- Email: [email protected]
Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst
Differentiation (2004) 72:224–229
r International Society of Differentiation 2004
OR IGI N A L A R T IC L E
Hossein Baharvand . Saeid Kazemi Ashtiani . Mojtaba Rezazadeh Valojerdi . Abdolhossein Shahverdi . Adeleh Taee . Davood Sabour
Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst
Received February 19, 2004; accepted in revised form May 7, 2004
Abstract Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. These cells have, therefore, potential for in vitro differentiation studies, gene function, and so on. The aim of this study was to produce a human embryonic stem cell line. An inner cell mass of a human blastocyst was separated and cultured on mouse embryonic fibroblasts in embryonic stem cell medium with related additives. The established line was evaluated by morphology; passaging; freezing and thawing; alkaline phosphatase; Oct-4 expression; anti-surface markers including Tra-1-60 and Tra-1-81; and karyotype and spontaneous differentiation. Differentiated cardiomyocytes and neurons were evaluated by transmission electron microscopy and immunocyto chemistry. Here, we report the derivation of a new
embryonic stem cell line (Royan H1) from a human blastocyst that remains undifferentiated in morphology during continuous passaging for more than 30 passages, maintains a normal XX karyotype, is viable after freezing and thawing, and expresses alkaline phosphatase, Oct-4, Tra-1-60, and Tra-1-81. These cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers in the presence or absence of recombinant human leukemia inhibitory factor. Royan H1 cells can differentiate in vitro in the absence of feeder cells and can produce embryoid bodies that can further differentiate into beating cardiomyocytes as well as neurons. These results define Royan H1 cells as a new human embryonic stem cell line.
. ) Hossein Baharvand (* Department for Biology of Stem Cells Royan Institute P.O. Box 19395-4644 Tehran, Iran Tel: 0098 21 2402486 Fax: 0098 21 2409314 E-mail: [email protected]
Introduction
Hossein Baharvand Saeid Kazemi Ashtiani Mojtaba Rezazadeh Valojerdi Abdolhossein Shahverdi Adeleh Taee Davood Sabour Department of Embryology Royan Institute P.O. Box 19395-4644 Tehran, Iran Mojtaba Rezazadeh Valojerdi Department of Anatomy Tarbiat Modarres University Tehran, Iran U.S. Copyright Clearance Center Code Statement:
Key words cardiomyocyte embryonic stem cells human neuron ultrastructure
Embryonic stem (ES) cells are most frequently derived from the inner cell mass (ICM) of blastocysts (Evans and Kaufman, 1981; Martin, 1981). In fact, the ICM is used to give rise to an ES cell line that remains undifferentiated and is pluripotent, meaning the cells have the potential to develop into any cell type from all three germ layers both in vivo and in vitro. ES cells or ES cell-likes have been produced in mice and other animal models, including chickens (Pain et al., 1996), hamsters (Doetschman et al., 1988), and pigs (Li et al., 2003). In primates, ES cell lines have been produced in the common marmoset (Thomson et al., 1996) and the rhesus monkey (Thomson et al., 1995). In 1998, Thomson and coworkers reported the first ES cell lines produced from human blastocyst stage embryos. Recently, the establishment of new human embryonic stem
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cell lines (Reubinoff et al., 2000; Lanzendorf et al., 2001; Mitalipova et al., 2003; Park et al., 2003; Pickering et al., 2003) has been reported. The ability to direct the differentiation of human ES cell lines into a population of a specific cell type such as insulin-producing pancreatic cells may provide a treatment for many diseases such as diabetes. The characteristics of ES cells indicate that these cells, in addition to use in developmental biology studies, have the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics applications. As a result, interest in producing ES cell lines in the human has increased over the past few years. In the present study, we report the establishment of a new human ES cell line: Royan H1. The pluripotency of this new cell line was evaluated in vitro by conventional tests.
Methods Cell line isolation A 6-day-old human blastocyst was donated to the study following the approval of the institutional review board and obtaining an informed consent from the couple undergoing in vitro fertilization treatment. The blastocyst was recovered through culturing in sequential culture media (Rs1 and Rs2; Vitrolife, Go¨teborg, Sweden) in accordance with the manufacturer’s instructions. Zona pelucida having been digested by acidic Tyrod’s solution (100 mM NaCl, 2.6 mM KCl, 1.6 mM CaCl21H2O, 4.9 mM MgCl212H2O, 5.4 mM glucose, 0.1 mM polyvinyl pyrolidone; Hogan et al., 1994), the blastocyst was cultured on a mitomycinC (Sigma, Taufkirchen, Germany) mitotically inactivated mouse embryonic fibroblast (MEF) feeder layer (isolated from day 13.5 post-coitum fetuses of NMRI outbred strain used at 75,000 cell cm 2) in gelatin (Sigma)-coated tissue culture dish (Falcon, Franklin Lakes, NJ, USA). Culture medium consisted of 80% knockout Dulbecco’s modified Eagle’s medium (Gibco, Karlsruhe, Germany) supplemented with 20% ES-qualified fetal calf serum (Gibco), 2 mM glutamine, (Gibco), 0.1 mM b-mercaptoethanol (Sigma), 1% nonessential amino acid stock (Gibco), 100 units ml 1 penicillin, and 100 mg ml 1 streptomycin (Gibco). After 1 day, the embryo attached on feeder cells and the inner cell mass was isolated with a mouth-controlled pipette and re-cultured on fresh MEF. During the isolation and early stages of ES cell cultivation, the medium was supplemented with human recombinant leukemia inhibitory factor (hLIF) at 1,000 units ml 1 (Chemicon, Hofheim/ TS, Germany), and hLIF was replaced by insulin-transferrin-selenite (Gibco). Ten days after the initial plating, an ES-like colony was dissociated with a combined approach of mechanical slicing with a pipette, followed by exposure to 10 mg ml 1 dispase (Gibco). The resulting colonies were grown in 5% CO2 and 95% humidity, and they were further propagated in clumps of 200–500 stem cell-like cells on MEF about every 7 days. Colonies were also periodically selected and cryopreserved by open-pulled straw method (Reubinoff et al., 2001) and stored in liquid nitrogen. Stem cell characterization Karyotype analysis: Karyotype analysis was carried out on the cells after passages 5 and 29. hES colonies were incubated with 10 mg/ml colcemid solution (Gibco) for 2 hr at 371C and in 5%
CO2 in air atmosphere. The cells were washed with PBS, trypsinized, and neutralized with ES medium. Then, pellets were resuspended and incubated with 0.075 M KCl for 17 min at 371C. Having been treated with hypotonic solution, the cells were fixed with 3:1 methanol:glacial acetic acid three times and dropped onto pre-cleaned chilled slides. Chromosome spreads were Giemsa banded and photographed. At least 20 metaphase spreads and five banded karyotypes were evaluated for chromosomal rearrangements. Cell surface markers: Colonies were evaluated for alkaline phosphatase (AP) activity using the alkaline phosphatase substrate kit (Sigma) in accordance with the manufacturer’s instructions. Tumor rejection antigens (Tra-1-60 and Tra-1-81) were detected by immunocytochemistry with specific primary antibodies (gifts of Peter Andrews, University of Sheffield, UK) and localized with rabbit anti-mouse immunoglobulins conjugated to fluorescein isothiocyanate (Sigma). Oct-4 expression: To evaluate expression of Oct-4 transcription factor, we used reverse transcriptase-polymerase chain reaction (RT-PCR) on colonies consisting predominantly of stem cells at passage 30. RNA was isolated from established ES cells using RNX-Plus (Fermentas, GmbH, Germany) and transcribed into cDNA using random hexamers and reverse transcriptase (Fermentas). Primer sets for Oct-4 were: forward primer, 5 0 -CTTGCTGCAGAAGTGGGTGGAGGAA-3 0 and reverse primer, 5 0 -CTGCAGTGTGGGTTTCGGGCA-3 0 (Reubinoff et al., 2000). Primers for aˆ-actin were used as positive control. Primer steps for b-actin were: 5 0 -CGCACCACTGGCATTGTCAT-3 0 (forward), 5 0 -TTCTCCTTGATGTCACGCAC-3 0 (reverse) (Reubinoff et al., 2000). PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized and photographed on a UV transluminator (UVItec, Cambridge, UK). In vitro differentiation: (1) Spontaneous differentiation into cardiomyocytes and neurons: The ES cells were cultured in the absence of MEF feeder layers in hanging drops to embryoid body (EB) formation for 2 days and in bacterial dishes for 5 or 20 days and grown in tissue culture plates for several days in human ES medium. The number of beatings of differentiating cardiomyocytes was counted under an inverted microscope. (1-a) Immunocytochemistry. Differentiated cells and neurons were fixed by 4% paraformaldehyde (Sigma) and stained with antibodies against microtubule-associated protein (MAP 2[a1b]; 1:200; Sigma) and neurofilament (DAKO, Glostrup, Denmark). The second antibody was fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Sigma). Direct immunocytochemistry was done with antibody against neuron-specific enolase labeled with horseradish peroxidase (DAKO). The antibody detected the use of 3,3-diaminobenzidine tetrahydrochlorides (DAKO). (1-b) Transmission electron microscopy (TEM). Forty days after plating EBs, we processed differentiated cardiomyocytes for TEM. Primary fixation: 2% glutaraldehyde in 0.1 M phosphate buffer saline (pH 7.4) for 2 hr and then two 30-min washes in the same buffer. Secondary fixation: 1% osmium tetroxide in the same buffer for 1.5 hr, followed by two washes in the buffer. The samples were washed three times in water (5 min each), dehydrated in an ethanol series—30%, 50%, 70%, 90%, and 95% for 15 min each, and then three times for 15 min each in 100% ethanol. Infiltration into Spurr’s resin was achieved by an ethanol:resin series: first in 50:50 (2 hr) then 100% resin (2 hr), and after that in 100% resin (overnight) and again in fresh resin (2 hr). The samples were then embedded in molds containing 100% resin and polymerized at 701C overnight. After polymerization, sections of about 80 nm were cut and stained with lead citrate for 8 min.Micrographs of transverse sections of ES and MEF cells were taken on plate films on a Zeiss EM900 (Oberkochen, Germany) transmission electron microscope. (2) Evaluation of human chorionic gonadotropin (hCG) and a-fetoprotein production. Colonies at passages level 15 and 19 were allowed to grow on mitotically inactivated MEFs to confluency (about 2 to 4 weeks). The medium was replaced every day after 2 weeks of differentiation, and medium in triplicate wells, conditioned
226 for 24 hr, was assayed by ELISA for a-fetoprotein and the hCG, markers of endoderm lineage and trophoblast differentiation, respectively. Conditioned medium of MEF was used as control group.
Results Stem cell characterization Isolation of ICM from a blastocyst yielded one cell line with ES morphological, biochemical, and/or immunocytochemical characteristics consistent with previously characterized pluripotent stem cell lines (Fig. 1). The line was named Royan H1 (Royan in Persian means embryo). The resulting cells had a high nucleus to cytoplasm ratio, prominent nucleoli, and colony morphology similar to that of rhesus monkey and human ES cells. The derived colonies were further propagated by mechanical disaggregation of clumps, which were re-plated onto fresh feeder cell layers. Whereas LIF was used during the early phase of the establishment of the cell lines, it was subsequently found to have no effect on the growth or differentiation of established culture (not shown). However, supplementation of culture medium with cultivation in the presence of insulin-transferrin-selenite increased undifferentiated colonies under standard growth conditions. Cell line Royan H1 was grown for 30 passages in vitro, and the cell line still consisted mainly of cells with the morphology of undifferentiated ES cells (Figs. 1A,1B). The cell line was successfully frozen and thawed. An evaluation of the cell line for cell surface markers that characterize undifferentiated human ES cell lines showed that the line stained positive for alkaline phosphatase (Fig. 1C). The line was also found to be positive for Tra-1-60 and Tra-1-81, both markers of undifferentiated human ES cell line (Figs. 1D,1E). Oct-4 is POU domain transcription factor, whose expression is limited in the mouse to pluripotent cells. Oct-4 is also expressed in human ES cells, and its expression is down-regulated when these cells are differentiated. Using RT-PCR to carry out mRNA analysis on isolated colonies consisting mainly of stem cells, we showed that Royan H1 also expressed Oct-4 (Fig. 1F). Karyotypic analysis, carried out at passages 5 and 27, indicated apparently normal XX human karyotype. In vitro differentiation Royan H1 maintained the potential to form derivatives of all three embryonic germ layers in vitro. The ES cells differentiated spontaneously when cultured in the absence of mouse embryonic fibroblast feeder layers, in hanging drops, and in bacterial culture dishes or grown in tissue culture plates for several days. We observed
cultures of cells or single cells with elongated processes that extended out from their cell bodies, forming networks as they contacted other cells in 25% (10/41) of embryoid bodies (Fig. 2A). These cells and the processes stained positively with antibodies against neuronspecific enolase (Fig. 2B), MAP-2(a1b) (Fig. 2C), and neurofilament proteins (Fig. 2D). Furthermore, we observed 10% (3/30) of embryoid bodies were differentiated into beating cells (42 2 beat/min; mean SEM). The ultrastructure characteristics of differentiated cardiomyocytes were studied. Transmission electron microscopy of these cells revealed mononuclear cells, with parallel arrays of myofibrillar bundles oriented in an irregular manner in some cells (Fig. 3A), whereas more mature sarcomeric organization was apparent in others (Figs. 3A,3B). In some areas, the formation of early and more developed and distinct Z bands, the I- and the A-band, could be observed (Fig. 3B). T-tubules or invaginations of the surface sarcolemma and H-band were observed (Fig. 3C). The diameter of the Z line corresponds to the diameter of the myofibrillar arrangements; the spacing between two Z lines was about 2 mm. The intercalated disc, another cellular structure that characteristically appears during in vivo cardiomyocyte differentiation, was observed in many of the differentiating cells (Fig. 3C). Therefore, gap junctions, macula adherence or spot desmosomes, and facia adherence were observed on the intercalated disc (Fig. 3C). After Royan H1 cells were allowed to differentiate for 2 weeks, both a-fetoprotein (41,000 IU/ml) and hCG (435 mIU/ml) were detected in conditioned cultured medium, indicating endoderm and trophoblast differentiation (Thomson et al., 1998). No hCG or afetoprotein was detected in conditioned medium from MEFs.
Discussion In 1998, Thomson and coworkers reported the first ES cell lines produced from human blastocyst stage embryos (Thomson et al., 1998). Within this report, they proposed criteria that true primate ES cell lines should meet. As in the mouse model, these criteria include derivation from the pre-implantaton embryo, prolonged undifferentiation in vitro, and the ability to form all three embryonic germ layers. However, the investigators did not feel it was ethical or feasible to produce human chimeras to determine that ES cell lines could contribute to the germ line. In the study presented here, we report the production of one new human ES line. This line was derived and grown as described. The derived ES cell line was successfully maintained for more than 30 passages, retain-
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Fig. 1 Stem cell characterizations: colony morphology (A). High magnification of hES cells (B). Detection of surface marker
expression, alkaline phosphatase (C), Tra-1-60 (D), Tra-1-81 (E), and Oct-4 (F).
Fig. 2 Phase contrast appearance and marker expression in cultures of neurons derived from hES. Phase contrast micrograph of differentiated cells (A). Direct immunocytochemistry (B) and indirect immunofluorescence (C, D) microscopy of differentiated cells decorated with antibodies against neuron-specific enolase (B), MAP2(a1b) (C), and neurofilament protein (D).
ing the morphology reported previously for both human (Thomson et al., 1998) and nonhuman (Thomson et al., 1995, 1996) primate ES cell lines. Human and nonhuman primate ES cells share a similar morpholo-
gy, which is distinct from mouse ES cells (Evans and Kaufman 1981; Thomson et al., 1995, 1998; Baharvand and Matthaei, 2003). Human ES cells form relatively flat, compact colonies that passage by a combined ap-
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Fig. 3 Ultrastructural analysis of hES-derived cardiomyocytes. Transmission electron microscopy of sectioned beating EB, 40 days after plating. Organized myofibrillar bundles can be seen in most myocytes (A). Higher magnification of an organized sarcomere
(B). Intercalated disc (C) and high-power electron micrograph showing a gap junction and a T-tubule. D, desmosome; F, myofibrillar bundle; G, gap junction; I, intercalated disk; M, mitochondria; N, nucleus; T, T-tubule. The scale bar represents 0.5 mm.
proach of mechanical slicing with a pipette followed by exposure to dispase (Reubinoff et al., 2000) or dissociate to patches of cells by collagenase (Thomson et al., 1998), whereas mouse ES cells form tight, more spindleshaped colonies that passage by single cells. Maintenance of human ES cell colony density and derivation of clonal cell lines are complicated by difficulties associated with the expansion of a single cell (Amit et al., 2000). Moreover, human ES cells grow more slowly than mouse ES cells. In addition, the lines express cell surface markers such as Tra-1-60 and Tra-1-81 as well as AP and transcription factor Oct-4, demonstrating that the line is undifferentiated (Thomson et al., 1995). Such a finding has also been previously reported for human ES cells (Thomson et al., 1998). The most useful and important property of these cells is their ability to differentiate in vitro into ectodermal, endodermal, and mesodermal derivatives. As with mouse ES cells, human pluripotent stem cells can form EBs. These structures appear to recapitulate the normal developmental processes of the early embryonic stage and promote the cell-cell interaction required for cell differentiation. Typically, mouse EBs are surrounded by a layer of endoderm and contain a heterogeneous
mixture of cell types. Morphologically, human EBs somehow resemble those generated from mouse ES cultures. Identification of cell derivatives of the three embryonic germ layers (such as neurons, cardiomyocytes, and expression of a-fetoprotein) in human EB cultures suggests that these cells are pluripotent and are capable of in vitro differentiation. Ultrastructural analysis of differentiating cardiomyocytes showed that these cells were mainly mononuclear and round or rod shaped, and that these contained different degrees of myofibrillar bundle organization and exhibited intercalated discs. These results are consistent with ultrastructural properties of early-stage cardiomyocytes. Previous studies demonstrated that, during in vivo cardiogenesis, myofibrils were initially distributed in sparse, irregular myofibrillar arrays, which gradually matured into parallel arrays of myofibrils and ultimately aligned into densely packed sarcomeres (Manasek, 1970; Chacko, 1976). In our study, the Z line, the I- and the A-band were distinct. T-tubules and H-band were distinguishable in some cells; however, in comparison with adult cardiomyocytes, the M-band was still missing. The M-band formation is considered the endpoint of myofibrillar
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maturation (Forsgren et al., 1982a, 1982b), and is presumably related to mechanical requirements in the heart pressure load. T-tubule and H-band have not been reported before. Similar mechanical functions are not required in the EBs and may explain the lack of the typical M-band formation. In comparison with the report of Kehat et al. (2001), the presence of these structures may be related to our long culture of differentiated cardiomyocytes (40 days). Human pluripotent stem cells with their potential to differentiate into a wide variety of cell types in culture would be invaluable for studies of some aspects of human embryogenesis and for transplantation medicine. These may serve to define the culture conditions and differential gene expression necessary for cell type-specific differentiation and for the isolation of lineage-restricted stem cells, which could serve as a source of cells for transplantation. Genetic modification of these pluripotent stem cells may allow the generation of universal donor cells or cells that have been customized to meet individual requirements. However, these goals warrant isolation studies and the use of human pluripotent stem cells. Acknowledgments We would like to thank Dr. Muhammad Ashraf and Dr. Safa Alhasani for the critical reading of the manuscript. Also, we gratefully acknowledge the assistance of Dr. Ahmad Vosugh and the staff of the Royan Institute in supporting this research and the staff in the electron microscopy facility of Shahid Beheshti University, in particular Abbas Piriaee and Mrs. Razieh Rohani. This work was supported by the Royan Institute.
References Amit, M., Carpenter, M.K., Inokuma, M.S., Chiu, C.P., Harris, C.P., Waknitz, M.A., Itskovitz-Eldor, J. and Thomson, J.A. (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolongated periods of culture. Dev Biol 227:271–278. Baharvand, H. and Matthaei, K.I. (2003) The ultrastructure of mouse embryonic stem cells. Reprod Biomed Online 7:330–335. Chacko, K.J. (1976) Observations on the ultrastructure of developing myocardium of rat embryos. J Morphol 150:681–709. Doetschman, T., Williams, P. and Maeda, N. (1988) Establishment of hamster blastocyst-derived embryonic stem (ES) cells. Dev Biol 127:224–227. Evans, M.J. and Kaufman, M.H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156. Forsgren, S., Carlsson, E., Strehler, E. and Thornell, L.E. (1982a) Ultrastructural identification of human fetal Purkinje fibres—a comparative immunocytochemical and electron microscopic study of composition and structure of myofibrillar M-regions. J Mol Cell Cardiol 14:437–449.
Forsgren, S., Strehler, E. and Thornell, L.E. (1982b) Differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in the bovine heart: an immuno- and enzyme histochemical study. Histochem J 14:929–942. Hogan, B., Constantini, F. and Lacy, E. (1994) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Kehat, I., Kenyagin-Karsenti, D., Snir, M., Segev, H., Amit, M., Gepstein, A., Livne, E., Binah, O., Itskovitz-Eldor, J. and Gepstein, L. (2001) Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 108:407–414. Lanzendorf, S.E., Boyd, C.A., Wright, D.L., Muasher, S., Oehninger, S. and Hodgen, G.D. (2001) Use of human gametes obtained from anonymous donors for the production of human embryonic stem cell lines. Fertil Steril 76:132–137. Li, M., Zhang, D., Hou, Y., Jiao, L., Zhang, X. and Wang, W.H. (2003) Isolation and culture of embryonic stem cells from porcine blastocysts. Mol Reprod Dev 65:429–434. Manasek, F.J. (1970) Histogenesis of the embryonic myocardium. Am J Cardiol 25:149–168. Martin, G.R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78:7634–7638. Mitalipova, M., Calhoun, J., Shin, S., Wininger, D., Scultz, T., Noggle, S., Venable, A., Lyons, I., Robins, A. and Stice, S. (2003) Human embryonic stem cell lines derived from discarded embryos. Stem Cells 21:521–526. Pain, B., Clark, M.E., Shen, M., Nakazawa, H., Sakurai, M., Samarut, J. and Etches, R.J. (1996) Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities. Development 122:2339–2348. Park, J.H., Kim, S.J., Oh, E.J., Moon, S.Y., Roh, S.I., Kim, C.G. and Yoon, H.S. (2003) Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 69:2007–2014. Pickering, S., Braude, P., Patel, M., Burns, C.J., Bolton, V. and Minger, S. (2003) Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. Reprod Biomed Online 7:353–364. Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. and Bongso, A. (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18: 399–404. Reubinoff, B.E., Pera, M.F., Vajta, G. and Trounson, A.O. (2001) Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 16:2187–2194. Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P., Becker, R.A. and Hearn, J.P. (1995) Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 92:7844–7848. Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P. and Hearn, J.P. (1996) Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 55:254–259. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S. and Jones, J.M. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282: 1145–1147.
r International Society of Differentiation 2004
OR IGI N A L A R T IC L E
Hossein Baharvand . Saeid Kazemi Ashtiani . Mojtaba Rezazadeh Valojerdi . Abdolhossein Shahverdi . Adeleh Taee . Davood Sabour
Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst
Received February 19, 2004; accepted in revised form May 7, 2004
Abstract Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. These cells have, therefore, potential for in vitro differentiation studies, gene function, and so on. The aim of this study was to produce a human embryonic stem cell line. An inner cell mass of a human blastocyst was separated and cultured on mouse embryonic fibroblasts in embryonic stem cell medium with related additives. The established line was evaluated by morphology; passaging; freezing and thawing; alkaline phosphatase; Oct-4 expression; anti-surface markers including Tra-1-60 and Tra-1-81; and karyotype and spontaneous differentiation. Differentiated cardiomyocytes and neurons were evaluated by transmission electron microscopy and immunocyto chemistry. Here, we report the derivation of a new
embryonic stem cell line (Royan H1) from a human blastocyst that remains undifferentiated in morphology during continuous passaging for more than 30 passages, maintains a normal XX karyotype, is viable after freezing and thawing, and expresses alkaline phosphatase, Oct-4, Tra-1-60, and Tra-1-81. These cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers in the presence or absence of recombinant human leukemia inhibitory factor. Royan H1 cells can differentiate in vitro in the absence of feeder cells and can produce embryoid bodies that can further differentiate into beating cardiomyocytes as well as neurons. These results define Royan H1 cells as a new human embryonic stem cell line.
. ) Hossein Baharvand (* Department for Biology of Stem Cells Royan Institute P.O. Box 19395-4644 Tehran, Iran Tel: 0098 21 2402486 Fax: 0098 21 2409314 E-mail: [email protected]
Introduction
Hossein Baharvand Saeid Kazemi Ashtiani Mojtaba Rezazadeh Valojerdi Abdolhossein Shahverdi Adeleh Taee Davood Sabour Department of Embryology Royan Institute P.O. Box 19395-4644 Tehran, Iran Mojtaba Rezazadeh Valojerdi Department of Anatomy Tarbiat Modarres University Tehran, Iran U.S. Copyright Clearance Center Code Statement:
Key words cardiomyocyte embryonic stem cells human neuron ultrastructure
Embryonic stem (ES) cells are most frequently derived from the inner cell mass (ICM) of blastocysts (Evans and Kaufman, 1981; Martin, 1981). In fact, the ICM is used to give rise to an ES cell line that remains undifferentiated and is pluripotent, meaning the cells have the potential to develop into any cell type from all three germ layers both in vivo and in vitro. ES cells or ES cell-likes have been produced in mice and other animal models, including chickens (Pain et al., 1996), hamsters (Doetschman et al., 1988), and pigs (Li et al., 2003). In primates, ES cell lines have been produced in the common marmoset (Thomson et al., 1996) and the rhesus monkey (Thomson et al., 1995). In 1998, Thomson and coworkers reported the first ES cell lines produced from human blastocyst stage embryos. Recently, the establishment of new human embryonic stem
0301–4681/2004/7205–224 $ 15.00/0
225
cell lines (Reubinoff et al., 2000; Lanzendorf et al., 2001; Mitalipova et al., 2003; Park et al., 2003; Pickering et al., 2003) has been reported. The ability to direct the differentiation of human ES cell lines into a population of a specific cell type such as insulin-producing pancreatic cells may provide a treatment for many diseases such as diabetes. The characteristics of ES cells indicate that these cells, in addition to use in developmental biology studies, have the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics applications. As a result, interest in producing ES cell lines in the human has increased over the past few years. In the present study, we report the establishment of a new human ES cell line: Royan H1. The pluripotency of this new cell line was evaluated in vitro by conventional tests.
Methods Cell line isolation A 6-day-old human blastocyst was donated to the study following the approval of the institutional review board and obtaining an informed consent from the couple undergoing in vitro fertilization treatment. The blastocyst was recovered through culturing in sequential culture media (Rs1 and Rs2; Vitrolife, Go¨teborg, Sweden) in accordance with the manufacturer’s instructions. Zona pelucida having been digested by acidic Tyrod’s solution (100 mM NaCl, 2.6 mM KCl, 1.6 mM CaCl21H2O, 4.9 mM MgCl212H2O, 5.4 mM glucose, 0.1 mM polyvinyl pyrolidone; Hogan et al., 1994), the blastocyst was cultured on a mitomycinC (Sigma, Taufkirchen, Germany) mitotically inactivated mouse embryonic fibroblast (MEF) feeder layer (isolated from day 13.5 post-coitum fetuses of NMRI outbred strain used at 75,000 cell cm 2) in gelatin (Sigma)-coated tissue culture dish (Falcon, Franklin Lakes, NJ, USA). Culture medium consisted of 80% knockout Dulbecco’s modified Eagle’s medium (Gibco, Karlsruhe, Germany) supplemented with 20% ES-qualified fetal calf serum (Gibco), 2 mM glutamine, (Gibco), 0.1 mM b-mercaptoethanol (Sigma), 1% nonessential amino acid stock (Gibco), 100 units ml 1 penicillin, and 100 mg ml 1 streptomycin (Gibco). After 1 day, the embryo attached on feeder cells and the inner cell mass was isolated with a mouth-controlled pipette and re-cultured on fresh MEF. During the isolation and early stages of ES cell cultivation, the medium was supplemented with human recombinant leukemia inhibitory factor (hLIF) at 1,000 units ml 1 (Chemicon, Hofheim/ TS, Germany), and hLIF was replaced by insulin-transferrin-selenite (Gibco). Ten days after the initial plating, an ES-like colony was dissociated with a combined approach of mechanical slicing with a pipette, followed by exposure to 10 mg ml 1 dispase (Gibco). The resulting colonies were grown in 5% CO2 and 95% humidity, and they were further propagated in clumps of 200–500 stem cell-like cells on MEF about every 7 days. Colonies were also periodically selected and cryopreserved by open-pulled straw method (Reubinoff et al., 2001) and stored in liquid nitrogen. Stem cell characterization Karyotype analysis: Karyotype analysis was carried out on the cells after passages 5 and 29. hES colonies were incubated with 10 mg/ml colcemid solution (Gibco) for 2 hr at 371C and in 5%
CO2 in air atmosphere. The cells were washed with PBS, trypsinized, and neutralized with ES medium. Then, pellets were resuspended and incubated with 0.075 M KCl for 17 min at 371C. Having been treated with hypotonic solution, the cells were fixed with 3:1 methanol:glacial acetic acid three times and dropped onto pre-cleaned chilled slides. Chromosome spreads were Giemsa banded and photographed. At least 20 metaphase spreads and five banded karyotypes were evaluated for chromosomal rearrangements. Cell surface markers: Colonies were evaluated for alkaline phosphatase (AP) activity using the alkaline phosphatase substrate kit (Sigma) in accordance with the manufacturer’s instructions. Tumor rejection antigens (Tra-1-60 and Tra-1-81) were detected by immunocytochemistry with specific primary antibodies (gifts of Peter Andrews, University of Sheffield, UK) and localized with rabbit anti-mouse immunoglobulins conjugated to fluorescein isothiocyanate (Sigma). Oct-4 expression: To evaluate expression of Oct-4 transcription factor, we used reverse transcriptase-polymerase chain reaction (RT-PCR) on colonies consisting predominantly of stem cells at passage 30. RNA was isolated from established ES cells using RNX-Plus (Fermentas, GmbH, Germany) and transcribed into cDNA using random hexamers and reverse transcriptase (Fermentas). Primer sets for Oct-4 were: forward primer, 5 0 -CTTGCTGCAGAAGTGGGTGGAGGAA-3 0 and reverse primer, 5 0 -CTGCAGTGTGGGTTTCGGGCA-3 0 (Reubinoff et al., 2000). Primers for aˆ-actin were used as positive control. Primer steps for b-actin were: 5 0 -CGCACCACTGGCATTGTCAT-3 0 (forward), 5 0 -TTCTCCTTGATGTCACGCAC-3 0 (reverse) (Reubinoff et al., 2000). PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized and photographed on a UV transluminator (UVItec, Cambridge, UK). In vitro differentiation: (1) Spontaneous differentiation into cardiomyocytes and neurons: The ES cells were cultured in the absence of MEF feeder layers in hanging drops to embryoid body (EB) formation for 2 days and in bacterial dishes for 5 or 20 days and grown in tissue culture plates for several days in human ES medium. The number of beatings of differentiating cardiomyocytes was counted under an inverted microscope. (1-a) Immunocytochemistry. Differentiated cells and neurons were fixed by 4% paraformaldehyde (Sigma) and stained with antibodies against microtubule-associated protein (MAP 2[a1b]; 1:200; Sigma) and neurofilament (DAKO, Glostrup, Denmark). The second antibody was fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Sigma). Direct immunocytochemistry was done with antibody against neuron-specific enolase labeled with horseradish peroxidase (DAKO). The antibody detected the use of 3,3-diaminobenzidine tetrahydrochlorides (DAKO). (1-b) Transmission electron microscopy (TEM). Forty days after plating EBs, we processed differentiated cardiomyocytes for TEM. Primary fixation: 2% glutaraldehyde in 0.1 M phosphate buffer saline (pH 7.4) for 2 hr and then two 30-min washes in the same buffer. Secondary fixation: 1% osmium tetroxide in the same buffer for 1.5 hr, followed by two washes in the buffer. The samples were washed three times in water (5 min each), dehydrated in an ethanol series—30%, 50%, 70%, 90%, and 95% for 15 min each, and then three times for 15 min each in 100% ethanol. Infiltration into Spurr’s resin was achieved by an ethanol:resin series: first in 50:50 (2 hr) then 100% resin (2 hr), and after that in 100% resin (overnight) and again in fresh resin (2 hr). The samples were then embedded in molds containing 100% resin and polymerized at 701C overnight. After polymerization, sections of about 80 nm were cut and stained with lead citrate for 8 min.Micrographs of transverse sections of ES and MEF cells were taken on plate films on a Zeiss EM900 (Oberkochen, Germany) transmission electron microscope. (2) Evaluation of human chorionic gonadotropin (hCG) and a-fetoprotein production. Colonies at passages level 15 and 19 were allowed to grow on mitotically inactivated MEFs to confluency (about 2 to 4 weeks). The medium was replaced every day after 2 weeks of differentiation, and medium in triplicate wells, conditioned
226 for 24 hr, was assayed by ELISA for a-fetoprotein and the hCG, markers of endoderm lineage and trophoblast differentiation, respectively. Conditioned medium of MEF was used as control group.
Results Stem cell characterization Isolation of ICM from a blastocyst yielded one cell line with ES morphological, biochemical, and/or immunocytochemical characteristics consistent with previously characterized pluripotent stem cell lines (Fig. 1). The line was named Royan H1 (Royan in Persian means embryo). The resulting cells had a high nucleus to cytoplasm ratio, prominent nucleoli, and colony morphology similar to that of rhesus monkey and human ES cells. The derived colonies were further propagated by mechanical disaggregation of clumps, which were re-plated onto fresh feeder cell layers. Whereas LIF was used during the early phase of the establishment of the cell lines, it was subsequently found to have no effect on the growth or differentiation of established culture (not shown). However, supplementation of culture medium with cultivation in the presence of insulin-transferrin-selenite increased undifferentiated colonies under standard growth conditions. Cell line Royan H1 was grown for 30 passages in vitro, and the cell line still consisted mainly of cells with the morphology of undifferentiated ES cells (Figs. 1A,1B). The cell line was successfully frozen and thawed. An evaluation of the cell line for cell surface markers that characterize undifferentiated human ES cell lines showed that the line stained positive for alkaline phosphatase (Fig. 1C). The line was also found to be positive for Tra-1-60 and Tra-1-81, both markers of undifferentiated human ES cell line (Figs. 1D,1E). Oct-4 is POU domain transcription factor, whose expression is limited in the mouse to pluripotent cells. Oct-4 is also expressed in human ES cells, and its expression is down-regulated when these cells are differentiated. Using RT-PCR to carry out mRNA analysis on isolated colonies consisting mainly of stem cells, we showed that Royan H1 also expressed Oct-4 (Fig. 1F). Karyotypic analysis, carried out at passages 5 and 27, indicated apparently normal XX human karyotype. In vitro differentiation Royan H1 maintained the potential to form derivatives of all three embryonic germ layers in vitro. The ES cells differentiated spontaneously when cultured in the absence of mouse embryonic fibroblast feeder layers, in hanging drops, and in bacterial culture dishes or grown in tissue culture plates for several days. We observed
cultures of cells or single cells with elongated processes that extended out from their cell bodies, forming networks as they contacted other cells in 25% (10/41) of embryoid bodies (Fig. 2A). These cells and the processes stained positively with antibodies against neuronspecific enolase (Fig. 2B), MAP-2(a1b) (Fig. 2C), and neurofilament proteins (Fig. 2D). Furthermore, we observed 10% (3/30) of embryoid bodies were differentiated into beating cells (42 2 beat/min; mean SEM). The ultrastructure characteristics of differentiated cardiomyocytes were studied. Transmission electron microscopy of these cells revealed mononuclear cells, with parallel arrays of myofibrillar bundles oriented in an irregular manner in some cells (Fig. 3A), whereas more mature sarcomeric organization was apparent in others (Figs. 3A,3B). In some areas, the formation of early and more developed and distinct Z bands, the I- and the A-band, could be observed (Fig. 3B). T-tubules or invaginations of the surface sarcolemma and H-band were observed (Fig. 3C). The diameter of the Z line corresponds to the diameter of the myofibrillar arrangements; the spacing between two Z lines was about 2 mm. The intercalated disc, another cellular structure that characteristically appears during in vivo cardiomyocyte differentiation, was observed in many of the differentiating cells (Fig. 3C). Therefore, gap junctions, macula adherence or spot desmosomes, and facia adherence were observed on the intercalated disc (Fig. 3C). After Royan H1 cells were allowed to differentiate for 2 weeks, both a-fetoprotein (41,000 IU/ml) and hCG (435 mIU/ml) were detected in conditioned cultured medium, indicating endoderm and trophoblast differentiation (Thomson et al., 1998). No hCG or afetoprotein was detected in conditioned medium from MEFs.
Discussion In 1998, Thomson and coworkers reported the first ES cell lines produced from human blastocyst stage embryos (Thomson et al., 1998). Within this report, they proposed criteria that true primate ES cell lines should meet. As in the mouse model, these criteria include derivation from the pre-implantaton embryo, prolonged undifferentiation in vitro, and the ability to form all three embryonic germ layers. However, the investigators did not feel it was ethical or feasible to produce human chimeras to determine that ES cell lines could contribute to the germ line. In the study presented here, we report the production of one new human ES line. This line was derived and grown as described. The derived ES cell line was successfully maintained for more than 30 passages, retain-
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Fig. 1 Stem cell characterizations: colony morphology (A). High magnification of hES cells (B). Detection of surface marker
expression, alkaline phosphatase (C), Tra-1-60 (D), Tra-1-81 (E), and Oct-4 (F).
Fig. 2 Phase contrast appearance and marker expression in cultures of neurons derived from hES. Phase contrast micrograph of differentiated cells (A). Direct immunocytochemistry (B) and indirect immunofluorescence (C, D) microscopy of differentiated cells decorated with antibodies against neuron-specific enolase (B), MAP2(a1b) (C), and neurofilament protein (D).
ing the morphology reported previously for both human (Thomson et al., 1998) and nonhuman (Thomson et al., 1995, 1996) primate ES cell lines. Human and nonhuman primate ES cells share a similar morpholo-
gy, which is distinct from mouse ES cells (Evans and Kaufman 1981; Thomson et al., 1995, 1998; Baharvand and Matthaei, 2003). Human ES cells form relatively flat, compact colonies that passage by a combined ap-
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Fig. 3 Ultrastructural analysis of hES-derived cardiomyocytes. Transmission electron microscopy of sectioned beating EB, 40 days after plating. Organized myofibrillar bundles can be seen in most myocytes (A). Higher magnification of an organized sarcomere
(B). Intercalated disc (C) and high-power electron micrograph showing a gap junction and a T-tubule. D, desmosome; F, myofibrillar bundle; G, gap junction; I, intercalated disk; M, mitochondria; N, nucleus; T, T-tubule. The scale bar represents 0.5 mm.
proach of mechanical slicing with a pipette followed by exposure to dispase (Reubinoff et al., 2000) or dissociate to patches of cells by collagenase (Thomson et al., 1998), whereas mouse ES cells form tight, more spindleshaped colonies that passage by single cells. Maintenance of human ES cell colony density and derivation of clonal cell lines are complicated by difficulties associated with the expansion of a single cell (Amit et al., 2000). Moreover, human ES cells grow more slowly than mouse ES cells. In addition, the lines express cell surface markers such as Tra-1-60 and Tra-1-81 as well as AP and transcription factor Oct-4, demonstrating that the line is undifferentiated (Thomson et al., 1995). Such a finding has also been previously reported for human ES cells (Thomson et al., 1998). The most useful and important property of these cells is their ability to differentiate in vitro into ectodermal, endodermal, and mesodermal derivatives. As with mouse ES cells, human pluripotent stem cells can form EBs. These structures appear to recapitulate the normal developmental processes of the early embryonic stage and promote the cell-cell interaction required for cell differentiation. Typically, mouse EBs are surrounded by a layer of endoderm and contain a heterogeneous
mixture of cell types. Morphologically, human EBs somehow resemble those generated from mouse ES cultures. Identification of cell derivatives of the three embryonic germ layers (such as neurons, cardiomyocytes, and expression of a-fetoprotein) in human EB cultures suggests that these cells are pluripotent and are capable of in vitro differentiation. Ultrastructural analysis of differentiating cardiomyocytes showed that these cells were mainly mononuclear and round or rod shaped, and that these contained different degrees of myofibrillar bundle organization and exhibited intercalated discs. These results are consistent with ultrastructural properties of early-stage cardiomyocytes. Previous studies demonstrated that, during in vivo cardiogenesis, myofibrils were initially distributed in sparse, irregular myofibrillar arrays, which gradually matured into parallel arrays of myofibrils and ultimately aligned into densely packed sarcomeres (Manasek, 1970; Chacko, 1976). In our study, the Z line, the I- and the A-band were distinct. T-tubules and H-band were distinguishable in some cells; however, in comparison with adult cardiomyocytes, the M-band was still missing. The M-band formation is considered the endpoint of myofibrillar
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maturation (Forsgren et al., 1982a, 1982b), and is presumably related to mechanical requirements in the heart pressure load. T-tubule and H-band have not been reported before. Similar mechanical functions are not required in the EBs and may explain the lack of the typical M-band formation. In comparison with the report of Kehat et al. (2001), the presence of these structures may be related to our long culture of differentiated cardiomyocytes (40 days). Human pluripotent stem cells with their potential to differentiate into a wide variety of cell types in culture would be invaluable for studies of some aspects of human embryogenesis and for transplantation medicine. These may serve to define the culture conditions and differential gene expression necessary for cell type-specific differentiation and for the isolation of lineage-restricted stem cells, which could serve as a source of cells for transplantation. Genetic modification of these pluripotent stem cells may allow the generation of universal donor cells or cells that have been customized to meet individual requirements. However, these goals warrant isolation studies and the use of human pluripotent stem cells. Acknowledgments We would like to thank Dr. Muhammad Ashraf and Dr. Safa Alhasani for the critical reading of the manuscript. Also, we gratefully acknowledge the assistance of Dr. Ahmad Vosugh and the staff of the Royan Institute in supporting this research and the staff in the electron microscopy facility of Shahid Beheshti University, in particular Abbas Piriaee and Mrs. Razieh Rohani. This work was supported by the Royan Institute.
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