IntroductionA recent clinical trial on a stem cell therapy was initiated using derivatives from embryonic stem cells (ESCs) . Human ESCs (hESCs) have a wide variety of possibilities in regenerative medicine, because of their two advantageous characteristics that distinguish them from adult stem cells: (1) embryonic stem cells can differentiate into any type of cell or tissue; and (2) embryonic stem cells have the capacity to proliferate indefinitely under optimal conditions. However, there are several obstacles in the use of hESCs that need to be overcome before they can be used in regenerative medicine. In addition to the scientific aspects, such as accurate control of proliferation and purity of differentiated cells, the reliability of the cell line is an ethical issue. This is because hESCs originate from human embryos. As the need for comprehensive information about the characteristics of hESC lines and provenance increased, several countries and groups established hESC registries [2,3]. The European registry, hESCreg (http://www.hescreg.eu/), was founded as a collaborative and interdisciplinary platform where researchers and the general public can access information about available hESC lines. The international stem cell registry at the University of Massachusetts Medical School (http://www.umassmed.edu/iscr/) has been continuously expanding its database since its launch in 2008, and currently has information on >400 hESCs and 120 induced pluripotent stem cells (iPSCs). In accordance with an order from the President of the United States. The National Institute of Health (NIH) has registered 91 hESC lines eligible for federal funding since 2009 (http://stemcells.nih.gov/research/registry). These registries provide information through their websites with features relevant to their own purposes.Several scientists have been active in the production of hESC lines in Korea [4,5]. Approximately 60 hESC lines have been known to be established in Korea. Although the Bioethics and Safety Act has been effective since 2005, the exact information about hESC lines was not available. To improve the credibility of hESC lines that have been established and used in Korea, an obligatory registration was initiated on January 1, 2010 according to the Bioethics and Safety Act 2008. Here, we report the first 1-year results of the registration.
2.1 Cell cultureThe hESC line H9 was cultured on STO (ATCC Manassas, VA, USA) feeder in 80% DMEM/F12 medium (Gibco, Carlsbad, CA, USA) supplemented with 20% KO serum (Gibco), 0.1 mM nonessential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), and 4 ng/mL bFGF (Invitrogen). The STO feeder cells were maintained in DMEM medium containing 10% FBS (Gibco), and used after mitotic inactivation by mitomycin C (Sigma, St. Louis, MO, USA). H9 cells were passaged every 5–7 days with 2 μL/mL ROCK inhibitor (Sigma), and the culture medium was changed daily. The submitted hESC lines were minimally cultured in their own media. The human fibroblast cell line IMR-90 (ATCC) was maintained in DMEM medium containing 10% FBS and 1% penicillin/streptomycin (Gibco).
2.2 DNA fingerprintingThe identity of the hESC line was analyzed by short tandem repeat (STR) analysis. The hESC colonies were removed from the surface of the culture dish and collected in a 15-ml conical tube. The STR analyses were performed by DowGene (Seoul, Korea) and Kogene (Seoul, Korea); both companies used a Powerplex 16 system (Promega). The STR data of the submitted hESC lines were compared with those of other lines with clustering software R and Microsoft Excel.
2.4 RT-PCRTotal RNA was isolated using Trizol (Invitrogen) according to the manufacturer’s protocol. Subsequently, cDNA was synthesized from 1 μg of the total RNA by PrimeScript 1st strand cDNA synthesis kit (TAKARA). Quantitative real-time PCR (qRT-PCR) was performed with TaqMan assays (Applied Biosystems) and Gene Expression probes (Applied Biosystems; GAPDH, Hs99999905_m1; NANOG, Hs02387400_g1; OCT4, Hs00742896_s1; SOX2, Hs00602736_s1; TERT, Hs00162669_m1). qRT-PCR was performed using TaqMan Universal PCR Master Mix and ABI 7500 Real-Time PCR System (Applied Biosystems). Expression levels were analyzed by ΔΔCt method. All reactions were duplicated.
2.5 Mycoplasma testsTwo methods were used to estimate mycoplasma contamination in the hESCs. Tests were performed immediately after receiving cells from applicants. For PCR detection, PCR Mycoplasma Detection Set (TARAKA) was used with 0.5 μL ES culture media. For enzymatic detection, MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland) was used with 100 μL ES culture media. Fresh ES culture media and positive control contained in the kit were used as controls. The amplified PCR products were analyzed by gel electrophoresis with 1% agarose gels. Enzymatic reactions were analyzed by Sirius L tube Luminometer (Berthold, Pforzheim, Germany).
3.1 Procedure for registrationThe registration procedure is shown in Figure 1. A stem cell scientist submitted application forms with copies of informed consent for use of surplus embryos. After a pre-review of the submitted files by the advisory review committee, we requested and analyzed the hESC lines. We analyzed DNA fingerprints, karyotypes, pluripotency markers, and mycoplasma contamination. The submitted files and our analyzed data were reviewed by the advisory review committee, which consisted of seven stem cell scientists, one bioethics professor, and two government employees. We had four committee meetings and two document reviews for the registration in 2010.
3.2 Informed consentThe Bioethics and Safety Act has been in effect since 2005. According to this act, only surplus embryos should be used for research. Therefore according to the provisions of this act, fertility clinics need to obtain informed consent from patients before the surplus embryos can be used for research. The registration indicated that 29 hESC lines were established from 917 surplus frozen embryos donated from 151 patients since 2005. It is now confirmed that all hESC lines were established from surplus embryos that have been provided by patients, with their informed consent, since 2005.
3.3 Analyses of hESC linesAmong 51 registered hESC lines, we analyzed the DNA fingerprint, karyotype, expression of pluripotency markers, and mycoplasma contamination of 27 lines. We harvested and analyzed hESCs as soon as the cells were submitted, but the cells were maintained for 1–2 days if the amount of cells available was insufficient for analyses. Another 25 lines were verified by the Ministry of Education, Science and Technology (MOEST) and were not additionally analyzed by us. A summary of the verified data is shown in Table. DNA fingerprinting using short tandem repeat (STR) analyses showed that all except analyzed lines except for one line are independent lines (Table). Among the 27 lines analyzed by us, three lines showed abnormal karyotypes (Figure 2A–D). Expression of pluripotency marker genes was detected in all the tested lines, although the expression levels of several pluripotency markers of an aneuploid hESC line (CHA-hES 16) were relatively lower than those of other hESC lines (Figure 2E). Mycoplasma contamination tests were performed twice if the first analysis yielded positive results. Eight lines were mycoplasma-positive in the first analysis and were resubmitted. Two lines were still mycoplasma-positive in the second analysis of submitted cells, while the other lines were negative in the second analysis of submitted cells, as shown in Table.
DiscussionThe Korea hESC registry was founded to ensure that hESC research in Korea is ethically responsible and conducted in accordance with the Bioethics and Safety Act by providing reliable information about hESC lines. Our registry is enforced by the Bioethics and Safety Act 2008; under this act, registration of hESC lines is obligatory in Korea. The obligatory registration system is feasible in Korea because researchers who want to use human surplus embryos are required to obtain a license from the MOHW, and the hESC line is thus a product of licensed research. The Bioethics and Safety Act has been in effect since 2005, and obligatory registration started in 2010.As a result of the registration, we confirmed that 29 hESC lines established after 2005 were from 917 surplus embryos obtained with informed consent. Among the 55 submitted lines, two lines could not be registered, and two other lines are under review. Dr. Hwang’s Sooam-hES1 (also known as NT-1) line was ineligible for registration because of the unethical aspects revealed by the report of the investigation committee of Seoul National University . In addition, NT-1 was revealed to be a product of parthenogenesis [6,7], which is not allowed under the Bioethics and Safety Act. Another hESC line that could not be registered is AMC-1, which showed the same DNA fingerprint as a previously established hESC line (Miz-hES4). When we requested the researchers to submit early passage of AMC-1, we were informed that it failed to grow. Among the 51 registered lines, 10 lines have abnormal karyotypes, as shown in Table. The hESC lines with abnormal karyotypes might be used as models of aneuploid chromosomal syndrome [8–10].Recently, methods for establishing an iPSC line were developed [11,12]. iPSCs have characteristics similar to hESCs with respect to their pluripotency and self-renewal capacity. Human iPSCs have been recognized as a good model system for genetic diseases and drug screening . We are considering the registration of human iPSC lines to provide information to the scientific community using the hESC registration system. The registration of human iPSC lines will be optional, not obligatory. We hope that this registry (kscr.nih.go.kr) promotes transparency of stem cell research in Korea.