Derivation of Human Embryonic Stem Cells from Day-8 Blastocysts Recovered after Three-Step In Vitro Culture
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《干细胞学杂志》
a Institute of Human Genetics, University of Newcastle, Newcastle upon Tyne, United Kingdom;
b School of Biological and Biomedical Sciences, University of Durham, Durham, United Kingdom;
c Newcastle Fertility Centre at Life, International Centre for Life, Newcastle upon Tyne, United Kingdom
Key Words. Human embryonic stem cells ? In vitro culture ? Pluripotency ? Differentiation
Correspondence: Miodrag Stojkovic, M.D., Institute of Human Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, U.K. Telephone: 44-191-219-4746; Fax: 44-191-219-4747; e-mail: miodrag.stojkovic@ncl.ac.uk
ABSTRACT
Human embryonic stem cells (hESCs) have the ability to generate cells from all three embryonic germ layers and represent a great potential source of cells for therapeutic uses . Pluripotent hESC lines have now been derived from the inner cell mass (ICM) of day-5 to day-7 surplus and donated human blastocysts after routinely using two-step in vitro culture . Previously we reported that more complex media for long-term in vitro culture of bovine embryos is necessary to improve the quality (i.e., cell number, as well as blastocyst and hatching rates) of in vitro recovered embryos . In humans, in vitro culture conditions are also suboptimal and result in low blastocyst rate or blastocysts with low cell number and lacking a distinct ICM .
Therefore, in this study we evaluated the effects of more complex three-step culture conditions on in vitro development of late human blastocysts (day 8), on the number of ICM cells, and whether these blastocysts could be used for derivation of hESCs.
MATERIALS AND METHODS
Traditionally, one- or two-step conditions for in vitro culture of human embryos are used to obtain day 5 or day 6 (IVF = day 0) early blastocysts. Then, these blastocysts have been used for the derivation of new hESC lines. We developed a three-step culture system that successfully supports the development of late (day-8) blastocysts. Analysis of cell numbers of ICMs revealed that day-8 blastocysts possess significantly (p<.01) more ICM cells than day-6 blastocysts (51.3 ± 9.6 versus 36.8 ± 11.9, respectively). In view of this result, we used day-8 blastocysts to derive hESCs. Of the 11 day-2 donated embryos, 7 (63.6%) blastocysts developed to day 6. All 7 of these blastocysts expanded or hatched on day-8 after transfer to the third step (GLCM). The representative pictures of day-6 and day-8 blastocysts are presented in Figures 1A and 1B, respectively. After isolation of ICMs by immunosurgery, three primary hESC colonies showed visible outgrowth (Fig. 1C), and one stable hESC line (hES-NCL1) with typical hESC morphology was successfully derived (Fig. 1D and 1E).
Figure 1. Morphology of human blastocysts and hES-NCL1 cells. (A): Day-6 blastocysts and (B) hatched day-8 blastocysts. Note the presence of very well-organized inner cell mass (*) in the day-8 blastocyst recovered after three-step in vitro culture. (C): Inner cell mass cells (arrow) grown on irradiated MEFs 10 days after immunosurgery. (D): Primary hESC colony grown on inactivated MEF cells. (E): Same colony at high magnification. Note the typical morphology of hESCs: They are small cells with large nucleoli. Scale bars: 100 μm (A, B, D); 200 μm (C); 50 μm (E). Abbreviations: hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast.
Our hES-NCL1 line has been cultured for more than 30 passages, and we found that both fresh and cryopreserved hESC colonies grown on different feeder cells or in feeder-free conditions were dense and compact and therefore suitable for enzymatic or mechanical passaging. Characterization studies demonstrated that hESCs expressed specific surface markers—GTCM-2, TG343, TRA1-60, and SSEA-4—and were positive for the expression of AP and OCT-4 (Fig. 2). Expressions of OCT-4, REX-1, NANOG, and TERT by RT-PCR are presented in Figure 3A. Karyotyping revealed that the hESCs have normal female karyotype (46, XX) when cultured on MEF cells (Fig. 3B).
Figure 2. (A, C, E, G): Phase contrast and (B, D, F, H) fluorescence microscopy images of hES-NCL1 cells. The human embryonic stem cells were stained with antibody recognizing the GTCM-2 (B), TG343 (D), TRA1-60 (F), SSEA-4 (H), OCT-4 (I), and alkaline phosphatase (K) epitopes. (J) Negative OCT-4 control. Scale bars: 200 μm (A, B, K); 100 μm (C, D, G–J); 50 μm (E, F).
Figure 3. (A): Reverse transcription PCR and (B) karyotype analysis of undifferentiated hES-NCL1 cells grown on mouse embryonic fibroblasts. The PCR products obtained used primers specific for OCT-4, REX-1, NANOG, and TERT. The housekeeping gene, GAPDH, was used to normalize the samples for the cDNA content. A plus sign (+) indicates the presence of reverse transcriptase, and a minus sign (–) indicates the lack of the enzyme. Normal female karyotype (46, XX) of hES-NCL1 cell line (B). Abbreviation: PCR, polymerase chain reaction.
Similar to hES-NCL1 cells cultured on MEF cells, hESCs in feeder-free conditions grow as compact undifferentiated colonies (Fig. 4A), expressing the TRA1-60 marker (Fig. 4B) and maintaining their normal karyotype after 11 passages (Fig. 4C).
Figure 4. (A): Morphology and (B) TRA1-60 staining of undifferentiated hES-NCL1 cells grown in feeder-free conditions (Matrigel and mouse embryonic fibroblast–conditioned medium) maintaining (C) normal karyotype (46, XX) after 11 passages.
Injection of hES-NCL1 cells into SCID mice resulted in consistent formation of teratomas that were primarily restricted to the site of injection. Gross analysis of excised tumor tissues showed solid teratomas and lesions containing fluid-filled cystic masses accompanied by solid tissues. Histological examination of teratomas revealed advanced differentiation of structures representative of all three embryonic germ layers, including cartilage, skin, muscle, primitive neuroectoderm, neural ganglia, secretory epithelia, and connective tissues (Fig. 5). Moreover, such tissues formed complex arrangements recapitulating the development of complex structures that no doubt require coordinated interactions between different cell types derived from different germ layers.
Figure 5. Histological analysis of teratomas formed from grafted colonies of hES-NCL1 cells in severe combined immunodeficient mice. (A): Neural epithelium (ne). (B): Structures of the skin including epidermis (ed), dermis (dm), and cornified layer (c). The stratum granulosum (arrow) is characterized by intracellular granules that contribute to the process of keratinization. (C–E):A wall of respiratory passage showing epithelium (ep), submucosa (sm), submucosal glands (sg), smooth muscle (mus), neural ganglia (ng), and supporting cartilage (cart). (F): High-magnification image of respiratory pseudostratified columnar epithelium containing occasional cells expressing cilia (arrow) and goblet cells secreting mucin (m). Histological staining: hematoxylin and eosin (A, D, E) and Weigert’s (B, C, F). Scale bars: 100 μm (A, D); 25 μm (B, C, E); 12.5 μm (F).
When hES-NCL1 cells were cultured in absence of feeders, spontaneous differentiation into smooth muscle cells (detected by the presence of alpha smooth muscle isoform actin; Fig. 6A) and to neuronal cells (expressing the neuronal progenitor marker, nestin; Fig. 6B) was observed.
Figure 6. Spontaneous differentiation of hES-NCL1 cells into (A) smooth muscle and (B) neuronal cells, demonstrating differentiation into cells of mesoderm and ectoderm, respectively. Human embryonic stem cells were stained with smooth muscle actin antibody (A) and nestin antibody (B). Scale bars: 100 μm.
DISCUSSION
We thank M. Choudhary, J. Fenwick, S. Harbottle, V. Lamb, C. Leary, S. Cassley, S. Parker, K. Yallop, and A. Elliott for their support and Drs. P. Andrews and M. Pera for their donation of the specific antibodies. This work was supported by Newcastle University Hospitals Special Trusties, Newcastle Health Charity, and Life Knowledge Park. Miodrag Stojkovic and Majlinda Lako contributed equally to this work.
REFERENCES
Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cells. STEM CELLS 2001;19:193–204.
Schuldiner M, Eiges R, Eden A et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res 2001;913:201–205.
Zhang S-C, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neuronal precursors from human embryonic stem cell. Nat Biotechnol 2001;19:1129–1133.
He J-Q, Ma Y, Lee Y et al. Human embryonic stem cells develop into multiple types of cardiac myocytes. Circ Res 2003;93:32–39.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell line from human blastocysts. Science 1998;282: 1145–1147.
Reubinoff BE, Pera MF, Fong C-Y et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Richards M, Fong C-Y, Chan W-K et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cell lines. Nat Biotechnol 2002;20:933–936.
Hovatta O, Mikkola M, Gertow K et al. A culture system using foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18: 1404–1409.
Mitalipova M, Calhoun J, Shin S et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526.
Park JH, Kim SJ, Oh EJ et al. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 2003;69:2007–2014.
Pickering SJ, Braude PR, Patel M et al. Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. RBM Online 2003;7:353–364.
Stojkovic M, Wolf E, Buttner M et al. Secretion of biologically active interferon tau by in vitro-derived bovine trophoblastic tissue. Biol Reprod 1995;53:1500–1507.
Stojkovic M, K?lle S, Peinl S et al. Effects of high concentration of hyaluronan in culture medium on development and survival rates of fresh and frozen/thawed in vitro produced bovine embryos. Reproduction 2002;124:141–153.
Spanos S, Becker DL, Winston RM et al. Anti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo development. Biol Reprod 2000;63: 1413–1420.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1353–1356.
Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev Biol 1987;121:1–9.
Marquardt H, Todaro GJ, Henderson LE et al. Purification and primary structure of a polypeptide with multiplication-stimulating activity from rat liver cell cultures: homology with human insulin-like growth factor II. J Biol Chem 1981;256:6859–6865.
Massague J, Kelly B, Mottola C. Stimulation by insulin-like growth factors is required for cellular transformation by type beta transforming growth factor. J Biol Chem 1985; 260:4551–4554.
Lavoir MC, Conaghan J, Pedersen RA. Culture of human embryos for studies on the derivation of human pluripotent cells: a preliminary investigation. Reprod Fertil Dev 1998; 10:557–561.
Fry RC. The effect of leukaemia inhibitory factor (LIF) on embryogenesis. Reprod Fertil Dev 1992;4:449–458.
Pera MF, Reubinoff B, Trounson A. Human embryonic stem cells. J Cell Sci 2000;113:5–10.(Miodrag Stojkovica, Majli)
b School of Biological and Biomedical Sciences, University of Durham, Durham, United Kingdom;
c Newcastle Fertility Centre at Life, International Centre for Life, Newcastle upon Tyne, United Kingdom
Key Words. Human embryonic stem cells ? In vitro culture ? Pluripotency ? Differentiation
Correspondence: Miodrag Stojkovic, M.D., Institute of Human Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, U.K. Telephone: 44-191-219-4746; Fax: 44-191-219-4747; e-mail: miodrag.stojkovic@ncl.ac.uk
ABSTRACT
Human embryonic stem cells (hESCs) have the ability to generate cells from all three embryonic germ layers and represent a great potential source of cells for therapeutic uses . Pluripotent hESC lines have now been derived from the inner cell mass (ICM) of day-5 to day-7 surplus and donated human blastocysts after routinely using two-step in vitro culture . Previously we reported that more complex media for long-term in vitro culture of bovine embryos is necessary to improve the quality (i.e., cell number, as well as blastocyst and hatching rates) of in vitro recovered embryos . In humans, in vitro culture conditions are also suboptimal and result in low blastocyst rate or blastocysts with low cell number and lacking a distinct ICM .
Therefore, in this study we evaluated the effects of more complex three-step culture conditions on in vitro development of late human blastocysts (day 8), on the number of ICM cells, and whether these blastocysts could be used for derivation of hESCs.
MATERIALS AND METHODS
Traditionally, one- or two-step conditions for in vitro culture of human embryos are used to obtain day 5 or day 6 (IVF = day 0) early blastocysts. Then, these blastocysts have been used for the derivation of new hESC lines. We developed a three-step culture system that successfully supports the development of late (day-8) blastocysts. Analysis of cell numbers of ICMs revealed that day-8 blastocysts possess significantly (p<.01) more ICM cells than day-6 blastocysts (51.3 ± 9.6 versus 36.8 ± 11.9, respectively). In view of this result, we used day-8 blastocysts to derive hESCs. Of the 11 day-2 donated embryos, 7 (63.6%) blastocysts developed to day 6. All 7 of these blastocysts expanded or hatched on day-8 after transfer to the third step (GLCM). The representative pictures of day-6 and day-8 blastocysts are presented in Figures 1A and 1B, respectively. After isolation of ICMs by immunosurgery, three primary hESC colonies showed visible outgrowth (Fig. 1C), and one stable hESC line (hES-NCL1) with typical hESC morphology was successfully derived (Fig. 1D and 1E).
Figure 1. Morphology of human blastocysts and hES-NCL1 cells. (A): Day-6 blastocysts and (B) hatched day-8 blastocysts. Note the presence of very well-organized inner cell mass (*) in the day-8 blastocyst recovered after three-step in vitro culture. (C): Inner cell mass cells (arrow) grown on irradiated MEFs 10 days after immunosurgery. (D): Primary hESC colony grown on inactivated MEF cells. (E): Same colony at high magnification. Note the typical morphology of hESCs: They are small cells with large nucleoli. Scale bars: 100 μm (A, B, D); 200 μm (C); 50 μm (E). Abbreviations: hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast.
Our hES-NCL1 line has been cultured for more than 30 passages, and we found that both fresh and cryopreserved hESC colonies grown on different feeder cells or in feeder-free conditions were dense and compact and therefore suitable for enzymatic or mechanical passaging. Characterization studies demonstrated that hESCs expressed specific surface markers—GTCM-2, TG343, TRA1-60, and SSEA-4—and were positive for the expression of AP and OCT-4 (Fig. 2). Expressions of OCT-4, REX-1, NANOG, and TERT by RT-PCR are presented in Figure 3A. Karyotyping revealed that the hESCs have normal female karyotype (46, XX) when cultured on MEF cells (Fig. 3B).
Figure 2. (A, C, E, G): Phase contrast and (B, D, F, H) fluorescence microscopy images of hES-NCL1 cells. The human embryonic stem cells were stained with antibody recognizing the GTCM-2 (B), TG343 (D), TRA1-60 (F), SSEA-4 (H), OCT-4 (I), and alkaline phosphatase (K) epitopes. (J) Negative OCT-4 control. Scale bars: 200 μm (A, B, K); 100 μm (C, D, G–J); 50 μm (E, F).
Figure 3. (A): Reverse transcription PCR and (B) karyotype analysis of undifferentiated hES-NCL1 cells grown on mouse embryonic fibroblasts. The PCR products obtained used primers specific for OCT-4, REX-1, NANOG, and TERT. The housekeeping gene, GAPDH, was used to normalize the samples for the cDNA content. A plus sign (+) indicates the presence of reverse transcriptase, and a minus sign (–) indicates the lack of the enzyme. Normal female karyotype (46, XX) of hES-NCL1 cell line (B). Abbreviation: PCR, polymerase chain reaction.
Similar to hES-NCL1 cells cultured on MEF cells, hESCs in feeder-free conditions grow as compact undifferentiated colonies (Fig. 4A), expressing the TRA1-60 marker (Fig. 4B) and maintaining their normal karyotype after 11 passages (Fig. 4C).
Figure 4. (A): Morphology and (B) TRA1-60 staining of undifferentiated hES-NCL1 cells grown in feeder-free conditions (Matrigel and mouse embryonic fibroblast–conditioned medium) maintaining (C) normal karyotype (46, XX) after 11 passages.
Injection of hES-NCL1 cells into SCID mice resulted in consistent formation of teratomas that were primarily restricted to the site of injection. Gross analysis of excised tumor tissues showed solid teratomas and lesions containing fluid-filled cystic masses accompanied by solid tissues. Histological examination of teratomas revealed advanced differentiation of structures representative of all three embryonic germ layers, including cartilage, skin, muscle, primitive neuroectoderm, neural ganglia, secretory epithelia, and connective tissues (Fig. 5). Moreover, such tissues formed complex arrangements recapitulating the development of complex structures that no doubt require coordinated interactions between different cell types derived from different germ layers.
Figure 5. Histological analysis of teratomas formed from grafted colonies of hES-NCL1 cells in severe combined immunodeficient mice. (A): Neural epithelium (ne). (B): Structures of the skin including epidermis (ed), dermis (dm), and cornified layer (c). The stratum granulosum (arrow) is characterized by intracellular granules that contribute to the process of keratinization. (C–E):A wall of respiratory passage showing epithelium (ep), submucosa (sm), submucosal glands (sg), smooth muscle (mus), neural ganglia (ng), and supporting cartilage (cart). (F): High-magnification image of respiratory pseudostratified columnar epithelium containing occasional cells expressing cilia (arrow) and goblet cells secreting mucin (m). Histological staining: hematoxylin and eosin (A, D, E) and Weigert’s (B, C, F). Scale bars: 100 μm (A, D); 25 μm (B, C, E); 12.5 μm (F).
When hES-NCL1 cells were cultured in absence of feeders, spontaneous differentiation into smooth muscle cells (detected by the presence of alpha smooth muscle isoform actin; Fig. 6A) and to neuronal cells (expressing the neuronal progenitor marker, nestin; Fig. 6B) was observed.
Figure 6. Spontaneous differentiation of hES-NCL1 cells into (A) smooth muscle and (B) neuronal cells, demonstrating differentiation into cells of mesoderm and ectoderm, respectively. Human embryonic stem cells were stained with smooth muscle actin antibody (A) and nestin antibody (B). Scale bars: 100 μm.
DISCUSSION
We thank M. Choudhary, J. Fenwick, S. Harbottle, V. Lamb, C. Leary, S. Cassley, S. Parker, K. Yallop, and A. Elliott for their support and Drs. P. Andrews and M. Pera for their donation of the specific antibodies. This work was supported by Newcastle University Hospitals Special Trusties, Newcastle Health Charity, and Life Knowledge Park. Miodrag Stojkovic and Majlinda Lako contributed equally to this work.
REFERENCES
Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cells. STEM CELLS 2001;19:193–204.
Schuldiner M, Eiges R, Eden A et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res 2001;913:201–205.
Zhang S-C, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neuronal precursors from human embryonic stem cell. Nat Biotechnol 2001;19:1129–1133.
He J-Q, Ma Y, Lee Y et al. Human embryonic stem cells develop into multiple types of cardiac myocytes. Circ Res 2003;93:32–39.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell line from human blastocysts. Science 1998;282: 1145–1147.
Reubinoff BE, Pera MF, Fong C-Y et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Richards M, Fong C-Y, Chan W-K et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cell lines. Nat Biotechnol 2002;20:933–936.
Hovatta O, Mikkola M, Gertow K et al. A culture system using foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18: 1404–1409.
Mitalipova M, Calhoun J, Shin S et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526.
Park JH, Kim SJ, Oh EJ et al. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 2003;69:2007–2014.
Pickering SJ, Braude PR, Patel M et al. Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. RBM Online 2003;7:353–364.
Stojkovic M, Wolf E, Buttner M et al. Secretion of biologically active interferon tau by in vitro-derived bovine trophoblastic tissue. Biol Reprod 1995;53:1500–1507.
Stojkovic M, K?lle S, Peinl S et al. Effects of high concentration of hyaluronan in culture medium on development and survival rates of fresh and frozen/thawed in vitro produced bovine embryos. Reproduction 2002;124:141–153.
Spanos S, Becker DL, Winston RM et al. Anti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo development. Biol Reprod 2000;63: 1413–1420.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1353–1356.
Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev Biol 1987;121:1–9.
Marquardt H, Todaro GJ, Henderson LE et al. Purification and primary structure of a polypeptide with multiplication-stimulating activity from rat liver cell cultures: homology with human insulin-like growth factor II. J Biol Chem 1981;256:6859–6865.
Massague J, Kelly B, Mottola C. Stimulation by insulin-like growth factors is required for cellular transformation by type beta transforming growth factor. J Biol Chem 1985; 260:4551–4554.
Lavoir MC, Conaghan J, Pedersen RA. Culture of human embryos for studies on the derivation of human pluripotent cells: a preliminary investigation. Reprod Fertil Dev 1998; 10:557–561.
Fry RC. The effect of leukaemia inhibitory factor (LIF) on embryogenesis. Reprod Fertil Dev 1992;4:449–458.
Pera MF, Reubinoff B, Trounson A. Human embryonic stem cells. J Cell Sci 2000;113:5–10.(Miodrag Stojkovica, Majli)