cell layers, SOX-1, ND-1, -cardiac actin and -fetoprotein, with GAPDH as the housekeeping control as revealed by reverse transcription–polymerase chain reaction.
The karyotype of line HS293 was normal male 46,XY at passage 9 and at passage 49. The karyotype of the line HS306 was normal female 46,XX at passage 32.
When using FCS in the derivations in 2002 to 2003, a total of 67 blastocysts resulted in four permanent lines (HS181, HS207, HS235, and HS237), and at the same time, nine additional lines grew from 2 to 9 passages and then faded off. When using 10 blastocysts in derivations with the SR medium, we obtained the present two permanent lines (HS293 and HS306), and from September to October 2004, a total of 27 blastocysts resulted in 10 early lines (HS346, HS351, HS356, HS360, HS361, HS362, HS363, HS364, HS366, and HS368) when using the same human foreskin feeder cells and SR medium. The line HS346 was obtained from an embryo that had been frozen 5 years ago, and it is presently at passage nine. The other early lines are at passages three through seven.
DISCUSSION
This study was supported by grants from the Swedish Research Council, the Juvenile Diabetes Research Foundation, the Finnish Academy, NorFa, and the Karolinska Institute. We thank Nicholas Bolton for revising the language of this manuscript, Marta Imreh for her culture work, and the laboratory personnel of the IVF Unit for their embryo cultures.
REFERENCES
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnol 2000;18:399–404.
Fishel SB, Edwards RG, Evans CJ. Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science 1984;223:816–818.
Keller G, Snodgrass HR. Human embryonic stem cells: the future is now. Nat Med 1999;5:151–152.
Edwards RG. Personal pathways to embryonic stem cells. Reprod Biomed Online 2002;4:263–278.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19: 971–974.
Carpenter MK, Rosler E, Rao MS. Characterization and differentiation of human embryonic stem cells. Cloning Stem Cells 2003;5:79–88.
Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.
Draper JS, Moore HD, Ruban LN et al. Culture and characterisation of human embryonic stem cell lines. Stem Cells Dev 2004;13:325–336.
Hovatta O, Mikkola M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409.
Amit M, Margulets V, Segev H et al. Human feeder layers for human embryonic stem cells. Biol Reprod 2003;68:2150–2156.
Amit M, Shariki C, Margulets V et al. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–845.
Imreh P WS, Unger C, Gertow K et al. Culture and expansion of the human embryonic stem cell line HS 181, evaluated in a double color system. Stem Cells Dev 2004;13:337–343.
Gertow KW, Rozell S, Sugars B et al. Organised development from human embryonic stem cells after injection into immunodeficient mice. Stem Cells Dev 2004;13:421–435.
Koivisto H, Hyvarinen M, Str?mberg A-M et al. Cultures of human embryonic stem cells: serum replacement medium or serum-containing media and the effect of basic fibroblast growth factor. Reprod Biomed Online 2004;9:330–337.
Bjuresten K, Hovatta O. Donation of embryos for stem cell research—how many couples consent? Hum Reprod 2003;18:1353–1355.
Mohr L, Trounson A. Cryopreservation of human embryos. Ann N Y Acad Sci 1985;442:536–543.
Inzunza J, Sahlen S, Holmberg K et al. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol Hum Reprod 2004;10:461–466.
Solter D, Knowles BB. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci U S A 1975;72:5099–5102.
Heins N, Englund MC, Sj?blom C et al. Derivation, characterization, and differentiation of human embryonic stem cells. STEM CELLS 2004;22:367–376.
Reubinoff BE, Pera MF, Vajta G et al. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 2001;16:2187–2194.
Mountford P, Nichols J, Zevnik B et al. Maintenance of pluripotential embryonic stem cells by stem cell selection. Reprod Fertil Dev 1998;10:527–533.
Henderson JK, Draper JS, Baillie HS et al. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. STEM CELLS 2002;20:329–337.
Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281–292.(José Inzunzaa,b, Karin Ge)
b Department of Medical Nutrition at Novum,
c Department of Laboratory Medicine, and
d Department of Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
Key Words. Human embryonic stem cells ? Serum replacement medium ? Foreskin fibroblasts ? Characterization ? Pluripotency
Correspondence: Outi Hovatta, M.D., Ph.D, Department of Clinical Sciences, Division of Obstetrics and Gynecology, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden; Telephone: 46-8-58580000; Fax: 46-8-58587575; e-mail: Outi.Hovatta@klinvet.ki.se
ABSTRACT
Derivation and culture of human embryonic stem cells (hESCs) from the inner cell mass (ICM) of blastocysts fertilized in vitro and establishment of permanent lines were originally carried out using media that contained fetal calf serum (FCS) and fetal mouse fibroblasts as feeder cells . The potential of transplanted hESCs in cases of severe degenerative diseases has been clearly recognized .
Animal-derived components, nonhuman sera, and feeder cells in the cultures bear a risk of transmitting animal pathogens to hESCs. Such materials are therefore not desirable in cell lines in regard to cell transplantation in humans.
Existing hESC lines, originally derived using fetal mouse fibroblasts and FCS, have been successfully cultured as nondifferentiated ESCs without feeder cells on Matrigel, using conditioned media from cultures of mouse feeder cells . These cultures were a step forward, although they were not entirely free of animal material. Human fetal fibroblasts and cells from fallopian tubes of women, obtained at surgery, have also enabled nondifferentiated growth of already existing hESCs, and the use of human serum in cultures has also been reported, with one line also derived in such conditions . Recently, derivation of an hESC line on fetal mouse feeder cells but using serum replacement (SR) was reported . We have derived and propagated new hESC lines using human foreskin fibroblasts as feeder cells .
Amit et al. described serum-free culture conditions for a line that had originally been derived using the conventional FCS-mouse fibroblast system. They used SR medium and postnatal human fibroblasts as feeder cells. The same group also described a system involving culture of hESCs on fibronectin and adding transforming growth factor ?1, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor to the culture medium . However, there are many differentiated cells at the margins of their colonies, as shown by their pictures. In these experiments, they used hESC lines that had been originally derived on mouse fibroblasts and in medium containing FCS.
We have also used SR medium for the culture of hESC lines that we originally derived in FCS-containing medium but with human foreskin fibroblasts as feeder cells . In a systematic survey, we found that adding bFGF at 8 ng/ml to culture medium containing 20% SR medium gave the best support for nondifferentiated growth of these hESCs .
We now describe fully characterized hESC lines (HS293 and HS306) that had been derived from the beginning using SR medium in the culture and human foreskin fibroblasts as feeder cells.
MATERIALS AND METHODS
The isolated ICM of the donated blastocyst received in October 2003 became attached to the feeder cells and grew to form a colony of typical hESCs (Fig. 2A). After 12 days, the first splitting onto new feeder cells was carried out, and thereafter the cells were passaged at 5- to 8-day intervals. This line, HS293, has been in continuous culture for 56 passages, with a doubling time of 24–36 hours. It was mechanically split and transferred to new dishes when the aggregate reached 2.5 to 6.4 x 103 cells. The size of the transferred aggregates has been 1 to 2 x 103 cells. Spontaneous differentiation was observed in 10%–20% of the cells in 1 out of 20 colonies during the 5- to 8-day culture period. The line has been frozen and thawed, and it grows well after thawing.
Figure 2. Colonies of lines (A) HS293 (passage level 12) and (B) HS306 (passage level 15) growing on the feeder layer. Magnification x40.
The other ESC line (HS306) from a blastocyst was received in February 2004. It has been similarly processed and is now at passage level 42 (Fig. 2B). It has similar characteristics to the line HS293 (Fig. 3). Its pluripotency in vitro has been analyzed, and the same markers were expressed as in line HS293.
Figure 3. Immunofluorescence staining for markers characterizing undifferentiated human embryonic stem cells and for markers of the three embryonic germ cell layers. Undifferentiated HS293 (passage 49) and HS306 (passage 40) expressed (A, F) Oct-4, (B, G) TRA-1-60, (C, H) TRA-1-81, and (D, I) SSEA-4 and did not express (E, J) SSEA-1. Embryoid bodies of HS293 (passages 49–51) and HS306 (passages 33–37) contained (K, N) ectoderm shown by Nestin expression, (L, O) mesoderm shown by bone morphogenetic protein-4 (BMP-4) expression, and (M, P) endoderm shown by alpha fetoprotein (AFP) expression. (A–E, K–M): HS293; (F–J, N–P): HS306. Specific staining is green (Alexa 488), and nuclear background staining is blue (DAPI). Original magnification (C–E, J, M–O) x10 and (A, B, F–I, K, L, P) x40.
The growing cells of both lines expressed markers characteristic of hESCs. Colonies of different sizes stained positively for Oct-4 (Figs. 3A, 3F), TRA-1-60 (Figs. 3B, 3G), TRA-1-81 (Figs. 3C, 3H), and SSEA-4 (Fig. 3D, 3I). In addition, undifferentiated colonies showed positive staining for GCTM-2 and alkaline phosphatase activity (data not shown). Staining for SSEA-1 was negative (Fig. 3E, 3J). Pluripotency in vivo of the line HS293 was shown by teratoma formation in beige/SCID mice. Tissue components could be identified representing ectodermal (Fig. 4A), mesodermal (Figs. 4B, 4C), and endodermal (Fig. 4D) tissue.
Figure 4. Teratomas formed after injection of the human embryonic stem cells to beige/severe combined immunodeficiency mice with tissue components of all three embryonic germ cell layers. Ectoderm , mesoderm , and endoderm . Original magnification x100.
The embryoid bodies formed from lines HS293 and HS306 expressed SOX-1, ND-1, alpha-cardiac actin, and AFP as constituents of the three germ cell layers (Fig. 5), as revealed by RT-PCR. The three germ layers were also confirmed using immunofluorescent staining against Nestin, BMP-4, and AFP, as seen in Figures 3K–3M, which correspond to HS293, and Figures 3N–3P, which correspond to HS306.
Figure 5. Embryoid bodies from lines HS293 and HS306 expressed markers for the three embryonic germ cell layers, SOX-1, ND-1, -cardiac actin and -fetoprotein, with GAPDH as the housekeeping control as revealed by reverse transcription–polymerase chain reaction.
The karyotype of line HS293 was normal male 46,XY at passage 9 and at passage 49. The karyotype of the line HS306 was normal female 46,XX at passage 32.
When using FCS in the derivations in 2002 to 2003, a total of 67 blastocysts resulted in four permanent lines (HS181, HS207, HS235, and HS237), and at the same time, nine additional lines grew from 2 to 9 passages and then faded off. When using 10 blastocysts in derivations with the SR medium, we obtained the present two permanent lines (HS293 and HS306), and from September to October 2004, a total of 27 blastocysts resulted in 10 early lines (HS346, HS351, HS356, HS360, HS361, HS362, HS363, HS364, HS366, and HS368) when using the same human foreskin feeder cells and SR medium. The line HS346 was obtained from an embryo that had been frozen 5 years ago, and it is presently at passage nine. The other early lines are at passages three through seven.
DISCUSSION
This study was supported by grants from the Swedish Research Council, the Juvenile Diabetes Research Foundation, the Finnish Academy, NorFa, and the Karolinska Institute. We thank Nicholas Bolton for revising the language of this manuscript, Marta Imreh for her culture work, and the laboratory personnel of the IVF Unit for their embryo cultures.
REFERENCES
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnol 2000;18:399–404.
Fishel SB, Edwards RG, Evans CJ. Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science 1984;223:816–818.
Keller G, Snodgrass HR. Human embryonic stem cells: the future is now. Nat Med 1999;5:151–152.
Edwards RG. Personal pathways to embryonic stem cells. Reprod Biomed Online 2002;4:263–278.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19: 971–974.
Carpenter MK, Rosler E, Rao MS. Characterization and differentiation of human embryonic stem cells. Cloning Stem Cells 2003;5:79–88.
Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.
Draper JS, Moore HD, Ruban LN et al. Culture and characterisation of human embryonic stem cell lines. Stem Cells Dev 2004;13:325–336.
Hovatta O, Mikkola M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409.
Amit M, Margulets V, Segev H et al. Human feeder layers for human embryonic stem cells. Biol Reprod 2003;68:2150–2156.
Amit M, Shariki C, Margulets V et al. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–845.
Imreh P WS, Unger C, Gertow K et al. Culture and expansion of the human embryonic stem cell line HS 181, evaluated in a double color system. Stem Cells Dev 2004;13:337–343.
Gertow KW, Rozell S, Sugars B et al. Organised development from human embryonic stem cells after injection into immunodeficient mice. Stem Cells Dev 2004;13:421–435.
Koivisto H, Hyvarinen M, Str?mberg A-M et al. Cultures of human embryonic stem cells: serum replacement medium or serum-containing media and the effect of basic fibroblast growth factor. Reprod Biomed Online 2004;9:330–337.
Bjuresten K, Hovatta O. Donation of embryos for stem cell research—how many couples consent? Hum Reprod 2003;18:1353–1355.
Mohr L, Trounson A. Cryopreservation of human embryos. Ann N Y Acad Sci 1985;442:536–543.
Inzunza J, Sahlen S, Holmberg K et al. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol Hum Reprod 2004;10:461–466.
Solter D, Knowles BB. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci U S A 1975;72:5099–5102.
Heins N, Englund MC, Sj?blom C et al. Derivation, characterization, and differentiation of human embryonic stem cells. STEM CELLS 2004;22:367–376.
Reubinoff BE, Pera MF, Vajta G et al. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 2001;16:2187–2194.
Mountford P, Nichols J, Zevnik B et al. Maintenance of pluripotential embryonic stem cells by stem cell selection. Reprod Fertil Dev 1998;10:527–533.
Henderson JK, Draper JS, Baillie HS et al. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. STEM CELLS 2002;20:329–337.
Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281–292.(José Inzunzaa,b, Karin Ge)