Human Cell 2006; 19: 65–70
doi: 10.1111/j.1749-0774.2006.00011.x
Blackwell Publishing, Ltd.
FEATURE: REGENERATIVE MEDICINE
Establishment and therapeutic use of human embryonic stem cell lines Hirofumi SUEMORI Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
Abstract Embryonic stem (ES) cell lines, which are derived from the inner cell mass of blastocysts, proliferate indefinitely in vitro, retaining their potency to differentiate into various cell types derived from all of the three embryonic germ layers: the ectoderm, mesoderm and endoderm. Establishment of human ES cell lines in 1998 has indicated the great potential of ES cells for applications in medical research and other purposes such as cell transplantation therapy. Careful assessment of safety and effectiveness using proper animal models is required before such therapies can be attempted on human patients. Monkey ES cell lines provide valuable models for such research. Key words: differentiation, human embryonic stem cells, regerative medicine.
INTRODUCTION Due to their ability to proliferate indefinitely and ongoing research that is making possible their in-vitro differentiation into an increasingly wide variety of mature cell types, human embryonic stem (ES) cell lines are beginning to show promise as an unlimited source of functional cells and tissue required for cell-transplantation therapy and regenerative medicine (Fig. 1). It is very important to determine reliable methods to establish and maintain ES cells, and also to devise various methods of inducing their differentiation into specific cell types. Already, mostly using mouse ES cells, there have been reports of the production of glia, cardiac muscle, hematopoietic cells, endothelial cells and various types of neurons, including dopamine-producing cells.1,2 All of these cell types have important medical applications. Recently, there have Correspondence: Hirofumi Suemori, Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Email:
[email protected] Received 7 January 2006; accepted 21 January 2006.
© 2006 The Author Journal compilation © 2006 Japan Human Cell Society
been increasing numbers of reports of production of such cell types using human ES cells.3 Clinical trials testing the efficacy of cells derived from human ES cells in the treatment of a variety of severe diseases are expected to commence in the near future. However, careful assessment of safety and effectiveness using proper animal models is required before such therapies can be attempted on human patients. Moreover, the nature of human ES cells should be investigated more closely, because human ES cells are different from mouse ES cells in many respects.
MOUSE, MONKEY AND HUMAN EMBRYONIC STEM CELLS Embryonic stem cell lines were established from blastocysts of the rhesus monkey and marmoset in 1995 and cynomolgus monkey in 2001.4–7 Because they are phylogenically close to humans, macaques such as rhesus and cynomolgus monkeys are thought to be more suitable as animal models than the marmoset. Due to its smaller size, however, the marmoset is significantly easier to handle and breed. Human ES cell lines were first established in 1998.8 The methods for establishment of non-human and human ES-cell lines were almost the same as those
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Figure 1 Therapeutic use of human embryonic stem (ES) cells.
used to derive mouse ES-cell lines.9 All three of these cell lines share the same general characteristics; however, some differences do exist between mouse and primate ES cells. First, primate ES cell colonies are flatter than the compact, domed mouse ES-cell colonies. Second, the expression of some stem-cell markers is different. Third, addition of leukemia inhibitory factor (LIF) in the culture medium has no effect on the maintenance of primate ES cells.6 Finally, mouse ES cells can grow after dissociation into single cells, whereas the efficiency of clonal expansion of primate ES cells is extremely low.10 These last two features are serious problems that make it difficult to maintain and manipulate primate ES cells. In addition, primate ES cells grow much more slowly, with a cell-cycle of about 30 h, compared to 12 h for mouse ES cells.
ESTABLISHMENT AND MAINTENANCE OF HUMAN EMBRYONIC STEM CELL LINES As reported in previous studies, the methods used to establish human and monkey ES cell lines are essentially
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the same as those used to generate mouse ES cells. Briefly, inner cell masses isolated from blastocysts are cultivated on a feeder layer of mouse embryonic fibroblasts or other cells. Stem-cell colonies are selected manually from differentiated derivatives. After consecutive selection of stem cells, undifferentiated ES cells can be maintained stably in culture. These procedures are identical in primates and mice. Under our conditions, the efficiency with which we can establish ES cell lines from cynomolgus monkey blastocysts is about 60–80%.7 This optimized rate is higher than that typically seen in mice. By using the protocol developed in monkey ES cell establishment, we derived three human ES cell lines from three blastocysts. Spontaneous differentiation of stem cells and low efficiency in subculturing were the major problems in developing and stably maintaining human ES cell lines. We had found that the spontaneous appearance of differentiated cells is reduced remarkably when fetal bovine serum (FBS) is replaced with Knockout Serum Replacement (KSR, Invitrogen, CA, USA) in the culture medium for monkey ES cells, and that KSR-containing medium performs well in the establishment of monkey ES cell lines. Human ES cells also can be established and maintained
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Therapeutic use of human ES cells
in their undifferentiated state for a longer period without periodical collection of the stem cell colonies when using KSR-containing medium. This is also valuable for the therapeutic use of human ES cells, because there is a concern regarding contamination of FBS in the culture medium with pathogenic agents. Similar to other primate ES cell lines, the human ES cell lines exhibit a very low plating efficiency when dissociated into single cells using a trypsin solution. Therefore, limited dissociation into cell clusters of 50–100 stem cells during subculturing was required to enable continued growth. We found a method for efficient subculturing using 0.25% trypsin partially disabled with 1 mM CaCl2. This enabled well-controlled and reproducible dissociation of the human ES cell colonies throughout the whole culture dish for routine and efficient subculturing. Human ES cells formed flat monolayer colonies with rather distinct cell borders (Fig. 2). Each cell had a bright nucleus with prominent nucleoli and a high nucleus/ cytoplasm ratio. Such morphology was very similar to that of human ES cell lines previously reported. They were stably maintained for more than 1 year without changes in their morphology or growth properties.
EXPRESSION OF STEM-CELL MARKERS OF HUMAN EMBRYONIC STEM CELLS Examination of cell-surface marker expression is useful for characterizing human ES cell lines. Cell-surface antigens such as SSEA-1, 3 and 4, TRA1–60 and –81, and a cell’s alkaline phosphatase activity are usually used, because these are detected easily by immunostaining or chromogenic enzyme detection assays (Fig. 2). Undifferentiated human ES cells express alkaline phosphatase activity, as well as SSEA-4, SSEA-3 TRA1-60 and TRA1-81 antigens, but not SSEA-1 antigen. Whereas human and rhesus ES cells have been reported to express SSEA-3 antigen, cynomolgus ES cells were negative for SSEA-3 by immunostaining. The expression in undifferentiated ES cells of genetic markers such as Oct-3/4, Cripto, Rex-1 and Nanog can be detected by RT-PCR. Embryonic stem cell lines had normal karyotype. Two cell lines had the female and one had the male karyotype. Cell lines were subjected to long-term cultivation using the enzymatic bulk transfer method, as some reports claimed that enzymatic dissociation of human ES cells results in rapid accumulation of chromosomal abnormalities. However, ES cell lines retained a normal karyotype after at least 100 passages in culture by bulk passage.
© 2006 The Author Journal compilation © 2006 Japan Human Cell Society
CRYOPRESERVATION OF HUMAN EMBRYONIC STEM CELL LINES Efficiency of cryopreservation of human ES cell lines has been reported to be very low and this makes it difficult to distribute cell lines. We previously reported an efficient cryopreservation method for primate ES cell lines based on vitrification method, and it showed high survival rates upon cryopreservation of a human ES cell line.11 We applied this improved method to the other two ES cell lines. Vitrification cryopreservation efficiency was much higher than that achieved by the conventional slow-cooling method. Survival rate by the slow-cooling method was less than 1%, whereas those by our modified vitrification method was 15–20%.
DIFFERENTIATION OF EMBRYONIC STEM CELLS IN V ITRO AND IN V IVO Pluripotency is the most important characteristic of ES cells. When ES cells were allowed to grow to higher densities to induce differentiation, several kinds of differentiated cells were observed, including vesicular epithelia resembling the visceral endoderm or yolk sac, mesenchymal cells showing outgrowth from cell clumps, and clusters of neurons and pigment cells. Formation of embryoid bodies (EB) is an effective method to induce the differentiation of ES cells into various cell types. EB are formed by culturing ES cell aggregates in Petri dishes. Since the proliferation of undifferentiated human ES cells is dependent on the feeder layer, and leukemia inhibitory factor (LIF) cannot support their growth, ES cells start differentiation immediately after detaching from the feeder layer. Thus, undifferentiated stem cells do not proliferate enough to form EB if the starting cell aggregates are too small. Therefore, it is important to start from larger ES cell aggregates to obtain good EB. ES-cell aggregates will form simple EB in a few days. They can be cultured in suspension until apparent cell differentiation is observed. In 2 to 3 weeks, beating heart muscle and hematopoietic cells may be observed. Alternatively, EB can be plated from the suspension culture to a tissue-culture dish at any time. EB will attach to the dish and undergo cell differentiation into various tissues such as neurons and cardiac muscles. Teratoma formation from ES cells is a simple and reliable method of analyzing the differentiation potency of ES cells. To produce teratomas, human ES cells are transplanted into SCID mice. About 107 cells are injected subcutaneously or intraperitoneally into SCID mice. Teratoma formation becomes apparent in 2 to 3 months.
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Figure 2 Human embryonic stem (ES) cells and expression of stem cell markers. (a, b) Phase contrast views of a human ES cell line, KhES-1 showing typical hES cell morphology. Cytochemical- and immunostaining of undifferentiated KhES-1 cells: (c) alkaline phosphatase; (d) SSEA-4; (e) SSEA-3; (f ) TRA-1-60.
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© 2006 The Author Journal compilation © 2006 Japan Human Cell Society
Therapeutic use of human ES cells
Figure 3 Differentiation in teratoma: (a) gross appearance of teratoma formed from human embryonic stem cells; (b) gutlike epithelia; (c) neural epithelia; and (d) muscle.
Histological examination of these teratomas can reveal various tissues derived from all three embryonic germ layers. Ectodermal tissues containing neurons, glia, glands, and epithelia are most commonly observed. Mesodermal tissues such as muscle, cartilage, and bone are also formed frequently. Endodermal tissues such as gut epithelium are formed relatively less frequent (Fig. 3).
GENETIC MODIFICATION OF HUMAN EMBRYONIC STEM CELLS Generally, transformation efficiency by electroporation or lipid-mediated gene transfer in primate ES cells is much lower than in mouse ES cells. Although efficient gene transfer into monkey ES cells using a lentiviral vector has been reported,12 an advantage of electroporation and lipofection is the avoidance of exposure to biohazards associated with viral gene transduction. Electroporation is preferable for gene targeting as it is difficult to achieve efficient homologous recombination using viral vectors
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and lipofection. Therefore, electroporation is considered to be the most suitable method for introducing genetic modifications into primate ES cells. For preliminary research, we optimized electroporation conditions to introduce foreign genes into cynomolgus monkey ES cells.13 Using an SV40-neo expression vector, about 100 stable clones can be consistently produced from 107 monkey ES cells after drug selection. Differences in efficiency, however, were observed for other ES cell lines. In addition, we compared the transcriptional activities of the PGK-1, CMV, and SV40 promoters in cynomolgus monkey ES cells. Although the PGK-1 and SV40 promoters work efficiently in monkey ES cells, the CMV promoter displayed significantly lower activity compared to the others. In-vitro genetic modification of ES cells is valuable for basic and preclinical research using primate ES cells. In mouse ES cells, it has been shown that constitutive expression of specific genes promotes their differentiation into defined cell types, as seen in the generation of
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dopaminergic neurons and insulin-producing cells.14–16 Modification of specific genes, such as the HLA genes, might also create ES cells that are not recognized by the immune system of the transplanted host.
PROBLEMS IN CLINICAL UTILIZATION OF HUMAN EMBRYONIC STEM CELLS Several problems have been identified in the clinical use of human ES cells. Control of tumorigenesity of ES cells, contamination of pathogenic agents during ES cell culture and immune rejection of grafted tissues are frequently cited. Because ES cells proliferate indefinitely, contamination of undifferentiated cells into transplants would cause formation of teratomas. Then, functional cells derived from ES cells should be purified enough that no stem cells remain in the grafts. Use of the feeder and medium containing animal-derived factors such as FBS has the potential to cause infectious diseases in patients and should be avoided. The development of a complete synthetic medium and feeder-free culture system is required. Rejection of grafts by the host immune system is recognized as one of the most serious problems in clinical application. As described above, genetic modification of ES cells is one way to solve this problem. Establishment of ES cell lines from embryos produced by patients’ somatic cell nuclear transfer into enucleated oocytes is considered an ideal method to create personalized ES cells that will not be rejected by patients. However, this therapeutic cloning seems to require highly refined techniques and is not practical so far. Instead of therapeutic cloning, reprogramming of somatic cells by fusion with ES cells may be used to create personalized ES cells.
CONCLUSIONS Human ES cells are believed to be powerful tools for celltransplantation therapies and also drug development. Although many problems remain to be solved before they can be used in therapeutic applications, recent progress in the field convinces us that clinical trials will be started in the not too distant future.
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