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a Cellular Neurobiology Research Branch, National Institute on Drug Abuse, Department of Health and Human Services (DHHS), Baltimore, Maryland, USA;
b Laboratory of Neuroscience, National Institute of Aging, DHHS, Baltimore, Maryland, USA;
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;
d BresaGen Inc., Athens, Georgia, USA
Key Words. Embryonic stem cells • Differentiation • Microarray
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{at}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{at}grc.nia.nih.gov
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein (endoderm). A large-scale microarray (16,659 genes) analysis identified 373 genes that were expressed at three-fold or higher levels in undifferentiated BG01 and BG02 cells as compared with pooled human RNA. Ninety-two of these genes were also highly expressed in four other hES lines (TE05, GE01, GE09, and pooled samples derived from GE01, GE09, and GE07). Included in the list are genes involved in cell signaling and development, metabolism, transcription regulation, and many hypothetical proteins. Two focused arrays designed to examine transcripts associated with stem cells and with the transforming growth factor-ß superfamily were employed to examine differentially expressed genes. Several growth factors, receptors, and components of signaling pathways that regulate embryonic development, in particular the nodal signaling pathway, were detected in both BG01 and BG02. These data provide a detailed characterization and an initial gene expression profile for the BG01 and BG02 human ES cell lines.
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INTRODUCTION |
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To 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 [4]. Eleven of these lines are currently available for research purposes, and limited data on the biology of 26 of these lines are available [5]. 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 [6]; however, there are no published data on molecular characterization of these cell lines.
Considerable additional information is, however, available on other hES cell lines, notably those derived by Thomson and colleagues [4]. 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 [7–11]. Genetic modification, including homologous recombination, has been reported [12, 13]. Differentiation into multiple phenotypes, including cardiac, hepatic, pancreatic, neural, and hematopoietic lineages, has been described [14–21]. Whether BG01 and BG02 lines have similar properties remains to be determined.
It is unlikely that all hES cell lines will be identical or equally stable in culture. Some differences in human cell lines have been described [5], 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 [22, 23]. Indeed, uniformly successful isolation is not possible in all mouse strains, and it has been difficult to generate rat ES cell lines [24].
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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Differentiation In Vitro
ES cell cultures were dissociated into small clumps by collagenase IV (Sigma) by incubating at 37°C for 5 minutes. The hES cell colonies were pelleted, resuspended in hES medium without bFGF (differentiation medium), and cultured in 6-well plates for 7 days with a medium change every second day. ES cell colonies grew in suspension as embryoid bodies (EBs), while remaining feeder cells adhered to the plate. The EBs were transferred into a new plate and were further cultured for 7 days before immunostaining.
Immunocytochemistry
Expression of stem cell markers was examined by immunocytochemistry, and staining procedures were as described previously [25]. Briefly, the ES cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking, the cells were incubated with primary antibody. The primary antibodies and the dilutions used are stage-specific embryonic antigen (SSEA)-1, SSEA-3, and SSEA-4 (Developmental Studies Hybridoma Bank, University of Iowa; http://www.uiowa.edu; 1:50); tumor recognition antigen (TRA)-1-60 and TRA-1-81 (Chemicon; Temecula, CA; http://www.chemicon.com; 1:100); octamer-binding transcription factor (OCT)-3/4 and cTnI (Santa Cruz Biotechnology; Santa Cruz, CA; http://www.scbt.com, 1:100); TuJ1 and -fetoprotein (Sigma; 1:2000); smooth muscle actin (Sigma; 1:200); and nestin (BD Biosciences; San Jose, CA, http://www.bdbiosciences.com; 1:500). Localization of antigens was visualized by using appropriate secondary antibodies (Alexa fluor 594 or 488, Molecular Probes; Eugene, OR; http://www.probes.com).
RT-PCR Analysis
Total RNA was isolated with TRIzol (Invitrogen), a modification of the guanidine isothiocyanate-phenol-chloroform extraction method. The undifferentiated state of cultured cells was verified by immunostaining of ES markers such as OCT-3/4, SSEA-4, and TRA-1-60. cDNA was synthesized using 2.5 µg total RNA in a 20-µl reaction with Superscript II (Invitrogen) and oligo (dT)12–18 (Promega; Madison, WI; http://www.promega.com). One microliter RNase H (Invitrogen) was added to each tube and incubated for 20 minutes at 37°C before proceeding to the RT-PCR analysis. The PCR primers for OCT-3/4, SOX-2, REX-1, UTF1, hTERT, Dppa5, Cx43, Cx45, and ABCG2 were described by Ginis et al. [26]; the primers for Nanog, FLJ13072 KIAA1265, MGC27165 ZNF342, DNMT3L, DAX-1, Eras, TUBB5, KRT8, KRT18, cardiac actin, and galanin are listed in Table 1
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Large-Scale Oligonucleotide Microarray
The microarray used in this analysis contained 16,659 70-bp oligonucleotides designed from 750 bases of the 3' end of each open reading frame that represents the largest verified set available. Twenty micrograms of total RNA from each of BG01 and BG02 and universal human RNA (huRNA, Clontech; Palo Alto, CA; http://www.clontech.com) were labeled with Cy5 and Cy3, respectively, and duplicate arrays were hybridized and processed using the modified procedure developed at the Center for Biologics Evaluation and Research (CBER) microarray program under an interagency agreement between CBER and the Advanced Technology Center of the National Cancer Institute. The images were captured under wavelengths appropriate for both Cy3 and Cy5 photomultiplier tubes (PMTs; 600V-750V) using a GenePix 4000B scanner (Axon Instruments, Inc.; Union City, CA; http://www.axon.com). At this PMT range, the images gave the best signal-to-noise ratio. The data were initially analyzed using Gene Pix software. Both the scanned image and analyzed data files were uploaded into the Center for Information microarray database (mAdb) (http://www.nciarray.nci.nih.gov). The data were analyzed using two different analytic tools: single array viewer tools and extended data extraction tool. Scatter plot analysis of a control array with Cy5- and Cy3-labeled total RNA derived from huRNA showed equal hybridization of spots as indicated by a straight line from X to Y axis, indicating good quality of most spots and uniform hybridization (not shown).
Focused Microarray Analysis
The nonradioactive GEArray Q series cDNA expression array filters for human stem cell genes and transforming growth factor (TGF) ß/BMPl pathway genes (Hs601 and Hs023; SuperArray Inc.; Frederick, MD; http://www.superarray.com) [26] were used according to the manufacturer’s protocol. The biotin dUTP-labeled cDNA probes were specifically generated in the presence of a designed set of gene-specific primers using total RNA (4 mg/filter) and 200 U Moloney murine leukemia virus reverse transcriptase (Promega). The array filters were hybridized with biotin-labeled probes at 60°C for 17 hours. After that, the filters were washed twice with 2 x SSC/1% SDS and then twice with 0.1 x SSC/1% SDS at 60°C for 15 minutes each. Chemiluminescent detection steps were performed by incubation of the filters with alkaline phosphatase-conjugated streptavidin and CDP-Star substrate. Array membranes were exposed to x-ray film. Quantification of gene expression on the array was performed with ScionImage software. cDNA microarray experiments were done twice with new filters and RNA isolated at different times. Results from the focused array were independently confirmed, and the array itself was validated using procedures previously described [27].
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RESULTS |
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). 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 [6]. 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).
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Undifferentiated 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.
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). 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.
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-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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. 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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). 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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). 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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shows a hybridization profile of BG01 and BG02, and Figure 7B summarizes the genes detected in both BG01 and BG02.
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). 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.
Gene Expression Profile by Human Stem Cell Array
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.
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-ß 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 [27], are not visible in Figure 8.
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DISCUSSION |
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The potential of hES cells to contribute to the germline of chimeric organisms cannot be tested in humans and is not readily tested in nonhuman primates [2–4], and thus additional criteria for evaluating human and primate ES cells need to be used [4]. Human and subhuman primate ES cells have been defined as cells that are derived from preimplantation of peri-implantation embryos, and can be maintained in culture for prolonged periods in an undifferentiated state, while retaining the capacity to differentiate into cells of all three embryonic germ layers [4]. Our results show that like the hES cell lines, BG01 and BG02 appear morphologically and antigenically similar to the previously described hES cell lines. Like other hES cell lines [26] and unlike mouse ES cells, BG01 and BG02 cells are larger, grow more slowly, and grow in fewer layers. BG01 and BG02 express markers of undifferentiated ES cells like Sox-2, Oct-3/4, Nanog, TERT, SSEA-3, and SSEA-4; lack expression of markers of differentiation; can be maintained in culture for over 40 passages; and retain the ability to differentiate and express markers characteristic of ectoderm (TuJ1), endoderm (AFP), and mesoderm (cTnI). Like other cell lines tested [26, 28], BG01 and BG02 express galanin, Keratin 8 and 18, and several additional novel markers of the ES state such as Nanog and ZFN342. Similar to other ES lines, BG01 and BG02 can be cryopreserved and propagated extensively, and preliminary results suggest that clonal derivatives can be isolated. Like other ES cells that have been described, BG01 and BG02 do not appear to require LIF for their propagation and survival, and expression of LIFR or gp130 is low or absent (Fig. 7; Table 5; data not shown).
Thus BG01 and BG02, like the limited number of other hES lines, fulfill the minimal definition of an ES cell. It is important to note that although the NIH hES cell registry includes 78 derivations (http://www.stemcells.nih.gov/registry/eligibilityCriteria.asp), most have not been demonstrated to meet the minimal definition of stem cells. A subset of the derivations have, however, been described as hES cell lines in the literature, primarily those from Wicell and ESI, that can be cryopreserved and maintained in culture, undifferentiated, for several months.
Direct comparison with other lines and publicly available databases suggest that BG01 and BG02 have properties that are very similar to other hES cells [4, 26]. However, BG01 and BG02 cells in vitro require a much higher density of MEFs in the feeder layer in order to maintain in an undifferentiated state. In contrast to other hES cell lines, BG01 and BG02 can be passaged by the use of trypsin, which works as well as nonenzymatic cell dissociation buffers. More importantly, BG01 and BG02 recovered more rapidly from frozen vials (within 2–3 days) as compared with GE01 and GE04 cell lines. In general, these cells are easy to grow and maintain in vitro, which could be an advantage of these lines as compared with other hES cells that require mechanical dissociation or special handling.
Gene expression patterns for the BG01 and BG02 lines were analyzed by immunocytochemistry, RT-PCR, and two separate microarray platforms. Three hundred seventy-three genes were identified as being highly expressed in both BG01 and BG02. Included were 92 stemness genes known to be expressed in mES or hES cells such as OCT-3/4, NANOG, Cripto/TDGF1, Cx43, and galanin [28]. The same genes were also overexpressed in an additional four hES lines (TE06, GE01, GE09, and a pooled set of subclones derived from GE01, GE09, and GE07). Differentially expressed genes include Nanog (a recently identified protein FLJ12581critical for maintaining pluripotency of mouse ES cells) and other hypothetical proteins, KIAA1573 and MGC27165 which were all highly expressed in both BG01 and BG02. Both cell lines also expressed ZNF342, DNMT3L, and DAX-1, which were also confirmed by RT-PCR. Genes highly expressed in BG01 and BG02 also included numerous cell signaling/cell cycle/cell development-related genes, metabolism genes including DNA replication and DNA repair enzymes, RNA-related ribosomal genes, metabolic activity-related genes, transcription factors, and immune response genes. Also present were 19 novel genes with unknown function, illustrating the value of a large-scale gene expression analysis and the potential for identifying novel pathways of regulation.
Among the genes that were differentially expressed between BG01 and BG02 are members of the TGF-ß superfamily member Nodal and its antagonist Lefty. Nodal signaling plays important roles in early embryonic development, patterning, and left-right axis positioning, as well as in the early stages of ES cell development [29–32]. Expression of genes related to the Nodal signaling pathway in undifferentiated BG01 and BG02 cells was studied with a focused microarray containing probes for TGF-ß superfamily members and key proteins involved in the TGF-ß signal-transduction pathway. Nodal; LeftyA; LeftyB; Cripto-1 (TDGF-1); Cerberus; activin receptors ALK-3; and SMAD3, SMAD5 and SMAF6, all molecules of the Nodal signaling pathway [33, 34], were expressed in both BG01 and BG02. While all components are present, this pathway is actively inhibited by negative regulators such as TDGF-1 and Nodal. Surprisingly, noggin does not appear to play as important a role in hES cell differentiation as has been proposed in rodent and xenopus studies [35–37]. Overall, the expression patterns for the TGF-ß superfamily were virtually identical in BG01 and BG02, further reinforcing the similarity of the two lines.
Human ES cells are likely to be an important resource for biomedical research over the next decade, since these cells will allow studies of differentiation, gene expression, and biochemical pathways to be performed using human material. Cells that have been differentiated from hES cells are also likely to be useful for a wide range of mundane but valuable purposes such as screening drugs and antiviral agents. Thus, in addition to the therapeutic possibilities, there are important potential uses of hES cells for basic research. Although it is potentially possible to generate a large number of hES cell lines, the number of lines that will be needed to be representative is unclear. Human ES cell lines may differ from each other due to differences in conditions and developmental stage under which they were isolated, in addition to male-female differences and differences related to genetic variations. While it is expected that the similarities among hES cell lines would be much greater than their differences, even small differences between hES cell lines may be critical in determining their utility. Although the NIH registry contains 78 putative stem cell lines(http://stemcells.nih.gov/registry/eligibilityCriteria.asp), very few of these lines have been described in the literature.
Our results show that BG01 and BG02 are generally similar to the other hES cell lines for which data on characterization are available. Both cell lines can be easily maintained in an undifferentiated state, grow rapidly, and readily differentiate into all major phenotypes, suggesting that these lines can be added to the short list of validated, potentially useful hES cell lines.
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ACKNOWLEDGMENT |
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