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Derivation and characterization of new human embryonic stem cell lines in Czech Republic Aleš Hampl 1,2,3, Stanislava Košková 1, Martina Vodinská 1, and Petr Dvořák 1,2,3 1 Department of Molecular Embryology, Institute of Experimental Medicine Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic; 2 Mendel University Brno, 613 00 Brno, Czech Republic; 3 Center for Cell Therapy and Tissue Repair, Charles University, 150 06 Prague, Czech Republic Human embryonic stem cells (hESCs) are exceptionally useful tool for studies of human development and represent a potential source for transplantation therapies. At present, only limited number of hESC lines representing a very small sample of the genetic diversity of the human population is available. Here we report the derivation and characterization of 7 new hESC lines that are maintained in the undifferentiated state for more than 18 months. DERIVATION, CHARACTERIZATION, AND MAINTENANCE OF hESCs Human embryos For derivation of hESCs, human embryos produced by in vitro fertilization for medically assisted induction of pregnancy were used. Human embryos were obtained after informed consent of patients and employed for hESCs derivation upon the approval from 1 the ethical review board of Institute of Experimental Medicine. From the total number of 98 embryos that have been processed in this study, 10 embryos were at the morula stage and 88 were at the blastocyst stage. Importantly, according to embryological criteria routinely used in IVF clinic, majority of the blastocysts (n=63) was graded as quality I. Derivation and culture of hESCs All embryos were first treated with pronase to remove zona pellucida. Morula stage embryos (n=10) were then immediately placed onto feeder layers of mitotically inactivated mouse embryonic fibroblasts (MEFs). MEFs obtained from CF-1 mice were used for all hESC derivations and cultures. Immunosurgery [1] to isolate inner cell masses (ICMs) was then applied on early to expanded blastocysts (n=69) but not on hatching/hatched blastocysts (n=19). Together, 64 ICMs and 19 zona pellucida-free hatched blastocysts were plated onto feeder layers. None of the morula stage embryos became attached to the dish whereas 44 isolated ICMs (69%) and 13 hatched blastocysts (68%) were found firmly attached after 24 hours in culture. An initial outgrowth was observed in 11 isolated ICMs and 3 hatched blastocysts at an average of 7.5 days after plating (varied between 4 and 12 days). Importantly, all embryos that produced an initial outgrowth (14 in total) were of high quality. According to clinical embryological criteria they were graded as category I. In summary, well recognizable colonies of prospective hESCs were obtained from 2 early blastocysts, 9 expanded blastocysts, and 3 hatched blastocysts. Finally, from these 14 initial outgrowths, 7 independent hESC lines named CCTL6, 8, 9, 10, 12, 13, and 14 were successfully established, expanded, and cryopreserved. Typical progression of hESC derivation process is shown in Figure 1. It 2 is of note that from 7 embryos that gave rise to hESC lines, 3 embryos were produced by intracytoplasmic sperm injection (ICSI, see Table 1). Until now, each cell line has been propagated in culture for at least 5 months using mechanical dissociation. Then, 3 hESC lines (CCTL9, 12, and 14) were adapted to passaging with collagenase. Two of these hESC lines (CCTL12 and CCTL14) were also adapted to culture on feeder layer of mitotically inactivated human foreskin fibroblasts (hFF). Newly established line of hFFs (SCRC-1041; Dr. Jonathan Auerbach, Stem Cell Resource, American Type Culture Collection, Manassas, VA) was used for this experiment. Both CCTL12 and CCTL14 hESC lines cultured on hFF were expanded and cryopreserved to create separate sublines. All 7 hESC lines were derived and are maintained in D-MEM:F-12 media supplemented with serum replacement (15%) and basic fibroblast growth factor (bFGF, 4 ng/ml). The population doubling time for hESCs included in our panel is approximately 20-40 hours. Expression of molecular markers of undifferentiated hESCs All CCTL hESC lines grow in colonies with typical hESC morphology that is demonstrated by round shape, compactness, high nucleocytoplasmic ratio of cells, and the presence of several prominent nucleoli per cell. The cells show strong positivity for molecular markers of undifferentiated hESCs, including TRA-1-60, TRA-1-81, TRA-254, SSEA-3, SSEA-4, Thy-1, alkaline phosphatase, and Oct-4, while they are negative for SSEA-1. The example of expression analysis of undifferentiated hESCs is shown in Figure 2A. 3 In vitro differentiation Two protocols were adopted to assess the capacity of hESCs to differentiate in vitro: a) non-adherent culture to form embryoid bodies and b) two-step differentiation protocol involving aggregation step followed by adherent culture of predifferentiated cells [2]. Interestingly, it was noted that under standard conditions some hESC lines were not able to efficiently form well-organized embryoid bodies. Still, increasing the starting density of hESCs by two-fold (from 5 x 105 to 1 x 106 per ml) was usually enough to restore this ability. No significant differences were observed among the CCTL lines in their potential to produce various differentiated cell types in two-step protocol. Figure 2B documents the outcomes of applying both differentiation protocols to hESCs of the lines CCTL12 and CCTL14. Karyotype analysis It was determined using Giemsa banding that out of 7 hESC lines, 6 lines have normal 46XX (CCTL8, 9, and 14) or 46XY (CCTL6, 10, and 13) karyotypes. Only in CCTL12 hESC line (46XX) karyotypic change occurred after 20 passages that was characterized by haploid karyotype in about 30% of cells. Surprisingly, this karyotypic abnormality was no longer detectable at passage 32. However, further culture (to passage 42) of CCTL12 line have led to trisomy of chromosome 12 in about 80% of cells. Notably, gain of chromosome 12 was repeatedly observed also in other laboratories [3, 4] thus pointing to the risk of use of hESCs for therapeutic purposes and to the necessity of periodical testing. Still, in contrast to what was observed by other investigators, increased dosage of chromosome 12 in CCTL12 is not accompanied by any obvious proliferative advantage. 4 Karyotypes of hESC lines CCTL14 (46XX) and CCTL12 (47XX,+12) are shown in Figure 3A. HLA haplotype As the spectrum of HLA antigens expressed on hESCs and/or their derivatives belongs to the clinically relevant characteristics, PCR-based HLA typing was performed in some CCTL hESC lines. As shown in Figure 3B, the results document that CCTL hESC lines represent a range of HLA haplotypes with alleles A01 and A02 shared by 3 lines of 4 analyzed. Microsatellite markers Ability to identify the presence of hESCs and/or their derivatives in various experimental or clinical settings may prove to be very useful in future. Therefore, we created fingerprints of CCTL hESC lines by determining 16 short tandem repeat (STR) loci using automated fluorescent PCR technology. All STRs analyzed are listed in the legend to Table 1. CONCLUSION Most importantly, derivation of 7 new hESC lines described here significantly extends the list of publicly available hESCs. Still, besides establishing this hESC line panel, our experiments also suggest a) that morula stage embryos are not suitable for derivation of hESCs, at least using current standard technology, b) that blastocysts created by virtue of ICSI have no disadvantage compared to those 5 created by standard in vitro insemination, and c) that hatched blastocysts may represent an alternative source to isolated ICMs for derivation of hESC lines. Finally, our finding of progressive karyotypic changes in one of our hESC lines further calls for a maximal caution when manipulating hESCs to be used in cellbased therapies. Acknowledgments We are very grateful to Dr. Jonathan Auerbach for providing us with human foreskin fibroblasts, to Dr. Petr Draber for providing us with antibodies, and to Dr. Vendula Wernerova for karyotyping. This research was supported in part by the Academy of Sciences of the Czech Republic (AV 0Z5039906) and by the Ministry of Education, Youth, and Sports of the Czech Republic (MSM 432100001 and LN 00A065). References 1. Solter D, Knowles BB. Immunosurgery of mouse blastocysts. PROC NATL ACAD SCI USA 1975;72:5099-5102. 2. Schuldiner M, Yanuka O, Itskovitz-Eldor J et al. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. PROC NATL ACAD SCI USA 2000;97:11307-11312. 3. Draper JS, Smith K, Gokhale P et al. Recurrent gain of chromosome 17q and 12 in cultured human embryonic stem cells. NAT BIOTECHNOL 2004;22:53-54. 4. Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N ENGL J MED 2004;350:1354-1356. 6 Figures 7 8 Figure legends Figure 1. Establishment of hESC lines The same standard derivation technology has been used in all hESC lines. Briefly, human blastocyst (A) was freed of zona pellucida (B) by pronase (Protease, cat. no. P 8811; Sigma, St. Louis, MO) and then it was, except for more advanced hatching blastocysts, subjected to immunosurgery (Complement sera from guinea pig, cat. no. S 1639; Anti-Human Serum antibody produced in rabbit, cat. no. H 3383; both Sigma) to release ICM. Isolated ICM (or whole embryo in case of more advanced hatching/hatched blastocyst) was immediately placed onto feeder layer of MEFs. Twenty-four hours later, ICM was inspected for its attachment (C), and then in daily intervals for occurrence of an initial outgrowth (D – day 7). After reaching appropriate size (E – day 10), the initial cell colony was subjected to the first splitting using glass needle. This first passage gave rise to several colonies (usually 2 to 4) of various sizes (F – day 3 after passage). Typical example of derivation process is shown. Scale bar = 25 mm. Figure 2. Determination of undifferentiated state of hESCs and their ability to differentiate in vitro A. Human ES cells were plated onto Permanox chambers (Nunc, Inc., Naperville, IL) and grown in standard D-MEM:F-12 media supplemented by 15% serum replacement and 4 ng/ml bFGF. At day 3 after passage the cells were processed for determination of markers of undifferentiated state by cytochemistry, immunocytochemistry, and Western blotting. For immunocytochemical analysis the cells were fixed in 4% paraformaldehyde 9 for 30 minutes at 4°C and blocked in 5% normal goat serum and 0.01% Tween in PBS pH 7.4 for 1 hour at RT. Then the cells were incubated overnight at 4°C with primary antibody from the following selection: TRA-1-60 (MAB4360; CHEMICON International, Inc., Temecula, CA), TRA-1-81 (MAB4381; CHEMICON), TRA-2-54 (MAB4354; CHEMICON), SSEA-1(TEC-1; from Dr. Petr Draber, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague), SSEA-3 (MAB4303; CHEMICON), SSEA-4 (MAB4304; CHEMICON), and Thy-1 (CBL 415; CHEMICON). Antibody binding was visualized by incubation for 1 hour at RT with appropriate FITClabeled secondary antibody. Nuclei were visualized by staining with propidium iodide. Microscopical analysis was performed using an upright Olympus BX60 microscope equipped with a Fluoview confocal laser scanning unit (Olympus C&S Ltd., Prague, Czech Republic). Cytochemical analysis of the activity of alkaline phosphatase (AP) was accomplished by substrate kit (Alkaline Phosphatase Substrate Kit I, Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. For determination of expression of Oct-4 by Western blot analysis the cells were lysed in Laemmli sample buffer and boiled for 5 minutes. After being separated on 10% SDS PAGE, the proteins were electrotransferred onto Hybond-P membrane (Amersham, Aylesbury, UK), immunodetected using primary antibody against Oct-4 (sc-9081; Santa Cruz Biotechnology, Santa Cruz, CA) and appropriate secondary antibody, and visualized by ECL+Plus reagent (Amersham) according to the manufacturer’s instructions. Representative data are shown. Scale bar = 25 mm. B. For assessment of their differentiation potential, hESCs were collagenased and plated in small clusters onto non-adherent 24-well dishes (Costar 3473; Corning Inc., 10 Corning, NY) in D-MEM:F-12 media supplemented with 20% fetal calf serum. After 15 days of culture, hESCs produced cavitated embryoid bodies (EBs) documenting their ability to properly differentiate into outer endodermal and inner ectodermal layers. Simple compact EBs at day 8 of culture (8 days) and cavitated EBs at day15 of culture (15 days) originating from hESCs of CCTL12 and CCTL14 lines are shown. There were differences between CCTL hESC lines in their ability to form morphologically well organized EBs. White bordered box (CCTL14, 8 days) distinguishes EBs with improved morphology upon increasing the density of hECs. Two step differentiation protocol that includes formation of EBs (5 days of non-adherent culture) followed by further 10 days of differentiation as an adherent culture was also employed and representative examples are shown (5+10 days). Scale bar = 300 mm. Figure 3. Karyotype and HLA haplotype analysis A. For karyotyping, hESCs grown to subconfluency were exposed for 1 hour to 0.1 mg/ml colchicine (SERVA Electrophoresis GmbH, Heidelberg, Germany) in culture media, trypsinized, pelleted by centrifugation, and swollen by 10 minute treatment at RT with hypotonic solution of 75 mM KCl in water. After pelleting again, 10 ml of cold fixative (methanol and acetic acid, 3:1) was added dropwise to cells and let stand at –20°C for 10 minutes. After final pelleting, cells were resuspended in fresh fixative to a concentration of about 5x105 cells/ml and then they were used to make chromosomal spreads to be Giemsa banded. A minimum of 50 metaphases was analyzed for all karyotypes. Normal karyotype of line CCTL14 (Normal) and karyotype of line CCTL12 with trisomy for chromosome 12 (Trisomic) are shown. 11 B. For analysis of HLA class I and class II antigens, DNA samples were isolated from hESCs grown under standard undifferentiated conditions using Blood & Cell Culture DNA Mini Kit (QIAGEN GmbH, Hilden, Germany). PCR-based kits INNOLiPA HLA-A Update, HLA-B Update, HLA-C, HLA-DRB1, and HLA-DQB1 Update (INNOGENETICS, Ghent, Belgium) were used according to the manufacturer’s instructions to determine HLA haplotypes. All currently available data are shown. Table 1. Summary of current status and available characteristics of hESC lines Seven independent hESC lines (CCTL) are currently available in Brno, Czech Republic (Department of Molecular Embryology), with 3 lines originating from blastocysts prepared by ICSI. Staining for markers of undifferentiated hESCs (1) and formation of embryoid bodies (2) are described in legend to Figure 2. Experiments toward determining the potential of hESCs to produce teratomas in mice are under progression (3). Karyotyping (4) and determination of HLA haplotypes (5) are described in legend to Figure 3. For microsatellite characterization of hESC lines (6) the following short tandem repeat loci have been analyzed by PCR combined with capillary electrophoresis: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, Amelogenin, D5S818, and FGA. Freezing of hESCs in standard cryotubes in DMSO-containing media has been successfully achieved. All hESC lines were shown to resume their undifferentiated growth after being frozen stored (7). The number of cryotubes stored in liquid nitrogen is given for each hESC line (8). The number of passages achieved with each hESC line by the end of August 2004 is provided (9). 12 Correspondence: Petr Dvořák Aleš Hampl Department of Molecular Embryology Department of Molecular Embryology Institute of Experimental Medicine AS CR Institute of Experimental Medicine AS CR & Mendel University Brno & Mendel University Brno Zemědělská 1, 613 00 Brno Zemědělská 1, 613 00 Brno Czech Republic Czech Republic Tel: +420-545133298 Tel: +420-545133297 Fax: +420-545133357 Fax: +420-545133357 E-mail: [email protected] E-mail: [email protected] 13