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Cell Biology International (2007) 31, 861869 (Printed in Great Britain)
Conventional and molecular cytogenetic diagnostic methods in stem cell research: A concise review
Purificación Catalinaa*, Fernando Coboa, José L. Cortésa, Ana I. Nietoa, Carmen Cabreraa, Rosa Montesa, Ángel Conchab and Pablo Menendeza*
aAndalusian Stem Cell Bank (Bancelán), Instituto de Investigaciones Biomédicas, Parque Tecnológico de la Salud, Avda del Conocimiento s/n, 18014 Granada, Spain
bServicio de Anatomía Patológica, Hospital Virgen de las Nieves, Granada, Spain
Regenerative medicine and cell therapy are emerging clinical disciplines in the field of stem cell biology. The most important sources for cell transplantation are human embryonic and adult stem cells. The future use of these human stem cell lines in humans requires a guarantee of exhaustive control with respect to quality control, safety and traceability. Genetic instability and chromosomal abnormalities represent a potential weakness in basic studies and future therapeutic applications based on these stem cell lines, and may explain, at least in part, their usual tumourigenic properties. So, the introduction of the cytogenetic programme in the determination of the chromosomal stability is a key point in the establishment of the stem cell lines. The aim of this review is to provide readers with an up-to-date overview of all the cytogenetic techniques, both conventional methods and molecular fluorescence methods, to be used in a stem cell bank or other stem cell research centres. Thus, it is crucial to optimize and validate their use in the determination of the chromosomal stability of these stem cell lines, and assess the advantages and limitations of these cutting-edge cytogenetic technologies.
Keywords: Stem cell research, Cytogenetics, Fluorescence in situ hybridization, Karyotyping, Comparative genomic hybridization, Spectral karyotyping.
*Corresponding authors. Tel.: +34 697 956939; fax: +34 958 020132.
Human adult and embryonic stem cells (hESC) in transplant programmes has supposed a considerable advance not only in regenerative medicine and cell therapy but also in basic biology. The potential therapeutic use of hESC derivatives requires guarantee of their quality, safety and traceability with the implementation of exhaustive quality control programmes. One of the main problems with stem cell lines of different source, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability which will explain, at least in part, their in vivo tumourigenic potential (Ringden et al., 2003; Cowan et al., 2004; Draper et al., 2004; Menendez et al., 2005a,b, 2006). Therefore, the introduction and the application of a strict cytogenetic programme is necessary. The study of processes such as cell-renewal, pluripotency, specific differentiation, undifferentiated associated markers, telomerase, mitochondrial, epigenetic and chromosomal stability must be considered and implemented in this emerging area of stem cell biology (Mandal et al., 2006).
The chromosomal stability in a cell line consists, mainly, of maintaining a normal karyotype both numerically and structurally, because hESC lines develop chromosomal aberrations after long-term culture indicating an unstable genomic status due to the in vitro culture after successive passages have been carried out (Maitra et al., 2005; Caisander et al., 2006; Ludwig et al., 2006). Human ESCs may be prone to acquiring chromosomal anomalies while being cultured continuously in vitro. This could be due to the in vitro environment itself and to the hESCs are cultured. Some reports have revealed the possibility that this happens due to the way the cell culture passages are carried out. So, there is 12 and 17 trisomy in cell cultures whose colonies were enzymatically disaggregated, whereas mechanical slicing to subculture the colonies in long-term culture maintain normal karyotype for a long time (Cowan et al., 2004; Draper et al., 2004; Hoffman and Carpenter, 2005; Mitalipova et al., 2005). Other studies reveal the appearance of chromosomal abnormalities in approximately 20% of the cell cultures, but in the majority of them mosaicism appears, with subpopulations which accumulate alterations in the karyotype (Hoffman and Carpenter, 2005). Enzymatic trypsin- or collagenase-based methods most likely favour the selection of fast-growing cells, which may have undergone mutations or chromosomal rearrangements (Mikkola et al., 2006). Therefore, hESC lines are expanded by mechanical cutting and all those cultured for prolonged periods of time have been chromosomally stable after both freeze–thaw procedures and culture for up to 35
The existence of chromosomal abnormalities in the stem cells with potential to be used in cell therapy programmes will be a relevant problem since, in vivo, the chromosomal changes are often associated with carcinogenesis (Ringden et al., 2003; Buzzard et al., 2004). Some researchers have reported abnormal karyotypes in hESC that are similar to those abnormalities observed in human embryonic carcinoma cells, such as trisomy 12 and 17 (Brimble et al., 2001; Clark et al., 2004). This increased dosage of chromosomes 17q and 12, provides a selective advantage and clonal evolution for the propagation of the undifferentiated abnormal hESC clone (Draper et al., 2004). Moreover, it has been reported that accumulation of karyotyping abnormalities was observed with long-term culture, when colonies were disaggregated to single cells, from trisomy of chromosomes 12, 17, X with occasional trisomy of chromosomes 1, 7, 8 and 14 (Brimble et al., 2004). The genetic instability in hESCs may arise from the selection of cells with a growth advantage in culture. Specifically the presence of isochromosome 12p is also a common feature of human teratocarcinoma cells (hESCs), the malignant counterpart of hESCs (Clark et al., 2004; Draper et al., 2004; Skotheim et al., 2002) and the amplification of 17q is associated with neuroblastoma (Westermann and Schwab, 2002). Furthermore, surrogate genes that control self-renewal, pluripotency, differentiation and apoptosis, including STELLAR, NANOG, GDF3, GRB2, BIRC5 and NT3, are located on those chromosomes (Burdon et al., 2002; Clark et al., 2004) and in vitro constraint may provide the pressure to increase the gene copy number. The expression of the truncated form of CD30 is also a feature that is associated with the inhibition of apoptosis and increased cell survival (Herszfeld et al., 2006). It is our opinion that extensive cytogenetic studies should be performed in parallel to CD30 immunophenotyping testing to rule out potential cell transformation (Menendez et al., 2005b). Therefore, the cytogenetic control of these hESC lines to check the chromosomal content before the application of any potential allogenic transplant programme is crucial because some researchers have described chromosomal alterations in these, very similar to those which appear in embryonic carcinomas and other infant tumours which have been proposed to have a pre-natal origin in an ancestral embryonic/foetal cell in utero (Cowan et al., 2004; Draper et al., 2004; Menendez et al., 2006).
The most common cell karyotype study method used for years has been the conventional or classic cytogenetic techniques, where the cells are cultured, metaphases are obtained and the crossed chromosomes with an appropriate stain are studied. This technique, although it was first described some time ago, is still valid and informative. However, currently, there are other state-of-the-art methods of molecular cytogenetics that could resolve the main problem of the conventional technique, because it is not always possible to obtain an adequate number of metaphases in the process or the quality of these metaphases does not permit a detailed study of the chromosomes. In this review we attempt to elucidate the technology available for the determination of chromosomal stability in stem cell biology, Furthermore we attempt to provide the reader with an up-to-date and unbiased assessment of the indications for the use of these techniques and their applicability in stem cell research.
2 Potential methods for cytogenetic diagnosis of stem cell cultures
2.1 Conventional cytogenetics
A karyotype may be defined as the accurate organization (matching and alignment) of the chromosomal content of any given cell type. In a karyotype, chromosomes are arranged and numbered by size, from the largest to the smallest. Karyotype is the normal nomenclature used to describe the normal or abnormal, constitutional or acquired chromosomal complement of an individual, tissue or cell line (ISCN nomenclature; Shaffer and Tommerup, 2005). The conventional cytogenetic techniques are used routinely for the determination of numerical chromosomal abnormalities or structural rearrangements, mainly translocations, in cultured hESC lines; this helps scientists to quickly identify chromosomal alterations. Therefore, cell cultures should be maintained in culture medium from the time they are obtained until they are used from chromosome preparation. Some of the most commonly used culture media in cytogenetics laboratories are MEM, RPMI 1640 and Ham F10; however to culture hESCs a specific medium with the inclusion of additional components is necessary, such as growth factors, to promote cell proliferation whilst preventing spontaneous differentiation. Human ESC lines are usually established on either inactivated mouse embryonic cells (MEFs) or different types of human embryonic fibroblasts (HEFs) (Menendez et al., 2004; Suemori et al., 2006). The morphology of the hESC colonies does not vary remarkably between the two feeder systems, but the density of the colonies are apparently higher in MEF cultures than in cultures grown on HEFs. A cell density of about 1 Fig. 1 Representative picture of a male karyotype of HS 293 hESC line.
Representative picture of a male karyotype of HS 293 hESC line.
2.2 Analysis and data interpretation of conventional cytogenetics
The aforementioned section has described the process involved in getting metaphase divisions from a sample slide, ready for analysis. A 10× objective lens can be used for screening, and oil immersion 100× objective is usually required to study the metaphases. The best way to systematically screen the slide is up-and-down, moving across the slide in small increments. It is very important to remember, when selecting divisions, to bias towards those with poor morphology. Selection of only good metaphases can lead to the failure to detect the abnormal clone presence.
Direct analysis down the microscope is possible with experience, and is generally adequate when full analysis is not required or when any clonal abnormality is simple. Direct analysis is probably the most rapid way of working through a case, but there are drawbacks, such as the fact that considerable experience is required. Preparing a karyogram of each division analysed produces the most reliable and easily checked analysis. To do this, several computer-based, semi-automated systems that can make a digitized image of the metaphase and help to produce the karyogram within a few minutes are used. Most centres should analyse as many metaphases as possible (no fewer than 40) for diagnostic studies, unless it can be adequately defined with fewer cells for representing a well-established clone or a homogeneous cell line. These minimum guidelines are routinely done in our Andalusian Stem Cell Bank.
2.3 Molecular cytogenetics: fluorescence in situ hybridization (FISH), multicolour FISH (M-FISH), comparative genomic hybridization (CGH) and spectral karyotyping (SKY)
Although classical cytogenetic analysis is a powerful tool for the assessment of acquired chromosomal changes, it can only be performed on divided cells and cannot detect cryptic rearrangements. The introduction of molecular cytogenetic techniques, based on fluorescence in situ hybridization (FISH), has revolutionized the field of cytogenetics by allowing the identification of complex and cryptic chromosomal abnormalities, without prior knowledge of chromosomal loci involved. FISH allows the study of chromosome exchanges and gene rearrangements, amplification, and deletions at the single-cell level. Another FISH-based technique, comparative genomic hybridization (CGH), can identify chromosome losses and gains in stem cells. Furthermore, the capacity to hybridize simultaneously 24 or more DNA probes in the FISH-based karyotyping of chromosomes has resulted in several novel techniques, such as multicolour FISH (M-FISH) and spectral karyotyping (SKY). These superior methods such as FISH (Inzunza et al., 2004; Mitalipova et al., 2005) and CGH have recently been implemented in hESC chromosomal analysis and only a combination of different techniques can guarantee ultimately good coverage of all possible genetic abnormalities (Speicher and Carter, 2005).
2.3.1 Fluorescence in situ hybridization (FISH)
FISH is based on fluorescently labelled probes that hybridize to unique DNA sequences along the chromosomes. FISH can be performed on either metaphase preparations or interphase cells. There are wide applications of FISH, mainly in cancer research and molecular diagnosis, but our aim is the study in stem cell research as we discuss in detail below.
The FISH technique that is widely used in laboratories involves the hybridization of labelled DNA probe in situ chromosomal target. Probe and target DNAs are denatured using high-temperature incubation in a formamide or salt solution. The probe is applied in excess, so the kinetics ensure that the probe anneals to the target DNA. Probe detection is accomplished by ultraviolet-light excitement of a fluorochrome, such as fluorescein-5-thiocynate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC), which is directly attached to probe DNA, or by incubation of hapten labelled probe with a fluorescent conjugate. The majority of probes used for laboratory purposes are commercially available. Most FISH probes fall into four categories: (i) centromeric probes directed against the centromeric region of the chromosome; (ii) locus-specific probes directed against a unique and specific DNA sequence; (iii) telomeric and subtelomeric probes aimed at detecting the chromosome telomeres, that is, the very ends of the chromosome arms (telomeric probes are not as widely used as other kinds of probes, which provide more information); and (iv) chromosome-specific probes which are used to study in depth the integrity of a given chromosome.
The centromeric specific probes for the α-satellite sequences are located at the centromeres of human chromosomes. α-Satellite DNA is composed of repeated monomers; the sequences are present in several hundreds or thousands of copies, producing strong signals. The α-satellite sequence of each chromosome is more divergent to allow for the development of centromere-specific probes. These probes are useful for the detection of aneuploidy in both metaphase and interphase cells. In our laboratory, these probes in particular are used to study the chromosomes 12, 17 and 20 in hESC.
The other chromosome specific probes, also known as chromosome “painting” probes, are composed of unique and moderately repetitive sequences from an entire chromosome or chromosomal region. The use of this type of probe can result in the full identification of all the all chromosomes involved. Even apparently simple reciprocal translocations can sometimes be shown to be more complex by this technique; sometimes morphologically normal chromosomes can be shown to have cryptic rearrangement. It needs to be pointed out that the identification of cryptic rearrangements may vary depending on different preparation methods from 200
Representative fluorescence in situ hybridization for chromosomes 13, 18, 21, X and Y in HS 293 hESC line.
2.3.2 Multicolour FISH and spectral karyotyping (SKY)
The FISH-based karyotyping of chromosomes has resulted in several novel techniques, such as multicolour FISH (M-FISH) and SKY FISH. Spectral karyotyping methods use fluorescent dyes that bind to specific regions of chromosomes. By using a series of specific probes each with varying amounts of dyes, different pairs of chromosomes have unique spectral characteristics. Slight variations in colour, undetectable by the human eye, are detected by a computer program that then reassigns an easy-to-distinguish colour to each pair of chromosomes. The result is a digital image rather than film, in full colour. Pairing of the chromosomes is simpler because homologous pairs are the same colour, and aberrations and cross-overs are more easily recognizable. In addition, the spectral karyotype has been used to detect translocations not recognizable by traditional banding analysis (Speicher et al., 1996).
Multicolour technologies have been applied to chromosome painting probes. The initial requirement was for 24 different colours, as this allows the simultaneous visualization of all human chromosomes in a single experiment (Langer et al., 2004). The main difference between these two techniques is the way in which the coloured images are captured. M-FISH combines binary ratio labelling and colour-changing karyotyping. These methods offer the capacity to hybridize simultaneously 24 or more DNA probes, based on simultaneous hybridization of 24 chromosome specific painting probes labelled with different combinations of 5 fluorochromes (Schröck et al., 1996; Karhu et al., 2001). In M-FISH a highly sensitive monochrome camera captures a series of images that have passed through different filters. Specific computer software analysed the acquired data from the probes and pseudocolours the chromosomes for analysis. The SKY is based on the principles of spectral imaging and Fourier spectroscopy. Five pure dyes that are spectrally distinct are used in combination to create the unique chromosome cocktail of probe (Bayani and Squire, 2001). Image acquisition is accomplished by the use of a specially designed triple filter. Both the SKY and M-FISH methods require the use of human whole chromosomal paints that are differentially labelled, so that each chromosome has a unique combination of colours emitted following hybridization for identification purpose. Each method can be performed as a laboratory procedure and is capable of identifying the cytogenetic origins of all chromosomes in the complement in one image acquisition step. In our Stem Cell Bank the M-FISH methods are used in hESC lines, so analyses conducted on M-FISH help to evaluate the genomic integrity of expanding cultures (Fig. 3).
Representative karyotype image of an M-FISH determination.
2.3.3 Comparative genomic hybridization (CGH)
CGH is a method that gives an overview of the whole genome and allows the detection of DNA copy number changes. Since its first description (Kallioniemi et al., 1992, 1994) it has become a powerful alternative to chromosome banding and FISH. This technique allows a genome screening of chromosomal imbalances without prior knowledge of genomic regions of interest. CGH is an alternative method to reveal unbalanced chromosomal changes that may occur in hESCs lines during long-term cultures, especially when it is difficult to obtain good quality metaphases.
In hESC research, CGH may be applied as a molecular cytogenetic technique to search for potential anomalies in genomic DNA by comparing the target DNA (either DNA from any stem cell or patient-specific DNA) with a reference control DNA from either a healthy volunteer or commercially available standard DNA. DNA is extracted from the cells of interest and labelled with a green fluorophore (fluorescein-5-thiocynate; FITC), whereas control DNA is labelled with a red fluorophore (tetraethylrhodamine isothiocyanate; TRITC). These are mixed together in equal proportions and hybridized to normal metaphase preparations. The green:red ratio generated by the two samples of DNA is analysed by a computer software program that detects gains and losses of material from the DNA test. Fluorescence intensity ratio profiles are generated, and peaks and valleys indicating possible gains and losses of DNA tests are marked if they exceed ratios below 0.75 and above 1.25 (Lundsteen et al., 1995) (Fig. 4). If there is a normal amount of genetic material, the equal hybridization of green hESC DNA and red control DNA will appear yellow. If there is a loss of genetic material, the segment will appear in red and the gain of material will produce a green signal as shown in Fig. 4. The presence and localization of chromosomal imbalances can be detected and quantified by analysing the green:red ratio using digital image analysis. These ratios show which chromosomal regions in the test genome are over- or under-represented relative to the reference genome (healthy volunteer DNA or commercially available standard DNA). As ratio profiles are calculated along all chromosomes in the normal metaphase, an overview of the chromosomal imbalances throughout the total reference genome (either healthy volunteer DNA or commercially available standard DNA) is created (Jeuken et al., 2002) (Fig. 4).
Representative CGH ideogram profile for chromosome 1. The ideogram indicates a surplus of the whole chromosomal material (n
Inzunza et al. (2004) applied CGH for the analysis of chromosomal stability of hESC lines. CGH was applied in 48 out of 50 single cells isolated from three different hESC lines which were shown to have a normal chromosomal content when analysed by CGH. The hESC lines were reanalysed at subsequent passages and at that point an aberrant X chromosome was detected by karyotyping and confirmed by single-cell CGH. In addition, Caisander et al. (2006) used CGH to analyse the chromosomal integrity in hESC lines. Similar to Inzunza et al., they determined by CGH the chromosomal status of five well-characterized hESC lines that were monitored after a long period of time in culture. Taken together, it is important to test the hESC lines for chromosomal integrity before any potential cell replacement therapy, as there appears to be a risk of tumour development when these cells are transplanted in vivo (Rindgen et al., 2003).
2.4 Image acquisition and analysis of FISH
Visualization and analysis of FISH signals from larger probes, such as chromosome paints, α-satellite sequence probes, or locus probes, M-FISH and CGH can be successfully used with a simple epifluorescence microscope with appropriate filter sets. The detection of smaller signals may require the help of computer based image analysis systems that are commercially available. Criteria for successful image acquisition and analysis should be provided by the imaging equipment, and analytical software. Nevertheless, some general points should be considered of which method of image acquisition and analysis is used. The overall signal intensity along the chromosome should be high with minimal background. Interpretation of the analysis relies both on the familiarity of the researcher with the basic technicalities of FISH assays, chromosomal identification by G-banding methods and knowledge of fluorescent microscopy. CGH requires the use of a high-quality and quantitative FISH imaging system with a dedicated CGH suite.
Automated imaging systems dramatically reduce the time it takes to produce a karyotype, and therefore can be seen as one of the most important developments in automation of the cytogenetic laboratory. The growth in fluorescent techniques such as FISH, M-FISH, SKY and CGH can also be attributed to automatic imaging (Brenner and Dunlay, 2003).
2.5 Advantages and limitations of the different cytogenetic techniques in stem cell biology
It is helpful to be aware of the advantages and potential limitations of these techniques, and to know when the use of specific genetic assays may be applied more for the study of the chromosomal stability in hESCs. These advantages and limitations are summarized in Table 1.
Advantages and disadvantages of the different cytogenetic techniques potentially used in stem cell biology
A conventional cytogenetic study is still widely regarded as being the gold standard for genetic tests, since it is the best one currently available for assessing the whole karyotype at one time. Nevertheless, it is subject to limitations as only dividing cells can be assessed, and analyses are expensive because of the lack of automation in sample processing and the time needed to analyse each division. Moreover, there is no useful result from some hESC lines if these results are not analysable, or there are no cell divisions and so only a few divisions can be analysed.
FISH is a very rapid, sensitive, and cost-effective technique that offers the capability to detect both numerical and structural chromosomal abnormalities in interphase and metaphase nuclei, and also FISH permits rapid sex determination. However, FISH has some limitations like cross-hybridization of non-specific fluorescence signals, non-specific background, and suboptimal signal intensity. Small deletions, duplications and inversions cannot be detected with painting probes.
The great advantage of the CGH technique is that it requires only the genomic DNA; moreover, CGH does not require prior knowledge of the genomic region to be studied. CGH can detect copy number changes, gains and losses of chromosomal regions. Nevertheless, CGH is able to detect a wide range of quantitative genetic alterations including duplication or deletion of single chromosome bands. The CGH analysis also indicates the presence of genetic abnormalities that are not detected by other cytogenetic or molecular approaches (Nacheva et al., 1998). The sensitivity of this technique in detecting low copy number increases or decreases is in the range of 10–20
M-FISH and SKY can refine complex karyotypes and detect hidden structural abnormalities; this technique can be performed only on metaphase spreads but cannot detect small rearrangements or intrachromosomal rearrangements such as deletions, duplications, or inversions. Taken together, the combination of conventional cytogenetics with different molecular cytogenetic techniques improves the accuracy of the chromosomal stability of hESC lines.
3 Concluding remarks
Since the first hESC line was established in 1998 (Thomson et al., 1998) a great interest among the scientific community and the public domain has arisen because hESCs represent a starting stem cell population from which derive differentiated derivatives with potential cell replacement strategy for different degenerative disorders. Furthermore, stem cell banks need to guarantee the existence of appropriate sources of such cell lines in a standardized way for the development of therapies through clinical trials. To assure the quality of the cell lines we require authentication and characterization to confirm the absence of contaminants and to guarantee the chromosomal stability in hESC lines as well as in adult stem cell populations (Cobo et al., 2005; Menendez et al., 2005a,b, 2006). For this purpose it is important that resources be used appropriately to get thorough enough information about chromosomal integrity of hESC lines. These findings also emphasize the importance of frequent karyotype analyses to assure that results obtained in any studies with hESCs are not biased because of abnormal chromosome constitution. Superior methods, such as fluorescent in situ hybridization (FISH) and comparative genome hybridization (CGH), have recently been implemented in hESC chromosomal analysis and only a combination of different techniques can guarantee ultimately good coverage of all possible genetic abnormalities. These most common cytogenetic tools used today are FISH, karyotyping and CGH. When combined, the limitations of each of these techniques, i.e. the inability to screen all the chromosomes of the genome for chromosomal changes (FISH), analytical difficulties due to the low-resolution banding pattern in hESCs lines (karyotyping), and the inability to detect balanced translocations, mosaicims, and ploidy (CGH) may be circumvented. This study compares the several techniques for the assurance of chromosomal stability of hESC lines (Table 1).
Stem cell banks must assure the quality, the traceability and the safety of these products, this being particularly important in order to avoid chromosomal anomalies in hESC lines and their adult counterparts. This review has intended to provide readers with an overview of the methodology that, we believe, should be used to assess the genomic integrity and chromosomal stability of these cell lines in order to assure their quality and avoid chromosomal instability for use in cell therapy and regenerative medicine.
This work was partially funded by the Fundación Progreso y Salud (grant references 0028/2006 and 0029/2006 to P.M.), Consejería de Salud, Junta de Andalucía, Spain and the Jose Carreras International Foundation against the Leukemia (FIJC-05/EDThomas 2006 to P.M.). We are also grateful to The Hospital Virgen de las Nieves (Servicio Andaluz de Salud) and The Instituto de Salud Carlos III for their full support throughout the development of this work. We would like to thank Ms Angela Helen Barnie for the English correction of the manuscript. We are also indebted to Dr Angel Martínez (MD Anderson Hospital, Madrid, Spain) and Dr Jesús Mari Hernandez Rivas and Dr Juan Luis García (Hospital Clínico Universitario de Salamanca, Salamanca, Spain) for their helpful discussions.
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Received 3 January 2007/19 January 2007; accepted 11 March 2007doi:10.1016/j.cellbi.2007.03.012