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a Cellular Neurobiology Research Branch, National Institute on Drug Abuse, Department of Health and Human Services (DHHS), Baltimore, Maryland, USA;
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7 J6 O H; Z) H) }. ?# s0 ?2 N3 Tb Laboratory of Neuroscience, National Institute of Aging, DHHS, Baltimore, Maryland, USA;
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c Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA;
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d BresaGen Inc., Athens, Georgia, USA
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Key Words. Embryonic stem cells ? Differentiation ? Microarray
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Xianmin Zeng, Ph.D., Development and Plasticity Section, Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 333 Cassell Drive, Baltimore, Maryland 21224, USA. Telephone: 410-550-6565 (ext 138); Fax: 410-550-1621; e-mail: xzeng@intra.nida.nih.gov Mahendra S. Rao, Ph.D., Laboratory of Neuroscience, National Institute of Aging, 33 Cassell Drive, Baltimore, Maryland 21224, USA. Telephone: 410-558-8204; Fax: 410-558-8249; e-mail: raomah@grc.nia.nih.gov" q Y9 J: C4 A+ |
' B ~7 E4 Q7 cABSTRACT
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Embryonic stem (ES) cells, isolated from the blastocysts of preimplantation embryos, are pluripotent and have the capability to generate all the differentiating cells present in the embryo. ES cells were first described in mice and recently have been identified from multiple species including subhuman primates (rhesus and marmoset) and human . Because of their unique properties, human (h)ES cells could be used for repair and replacement of cells or tissues lost due to disease or trauma. A universal bank of well-characterized hES cells from which specific cells can be generated would potentially be invaluable for basic research and cell replacement therapy.
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4 i0 O5 z# S: G# BTo date, 78 different lines from the National Institutes of Health (NIH) registry have been identified and tentatively classified as ES cells based on general morphological similarity. Early experiments suggest that the properties of hES cells differ in some respects from mouse ES cells . Eleven of these lines are currently available for research purposes, and limited data on the biology of 26 of these lines are available . Two of these lines, BG01 and BG02, were generated from embryos whose poor development was such that in the course of usual IVF practice they would have been discarded because 6–7 days post fertilization, fully 1–2 days after the usual time of embryo transfer, they had not developed sufficiently to survive cryopreservation. A report of their basic biology is available ; however, there are no published data on molecular characterization of these cell lines.
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Considerable additional information is, however, available on other hES cell lines, notably those derived by Thomson and colleagues . Teratocarcinoma formation, long-term stability, derivation of feeder-free subclones, microarray analysis, genome scan, and serial analysis of gene expression analysis have been completed or initiated for several lines . Genetic modification, including homologous recombination, has been reported . Differentiation into multiple phenotypes, including cardiac, hepatic, pancreatic, neural, and hematopoietic lineages, has been described . Whether BG01 and BG02 lines have similar properties remains to be determined.
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" ]# c. @. K# `" ~3 bIt is unlikely that all hES cell lines will be identical or equally stable in culture. Some differences in human cell lines have been described , although whether they reflect differences in the methods of propagation or illustrate underlying differences in biology remains to be determined. We also note that when rodent ES lines have been examined, strain differences in isolation and propagation of lines have been described . Indeed, uniformly successful isolation is not possible in all mouse strains, and it has been difficult to generate rat ES cell lines .& e6 e: X* F2 ~, ]) p4 i
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In an effort to understand the properties of the BG lines, we have propagated BG01 and BG02 in culture and examined their growth; differentiation characteristics; and gene expression patterns using immunostaining, reverse transcriptase polymerase chain reaction (RT-PCR), and microarray analysis. We show that BG01 and BG02 have the capacity to differentiate into cells that express divergent tissue-specific antigens consistent with pluripotency and express markers similar to other pluripotent hES cells. No significant differences were observed in gene expression profiles between these two lines.
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MATERIALS AND METHODS
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Growth and Morphology
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BG01 and BG02 were cultured on MEF feeders and grew as colonies of tightly compacted undifferentiated cells resembling mouse and hES cells (Fig. 1). Like murine ES cells, BG01 and BG02 colonies had a high nuclear/cytoplasmic ratio. These cells were propagated in vitro for more than 40 passages and maintained a normal karyotype . Successful passage of cells was achieved by using either trypsin or nonenzymatic cell dispersal buffers, and we found that both worked equally well. In general, the cultures became confluent 4–5 days after seeding and the cells had a doubling time of 30–35 hours, similar to that reported for other hES cells grown on feeders or feeder-free conditioned medium. In addition, these cells recovered quickly from frozen vials within 2–3 days, and an undifferentiated state was easily and reliably maintained with no instances of spontaneous differentiation. Spontaneous differentiation was, however, observed when BG01 or BG02 cells were seeded on MEFs at lower density (e.g., 1 x 105 cells/35 mm dish).& p+ o4 T2 L' K3 o$ W* q* r
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Figure 1. Morphology of undifferentiated BG01 and BG02 cells. Phase contrast microscopy of undifferentiated BG01 and BG02 cells grown on an MEF feeder layer. The scale bar is 20 μm.
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Cell Surface and ES Cell Marker Expression
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Immunocytochemistry was used to analyze whether BG01 and BG02 were similar to other hES cell lines in expressing cell surface markers that characterize undifferentiated pluripotent stem cells. These include SSEA-3 and SSEA-4; tumor recognition antigen, TRA-1-60 and TRA-1-81; and the POU transcription factor, OCT-3/4.
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4 l) f% U3 Y# DUndifferentiated BG01 and BG02 cells were strongly positive for TRA-1-60, TRA-1-81, SSEA-4, and OCT-3/4, but negative for SSEA-1. Almost all of the colonies were positive for TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and OCT-3/4, and the majority of cells in the colonies were stained for these markers (Fig. 2). Almost no positive staining was observed outside the ES colonies or in the feeder cells for TRA-1-60, TRA-1-81, SSEA-4, and OCT-3/4, but some positive cells were found outside the colonies for SSEA-3 (Fig. 2). Staining intensity for SSEA-4, TRA-1-60, TRA-1-81, and OCT-3/4 was consistently strong both within individual colonies and among the colonies, but staining intensity was weaker for SSEA-3.0 K7 X8 c0 W' _% w' H; P0 f' c
( p( } ~% y5 p7 M4 U. v3 h! D" yFigure 2. Morphology of undifferentiated BG01 and BG02 cells and expression of markers by immunocytochemistry. Both BG01 and BG02 are strongly positive for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-3/4. Antibody staining is in red (SSEA-3 and SSEA-4) or green (TRA-1-60, TRA-1-81, and OCT-3/4), while nuclear DAPI staining is in blue. The scale bar is 20 μm for SSEA-4 and OCT-3/4 and 10 μm for the others.
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) X9 i* [5 t" p2 ?+ D1 w3 S, F5 \2 xRT-PCR was used to confirm some of the markers analyzed by immunochemistry and to examine additional markers that are associated with stem cells. Primers that span intron-exon boundaries for the TERT gene were used to assess possible genomic contamination. As expected, messages for telomerase reverse transcriptase TERT, homeobox-domain transcription factor SOX-2, zinc finger protein REX-1, and a gene similar to developmental pluripotency-associated 5 (Dppa 5) were detected in undifferentiated BG01 and BG02 cells (Fig. 3). Transcriptional coactivator UTF1 was also expressed in BG01 and BG02. Transcripts for some cell surface markers reported for blastocysts or stem cells, such as the gap junction proteins connexin-43 and connexins-45 (Cx-43 and Cx-45), and ABC transporter ABCG were also detected in undifferentiated BG01 and BG02 cells (Fig. 3). Table 2 summarizes the markers expressed by BG01 and BG02 using either immunocytochemistry, RT-PCR, or both.7 j+ y0 G9 K L2 P
1 @& H7 f" I% LFigure 3. Expression of markers associated with ES cells in BG01 and BG02 by RT-PCR. Genes known to be associated with the pluripotent state (hTERT, OCT-3/4, SOX-2, and REX-1) are expressed in both BG01 and BG02; five additional genes, Dppa5, UTF1, ABCG2, Cx43, and Cx45 are also expressed by BG01 and BG02. PCR was performed using gene-specific primers (Table 1) with glyceraldehyde-3-phosphate dehydrogenase as a control. Markers are a 100-bp ladder with the lowest band being 100 bp.6 @0 D5 T# V, I% ^4 b* l
& k& B! ]! @; W1 a0 ]6 _7 vTable 2. Expression of markers is associated with stem cells in undifferentiated BG01 and BG02 cells& N- R' N: p8 y Y( R
: o: S0 ?3 m% rAbility to Differentiate into Cell Types from All Three Germ Layers
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The capacity of BG01 and BG02 to differentiate in vitro was evaluated. Differentiation of hES cells was initiated by culturing in the absence of an MEF feeder layer and bFGF. Under these conditions, hES cells formed embryoid bodies (EBs) of heterogeneous cells. TuJ1-positive neurons, cardiac troponin I-positive cardiomyocytes, and -fetoprotein-positive cells (Fig. 4) were identified in EBs by immunocytochemistry. In addition, cells positive for nestin and smooth muscle actin were identified (Fig. 4). This result indicated that BG01 and BG02 were able to differentiate in vitro into ectodermal, mesodermal, and endodermal derivatives.
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4 V2 b/ v* C7 n( f( tFigure 4. In vitro differentiation of BG01 and BG02 via embryoid bodies. Differentiation of hES cells was initiated by forming EBs in the absence of MEFs and bFGF. Positive immunostaining was identified for AFP, cTnI, SMA, nestin, and TuJ1, indicating that both lines can differentiate to express markers of ectoderm, mesoderm, and endoderm. The scale bar is 20 μm.
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Microarray Analysis of Gene Expression in Undifferentiated Cells
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& u( r0 {; U+ h1 Y0 R; FThe large-scale oligonucleotide microarray was employed to examine the overall gene expression profile of undifferentiated BG01 and BG02 cells. The probes used for this array included 1,987 hypothetical proteins and 72 expressed sequence tags (ESTs) and span approximately 50% of the human genome. RNA from undifferentiated BG01 and BG02 cells was compared with pooled huRNA that hybridizes with most genes on the array. The array results are shown in Figure 5. Figure 5A and B show the image profiles of BG01 and BG02, Figure 5C and D show scatter plot analyses of BG01 and BG02, and Figure 5E shows a comparison of highly expressed genes (threefold or higher level) among BG01, BG02, and other ES cell lines.
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/ o. S* w* @3 {; l) fFigure 5. Expression profiling of BG01 and BG02 hES cell lines. A and B): Representative images after competitive hybridization of oligonucleotide arrays using cy5-labeled RNA from BG01 (A) and BG02 (B) hES cell lines and cy3-labeled huRNA. Total RNA (5 μg) isolated from hES cell lines and huRNA was labeled with cy5 and cy3 dyes, respectively, and used to hybridize oligonucleotide arrays containing ~17,000 features. The images were collected using a GenePix scanner. C and D): Scatter plot analysis of cy5-labeled genes in BG01 (C) and BG02 (D) and cy3-labeled genes in hURNA to show differential gene expression. E). Venn diagram showing similarity of highly expressed genes (threefold and higher) in BG01 and BG02 cell lines at 99% confidence interval. A total of 1,006 genes were highly expressed in BG01, 406 were highly expressed in BG02, and 373 of these were common to both cell lines. Of the 373 transcripts common to BG01 and BG02, 92 were also found to be highly expressed in other hES cell lines and 281 were found only in BG01 and BG02.6 \* z+ T9 v- Y, U8 ^* F* n3 p
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A total of 373 genes common to both BG01 and BG02 were identified as being differentially expressed, defined as a threefold or greater increase as compared with huRNA (Table 3). Among them, 92 genes, including several stemness genes known to be expressed in mES or hES cells such as OCT-3/4, NANOG, Cripto/TDGF1, Cx43, and galanin, are also differentially expressed in an additional four hES lines (TE06, GE01, GE09, and a pooled set of subclones derived from GE01, GE09, and GE07). Expression of several of these 92 genes in BG01 and BG02 was confirmed by RT-PCR (Fig. 6). Nanog (a recently identified hypothetical protein FLJ12581critical for maintaining pluripotency of mES cells) and three other hypothetical proteins, FLJ13072 KIAA1265, and MGC27165 were all expressed in both BG01 and BG02 (Fig. 6A). ZNF342, DNMT3L, DAX-1, and Eras transcripts were detected by RT-PCR (Fig. 6B).
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Table 3. Highly expressed genes in BG01 and BG02 cells (> threefold)
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Figure 6. RT-PCR confirmation of representative genes detected by the large-scale microarray analysis of BG01 and BG02. A) Nanog, a recently identified hypothetical protein FLJ12581 that is critical for maintaining pluripotency of mES cells and three other hypothetical proteins, FLJ13072 KIAA1265, and MGC27165 were expressed in both BG01 and BG02. B) ZNF342, DNMT3L, DAX-1, and Eras were also expressed. C) Expression of keratin 8, 14, and 19; cardiac actin; and tubulin (genes associated with differentiation) were also detected by RT-PCR.
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P1 ~& T( k' qThe remaining 281 of the 373 genes that were found to be differentially expressed by threefold or greater in BG01 and BG02 were not found to be differentially expressed in the other hES cell lines. These included metabolic-related genes, ribosomal proteins, histone proteins, and many hypothetical proteins. In addition, 19 novel genes were also identified and are listed separately (Table 4). We noted that a few genes that are considered as markers of differentiation were expressed at high levels in both undifferentiated BG01 and BG02 cells. These included keratin 8, 14, and 19; cardiac actin; and tubulin. Expression of these genes in undifferentiated ES cells was also found by RT-PCR (Fig. 6C).
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Table 4. Nineteen novel genes highly expressed (> threefold) identified by a large-scale microarray" E) s' R, p' c7 J# f+ W
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Among the genes that were differentially expressed are the TGF-? superfamily member Nodal and its antagonist Lefty. Nodal signaling is known to play an important role in endoderm formation, early embryonic patterning, and left-right axis positioning. To further examine expression of genes related to the Nodal signaling pathway in undifferentiated cells, we employed a focused microarray (96 genes) containing probes of the TGF-? superfamily and key proteins involved in the TGF-? signal-transduction pathway. Figure 7A shows a hybridization profile of BG01 and BG02, and Figure 7B summarizes the genes detected in both BG01 and BG02.# i8 d& G: k7 s6 d: k2 i" O
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Figure 7. Gene expression profile of BG01 and BG02 by a focused human stem cell microarray. A) Images of arrays hybridized using BG01 and BG02 RNA. B) Summary of genes expressed by BG01 and BG02.: h) D( X' [1 _+ F4 J
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Of the 96 transcripts examined, 31 transcripts were detected in both BG01 and BG02 and 65 transcripts were not detected in either line (Fig. 7B). Nodal, LeftyA, LeftyB, Cripto-1 (TDGF-1), Cerberus, activin receptor ALK-3, and SMAD (3, 5, and 6), all involved in the Nodal signaling pathway, were expressed in both BG01 and BG02. Several bone morphogenetic proteins (BMPs), BMP2, BMP4, and BMP9 were detected in undifferentiated ES cells; however, BMP1, BMP3, BMP5, BMP6, BMP7, BMP8, BMP10, and BMP11 were not expressed in BG01 or BG02. Basic helix-loop-helix transcription factor ID2, ID3, and ID4, but not ID1 were also found to be expressed in BG01 and BG02.
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0 p: m% p, {9 f6 _3 sGene Expression Profile by Human Stem Cell Array% W# e! j, b/ H; O
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A focused array with genes related to human stem cell populations was used to analyze gene expression in undifferentiated BG01 and BG02 cells. The array contains 266 known genes, including 86 that encode markers expressed by stem cells at various stages of differentiation, 96 growth factors and cytokines known to regulate stem cell growth, 36 genes encoding extracellular matrix molecules expressed at appropriate developmental stages, and genes encoding proteins such as cell cycle regulators that are thought to be involved in stem cell division. Positive controls and housekeeping genes were also included for normalization to generate relative expression profiles.7 w# a) o" ^2 K' E8 u
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The results of the human stem cell array analysis are summarized in Figure 8. Of the 266 genes represented by the array, 102 genes were detected in BG01 and BG02. Genes highly expressed included 12 transcription factors, 7 cell cycle-related genes, 25 markers for stem and differentiated cells, 44 growth factors, adhesion molecules and cytokines, and 14 others. In particular, telomerase (TERT), telomerase-associated protein 1, sonic hedgehog, five members of the TGF-? superfamily and receptors, seven members of the FGF family and receptors, 3 WNT genes, and ESG1, an expressed sequence tag that is weakly similar to embryonic stem cell-specific gene 1, were highly expressed. Members of the FGF and leukemia inhibitory factor (LIF) families and their receptors (LIFR) that were expressed are listed in Table 5. Expression of LIF and LIFR was below the limit of detection, and expression of gp130 was very low. The spots for these transcripts, which can be identified in the array , are not visible in Figure 8.
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: E: R, J! C1 Z5 i8 hFigure 8. Gene expression profile of BG01 and BG02 by a focused TGF-?/BMP microarray. A) Images of arrays hybridized using BG01 and BG02 RNA. B) Summary of genes expressed by BG01 and BG02.
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* G# R9 H3 Y$ X9 q, i/ r. F! BTable 5. BG01 and BG02 cells express several FGFs and their receptors but not LIF and its receptor LIFR using a human stem cell array
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DISCUSSION
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* x; h' U8 n( q& R YWe thank members of our laboratories for constant stimulating discussions. We acknowledge the support of NIH grant R24DK063689 to BresaGen Inc.) s, P! r+ f2 D1 b" ]
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Received August 22, 2003; accepted for publication October 28, 2003(Xianmin Zenga, Takumi Miu) |
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发表于 2009-3-5 10:38
发表于 2015-5-28 17:18
