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a Department of Obstetrics and Gynecology and
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& Y4 P- a n3 }/ Z" l- Ib Institute of Reproductive Medicine and Population, Medical Research Center, College of Medicine, Seoul National University, Seoul, Korea;. y. X5 y% M" W8 [+ [, o# x
7 R! Z# A- b+ D! k9 [$ y* {c Laboratory of Electron Microscope, Seoul National University Children’s Hospital, Seoul, Korea;
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) X2 q4 x" T7 a. B+ t: ad Department of Physiology, Yonsei University College of Medicine, Seoul, Korea
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; O) m4 B& y% E: M+ wKey Words. Cell replacement therapy ? Derivation of hESCs ? Differentiation into cardiomyocytes ? EM analysis of hESCs ? Human embryonic stem cells
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Correspondence: Shin Yong Moon, M.D., Ph. D., Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. Telephone: 82-2-2072-2384; Fax: 82-2-3672-7601; e-mail: shmoon@plaza.snu.ac.kr; and Dong-Wook Kim, Ph.D., Yonsei University College of Medicine, Department of Physiology, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea. Telephone: 82-2-361-5208; Fax: 82-2-393-0203; e-mail: dwkim2@yumc.yonsei.ac.kr
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: F( T+ x9 l( ~4 Q) Z/ t8 wABSTRACT
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Since mouse embryonic stem cells (mESCs) were isolated and cultured in vitro two decades ago , the research of ESCs has made outstanding contributions to our understanding of developmental biology. ESCs, derived from the inner cell mass (ICM) in preimplantation embryos, can proliferate extensively in vitro while maintaining an undifferentiated state and differentiate into most cell types under certain conditions . This ability of ESCs makes them a good source for cell replacement therapy . In addition, ESCs can be used as a source for study of basic developmental biology, identification of factors that are involved in regulation of developmental processes and differentiation into certain cells or tissue, and screening for drugs or toxins .
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Human ESC (hESC) lines have been successfully derived from human blastocysts . Derivation and characterization of hESCs is very important in terms of the direct application to human diseases. Like mESCs, the essential characteristics of hESCs include (a) derivation from the preimplantation embryos, (b) prolonged proliferation in vitro, and (c) stable developmental potentials to form derivatives of all three embryonic germ layers even after prolonged culture. However, hESCs are different from mESCs in the expression of markers. Stage-specific embryonic antigen-1 (SSEA-1), a cell surface marker, is expressed in mESCs but not in hESCs . In contrast, SSEA-3, SSEA-4, TRA-1-60, and TRA-1–81 are markers that are expressed only in hESCs .
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Recently, several studies have shown that neuronal cells , cardiomyocytes , and pancreatic ? cells can be induced from hESCs. These results give promise to the clinical application of hESCs for the treatment of diseases such as Parkinson’s disease, diabetes, and heart disease. However, ESCs can display different differentiation potentials under the same conditions . Thus, testing the differentiation potentials of existing ESC lines is critical in the selection of the appropriate cell line for each experimental purpose. For cell replacement therapy in the field of neurological disorders, the cell lines that most effectively give rise to neuronal populations will be useful. Accordingly, establishment and characterization of many hESC lines are important in this respect.# ?7 {7 J, B/ s$ [, j
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Here we report the establishment of new hESC lines, SNUhES1, 2, and 3. We observed that these cells have the same characteristics as the existing hESC lines in the undifferentiated state and can differentiate into cardiomyocyte lineage in vitro. In addition, our analysis by electron microscopy (EM) shows that the undifferentiated hESCs and differentiated cells are clearly different in their cellular structure.8 S9 y* q4 A6 ~1 A2 G, \" R
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MATERIALS AND METHODS- |) S$ k4 V T* a+ ?
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Derivation of Three hESC Lines: SNUhES1, SNUhES2, and SNUhES30 l* J8 C# y9 z* G' v6 h
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Blastocysts cultured from cryopreserved pronuclear stage embryos were used for establishment of hESC lines. Ten healthy blastocysts that showed both clear ICM and trophectoderm under the microscope were obtained from 73 embryos. The ICM was immunosurgically isolated from nine blastocysts containing a large ICM . The remaining blastocyst had a relatively small ICM, and thus the ICM was separated by the whole-embryo culture method to reduce the risk of cell loss. ICM isolated by both methods was plated onto fresh mouse STO feeder layers. After 5–7 days of culture, clumps of small, tightly packed cells proliferated from three (two of nine ICM isolated by immunosurgery and one isolated by whole-embryo culture) of the 10 ICM. These clumps were mechanically dissociated and replated onto fresh feeder layers. The replated cell clumps after several passages gave rise to flat colonies of cells with defined borders that morphologically resembled human or primate ESCs (Fig. 1A, C, and E). Under high magnification (x200), these cells showed a high ratio of nucleus to cytoplasm and prominent nucleoli (Fig. 1B, D, and F; Fig. 2A), as described previously. Each of SNUhES1, 2, and 3 cell lines was passaged for more than 90, 120, and 100 passages, respectively, while maintaining an undifferentiated state in the presence of the STO feeder layer. During routine passage of the cells, spontaneous differentiation was observed in some colonies even in the presence of the STO feeder layer. Differentiation usually happened in the central part of the colony or in its periphery, and the differentiated portions were manually removed before passaging undifferentiated cells.
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) E/ p- ]4 r1 DFigure 1. Derivation of human embryonic stem cell lines. Representative photographs of (A, B) SNUhES1, (C, D) SNUhES2, and (E, F) SNUhES3, respectively, are shown. SNUhES1 and 2 were derived from nine blastocysts using an immunosurgical method, while SNUhES3 was derived from a blastocyst using the whole-embryo culture method. Photographs were taken at low (x40; A, C, E) or high (x200; B, D, F) magnification.# g2 M9 N9 p/ R
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Figure 2. Fine structures of undifferentiated and differentiated hESCs. Morphological comparison between (A) undifferentiated and (B, C) differentiated hESCs analyzed by electron microscopy. The two types of cells clearly showed a big morphological difference. Undifferentiated hESCs have large nuclei with prominent nucleoli, whereas differentiated hESCs exhibit mature cellular organelles. Magnification: A, x3.5K; B, x8.0K; C, x12K. Abbreviations: hESC, human embryonic stem cell; RER, rough endoplasmic reticulum." ^" X3 b1 ]. t; _' E% D
/ r( z A# @9 r6 e; y" V7 E& v" _Marker Expression, Karyotyping and DNA Fingerprinting of hESC Lines
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Pluripotent hESCs have unique characteristics. In general, they show high expression levels of AP, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and telomerase . We began to investigate whether our hESC lines fit these criteria. As shown in Figure 3A, our cell lines showed a high level of AP activity. Elevated expression of this enzyme is associated with undifferentiated pluripotent stem cells . Immunophenotyping of the hESCs was performed using a series of antibodies that detect cell surface markers. Our hESCs stained positively for SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (Fig. 3C–F) but not SSEA-1 (Fig. 3B), a marker for mESCs. Staining intensity for SSEA-4 was consistently strong, but the intensity for SSEA-3 was relatively weak and variable among colonies, as reported previously . SSEA-3 and SSEA-4 are glycoproteins specifically expressed in early embryonic development and by undifferentiated hESCs . TRA-1–60 and -81 are tumor-related antigens that are normally synthesized by undifferentiated hESCs . In addition, Oct-4 expression was observed only in undifferentiated hESCs, and its expression disappeared when hESCs differentiated (Fig. 4A). Oct-4 is a transcription factor that is essential for establishment and maintenance of undifferentiated hESCs and mESCs .4 a$ D m! h) q9 H1 }8 h! A
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Figure 3. Marker analyses of hESCs. Staining of ESC markers such as (A) alkaline phosphatase, (B) SSEA-1, (C) SSEA-3, (D) SSEA-4, (E) TRA-1-60, and (F) TRA-1-81 are shown in SNUhES3 cells (x150 magnification). Similar results were obtained for the cell lines SNUhES1 and 2. Abbreviation: hESCs, human embryonic stem cells.2 m" I5 n8 ]0 {! k m% _( N! M3 F5 n
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Figure 4. Expression of Oct-4 and telomerase in hESC lines. (A): Oct-4 expression in hESC lines: lane 1, undifferentiated SNUhES1; lane 2, differentiated SNUhES1; lane 3, undifferentiated SNUhES2; lane 4, differentiated SNUhES2; lane 5, undifferentiated SNUhES3; lane 6, differentiated SNUhES3; lane 7, STO feeder layer. (B): SNUhES cell lines express high levels of telomerase activity. A 36-bp internal control was used for amplification efficiency and quantification, as indicated by the arrow. A ladder of telomerase products amplified by PCR is shown with six base increments starting at 50 nucleotides at the portion indicated by the asterisk. Lane 1, positive control provided by kit; lane 2, heat-inactivated positive control; lane 3, PCR control without addition of template; lane 4, undifferentiated SNUhES1; lane 5, heat-inactivated SNUhES1; lane 6, undifferentiatedSNUhES2;lane7, heat-inactivatedSNUhES2; lane 8, undifferentiated SNUhES3; lane 9, heat-inactivated SNUhES3; lane 10, STO feeder layer; lane 11, heat-inactivated STO feeder layer. Abbreviations: hESC, human embryonic stem cell; PCR, polymerase chain reaction.$ X9 @' z, P( o; g. @
* D6 |; Y* r6 G# zHigh levels of telomerase activity, a useful marker for identifying undifferentiated hESCs , were also observed in the three cell lines (Fig. 4B). Undifferentiated SNUhES1 (lane 4), 2 (lane 6), and 3 (lane 8) showed the same high activity as the positive control (lane 1) provided by the kit, but heat-inactivated samples (lanes 2, 5, 7, 9, and 11) and the STO feeder layer (lane 10) did not retain any telomerase activity. The high level of telomerase activity in these cell lines indicates that they have a potential to infinitely proliferate . Thus, from this pattern of marker expression, these cell lines satisfy the criteria that characterize existing, pluripotent hESCs. Karyotyping was performed at passages 12 to 15, and all three cell lines retained normal karyotypes (Fig. 5). In this assay, SNUhES1 and SNUhES3 had 46, XY karyotypes (Fig. 5A and C), whereas SNUhES2 showed a 46, XX karyotype (Fig. 5B). DNA fingerprinting was performed for these cell lines (Table 1). From the study of the nine STR loci , it is clear that these three cell lines were derived from different embryos. These fingerprinting results also provide useful information for identification of each cell line after cell distribution.9 K/ }4 K1 V) r6 ]& O8 C
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Figure 5. Karyotypes of SNUhES cell lines. When analyzed by G-staining method, karyotypes of after 12–15 passages were found to be normal: (A) SNUhES1, 46, XY, (B) SNUhES2, 46, XX, and (C) SNUhES3, 46, XY.5 a0 g4 a/ B& {( w# j" K
. Q2 k5 S9 y- s6 aTable 1. DNA fingerprinting for SNUhES cell lines: distribution of alleles for the nine short tandem repeat loci in the three human embryonic stem cell lines
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' p0 K2 k3 T+ ~; C1 i3 ?Differentiation Potentials of hESCs in SCID Mice
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An important property of ESCs is their ability to differentiate into all kinds of somatic cell types. To test this potential in vivo, the hESCs were injected into SCID mice . As shown in Figure 6, these cells produced teratomas in each injected SCID mouse. Teratomas were found to contain tissues of the three embryonic germ layers: endoderm (gut-like structure and gut epithelium ), mesoderm (cartilage ), and ectoderm (neural rosettes ). When these cell lines were cultured in a feeder-free condition, differentiation occurred rapidly in vitro (data not shown). When cultured on bacterial Petri dishes, these cell lines also showed a potential to make EBs, intermediates during the process of differentiation. Thus, these results suggest that the established cell lines are pluripotent even after prolonged proliferation.
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& T$ P }5 _+ j& wFigure 6. Teratoma formation after injection of human embryonic stem cells into severe combined immunodeficient mice. The tissues were stained with hematoxylin and eosin. (A): Gut-like structure (endoderm) from SNUhES1. (B): Gut epithelium-like tissue (endoderm) from SNUhES2. (C): Cartilage-like tissue (mesoderm) from SNUhES3. (D): Neural rosettes-like structure (ectoderm) from SNUhES1.
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; F0 c6 w; R E. |Structural Differences between Undifferentiated and Differentiated hESCs+ q% Q# |3 a E
6 f% l9 n# M. A2 q [ i( vIt was reported that undifferentiated hESCs have a high ratio of nucleus to cytoplasm . This fact prompted us to investigate the structural differences between undifferentiated and differentiated hESCs in more detail using EM. Expanded undifferentiated colonies and EBs (8 weeks old) were used for analyses by transmission EM (TEM). As expected , the nucleus to cytoplasm ratio was high in undifferentiated hESCs (Fig. 2A). In addition, several other features were observed in these cells: They had indistinct cell membranes, free ribosomes, and ovoid nuclei with one to three reticulated nucleoli. Among the cellular organelles, small mitochondria with a few crista, a characteristic of premature cells, were occasionally found, but others such as rough endoplasmic reticulum (RER), Golgi complex, and lipid droplet were not observed. In contrast, differentiated cells showed highly developed cellular organelles such as extensive Golgi complexes associated with small secretory vesicles and ER studded with ribosomes (Fig. 2B, C), indicating that cells are actively synthesizing secretory proteins as in somatic tissues. Organelles like lipid droplet and large mitochondria were also evident. Cytoplasmic membranes were irregular and extensively developed to enlarge the interface between cells. The existence of desmosomes and tonofilaments suggests that these cells differentiated into epithelial cells. Microvilli (shown in Fig. 2C) are similar (e.g., columnar and dense) to those of gastric epithelial cells. Cyto-skeleton components such as actin and tonofilaments were also shown. Taken together, these results clearly show that undifferentiated hESCs have a relatively simple structure during proliferation, whereas the differentiated cells resemble epithelial cells and display all kinds of cellular organelles for intracellular and intercellular activities such as protein transport, as shown in adult somatic cells.
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, _5 S3 B5 |. _2 qIn Vitro Differentiation into Cardiomyocytes- V; {3 D' [ l, Q8 K: j
1 p/ }& f4 T2 P2 `. tTo examine the differentiation potentials of three cell lines into cardiomyocytes, EBs formed from hESC colonies were first induced into mesodermal fate in a suspension culture and then differentiated into cardiomyocytes after attachment onto culture dishes. In general, mesodermal markers (e.g., enolase, cartilage matrix protein) began to be expressed approximately 18 days after suspension culture of EBs in all three cell lines. Thus, based on the expression of mesodermal markers, we attached 20-, 25-, and 30-day-old EBs onto gelatin-coated culture dishes after suspension culture. After further differentiation (~20 days) of attached EBs, contracting clusters were found from 30-day-old EBs but not from 20- or 25-day-old EBs. Contracting EBs were made from SNUhES3 most efficiently (~20% of total clusters), but they were seldom found in SNUhES1 and 2. These contractions continued for up to 4 weeks.! D& ?# k5 }. s8 ]! Z
* d+ @' D) e5 z0 q: R& d4 G- E% \Using immunocytochemistry, the presence of cTnI, a cardiac-specific protein that is involved in the regulation of cardiac muscle contraction , was studied in differentiated EBs. As shown in Figure 7A–C, all three SNUhES cell lines expressed cTnI. However, among three cell lines, SNUhES1 and 3 showed a relatively strong expression of cTnI in comparison with SNUhES2, which revealed a weak expression of cTnI. SNUhES1, 2, and 3 cells gave rise to about 40%, 19%, and 60%, respectively, in the number of cTnI-positive cells among 4,6-diamidino-2-phenylindole–positive total cells (data not shown). These results indicate that of the three cell lines, SNUhES3 differentiates into cardiomyocytes most effectively.9 ~- q) k, U; ~$ ~. ]2 v
% |! ~- V1 n' LFigure 7. Analyses of embryonic stem cell–derived cardio-myocytes by immunostaining and reverse transcription polymerase chain reaction. (A–C): Immunostaining of differentiated EBs with mouse anti-cTnI antibody and Alexa Fluor 488 (green)–labeled donkey anti-mouse immunoglobulin G on SNUhES1, 2, and 3, respectively. (D): Expression of cardiac-specific markers in EBs (lane 2) differentiated from SNUhES3 compared with undifferentiated SNUhES3 (lane 1). Abbreviation: EB, embryoid body.
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: S" t0 u$ z1 DSeveral other cardiac markers were analyzed from the SNUhES3 by RT-PCR. Figure 7D shows that GATA4, ANF, and cardiac actin (cACT) were also more highly expressed in cells differentiated from SNUhES3 (lane 2) than in undifferentiated cells (lane 1). GATA4 is known to be expressed in precardiac mesoderm of the developing heart , and ANF is a hormone that is expressed in ventricular cardiomyocytes .
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DISCUSSION
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We thank Dr. Jung Bin Lee (Forensic Medicine, College of Medicine, Seoul National University) for performing DNA fingerprinting. This research was supported by grant numbers SC11011 and SC12060 from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea.
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发表于 2009-3-5 10:52
发表于 2015-5-29 21:18
