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Improved cryopreservation of human embryonic stem cells with trehalose
RBMOnline - Vol 11. No 6. 2005 733–739 Reproductive BioMedicine Online; www.rbmonline.com/Article/1944 on web 10 October 2005
Article Improved cryopreservation of human embryonic stem cells with trehalose Wen Jie Zhang graduated from Shanghai 2nd Medical University (China) and was awarded his MD degree in 1992. He did his PhD research in Japan and took 4 years of post-doctoral training at Washington University School of Medicine in St Louis, USA. He worked with Dr Kyunghee Choi on the early development of hematopoietic and endothelial cells utilizing an in-vitro differentiation model of embryonic stem (ES) cells. He returned to China in 2004 and is now working on the differentiation of human ES cells. The ultimate goal of his current work is to provide seed cells (such as endothelial cells, chondrocytes) from differentiated ES cells for tissue engineering.
Dr Wen Jie Zhang Chun Fang Wu2, Hsiao Chien Tsung2, Wen Jie Zhang2, Yan Wang, Jun Hong Lu, Zheng Ya Tang, Yan Pin Kuang, Wei Jin, Lei Cui, Wei Liu, Yi Lin Cao1 Shanghai Key Laboratory of Tissue Engineering, Shanghai 9th People’s Hospital, Shanghai 2nd Medical University, Shanghai 200011, China 1 Correspondence: Tel: +86 21 63138341, ext. 5192; Fax: +86 21 53078128; e-mail: [email protected] 2 These authors contributed equally to this work
Abstract Human embryonic stem (ES) cells have been established either from fresh or frozen embryos. The recovery rates of undifferentiated human ES cells after cryopreservation with conventional slow-rate freezing and rapid-thawing methods are relatively low. The purpose of this study was to improve cryopreservation efficiency by modifying conventional methods with addition of trehalose. Immature oocytes donated from patients undergoing IVF treatment were utilized to generate blastocysts. One human ES cell line (named hES1) was established and characterized in detail. The hES1 cells expressed regular human ES cell markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, tumour rejection antigens TRA-1–60, TRA1–81 and octamer-binding transcription factor Oct-4 with high levels of alkaline phosphatase and telomerase activities. Cells could be differentiated to form teratomas in vivo. With slow-rate freezing and rapid-thawing methods modified by adding trehalose, the recovery rate of undifferentiated hES1 cells has been greatly improved from 15 to 48%. Cells retained pluripotency with normal karyotype after thawing. The results indicated that the use of trehalose is efficient and convenient for cryopreservation of human ES cells. Keywords: cryopreservation, human embryonic stem cells, pluripotency, trehalose
Embryonic stem (ES) cells are derived from the inner cell mass (ICM) cells of the preimplantation blastocyst. Cells are able to self-renew indefinitely in vitro, yet still retain a normal karyotype and the capacity for differentiation into a wide variety of somatic tissues of all three germ layers. Given the selfrenewal potential of ES cells, combined with their pluripotent differentiation capacity, ES cells can potentially provide an alternative cell source for cell therapy, gene therapy and tissue engineering.
(Reubinoff et al., 2000; Richards et al., 2002; Mitalipova et al., 2003; Park et al., 2003, 2004; Baharvand, 2004). To date, there are probably more than 120 human ES cell lines worldwide, 78 of which have been included in the National Institutes of Health database (for more information see http://stemcells.nih. gov/research/registry/). Unfortunately, numerous newly derived human ES cell lines are stored at early passage and are not fully characterized. Fewer than 20 cell lines are currently available for scientists. In addition, it is important to note that there are still no very efficient protocols for cryopreservation of human ES cells, which would prevent the widespread use of these cells in research and clinical applications.
Since the first establishment of human ES cells by Thomson and colleagues (Thomson et al., 1998), more new human ES cell lines have been established from either fresh or frozen embryos
Cryopreservation of human ES cells has been reported to be difficult. Slow-rate freezing and rapid-thawing methods are commonly used for cryopreservation of cell lines (Freshney,
Introduction
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Article - Cryopreservation of human embryonic stem cells - CF Wu et al. 1994). These methods are efficient for the cryopreservation of mouse ES cells, but unsuited for human ES cells. Following these methods, the survival of undifferentiated human ES cells after thawing is usually very poor (~10%); most of the cells either differentiate or die (Reubinoff et al., 2000). Kim et al. reported that the recovery rate of human ES cells after slow-rate freezing and rapid thawing could be improved by adding type IV collagen or laminin to the freezing medium (Kim et al., 2004). Another means of preserving human ES cells is the vitrification method. Reubinoff et al. have improved cryopreservation efficiency by using the open pulled vitrification method (Reubinoff et al., 2001). Recently, Richards et al. described a safe, xeno-free cryopreservation protocol for human ES cells involving vitrification in closed sealed straws. Cells exhibited high survival rates and low differentiation rates after thawing (Richards et al., 2004). However, vitrification protocols are extremely labour intensive. It is difficult to handle bulk quantities of cells using this technique. In the present study, one human ES cell line (named hES1) has been established and characterized in detail. An efficient and convenient cryopreservation protocol for human ES cells has been established by modifying conventional slow-rate freezing and rapid-thawing methods by adding trehalose.
Materials and methods Establishment of hES1 cells Spermatozoa and immature oocytes were donated by couples undergoing IVF treatment with informed consent. Approval was obtained for this study from the Internal Review Board on Human Subjects Research and Ethics Committees (Shanghai 9th People’s Hospital, Shanghai, China). The mature oocytes resulted from the culturing of immature oocytes in Global medium (LifeGlobal, Guelph, Ontario, Canada) overnight were fertilized by intracytoplasmatic sperm injection, and then cultured to blastocyst stage in Global medium. ICM cells were isolated from day 6 blastocysts by immunosurgery as previously described (Solter and Knowles, 1975). Cells were then transferred onto a feeder layer of mitomycin-C (Sigma, St Louis, MO, USA) inactivated mouse embryonic fibroblasts (MEF) in human ES cell culture medium. The medium consisted of knockout Dulbecco’s modified Eagle’s medium (KO-DMEM; Invitrogen, Carlsbad, CA, USA), supplemented with 20% knockout serum replacement (Invitrogen) or ES culture grade fetal bovine serum (Hyclone, Logan, UT, USA), 1% non-essential amino acid, 50 IU/ml penicillin and 50 μg/ml streptomycin, 2 mmol/l L-glutamine, 0.1 mmol/l β-mercaptoethanol (all from Invitrogen), and 4 ng/ml human recombinant β-fibroblast growth factor (R and D Systems, Minneapolis, MN, USA). After 10–15 days, the ICMderived mass was physically split into 4–5 clumps using microneedles. The clumps were then transferred to a new well that was covered with fresh MEF feeder layer. Physical splitting was carried out for routine passaging.
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Characterization of hES1 cells Alkaline phosphatase (AKP) staining AKP activity was determined using the method described by Tsung et al. (2003). Nitrobluetetrazolium and s-bromo-4chloro-3-indolyl phosphate were purchased from Sigma.
Cell surface markers for human ES cells Immunofluorescent staining was performed as described by Solter and Knowles (1987). Monoclonal antibodies against stage specific embryonic antigens SSEA-1, SSEA-3 and SSEA4 were purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA). Antibodies against tumour rejection antigens TRA-1–60 and TRA-1–81 were obtained from Chemicon. Rabbit polyclonal antibody against Oct-4 was made in the laboratory (Tsung et al., 2003). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody and FITCconjugated goat anti-rabbit IgG antibody were purchased from Dakocytomation (Carpenteria, CA, USA).
Reverse transcriptase-polymerase chain reaction (RTPCR) analysis Total RNA was purified from 1.6 × 105 cells, reverse transcribed into cDNA with an RT-PCR kit (TaKaRa, Shiga, Japan). Human embryonic fibroblast (hEF) cells and Tera-2 cells were used as negative and positive controls respectively. Primer sequences of octamer-binding transcription factor Oct-4 used in the study were as follows: forward, CGR GAA GCT GGA GAA GGA GAA GCTG; reverse, AAG GGC CGC AGC TTA CAC ATG TTC. The PCR cycles consisted of an initial denaturation step at 95°C for 2 min, followed by 35 amplification cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 60 s, and a final extension at 72°C for 10 min. Internal standard primers of β-actin (BD Biosciences, Franklin Lakes, NJ, USA) were used as controls. The amplified products were separated on 1.2% agarose gel and visualized with ethidium bromide.
Telomerase activity assay A telomerase PCR-enzyme-linked immunosorbent assay (ELISA) kit was purchased from Roche Molecular Biochemical (Mannheim, Germany). Telomerase activity was detected according to the manufacturer’s protocol. Tumour cell line 293 and hEF cells were used as positive and negative controls respectively.
Teratoma formation To test the pluripotency of hES1 cells in vivo, cells were dissected into clumps of about 300–400 cells each. About 1.6 × 105 hES1 cells were injected subcutaneously into the SCID (severe combined immunodeficiency) mice. After 6–8 weeks, the resulting tumours were dissected out, fixed, embedded in paraffin, and examined histologically with haematoxylin and eosin staining.
Article - Cryopreservation of human embryonic stem cells - CF Wu et al.
Cryopreservation of hES1 cells The hES1 cells were cryopreserved in clumps by conventional slow-rate freezing and rapid thawing methods (Freshney, 1994) with some modifications. Briefly, about 20–25 clumps (over 100 cells per clump) of hES1 cells were collected and transferred into a cryo-vial (Nalge Nunc; Naperville, IL, USA) containing 0.5 ml of pre-cooled (0°C) freezing medium. The freezing medium consisted of 90% knockout serum replacement, 10% dimethyl sulphoxide (DMSO; Sigma) and 0.2 mol/l of trehalose (Sigma). The vials were slowly cooled (~1°C/min) in a freezing container (Nagle Nunc) to –80°C overnight, and then plunged into and stored in liquid nitrogen. For thawing cells, the vials were rapidly thawed in a 37°C water bath. The cell drops were then gradually diluted with 2 ml of ES cell culture medium supplemented with 0.2 mol/l of trehalose. Cells were spun down at 1000 g for 5 min and plated onto a fresh MEF feeder layer with regular ES cell culture medium supplemented with 0.1 mol/l trehalose. The medium was replaced with regular ES cell culture medium after 1 h. Recovered colonies were scored after 7 days by colony morphology and AKP staining. In control groups, regular ES cell freezing medium (90% knockout serum replacement, 10% DMSO) and culture medium were used during the freezing and thawing processes respectively. Cells from passage 22, 28, and 38, which had been cryopreserved for over 16, 10 and 1 weeks respectively were tested. Three vials of each passage were repeated. The characteristics and pluripotency of hES1 were reconfirmed after thawing.
Results Establishment and maintenance of hES1 cells Fifteen blastocysts were generated from 43 fertilized oocytes. ICM cells were isolated and plated separately onto MEF feeder layers. After 10–15 days, four ICM-derived cell masses were obtained. The cell masses consisted of small cells (Figure 1A). In serum free culture conditions, one cell line named hES1 was established and had been maintained for over 70 passages in 14 months. However, another three colonies cultivated in serum containing media all differentiated and died at passages 4, 6, and 16 respectively. Morphologically, undifferentiated hES1 colonies showed flat nest like structure with distinct cell borders in monolayer culture (Figure 1B and C). Spontaneous differentiation was routinely observed (around 15%) during cell passaging (data not shown). The colonies were split every 6–7 days.
Characterization of hES1 cells To characterize the hES1 cells, specific staining for human ES cells was checked at passages 6, 15, 38 and 70. Similar results were obtained from all passages. Thus, representative data from passage 38 are presented. AKP is known to be strongly expressed in pluripotent stem cells and has been used as a routine marker
for undifferentiated ES cells (Pera et al., 2003). As shown in Figure 2A, most of the hES1 colonies were positive for AKP staining. The hES1 cells also showed positive staining for SSEA-3 and SSEA-4 (Figure 2B and C), which are the specific markers for human ES cells (Pera, 2003). Cells also expressed TRA-1–60 and TRA-1–81 (Figure 2D and E), but not SSEA-1 (Figure 2F), which is the specific marker for mouse ES cells (Pera et al., 2003). Oct-4 is a transcription factor and has been considered as a marker for the pluripotent stem cells (Pesce and Scholer, 2001). Immunofluorescent staining showed that hES1 cells expressed Oct-4 (Figure 2G), which was confirmed by RT-PCR analysis as well (Figure 2H). Normally, telomerase is highly expressed in tumour cells or stem cells. The telomerase activity detected by telomerase PCR-ELISA Kit showed that hES1 cells expressed a high level of telomerase activity (Figure 2I). Karyotype analysis of hES1 cells was carried out at passages 18, 38 and 70. All three tests showed normal karyotype of 46, XY (data not shown). In order to testify the pluripotency of hES1 cells, cells were injected subcutaneously into SCID mice. After 6–8 weeks, all mice developed teratoma that contained tissues representative of all three germ layers including neural tube like structures, muscles, cartilages, and glandular structures (Figure 3). Similar results were observed in both early and late passages.
Cryopreservation of hES1 cells Undifferentiated and differentiated hES1 colonies were scored by colony morphology and AKP staining on day 7 after thaw (Figure 4A–C). Following conventional slow-rate freezing and rapid thawing methods, the recover rate of undifferentiated hES1 cells was lower than 15% (Table 1, group A). In order to improve the cryopreservation efficiency, the conventional method was modified by adding trehalose during cell freezing and thawing processes. By adding trehalose in the freezing medium alone, the recovery rate of undifferentiated cells could be improved to 37% (Table 1, group B). By gradually decreasing the concentration of trehalose in the cell thawing process, the average recovery rate of undifferentiated hES1 cell achieved was 42% (Table 1, group C). The best recovery rate (48%) was achieved from the group in which trehalose was utilized in both freezing and thawing processes (Table 1, group D). Meanwhile, the pluripotency of hES1 cells was sustained after thawing. Cells were still positive for AKP and SSEA-4 staining (Figure 4C and D), could still form teratoma in vivo (Figure 4E and F), and kept normal karyotype of 46,XY (data not shown).
Discussion Human ES cell lines have been established from either fresh or frozen embryos donated by couples undergoing IVF treatment. The sources of embryos are very limited. This study utilized immature oocytes donated by patients undergoing IVF treatment, which would normally be discarded. Although it is less efficient for obtaining high quality embryos than the regular method, which starts with mature oocytes, it is still valuable to use these oocytes for research purposes.
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Article - Cryopreservation of human embryonic stem cells - CF Wu et al.
Figure 1. Morphology of human embryonic stem (hES)1 cells. (A (A) Primary inner cell mass-derived cell mass developed from a blastocyst after 15 days culture on mouse embryonic fibroblasts. (B) A typical hES1 colony at passage 6. (C) A typical hES1 colony at passage 38. Scale bars: 200 μm.
Figure 2. Characterization of human embryonic stem (hES)1 cells at passage 38. (A) Alkaline phosphatase staining of hES1 colonies. (B–G) Immunofluorescent staining of hES1 colonies with monoclonal antibodies against stage-specific embryonic antigens SSEA-3 (B), SSEA-4 (C), tumour rejection antigens TRA-1–60 (D), TRA-1–81 (E), SSEA-1 (F), and octamer-binding transcription factor Oct-4 (G). (H) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Oct-4 expression: Lane 1, DNA ladder (100 bp); lane 2, human embryonic fibroblast (hEF) cells as negative control; lanes 3–5, hES1 cells at passage 13, 26 and 38 respectively; lane 6, terato-2 cells as positive control. (I) Telomerase activities analysed by Telo/PCR-enzyme-linked immunosorbent assay kit. hEF and 293 cells were used as negative and positive controls, respectively. Scale bars: 200 μm.
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Article - Cryopreservation of human embryonic stem cells - CF Wu et al.
Figure 3. In-vivo differentiation of human embryonic stem (hES)1 cells. hES1 cells at passage 38 were injected subcutaneously into SCID (severe combined immunodeficiency) mice. After 6 weeks, tumours were collected, fixed and stained with haematoxylin and eosin. Neural tube-like structures (A), muscles (B; arrow), cartilage (B; arrowhead), and glandular structures (C) were observed. Scale bars: 200 μm.
Figure 4. Characterization of human embryonic stem (hES)1 cells after cryopreservation. Undifferentiated (A) and differentiated (B) hES1 cells were judged by colony morphology 7 days after thawing. Undifferentiated cells were positive for alkaline phosphatase (C) and stage-specific embryonic antigen-4 (D) staining. When recovered cells were injected into SCID mouse, they could form teratoma, which contained tissues from three germ layers (E and F). Scale bars: 200 μm.
Table 1. Recovery rates of human embryonic stem (hES)1 cells after cryopreservation. Recovered cells were scored 7 days after thawing. Cell passage
No. colonies
No. colonies survived Group Ab Undifferen- Differentiated (%) tiated (%)
Group Bb Group Cb Group Db Undifferen- Differen- Undifferen- Differen- Undifferen- Differentiated (%) tiated (%) tiated (%) tiated (%) tiated (%) tiated (%)
P 22 P 28 P 38 Total
60 75 75 210
8 (13) 12 (16) 10 (13) 30 (14)
21 (35) 31 (41) 26 (35) 78 (37)
4 (7) 5 (7) 4 (5) 13 (6)
8 (13) 9 (12) 8 (11) 25 (12)
23 (38) 32 (43) 33 (44) 88 (42)
10 (17) 12 (16) 14 (19) 36 (17)
29 (48) 38 (51) 34 (45) 101 (48)
13 (22) 15 (20) 15 (20) 43 (20)
Three vials of each passage were tested. Group A: cells were frozen and thawed without trehalose. Group B: cells were frozen with trehalose but thawed without trehalose. Group C: cells were frozen without trehalose but thawed with trehalose. Group D: cells were frozen and thawed with trehalose. a
b
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Article - Cryopreservation of human embryonic stem cells - CF Wu et al. Four ICM-derived cell masses were initially obtained. Unfortunately, three of those cultivated in serum-containing media were terminated with cell differentiation. Only one clone cultivated in serum-free medium was able to continue propagating. It is well known that serum contains many growth factors that may induce ES cell differentiation. In addition, the quality of serum varies from batch to batch, and even ES culture grade serum needs to be pre-selected before use. Therefore, using serum replacement is a much easier way to maintain stable culture conditions and avoid cell differentiation (Koivisto et al., 2004). The new established hES1 cell line has been well characterized by setting standards for human ES cells, including positive staining of specific cell-surface markers (SSEA-3, SSEA-4, Oct-4, TRA-1–60 and TRA-1–81), high AKP and telomerase activities, as well as normal karyotyping, and multi-lineage differentiation potential. The characteristics of hES1 cells are in good agreement with those other groups reported (Reubinoff et al., 2000; Richards et al., 2002), suggesting that the cell line established in this study is a new human ES cell line. Current protocols for cryopreservation of human ES cells are not satisfactory. Vitrification protocols have been demonstrated to be much more efficient than the standard slow-rate freezing and rapid thawing protocols (Reubinoff et al., 2001; Richards et al., 2004). However, it must be noted that vitrification protocols are extremely labour intensive. The protocols might be suitable for establishing human ES cell banks, but not suited for research purposes that normally need large amounts of cells. Thus, this study has aimed to improve cryopreservation efficiency by modifying the standard slow-rate freezing and rapid thawing protocols. Using regular freezing-medium combined with regular thawing, it was found that many cells survived right after thawing, but most cells died within 24 h (data not shown), indicating that cell death happened after thawing. ‘Stem cells are very finicky – when they come under stress they usually kill themselves’ (Brumfiel, 2004). Osmotic change could be one type of stress during the thawing procedure. In order to minimize such stress, a series dilution of trehalose was added after thawing. Surprisingly, the recovery rates of undifferentiated hES1 cells appeared to be increased with such a modification alone.
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Trehalose is a disaccharide of glucose commonly found in high concentrations in many organisms capable of surviving complete dehydration. It is assumed that trehalose can stabilize cell membranes. It has been widely used as a cryoprotectant in the freezing medium for cryopreservation of cells such as spermatozoa, oocytes, and haematopoietic cells, as well as tissues (Erdag et al., 2002; Aboagla and Terada, 2003; Buchanan et al., 2004). Ji et al. (2004) have used trehalose in the cryopreservation of adherent human ES cells. However, in all these studies, trehalose was used in the freezing medium as a cryoprotectant. In the current study, 0.2 mol/l of trehalose was used, which is the concentration used for the cryopreservation of human haematopoietic cells (Buchanan et al., 2004). Adding trehalose at the thawing stage alone appeared to increase the recovery rate (42%). The effects were even better than using it in the freezing medium alone (37%). The overall effect was slightly enhanced by adding trehalose in both freezing and thawing process (48%). These results indicate that adding
trehalose is beneficial for preserving human ES cells. Although the preserving method was only tested on the ES cell line established in the authors’ laboratory, further studies with H1 cells [the human ES cell line from WiCell (Madison, WI, USA)] are currently in progress. For those cell lines already been frozen in regular medium, adding trehalose at the thawing stage alone could hopefully increase the recovery rates of those cells. Besides trehalose, sucrose was also tested in this study. Sucrose was efficiently used in the cryopreservation of oocytes (Wright et al., 2004). Unfortunately, no significant improvement was observed with human ES cells. The protecting mechanism of adding trehalose is not so clear, it is probably due to its stabilizing effects on cell membrane, thus minimizing the osmotic changes during the freezing and thawing process. In summary, a single human ES cell line has been successfully established and characterized, thereby setting standards for human embryonic stem cells. More importantly, an effective and convenient protocol for cryopreservation of human ES cells has been developed. The protocol will also be beneficial for recovering human ES cells already frozen without trehalose.
Acknowledgements This work was supported by the National Basic Research Program of China (G1999054300, 2005CB522700), Shanghai Science and Technology Development Foundation.
References Aboagla EM, Terada T 2003 Trehalose-enhanced fluidity of the goat sperm membrane and its protection during freezing. Biology of Reproduction 69, 1245–1250. Baharvand H, Ashtiani SK, Valojerdi MR et al. 2004 Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst. Differentiation 72, 224–229. Brumfiel G 2004 Cell biology: just add water. Nature 428, 14–15. Buchanan SS, Gross SA, Acker JP et al. 2004 Cryopreservation of stem cells using trehalose: evaluation of the method using a human hematopoietic cell line. Stem Cells and Development 13, 295–305. Erdag G, Eroglu A, Morgan J et al. 2002 Cryopreservation of fetal skin is improved by extracellular trehalose. Cryobiology 44, 218–228. Freshney RI 1994 Culture of Animal Cells; a Manual of Basic Technique. Wiley-Liss, New York, pp. 255–265. Ji L, de Pablo JJ, Palecek SP 2004 Cryopreservation of adherent human embryonic stem cells. Biotechnology Bioengineering 88, 299–312. Kim SJ, Park JH, Lee JE et al. 2004 Effects of type IV collagen and laminin on the cryopreservation of human embryonic stem cells. Stem Cells 22, 950–961. Koivisto H, Hyvarinen M, Stromberg AM et al. 2004 Cultures of human embryonic stem cells: serum replacement medium or serum-containing media and the effect of basic fibroblast growth factor. Reproductive BioMedicine Online 9, 330–337. Mitalipova M, Calhoun J, Shin S et al. 2003 Human embryonic stem cell lines derived from discarded embryos. Stem Cells 21, 521–526. Park SP, Lee YJ, Lee KS et al. 2004 Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers. Human Reproduction 19, 676–684. Park JH, Kim SJ, Oh EJ et al. 2003 Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biology of Reproduction 69, 2007–2014. Pera MF, Filipczyk AA, Hawes SM et al. 2003 Isolation, characterization, and differentiation of human embryonic stem cells. Methods in Enzymology 365, 429–446.
Article - Cryopreservation of human embryonic stem cells - CF Wu et al. Pesce M, Scholer HR 2001 Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19, 271–278. Reubinoff BE, Pera MF, Vajta G et al. 2001 Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Human Reproduction 16, 2187–2194. Reubinoff BE, Pera MF, Fong CY et al. 2000 Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnology 18, 399–404. Richards M, Fong CY, Tan S et al. 2004 An efficient and safe xenofree cryopreservation method for the storage of human embryonic stem cells. Stem Cells 22, 779–789. Richards M, Fong CY, Chan WK et al. 2002 Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nature Biotechnology 20, 933–936. Solter D, Knowles B 1987 Monoclonal antibody defining a stagespecific mouse embryonic anigen (SSEA-1). Proceedings of the National Academy of Sciences of the USA 75, 5565–5569.
Solter D, Knowles B 1975 Immunosurgery of mouse blastocyst. Proceedings of the National Academy of Sciences of the USA 72, 5099–5102. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. 1998 Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147. Tsung HC, Du ZW, Rui R et al. 2003 The culture and establishment of embryonic germ (EG) cell lines from Chinese mini swine. Cell Research 13, 195–202. Wright DL, Eroglu A, Toner M et al. 2004 Use of sugars in cryopreserving human oocytes. Reproductive BioMedicine Online 9, 179–186. Received 6 July 2005; refereed 25 July 2005; accepted 26 August 2005.
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Article Improved cryopreservation of human embryonic stem cells with trehalose Wen Jie Zhang graduated from Shanghai 2nd Medical University (China) and was awarded his MD degree in 1992. He did his PhD research in Japan and took 4 years of post-doctoral training at Washington University School of Medicine in St Louis, USA. He worked with Dr Kyunghee Choi on the early development of hematopoietic and endothelial cells utilizing an in-vitro differentiation model of embryonic stem (ES) cells. He returned to China in 2004 and is now working on the differentiation of human ES cells. The ultimate goal of his current work is to provide seed cells (such as endothelial cells, chondrocytes) from differentiated ES cells for tissue engineering.
Dr Wen Jie Zhang Chun Fang Wu2, Hsiao Chien Tsung2, Wen Jie Zhang2, Yan Wang, Jun Hong Lu, Zheng Ya Tang, Yan Pin Kuang, Wei Jin, Lei Cui, Wei Liu, Yi Lin Cao1 Shanghai Key Laboratory of Tissue Engineering, Shanghai 9th People’s Hospital, Shanghai 2nd Medical University, Shanghai 200011, China 1 Correspondence: Tel: +86 21 63138341, ext. 5192; Fax: +86 21 53078128; e-mail: [email protected] 2 These authors contributed equally to this work
Abstract Human embryonic stem (ES) cells have been established either from fresh or frozen embryos. The recovery rates of undifferentiated human ES cells after cryopreservation with conventional slow-rate freezing and rapid-thawing methods are relatively low. The purpose of this study was to improve cryopreservation efficiency by modifying conventional methods with addition of trehalose. Immature oocytes donated from patients undergoing IVF treatment were utilized to generate blastocysts. One human ES cell line (named hES1) was established and characterized in detail. The hES1 cells expressed regular human ES cell markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, tumour rejection antigens TRA-1–60, TRA1–81 and octamer-binding transcription factor Oct-4 with high levels of alkaline phosphatase and telomerase activities. Cells could be differentiated to form teratomas in vivo. With slow-rate freezing and rapid-thawing methods modified by adding trehalose, the recovery rate of undifferentiated hES1 cells has been greatly improved from 15 to 48%. Cells retained pluripotency with normal karyotype after thawing. The results indicated that the use of trehalose is efficient and convenient for cryopreservation of human ES cells. Keywords: cryopreservation, human embryonic stem cells, pluripotency, trehalose
Embryonic stem (ES) cells are derived from the inner cell mass (ICM) cells of the preimplantation blastocyst. Cells are able to self-renew indefinitely in vitro, yet still retain a normal karyotype and the capacity for differentiation into a wide variety of somatic tissues of all three germ layers. Given the selfrenewal potential of ES cells, combined with their pluripotent differentiation capacity, ES cells can potentially provide an alternative cell source for cell therapy, gene therapy and tissue engineering.
(Reubinoff et al., 2000; Richards et al., 2002; Mitalipova et al., 2003; Park et al., 2003, 2004; Baharvand, 2004). To date, there are probably more than 120 human ES cell lines worldwide, 78 of which have been included in the National Institutes of Health database (for more information see http://stemcells.nih. gov/research/registry/). Unfortunately, numerous newly derived human ES cell lines are stored at early passage and are not fully characterized. Fewer than 20 cell lines are currently available for scientists. In addition, it is important to note that there are still no very efficient protocols for cryopreservation of human ES cells, which would prevent the widespread use of these cells in research and clinical applications.
Since the first establishment of human ES cells by Thomson and colleagues (Thomson et al., 1998), more new human ES cell lines have been established from either fresh or frozen embryos
Cryopreservation of human ES cells has been reported to be difficult. Slow-rate freezing and rapid-thawing methods are commonly used for cryopreservation of cell lines (Freshney,
Introduction
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Article - Cryopreservation of human embryonic stem cells - CF Wu et al. 1994). These methods are efficient for the cryopreservation of mouse ES cells, but unsuited for human ES cells. Following these methods, the survival of undifferentiated human ES cells after thawing is usually very poor (~10%); most of the cells either differentiate or die (Reubinoff et al., 2000). Kim et al. reported that the recovery rate of human ES cells after slow-rate freezing and rapid thawing could be improved by adding type IV collagen or laminin to the freezing medium (Kim et al., 2004). Another means of preserving human ES cells is the vitrification method. Reubinoff et al. have improved cryopreservation efficiency by using the open pulled vitrification method (Reubinoff et al., 2001). Recently, Richards et al. described a safe, xeno-free cryopreservation protocol for human ES cells involving vitrification in closed sealed straws. Cells exhibited high survival rates and low differentiation rates after thawing (Richards et al., 2004). However, vitrification protocols are extremely labour intensive. It is difficult to handle bulk quantities of cells using this technique. In the present study, one human ES cell line (named hES1) has been established and characterized in detail. An efficient and convenient cryopreservation protocol for human ES cells has been established by modifying conventional slow-rate freezing and rapid-thawing methods by adding trehalose.
Materials and methods Establishment of hES1 cells Spermatozoa and immature oocytes were donated by couples undergoing IVF treatment with informed consent. Approval was obtained for this study from the Internal Review Board on Human Subjects Research and Ethics Committees (Shanghai 9th People’s Hospital, Shanghai, China). The mature oocytes resulted from the culturing of immature oocytes in Global medium (LifeGlobal, Guelph, Ontario, Canada) overnight were fertilized by intracytoplasmatic sperm injection, and then cultured to blastocyst stage in Global medium. ICM cells were isolated from day 6 blastocysts by immunosurgery as previously described (Solter and Knowles, 1975). Cells were then transferred onto a feeder layer of mitomycin-C (Sigma, St Louis, MO, USA) inactivated mouse embryonic fibroblasts (MEF) in human ES cell culture medium. The medium consisted of knockout Dulbecco’s modified Eagle’s medium (KO-DMEM; Invitrogen, Carlsbad, CA, USA), supplemented with 20% knockout serum replacement (Invitrogen) or ES culture grade fetal bovine serum (Hyclone, Logan, UT, USA), 1% non-essential amino acid, 50 IU/ml penicillin and 50 μg/ml streptomycin, 2 mmol/l L-glutamine, 0.1 mmol/l β-mercaptoethanol (all from Invitrogen), and 4 ng/ml human recombinant β-fibroblast growth factor (R and D Systems, Minneapolis, MN, USA). After 10–15 days, the ICMderived mass was physically split into 4–5 clumps using microneedles. The clumps were then transferred to a new well that was covered with fresh MEF feeder layer. Physical splitting was carried out for routine passaging.
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Characterization of hES1 cells Alkaline phosphatase (AKP) staining AKP activity was determined using the method described by Tsung et al. (2003). Nitrobluetetrazolium and s-bromo-4chloro-3-indolyl phosphate were purchased from Sigma.
Cell surface markers for human ES cells Immunofluorescent staining was performed as described by Solter and Knowles (1987). Monoclonal antibodies against stage specific embryonic antigens SSEA-1, SSEA-3 and SSEA4 were purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA). Antibodies against tumour rejection antigens TRA-1–60 and TRA-1–81 were obtained from Chemicon. Rabbit polyclonal antibody against Oct-4 was made in the laboratory (Tsung et al., 2003). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody and FITCconjugated goat anti-rabbit IgG antibody were purchased from Dakocytomation (Carpenteria, CA, USA).
Reverse transcriptase-polymerase chain reaction (RTPCR) analysis Total RNA was purified from 1.6 × 105 cells, reverse transcribed into cDNA with an RT-PCR kit (TaKaRa, Shiga, Japan). Human embryonic fibroblast (hEF) cells and Tera-2 cells were used as negative and positive controls respectively. Primer sequences of octamer-binding transcription factor Oct-4 used in the study were as follows: forward, CGR GAA GCT GGA GAA GGA GAA GCTG; reverse, AAG GGC CGC AGC TTA CAC ATG TTC. The PCR cycles consisted of an initial denaturation step at 95°C for 2 min, followed by 35 amplification cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 60 s, and a final extension at 72°C for 10 min. Internal standard primers of β-actin (BD Biosciences, Franklin Lakes, NJ, USA) were used as controls. The amplified products were separated on 1.2% agarose gel and visualized with ethidium bromide.
Telomerase activity assay A telomerase PCR-enzyme-linked immunosorbent assay (ELISA) kit was purchased from Roche Molecular Biochemical (Mannheim, Germany). Telomerase activity was detected according to the manufacturer’s protocol. Tumour cell line 293 and hEF cells were used as positive and negative controls respectively.
Teratoma formation To test the pluripotency of hES1 cells in vivo, cells were dissected into clumps of about 300–400 cells each. About 1.6 × 105 hES1 cells were injected subcutaneously into the SCID (severe combined immunodeficiency) mice. After 6–8 weeks, the resulting tumours were dissected out, fixed, embedded in paraffin, and examined histologically with haematoxylin and eosin staining.
Article - Cryopreservation of human embryonic stem cells - CF Wu et al.
Cryopreservation of hES1 cells The hES1 cells were cryopreserved in clumps by conventional slow-rate freezing and rapid thawing methods (Freshney, 1994) with some modifications. Briefly, about 20–25 clumps (over 100 cells per clump) of hES1 cells were collected and transferred into a cryo-vial (Nalge Nunc; Naperville, IL, USA) containing 0.5 ml of pre-cooled (0°C) freezing medium. The freezing medium consisted of 90% knockout serum replacement, 10% dimethyl sulphoxide (DMSO; Sigma) and 0.2 mol/l of trehalose (Sigma). The vials were slowly cooled (~1°C/min) in a freezing container (Nagle Nunc) to –80°C overnight, and then plunged into and stored in liquid nitrogen. For thawing cells, the vials were rapidly thawed in a 37°C water bath. The cell drops were then gradually diluted with 2 ml of ES cell culture medium supplemented with 0.2 mol/l of trehalose. Cells were spun down at 1000 g for 5 min and plated onto a fresh MEF feeder layer with regular ES cell culture medium supplemented with 0.1 mol/l trehalose. The medium was replaced with regular ES cell culture medium after 1 h. Recovered colonies were scored after 7 days by colony morphology and AKP staining. In control groups, regular ES cell freezing medium (90% knockout serum replacement, 10% DMSO) and culture medium were used during the freezing and thawing processes respectively. Cells from passage 22, 28, and 38, which had been cryopreserved for over 16, 10 and 1 weeks respectively were tested. Three vials of each passage were repeated. The characteristics and pluripotency of hES1 were reconfirmed after thawing.
Results Establishment and maintenance of hES1 cells Fifteen blastocysts were generated from 43 fertilized oocytes. ICM cells were isolated and plated separately onto MEF feeder layers. After 10–15 days, four ICM-derived cell masses were obtained. The cell masses consisted of small cells (Figure 1A). In serum free culture conditions, one cell line named hES1 was established and had been maintained for over 70 passages in 14 months. However, another three colonies cultivated in serum containing media all differentiated and died at passages 4, 6, and 16 respectively. Morphologically, undifferentiated hES1 colonies showed flat nest like structure with distinct cell borders in monolayer culture (Figure 1B and C). Spontaneous differentiation was routinely observed (around 15%) during cell passaging (data not shown). The colonies were split every 6–7 days.
Characterization of hES1 cells To characterize the hES1 cells, specific staining for human ES cells was checked at passages 6, 15, 38 and 70. Similar results were obtained from all passages. Thus, representative data from passage 38 are presented. AKP is known to be strongly expressed in pluripotent stem cells and has been used as a routine marker
for undifferentiated ES cells (Pera et al., 2003). As shown in Figure 2A, most of the hES1 colonies were positive for AKP staining. The hES1 cells also showed positive staining for SSEA-3 and SSEA-4 (Figure 2B and C), which are the specific markers for human ES cells (Pera, 2003). Cells also expressed TRA-1–60 and TRA-1–81 (Figure 2D and E), but not SSEA-1 (Figure 2F), which is the specific marker for mouse ES cells (Pera et al., 2003). Oct-4 is a transcription factor and has been considered as a marker for the pluripotent stem cells (Pesce and Scholer, 2001). Immunofluorescent staining showed that hES1 cells expressed Oct-4 (Figure 2G), which was confirmed by RT-PCR analysis as well (Figure 2H). Normally, telomerase is highly expressed in tumour cells or stem cells. The telomerase activity detected by telomerase PCR-ELISA Kit showed that hES1 cells expressed a high level of telomerase activity (Figure 2I). Karyotype analysis of hES1 cells was carried out at passages 18, 38 and 70. All three tests showed normal karyotype of 46, XY (data not shown). In order to testify the pluripotency of hES1 cells, cells were injected subcutaneously into SCID mice. After 6–8 weeks, all mice developed teratoma that contained tissues representative of all three germ layers including neural tube like structures, muscles, cartilages, and glandular structures (Figure 3). Similar results were observed in both early and late passages.
Cryopreservation of hES1 cells Undifferentiated and differentiated hES1 colonies were scored by colony morphology and AKP staining on day 7 after thaw (Figure 4A–C). Following conventional slow-rate freezing and rapid thawing methods, the recover rate of undifferentiated hES1 cells was lower than 15% (Table 1, group A). In order to improve the cryopreservation efficiency, the conventional method was modified by adding trehalose during cell freezing and thawing processes. By adding trehalose in the freezing medium alone, the recovery rate of undifferentiated cells could be improved to 37% (Table 1, group B). By gradually decreasing the concentration of trehalose in the cell thawing process, the average recovery rate of undifferentiated hES1 cell achieved was 42% (Table 1, group C). The best recovery rate (48%) was achieved from the group in which trehalose was utilized in both freezing and thawing processes (Table 1, group D). Meanwhile, the pluripotency of hES1 cells was sustained after thawing. Cells were still positive for AKP and SSEA-4 staining (Figure 4C and D), could still form teratoma in vivo (Figure 4E and F), and kept normal karyotype of 46,XY (data not shown).
Discussion Human ES cell lines have been established from either fresh or frozen embryos donated by couples undergoing IVF treatment. The sources of embryos are very limited. This study utilized immature oocytes donated by patients undergoing IVF treatment, which would normally be discarded. Although it is less efficient for obtaining high quality embryos than the regular method, which starts with mature oocytes, it is still valuable to use these oocytes for research purposes.
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Article - Cryopreservation of human embryonic stem cells - CF Wu et al.
Figure 1. Morphology of human embryonic stem (hES)1 cells. (A (A) Primary inner cell mass-derived cell mass developed from a blastocyst after 15 days culture on mouse embryonic fibroblasts. (B) A typical hES1 colony at passage 6. (C) A typical hES1 colony at passage 38. Scale bars: 200 μm.
Figure 2. Characterization of human embryonic stem (hES)1 cells at passage 38. (A) Alkaline phosphatase staining of hES1 colonies. (B–G) Immunofluorescent staining of hES1 colonies with monoclonal antibodies against stage-specific embryonic antigens SSEA-3 (B), SSEA-4 (C), tumour rejection antigens TRA-1–60 (D), TRA-1–81 (E), SSEA-1 (F), and octamer-binding transcription factor Oct-4 (G). (H) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Oct-4 expression: Lane 1, DNA ladder (100 bp); lane 2, human embryonic fibroblast (hEF) cells as negative control; lanes 3–5, hES1 cells at passage 13, 26 and 38 respectively; lane 6, terato-2 cells as positive control. (I) Telomerase activities analysed by Telo/PCR-enzyme-linked immunosorbent assay kit. hEF and 293 cells were used as negative and positive controls, respectively. Scale bars: 200 μm.
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Article - Cryopreservation of human embryonic stem cells - CF Wu et al.
Figure 3. In-vivo differentiation of human embryonic stem (hES)1 cells. hES1 cells at passage 38 were injected subcutaneously into SCID (severe combined immunodeficiency) mice. After 6 weeks, tumours were collected, fixed and stained with haematoxylin and eosin. Neural tube-like structures (A), muscles (B; arrow), cartilage (B; arrowhead), and glandular structures (C) were observed. Scale bars: 200 μm.
Figure 4. Characterization of human embryonic stem (hES)1 cells after cryopreservation. Undifferentiated (A) and differentiated (B) hES1 cells were judged by colony morphology 7 days after thawing. Undifferentiated cells were positive for alkaline phosphatase (C) and stage-specific embryonic antigen-4 (D) staining. When recovered cells were injected into SCID mouse, they could form teratoma, which contained tissues from three germ layers (E and F). Scale bars: 200 μm.
Table 1. Recovery rates of human embryonic stem (hES)1 cells after cryopreservation. Recovered cells were scored 7 days after thawing. Cell passage
No. colonies
No. colonies survived Group Ab Undifferen- Differentiated (%) tiated (%)
Group Bb Group Cb Group Db Undifferen- Differen- Undifferen- Differen- Undifferen- Differentiated (%) tiated (%) tiated (%) tiated (%) tiated (%) tiated (%)
P 22 P 28 P 38 Total
60 75 75 210
8 (13) 12 (16) 10 (13) 30 (14)
21 (35) 31 (41) 26 (35) 78 (37)
4 (7) 5 (7) 4 (5) 13 (6)
8 (13) 9 (12) 8 (11) 25 (12)
23 (38) 32 (43) 33 (44) 88 (42)
10 (17) 12 (16) 14 (19) 36 (17)
29 (48) 38 (51) 34 (45) 101 (48)
13 (22) 15 (20) 15 (20) 43 (20)
Three vials of each passage were tested. Group A: cells were frozen and thawed without trehalose. Group B: cells were frozen with trehalose but thawed without trehalose. Group C: cells were frozen without trehalose but thawed with trehalose. Group D: cells were frozen and thawed with trehalose. a
b
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Article - Cryopreservation of human embryonic stem cells - CF Wu et al. Four ICM-derived cell masses were initially obtained. Unfortunately, three of those cultivated in serum-containing media were terminated with cell differentiation. Only one clone cultivated in serum-free medium was able to continue propagating. It is well known that serum contains many growth factors that may induce ES cell differentiation. In addition, the quality of serum varies from batch to batch, and even ES culture grade serum needs to be pre-selected before use. Therefore, using serum replacement is a much easier way to maintain stable culture conditions and avoid cell differentiation (Koivisto et al., 2004). The new established hES1 cell line has been well characterized by setting standards for human ES cells, including positive staining of specific cell-surface markers (SSEA-3, SSEA-4, Oct-4, TRA-1–60 and TRA-1–81), high AKP and telomerase activities, as well as normal karyotyping, and multi-lineage differentiation potential. The characteristics of hES1 cells are in good agreement with those other groups reported (Reubinoff et al., 2000; Richards et al., 2002), suggesting that the cell line established in this study is a new human ES cell line. Current protocols for cryopreservation of human ES cells are not satisfactory. Vitrification protocols have been demonstrated to be much more efficient than the standard slow-rate freezing and rapid thawing protocols (Reubinoff et al., 2001; Richards et al., 2004). However, it must be noted that vitrification protocols are extremely labour intensive. The protocols might be suitable for establishing human ES cell banks, but not suited for research purposes that normally need large amounts of cells. Thus, this study has aimed to improve cryopreservation efficiency by modifying the standard slow-rate freezing and rapid thawing protocols. Using regular freezing-medium combined with regular thawing, it was found that many cells survived right after thawing, but most cells died within 24 h (data not shown), indicating that cell death happened after thawing. ‘Stem cells are very finicky – when they come under stress they usually kill themselves’ (Brumfiel, 2004). Osmotic change could be one type of stress during the thawing procedure. In order to minimize such stress, a series dilution of trehalose was added after thawing. Surprisingly, the recovery rates of undifferentiated hES1 cells appeared to be increased with such a modification alone.
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Trehalose is a disaccharide of glucose commonly found in high concentrations in many organisms capable of surviving complete dehydration. It is assumed that trehalose can stabilize cell membranes. It has been widely used as a cryoprotectant in the freezing medium for cryopreservation of cells such as spermatozoa, oocytes, and haematopoietic cells, as well as tissues (Erdag et al., 2002; Aboagla and Terada, 2003; Buchanan et al., 2004). Ji et al. (2004) have used trehalose in the cryopreservation of adherent human ES cells. However, in all these studies, trehalose was used in the freezing medium as a cryoprotectant. In the current study, 0.2 mol/l of trehalose was used, which is the concentration used for the cryopreservation of human haematopoietic cells (Buchanan et al., 2004). Adding trehalose at the thawing stage alone appeared to increase the recovery rate (42%). The effects were even better than using it in the freezing medium alone (37%). The overall effect was slightly enhanced by adding trehalose in both freezing and thawing process (48%). These results indicate that adding
trehalose is beneficial for preserving human ES cells. Although the preserving method was only tested on the ES cell line established in the authors’ laboratory, further studies with H1 cells [the human ES cell line from WiCell (Madison, WI, USA)] are currently in progress. For those cell lines already been frozen in regular medium, adding trehalose at the thawing stage alone could hopefully increase the recovery rates of those cells. Besides trehalose, sucrose was also tested in this study. Sucrose was efficiently used in the cryopreservation of oocytes (Wright et al., 2004). Unfortunately, no significant improvement was observed with human ES cells. The protecting mechanism of adding trehalose is not so clear, it is probably due to its stabilizing effects on cell membrane, thus minimizing the osmotic changes during the freezing and thawing process. In summary, a single human ES cell line has been successfully established and characterized, thereby setting standards for human embryonic stem cells. More importantly, an effective and convenient protocol for cryopreservation of human ES cells has been developed. The protocol will also be beneficial for recovering human ES cells already frozen without trehalose.
Acknowledgements This work was supported by the National Basic Research Program of China (G1999054300, 2005CB522700), Shanghai Science and Technology Development Foundation.
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