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RBM V10.5 Text RBMOnline - Vol 10. No 5. 2005 617–627 Reproductive BioMedicine Online; www.rbmonline.com/Article/1658 on web 3 March 2005 Article Establishment and characterization of new human embryonic stem cell lines Born in 1973, Necati Findikli obtained his BSc degree from the Bosphorus University Molecular Biology and Genetics Department in Turkey. He obtained his MSc degree from Bilkent University, Turkey in 1998, specializing in p53-regulated genes and cell cycle control in cancer. He worked as a research fellow in the Institute of Cancer Research (London, UK) between 1998 and 1999 and joined the Istanbul Memorial Hospital ART and Genetics Centre in October 2000. He is now working in the research and development section of this centre. His current research interests are somatic cell nuclear transfer and human embryonic stem cells. Dr Necati Findikli Necati Findikli1, Semra Kahraman, Oya Akcin, Semra Sertyel, Zafer Candan Istanbul Memorial Hospital, ART and Reproductive Genetics Centre, R&D Laboratory, Piyale Pasa Bulv. 80270 Okmeydani-Sisli, Istanbul, Turkey 1Correspondence: Tel: +90 212 2106666/3405; Fax: +90 212 2107140; e-mail:[email protected] Abstract Human embryonic stem cells (hESC), with their ability to differentiate into all cell types in the human body, are likely to play a very important therapeutic role in a variety of neurodegenerative and life-threatening disorders in the near future. Although more than 120 different human embryonic stem cell lines have been reported worldwide, only a handful are currently available for researchers, which limits the number of studies that can be performed. This study reports the isolation, establishment and characterization of new human embryonic stem cell lines, as well as their differentiation potential into variety of somatic cell types. Blastocyst-stage embryos donated for research after assisted reproductive techniques were used for embryonic stem cell isolation. A total of 31 blastocysts were processed either for immunosurgery or direct culture methods for inner cell mass isolation. A total of nine primary stem cell colonies were isolated and of these, seven cell lines were further expanded and passaged. Established lines were characterized by their cellular and colony morphology, karyotypes and immunocytochemical properties. They were also successfully cryopreserved/thawed and showed similar growth and cellular properties upon thawing. When induced to differentiate in vitro, these cells formed a variety of somatic cell lineages including cells of endoderm, ectoderm and mesoderm origin. There is now an exponentially growing interest in stem cell biology as well as its therapeutic applications for life-threatening human diseases. However, limited availability of stem cell lines as well as financial or ethical limitations restrict the number of research projects. The establishment of new hESC lines may create additional potential sources for further worldwide and nationwide research on stem cells. Keywords: blastocyst culture, cryopreservation, embryonic stem cells, immunosurgery, in-vitro differentiation Introduction isolation and clinical use, immune rejection is another major issue to be resolved. Although it has recently been shown that Since their first establishment in 1998, several studies have it is possible to isolate human embryonic stem cells via reported the derivation and characterization of different human ‘therapeutic cloning’, extremely low efficiency as well as embryonic stem cells (Thomson et al., 1998; Reubinoff et al., moral acceptability of this technique makes it inapplicable in 2000; Lanzendorf et al., 2001; Park et al., 2003; Heins et al., clinical cases for the time being (Hwang et al., 2004). Another 2004; Pickering et al., 2004). Although more than 120 human approach proposed is to establish a ‘stem cell bank’ which embryonic stem cell lines exist at present, only a handful meet contains hundreds of thousands immunophenotyped stem cell the criteria defined by the National Institutes of Health (NIH). lines in order to match the recipient’s HLA profile for Therefore, most of the research and findings on embryonic immunocompetency (Trounson, 2002). stem cells so far comes from reports utilizing only a couple of embryonic stem cell lines established by these groups. From this perspective, isolation and characterization of novel human embryonic stem cell lines is extremely important not However, besides the ethical and technical constraints on only for expansion of the number of studies, but also for 617 Article - New human embryonic stem cell lines - N Findikli et al. increasing understanding of stem cell biology, which can lead published protocol (Solter and Knowles, 1975). Antibody to establishment of alternative cell therapy protocols. Besides against human albumin (Sigma) and guinea pig complement other properties, embryonic stem cells are likely to prove a (Accurate Chemical, Westbury, NY, USA) were used for very valuable source for research involving mainly lysing and removing trophoblastic cells. Further cell and embryology, oncology, toxicology and pharmacology debris removal was performed by serial pipetting with (Edwards, 2004). decreasing inner diameters. Intact inner cell mass clumps were then transferred onto MEF culture containing embryonic stem This study reports the isolation and characterization of seven cell culture media and cultured for 7–10 days. new human embryonic stem cell lines. These cell lines were also evaluated for their in-vitro differentiation properties into a Alternatively, after the zona removal, direct culture was variety of somatic cell types. performed by transferring zona-free human blastocysts onto previously prepared feeder cells in the presence of complete Materials and methods stem cell media. Culture media was changed every day and cellular and colony morphology was recorded until the The study was performed between January 2003 and May appearance of the first stem-cell like colonies, which was 2004 in Istanbul Memorial Hospital ART and Reproductive around 7 days after initial plating. Genetics Centre. Thirty-one blastocyst-stage human embryos donated for research after assisted reproduction treatment were Embryonic stem cell culture used. The study was approved by the IRB of Istanbul Memorial Hospital and informed consent was obtained from Stem cell culture media included Ko-DMEM (Gibco BRL), each donor couple. 15% FBS (Hyclone, South Logan, UT, USA), 2 mmol/l L-glutamine (Gibco BRL), penicillin (50 IU/ml)/streptomycin Blastocyst-stage embryos included in the study were (50 µg/ml; Gibco BRL), non-essential amino acids (×1; anonymously obtained from the embryology laboratory. NEAA, Sigma), β-mercaptoethanol (0.1 mmol/l; Sigma), Before they were processed for inner cell mass isolation or insulin–transferrin–-selenium complex (×1; ITS; Gibco BRL) direct culture, they were morphologically evaluated according and human recombinant leukaemia inhibitory factor (LIF) to scoring criteria reported elsewhere (Gardner et al., 2000). (12 ng/ml; Chemicon, Temecula, CA, USA). After an initial eight passages, LIF was omitted from culture media. Feeder cell preparation Karyotyping and immunocytochemistry Mouse embryonic fibroblast (MEF) cells isolated from inbred 12- to 14-day pregnant BALb/c mice were used as feeder cells. Stem cell colonies were cultured in media containing In all manipulations with MEF cells, media containing high 0.1 µg/ml colcemide (Biological Industries Ltd., Kibbutz Beit ° glucose DMEM, 10% FBS (Gibco BRL; Invitrogen, Haemek, Israel) for 3 h at 37 C, 6% CO2. Colonies were then Gaithersburg, MD, USA), 2 mmol/l L-glutamine (Gibco BRL), mechanically isolated from MEF feeders and transferred into penicillin (50 IU/ml)/streptomycin (50 µg/ml; Gibco BRL) 15 ml conical Falcon tubes containing cell culture media. After and mercaptoethanol (0.1 mmol/l; Sigma, Poole, Dorset, UK) centrifugation at 145 g for 10 min, the pellet was resuspended were used. Cells between passages 2 and 6 were used for stem with hypotonic medium (0.075 mol/l KCl). Cells were then cell culture. Mitotic inactivation was performed by exposing incubated for 17 min at 37°C, fixed with methanol/acetic acid the cells to mitomycin-C (10 µg/ml; Sigma) containing media (3:1) and processed for G banding. for 3 h. Inactivated cells were trypsinized, seeded into culture plates covered with 0.1% gelatine (Sigma) and used as feeders For immunocytochemistry, the cells were fixed in 4% after 48 h of incubation. paraformaldehyde in phosphate-buffered saline (PBS; Gibco BRL) for at least 15 min. Antibodies raised against surface Isolation and preparation of human foreskin cells were as antigens SSEA-4, TRA-1–60, TRA-1–81 and alkaline previously reported (Hovatta et al., 2003) phosphatase substrates was obtained commercially as a stem cell characterization kit (Chemicon). Analysis was performed Inner cell mass isolation according to manufacturer’s instructions by using FITC- conjugated goat anti-mouse secondary antibody (Santa Cruz It is generally agreed that the embryonic stage at which the Biotechnology Inc., Santa Cruz, CA, USA) and immunosurgery procedure can be applied is mainly epifluorescence microscope (BX50; Olympus, Tokyo, Japan). determined by the degree of trophectoderm expansion with Other antibodies such as OCT-4, nestin, microtubule intact and apparent inner cell mass, which usually occurs associated protein (MAP) 2A and B and cardiac-specific between days 5 and 7 after insemination. Moreover, culture of troponin I used in this study were
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