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Designing, Testing, and Validating a Focused Stem Cell Microarray for Characterization of Neural Stem Cells and Progenitor Cells YONGQUAN LUO,a JINGLI CAI,a IRENE GINIS,a YANYANG SUN,b SIULAN LEE,b SEAN X. YU,b AHMET HOKE,c MAHENDRA RAOa a
Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, Baltimore, Maryland, USA; bSuperArray Bioscience Corporation, Frederick, Maryland, USA; cDepartment of Neurology, Johns Hopkins University, Baltimore, Maryland, USA Key Words. Embryonic stem cells · Neural stem cells · Focus array · Microarray · Functional genomics
A BSTRACT Fetal neural stem cells (NSCs) have received great attention not only for their roles in normal development but also for their potential use in the treatment of neurodegenerative disorders. To develop a robust method of assessing the state of stem cells, we have designed, tested, and validated a rodent NSC array. This array consists of 260 genes that include cell typespecific markers for embryonic stem (ES) cells and neural progenitor cells as well as growth factors, cell cycle-related genes, and extracellular matrix molecules known to regulate NSC biology. The 500-bp polymerase chain reaction products amplified and validated by using gene-specific primers were arrayed along with positive controls. Blanks were included for quality control, and some genes were arrayed in duplicate. No cross-hybridization was detected. The quality of the arrays and their sensitivity were also examined
by using probes prepared by conventional reverse transcriptase or by using amplified probes prepared by linear polymerase replication (LPR). Both methods showed good reproducibility, and probes prepared by LPR labeling appeared to detect expression of a larger proportion of expressed genes. Expression detected by either method could be verified by RT-PCR with high reproducibility. Using these stem cell chips, we have profiled liver, ES, and neural cells. The cell types could be readily distinguished from each other. Nine markers specific to mouse ES cells and 17 markers found in neural cells were verified as robust markers of the stem cell state. Thus, this focused neural stem array provides a convenient and useful tool for detection and assessment of NSCs and progenitor cells and can reliably distinguish them from other cell populations. Stem Cells 2003;21:575-587
INTRODUCTION Neural stem cells (NSCs) and progenitor cells are present throughout development and persist in the adult [1-3]. Multiple classes of NSCs and progenitors have been identified, and these cells differ from each other in their differentiation
ability, cytokine response, and antigens expressed [3, 4]. Moreover, as cells have been evaluated for therapy it has become clear that non-neural cells may also transdifferentiate or dedifferentiate into neural derivatives upon exposure to appropriate differentiating agents [4, 5]. Furthermore,
Correspondence: Mahendra Rao, M.D., Johns Hopkins University, Baltimore, Maryland 21224, USA. Telephone: 410-5588204; Fax: 410-558-8249; e-mail:
[email protected] Received April 11, 2003; accepted for publication July 17, 2003. ©AlphaMed Press 1066-5099/2003/$12.00/0
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embryonic stem (ES) cells may be used to obtain NSC and progenitor cells, and these differentiated cells must be distinguished from ES cells to prevent transplantation of potentially teratocarcinoma-forming cells [5, 6]. Finally, NSCs differ from many other stem cell populations in that investigators have developed techniques to propagate them in continuous culture. However, it has been noted that cultures may spontaneously differentiate or alter characteristics sufficiently, such that their stem cell character is altered or lost. These observations highlight the importance of careful monitoring of the properties of the cells harvested and the quality of the cells maintained in culture using a reliable and reproducible method that is low cost and readily available to most laboratories involved in stem cell research. What is also clear is that few unique markers for NSCs exist. Many of the markers previously thought to be specific for stem cells have been shown to be expressed by differentiated cells (nestin, nucleostenin, musashi, etc.) or shared with other stem cell populations (e.g., ABCG2, telomerase reverse transcriptase [TERT], telomerase activity), and no single positive marker specific for NSCs is currently available. Rather, laboratories have successfully isolated stem cell populations from mixed cultures using a combination of markers. Presence of candidate stem cell markers, such as AC133, Hoechst dye labeling, Sox-1, Sox-2, and TERT, have been combined with negative selection using the absence of a battery of markers to detect and enrich stem cell populations [7]. Evaluating the purity and state of NSC populations therefore relies on using a battery of markers. Different laboratories utilize some subset, often nonoverlapping, of known markers. Given the amount of tissue or cells required for the variety of tests and the difficulty in obtaining, maintaining, and validating antibody, Western blot, polymerase chain reaction (PCR), or Northern blot markers, consistent evaluation of neural populations across laboratories and even between one batch of cells and another has been difficult. Traditional assay techniques such as Northern blot, ribonuclease protection assay, or more recent techniques such as quantitative real-time reverse transcriptase-PCR (RT-PCR), are suitable for studying one or a few genes in a single assay. The ideal tool required would be one that is capable of studying gene expression of related gene sets simultaneously. If such a sufficiently reliable tool existed, then not only could cell type-specific markers be studied, but additional candidate genes known to be expressed at the appropriate stage of development could be rapidly evaluated as well. Such genes could be growth factors, extracellular matrix (ECM) molecules, chemokines, or key regulators of cell proliferation or apoptosis. Thus, developing a reliable gene expression analysis tool for stem cell-specific gene sets
Making a Focused Array for Accessing Neural Stem Cells will not only enhance the ability to characterize stem cells but also help in understanding the mechanisms that regulate stem cell differentiation. cDNA microarray technology, a relatively new technology that allows simultaneous assessment of the expression of thousands of genes, is a potential candidate assay method. This method has been successfully used to identify gene expression patterns associated with specific biological functions [5, 8] and has been proven to be reliable and reproducible [5, 8]. However, there are a number of technical limitations and problems that are associated with most commercially available microarrays that limit their use for assays, such as the one we have proposed. The first is the limitation of gene coverage. Although high-density microarrays contain thousands of genes on a single glass slide, they usually fail to provide full gene coverage of specific gene groups necessary for a particular application. The incomplete coverage of the array may come from the fact that: A) collection of genes in high-density cDNA arrays is usually generated without specific application in mind, and B) the majority of genes involved in regulation of cellular activities and cell differentiation are often only expressed for a very short period of development [9]. It is therefore not unusual that many of those cell development and differentiation importance genes are not included in high-density microarrays. For example, only 49 of the 70 known interleukin and receptor-related genes are present on one commercial array. Likewise, few of the 23 known fibroblast growth factor (FGF) genes are present on any of the large microarrays that are available. The second problem with large-scale arrays is cDNA fragment selection and quality. The cDNA fragments used in the microarrays are usually 3′ end biased since they are generated from oligo(dT)-primed cDNA synthesis. The 3′ end region is not necessarily the most gene-specific region for some genes and is often a poor choice for a gene-specific probe. The third problem is its complexity of data collection and analysis. In fact, the cost and complexity involved in special experiment equipment acquirement and complex data analysis are two barriers that prohibit most research laboratories from using microarray technology as a routine research tool. Thus, a general high-density cDNA array may not be the best tool for gene-expression profiling in stem cell research. In this paper, we report a mouse stem cell array containing a specific set of genes related to stem cell proliferation and differentiation. We have collected, cloned, and validated 260 genes for this array. The chip is composed of known molecular markers for ES cells, neural progenitor cells, growth factors, cell cycle-related genes, and ECM molecules. The genes were printed as a validated set of 500-bp (approximate) PCR
Luo, Cai, Ginis et al. products. Testing these chips using either conventional Moloney murine leukemia virus (MMLV) RT or the linear polymerase replication (LPR) method showed that the arrays were of high quality and provided reliable detection and reproducibility. Using these stem cell chips, we can identify different gene expression patterns in tissues formed by different cell types, demonstrating the potential use. Overall, we conclude that this chip provides a convenient and useful tool for research in the stem cell field.
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middle of regions of cDNA fragments of the genes replicated by forward and reverse primers. PCR reactions were performed by using the clones as templates in the presence of the forward and QC primers. If the PCR products were obtained with expected fragment lengths, the cloning was deemed successful. When the identity of the cDNA fragment was in doubt, DNA sequencing was used for verification. Forward primers 5′
MATERIALS AND METHODS Primer Design, Clone Preparation, and Verification Primers to amplify genes were designed by using a computer program developed by SuperArray Bioscience Corporation (Frederick, MD; http://www.superarray.com). Briefly, a list of candidate genes was prepared. The National Center for Biotechnology Information (NCBI) Unigene number was obtained from the NCBI database (http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?db=unigene). The longest cDNA sequence among the Unigene cluster was then picked for cDNA fragment selection. From the longest cDNA sequence, PCR primer pairs were then designed by using Primer 3 software to obtain 300-600 base products. The resulting candidate cDNA fragment set was subjected to a basic local alignment search against all Genbank sequences. The fragment with the least sequence similarity against other genes was then chosen as the cDNA probe. Corresponding primers for syntheses of these gene-specific cDNA fragments were chosen and used in processes of cloning, RT-PCR, and labeling of probes with the AmpoLabeling kit (SuperArray) in array hybridization. The reverse gene-specific primers were also used in preparation of labeling of probes with a conventional method for array hybridization. An internal primer was used for quality control purposes. Clones for verified cDNA fragments of candidate genes were prepared by following the procedures described in the TA Cloning Dual Promoter Kit (Cat# K2050-01, Invitrogen; Carlsbad, CA; http://www.invitrogen.com). The cDNA was synthesized with RNA from various tissues, including mouse D3 ES cells, by following the procedures described in the RT-PCR section. PCR reactions were performed and PCR fragments with the expected lengths were obtained and then ligated into the vectors in the presence of T4 DNA ligase. These completed ligation reactions were transformed into INVαF′-competent cells, plated on X-Gal plates, and grown overnight at 37°C. The white clones were picked to verify cDNA fragment cloning. Validation of cDNA fragment clones were performed as follows: as indicated in the following pictures, quality control primers (3′ ≥ 5′, QC primer) were designed in the
3′ Gene of interest
QC primer
Reverse primer
PREPARATION OF ARRAYS Arrays were made by PCR-amplified fragments from plasmids containing the cloned inserts and forward and QC primers. The resulting PCR products were then concentrated and adjusted to 150 µg/ml in 0.08 N freshly prepared NaOH. Bromophenol blue (0.001%) was added into the source plate as the tracking dye to monitor the array printing quality. A Cartesian SynQuad Prosys dispensor (Genomic Solutions; Ann Arbor, MI; http://www.genomicsolutions.com) was used to dispense between 10-15 nl cDNA solution onto nylon membrane (Nytran, Amersham Bioscience; Buckinghamshire, UK; www.amershambioscience.com). All array spots were arranged in a rectangular area (23 × 35 mm). Spot diameter was between 0.7-0.9 mm. The spot-to-spot distance was 1.25 mm. The printed membrane was air dried at room temperature overnight and then subjected to 1200 J ultraviolet cross-linking. The array was stored at -20ºC until used. cDNA Microarray and Data Analysis Total RNA from indicated tissues or cells was isolated by using TRIzol (Invitrogen). The biotin deoxyuridine triphosphate (dUTP)-labeled cDNA probes were specifically generated by following protocols of either the AmpoLabeling (LPR kit; Cat#: L-03N) or conventional MMLV RT method (Cat#: MM-601N, SuperArray Bioscience Corp.). For LPR amplification, RNAs were first annealed with a random primer at 70°C for 3 minutes, then reverse transcribed to cDNA at 37°C for 25 minutes. These cDNAs were amplified by PCR with gene-specific primers and presence of biotin-16-dUTP. The PCR cycle was 85°C for 5 minutes; 30 cycles (85°C, 1 minute; 50°C, 1 minute; 72°C, 1 minute); and 72°C for 5 minutes. For MMLV RT probe preparation, the RNAs were simply reverse transcribed to cDNAs in the presence of gene-specific primers and biotin-16dUTP at 42°C for 90 minutes. The mouse stem array filters were hybridized with the biotin-labeled probes at 60°C for 17 hours. After that, the filters were first washed twice with 2 × standard saline citrate (SSC)/1% SDS and then twice with
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0.1 × SSC/1% SDS at 60°C for 15 minutes each. Chemiluminescent detection steps were performed by subsequent incubation of the filters with alkaline phosphatase-conjugated streptavidin and CDP-Star (Applied Biosystems; Salt Lake City, UT; http://www.appliedbiosystems.com) substrate and exposure to electrochemiluminescence film. For data analysis, the positive and negative spots were independently identified and verified by at least two people. Only the matched positive and negative results of two experiments are presented. For quantification, intensity of spots was first measured by ImageQuant 5.2 software (Amersham Biosciences), and then the average intensities derived from 10 blank spots were subtracted. These subtracted intensities were divided by an average of intensities from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (three spots in each array), to obtain a relative intensity for each spot. The relative intensities were used to generate scatter plots in Figures 1 and 2 using Excel software. Cross-Hybridization Assessment Seventeen genes were randomly selected (fgf4, Gcm2, Mtap1b, Mtab2, Tubb3, Prox1, Neurofilament [Nfl], neural cell adhesion molecule [NCAM]2, Cst3, Ccng2, CDKn1b, DNMT1, Cdh4, Itga6, Rpl13a, Actb, and GAPDH). Biotinlabeling probes were constructed by PCR. Each reaction of 10 µl volume contained 1 µl 10× PCR buffer, 150 µmol MgCl2, 0.8 µl buffer BN (SuperArray Bioscience Corp.), 20 pmol primer, 1 µl 100× diluted specific gene inserted plasmid, 1 U RedTaq DNA polymerase (Sigma; St. Louis, MO; http://www.sigmaaldrich.com), and 0.2 pmol biotin-dUTP. The reaction was performed in 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final extension for 10 minutes at 72°C. Three microliters of each labeled cDNA fragment were used to check probe quality by running it in 1.2% agarose gel. The labeled probe for each gene was then pooled and used to perform an array hybridization following procedures described in the section on cDNA microarray and data analysis. Isolation of Embryonic Neural Tubes, Hippocampus, and Liver Timed pregnant Sprague-Dawley rats at E14.5 and young male Sprague-Dawley rats (6 months) were purchased from Harlan Sprague-Dawley (Indianapolis, IN; http://www.harlan.com) and housed individually in standard cages at the National Institute on Aging (NIA) National Institutes of Health (NIH). They were maintained at 22°C on a 12/12h light/dark cycle with free access to food (NIH-07) and water. The rats were allowed to acclimate to the vivarium until the day of the experiment. Experimental handling and experimental procedures were approved by the NIA Animal Care and Use Committee.
E14.5 Sprague-Dawley rat embryos were used to isolate neural progenitor cells as previously described [10]. Briefly, the rat embryos were removed and placed in a Petri dish containing ice-cold phosphate-buffered saline (PBS; Invitrogen). The trunk segments of the embryos (last 10 somites) were dissected, rinsed, and then transferred to fresh cold PBS. The hippocampus and liver tissues from 6-monthold male Sprague-Dawley rats were dissected. All isolated embryonic neural tubes and tissues derived from adult rats were stored in an RNAlater solution (Ambion; Austin, TX; http://www.ambion.com) at 4°C for late RNA isolation. Mouse D3 ES Cell Line Undifferentiated D3 ES cells (American Type Culture Collection [ATCC]) were first expanded on STO-1 feeder cells (ATCC) that were treated with 1 mg/ml mitomycin C (Sigma) for 3 hours to arrest cell division. Subconfluent D3 cultures were then trypsinized and replated onto gelatin-coated tissue culture plates in the presence of 1,400 U/ml of leukemia inhibitory factor ([LIF] Chemicon; Temecula, CA; http://www.chemicon.com) in ES cell medium consisting of knockout Dulbecco’s minimal essential medium ([MEM] Invitrogen/GIBCO) supplemented with 15% ES-qualified fetal bovine serum (Invitrogen/GIBCO), 100 mM MEM nonessential amino acids, 0.55 mM β-mercaptoethanol, 2 mM L-glutamine, and antibiotics (all from Invitrogen/GIBCO). When confluent, the D3 cells were harvested and stored at 80°C for late RNA isolation. A separate analysis of this D3 cell line grown under feeder-free conditions by RT-PCR and immunocytochemistry showed expression of OCT-3/4, Sox-2, Rex1, and Tert without detection of differentiated cell markers such as NCAM, Nfl, and glial fibrillary acidic protein (GFAP) (I. Ginis, personal communication). RT-PCR cDNA was synthesized using 1 µg total RNA in the presence of Superscript II and oligo(dT) 12-18 (both from Invitrogen). The PCR was performed in a 20-µl reaction solution containing 2 µl 10× PCR buffer, 150 µmol MgCl2, 10 nmol deoxyribonucleoside-triphosphate , 20 pmol primer, 1 µl 10× diluted cDNA, and 1 U RedTaq DNA polymerase (Sigma). The PCR conditions were as follows: 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final extension for 10 minutes at 72°C. Primer sequences are available upon request. RESULTS Developing a Rodent Neural Stem Gene Array A total of 260 known genes was chosen (Table 1). Genes were selected on the basis of their known distribution and
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Image profiles
Confirmation by PCR
LPR
MMLV RT
Array+ PCR+
Array– PCR+
Array– PCR–
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156
24
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D Reproducibility in quantification
Reproducibility in quantity MMLV RT method Exp. 1 n of positives
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Exp. 2
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n of negatives 1.5
Blanks
10 12
Exp 2
Positive and housekeeping controls
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1 0.5
Samples Duplicated genes
184
72
180
76
0
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4
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4
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n of n of negatives positives 10
Positive and housekeeping controls
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Blanks
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LPR method
Exp. 1 n of positives
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0.5 Exp. 1
LPR method
Groups
r2 = 0.9911
12
1 0.5
Samples Duplicated genes
184
72
180
76
0
6
4
6
4
-0.5
Total
202
86
198
90
r2 = 0.7272
1
0.5
1.5
Exp. 1
Figure 1. Quality control of the chips and comparison of results derived from MMLV RT and LPR methods. Total RNA from mouse D3 ES cells and adult rat hippocampus was first isolated and then mixed together with a ratio of 3 over 1. This mixed RNA (0.8 mg/membrane) was used to prepare probes using MMLV RT Semi-quantitative RT-PCR or LPR methods. Labeled probes were then used to perform array hybridization. A) shows a distinct image profile obtained by these two methods. The LPR method (71%, 184/260) also reveals a higher detection rate than the MMLV RT method M bp Cycle (31%, 80/260). Short arrow: spot for integrin b5. Underline: four positive controls 600 in triplicate. B) is a summarized result comparing array data and RT-PCR ampli25 fication. Both methods show high agreements (96%-98%) between positive results 600 30 in array and RT-PCR. The false-positive rate is from 2.5%-4.3%. Tables in (C) 600 summarize the results of positive and negative hybridization from two experi35 ments. A relative intensity of each spot was calculated and plotted for these repeated experiments. These scatter plots are shown on (D). The data suggest a larger variability for probe prepared by the LPR method compared with the MMLV RT-labeled probe. Further analysis of expression of five genes by semiquantitative RT-PCR is shown on E. Results from this RT-PCR are in agreement with those obtained by MMLV RT method (see underline in A). Itg ae Pp ia R pl 13 a Ac tb G AP D H
E
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A
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Image profiles Mouse D3 ES cells
E14.5 Rat NTs
Comparison between tissues
4 3 Liver
2
r = 0.5563
2 1 0
Adult rat liver
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4 3 Liver
E14.5 NT
RNA relative intensity
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r = 0.4603
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Liver
2
r2 = 0.703
1 0 1
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2 E14.5 NT
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RNA relative intensity
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D RT-PCR
Markers detected by array
Gene names Dnmt1 ES Fgf4 Itga6 Pou5f1 Sox2 Lifr Zfp42 Gcm2 Prdc Nkx2-2 Neural progenitors Mtap1b Tubb3 Cdkn1b Nfl Ccng2 Cdh4 Ncam2 Ncam1 Mtab2 Cdh2 Fabp7 Vim Syn1 Cst3 Cdh5 Prox1
PCR
Array
Markers
D3 ES
E14.5 NT
Liver
D3 ES
E14.5 NT
Liver
0.40 0.37 0.39 0.29 0.15 0.08 0.40 0.22 0.16 0.38 0.10 0.07 0.07 0.04 0.00 0.01 0.00 0.00 0.04 0.00 0.00 0.04 0.04 0.01 0.09 0.00
0.05 0.11 0.01 0.21 0.13 0.07 0.06 0.15 0.23 0.83 0.77 0.73 0.24 0.20 0.13 0.01 0.01 0.42 0.34 0.24 0.68 0.05 0.10 0.05 0.02 0.20
0.00 0.19 0.10 0.14 0.07 0.29 0.03 0.25 0.09 0.64 0.30 0.17 0.06 0.15 0.03 0.08 0.00 0.13 0.28 0.24 0.00 0.11 0.14 0.07 0.04 0.38
0.62 0.80 0.67 0.23 0.98
0.20 0.37 0.21 0.15 0.71
0.13 0.43 0.19 0.21 0.13
FGF4
POU5f1
DNMT1
ABCG2
M bp
Lif
600
Tebp
Tert
Terf1
Itga6
M
Sox2
600
Mtap1b
Nfl
Tubb3
NCAM2
NKX2.2
M 600
0.10 0.09 0.53 0.22 0.12 0.34 0.23 0.04
0.14 0.19 0.85 0.40 0.48 0.63 0.49 0.12
0.07 0.04 0.07 0.14 0.09 0.13 0.08 0.07
p27Kip1
Cdh4
Cyclin G2
PTEN
GAPDH
M 600
1
2
3
1
2
3
1
2
3
1
2
3
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Figure 2. Array and RT-PCR analyses for different types of tissues. Total RNAs (3 µg/membrane) derived from mouse D3 ES cells, rat E14.5 neural tubes, and adult rat liver were used to make biotin dUTP-labeled cDNA probes with the MMLV RT method. The mouse stem cell array filters were hybridized with these probes and their images were recorded. A) shows a representative image profile of three experiments. A short arrow indicates ES markers in the first row, whereas a longer arrow points to neural markers in the fifth row. A majority of ES markers can be detected in undifferentiated D3 cells but not in partially differentiated neural progenitors and adult liver. Neural markers are detected relatively higher in E14.5 rat neural tubes compared with the expression in D3 and adult liver. A relative intensity of each spot was calculated and used to make scatter plot as shown in (B). Low r2 values indicate variability of gene expression among these tissues. The relative intensities (mean of three experiments with CV
