Stem Cell Reviews Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1550–8943/06/2:117–126/$30.00 (Online) 1558–6804

Original Article Embryonic Stem Cell Bank A Work Proposal A. Nieto,* F. Cobo, A. Barroso-delJesús, A. H. Barnie, P. Catalina, C. M. Cabrera, J. L. Cortes, R. M. Montes, and A. Concha Andalusian Stem Cells,Andalusian Stem Cell, Bank, Granada, Spain Abstract Human embryonic stem cells (hESCs) have an unlimited capacity to proliferate by a self-renewal process and can be differentiated in the three germ layers, opening doors to new clinical therapies to replace missing or damaged cells. The number of research groups and projects using human stem cells has increased largely in the last 5 yr. The creation of stem cell banks is another important step to support the advance of research in this field. Banks must be operated within the strict regulatory famework of good manufacturing practices and good laboratory practices that assure the highest quality standards and must implement a quality system that complies with international quality systems standards. It may also be appropriate to aim at an accreditation in order to assure correct laboratory practices at all times. Stem cell banks should receive the lines previously derived by other groups and hESCs should be provided for groups that justify their use in a research project previously approved by an ethical committee. The assays generally accepted as typical of hESCs together with the microbiological analysis should be performed in order to assure a consistent, reliable, and safe line for the researchers. In this article, the Andalusian Stem Cell Bank proposes a model of a stem cell banking process in order to create a flow diagram of hESC lines and, following the international initiatives in stem cells research, to achieve the full characterization of cells and a standardization of protocols that would simplify the hESCs culture. Index Entries: Bank; characterization; flow diagram; hESC; quality controls; microbiological controls.

Introduction *Correspondence and reprint requests to: Ana I. Nieto, Andalusian Stem Cell Bank, Hospital Universitario Virgen de las Nieves, Avda. Fuerzas Armadas 2, 18014 Granada, Spain. E-mail: [email protected]

Since Thomson et al. (1) obtained the first successful derivation of human embryonic stem cells (hESCs) from the pluripotent inner cell mass of embryos, a new and exciting field in biomedical research has been opened. In preliminary studies done with mouse embryonic stem cells, diseases such as diabetes, myocardical infartus, neurological disorders or traumatic injuries of spinal cord have been treated, obtaining encouraging results (2–4). Obviously, in human medicine, the experiments are not achieving the same degree of development but the advances in the past few years have been enormous. Moreover, hESCs can be used as models of human embryonic development, to study human embryonic gene expression

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and by the pharmaceutical companies to create models in vitro for research in drug discovery and toxicology (5). In spite of scientific applications and commercial potential, working with embryonic stem cells has ethical consideration regarding the use of in vitro fertilized embryos. Furthermore, logistical difficulties such as the lack of standard protocols in the management of the different hESC lines are annoying obstacles in an efficient study of embryonic stem cells in the laboratory. In this sense, the research groups that derive ES lines have used different techniques such as immunosurgery, plating whole blastocysts, coculture in different feeder cells layers, or in an extracelullar matrix (6–8). The culture medium is also different among

118 ___________________________________________________________________________________________________Nieto et al. the groups. Standardization in the characterization of hESCs to normalize data across different hESC lines and different laboratories has been proposed (9) www.stemcellforum.org. A similar standardization regarding derivation and culture methods is also desirable. The creation of stem cell banks could help in the development of standardized protocols of culture conditions that could be tested in hESC lines deposited. When the International Research Community establishes standardized protocols for hESC lines, the Bank will distribute these protocols between the research groups interested in them. The UK stem cell bank is one of the pioneer banks in Europe, which has proposed the grounds on which a bank should be established and a program of research that improves the banking of stem cells including cryopreservation and cell characterization (10,11). The origin of hESC lines in the bank will be lines derived for research groups that have decided to deposit them. Then, the bank could offer the lines to the research community reducing the need for individual research teams to generate their own stem cell lines and the number of tissues and embryos used for the projects. During the banking, all the processes should be developed following the European Union good manufacturing practices related to validation, screening, processing, storing, and delivering stem cell lines to the users included in the International Quality standards. In this sense, the bank must have a code of practice which broadly outlines the criteria to be observed for working with human stem cell lines and that have to be similar to the standards set out in the European Tissue Directive and Codes on Tissue Therapies. Regarding the facilities, the stem cell banks count on specific GMPfacilities for a future clinical use, where the cells are required to be produced under the same rules applied for pharmaceutical manufacturers (9). Different cleanrooms with monitorized environmental controls that minimize microbial and particulate contamination are necessary to process the cells (Fig. 1). The equipment has to be subject to schedules of maintenance and calibration to assure the acceptable criteria of quality. The level of monitoring must be very strict. Detailed documents should be provided to deposit the hESC lines in the bank. These documents have to include information about the approved project based on hESC lines that have been derived, information about the informed consent and the clinical records of the donor and the laboratory protocols used in the culture and characterization of the line. The research groups that apply for the hESC lines must present a project describing the purpose of the use of the cells that must be approved by a Regulatory Committee. The Stem Cell Bank Code of Practice envisages that the Property and Intellectual Property Rights of cell lines belong to the depositor of the line. In this article, we describe a banking proposal, which includes the subdivisions of the bank, microbiological controls applied in the bank and the tests that have to be carried out during the banking process to assure the quality of hESCs, their pluripotency and genetic stability.

Banking of Human Embryonic Stem Cells Lines Feeder Stock The majority of embryonic stem cell lines available at present have been derived or cultured using mitotic inactivated

cells from mouse or human as feeders cells but also using serum, medium or conditioned medium with animal components (1,7,12). The attempts to derive or culture cell lines without using feeders or only with human component mediums has given rise to colonies of cells that differentiate quickly and whose expansion is rather complicated (13). Although Xu et al. (14) reached prolonged feeder-free growth of undifferentiated hESCs on an extracellular matrix (Matrigel™) or laminin that coated plastic plates or flasks using additionally a medium conditioned by inactivated mouse embryonic fibroblats, Richards et al. (13) demonstrated that feeders were superior to feeder-free matrices for prolonged undifferentiated hESCs growth. When analyzing cells from different origins, the best results corresponded to feeders from human fetal muscle and skin, human adult skin, and MEF. Some of these cells can be obtained from commercial support such as American-Type Culture Collection (ATCC, LGC Promochem) D551/CCL-10 (skin) or CCL-2552 (neonatal foreskin), whereas in other cases, they have been derived from mouse embryos or biopsy tissue. New feeder-independent hESC culture using only recombinant sources or purified from human material are being developed (15). Although karyotypic changes were observed in the lines derived and cultured on this system, it is a promising step for a future clinical application of hESC. Nowadays the culture of hESC lines only with clinical ends has not been performed. The bank needs a stock of cells that can be used as feeders after inactivation and whose origin should preferably be the same as those used for the growth of the hESC line deposited. This stock should make up a master feeder cell bank, where the ampules with the cryopreserved cells should be stored and in which the quality controls necessary for stem cell banking should be applied. On the other hand, the bank could receive lines derived and cultured on feeders or in feeder-free conditions. The banking process of hESC lines should be developed separately. When the banking process of hESC lines that have been grown on feeders has been completed, culturing the line in a feeder and animal product free conditions should be attempted in order to know its ability to grow and maintain its undifferentiated state with these conditions.

Human Embryonic Stem Cell Line Stock The aim of the bank is to achieve the highest number of frozen hESCs with the lowest number of passages in order to avoid the appearance of genetic changes that can emerge when the subcultures are increased and the cells are adapted to the in vitro growth conditions (16). As the hESC lines can have different number of passages before being deposited and for practical reasons, we will consider that the ampules deposited will be in bank passage (BP0) and we will continue with this nomenclature. Following the guidelines of the UK stem cell bank (10,11), the bank should count on 1. An archive with at least four ampules of each line and whose number of passages should be the same as that of the frozen ampules in each section. 2. A premaster bank with at least 10 ampules with five subcultures carried out in the bank itself (BP5).

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Fig. 1. Clean rooms in Andalucian stem cell bank facilities.

3. A master bank with at least 10 ampules with 10 passages (BP10). 4. A distribution bank with at least 10 ampules at passage 15 (BP15). This subsection of the bank should be in charge of the cell expansion and should maintain frozen ampules at passages 20, 25, and 30.

Setting Up a Premaster Bank Once the application to deposit of the cell lines in the bank has been passed, the research group that has donated the cells should deposit at least four frozen ampules of the cell line. In addition to the cell line, the depositor should hand over the protocols used for the cell culture, especially the protocols for freezing and thawing, the use of feeder or extracellular matrix, the culture medium used, when to carry out the passages, and the way in which to carry them out (mechanical or enzymatical). The transfer of protocols is essential because, as previously mentioned, at present there is no standardization and the conditions and the culture mediums are specific for each hESC line (Fig. 2. Modified version of UK stem cell bank flowchart 11). Two of the ampules should be maintained frozen in the archive while the other two should be thawed and cultured with the culture conditions specified in the protocols indicated by the depositor of the line. Immediately after thawing, samples from the two thawed ampules are used for

microbiological and viability tests and cross-contamination analysis. The culture medium that should be used after the first thaw of the cells should contain the antibiotics indicated in the protocol referring to medium preparation. When the culture has reached the point of subculture (BP1), half of the cells should be frozen and the ampule should then form part of the archive. The rest of the cells should be subcultured in medium without antibiotics until the number of flasks or cell plates is multiplied by 16 (BP5). At this point, a culture flask should be used to carry out a microbiological test (including mycoplasm tests), a prefreezing viability test, a karyotype analysis to check the genetic stability of the line, and a cross-contamination analysis to check the authenticity of the cells. Another culture flask should be used to carry out a phenotypic characterization in order to assess the differentiation state of the cell line and its differentiation capacity ex vivo. This study should include: (1) the determination of surface antigen markers of pluripotency, (2) the measurement of the expression level of genes associated to the undifferentiated phenotype, and (3) the ability of cells to form embryoid bodies and differentiate ex vivo. The rest of the cells should be frozen generating 14 ampules from which 11 ampules would make up the premaster bank, one would be introduced into the archive (BP5) and the remaining two ampules would be used to set up the master bank. We consider that the minimum number of frozen ampules in the premaster should be 10,

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Fig. 2. Setting up a premaster bank. (Modified version of UK stem cell bank flowchart; 11.)

the extra frozen ampule resulting from the described procedure could resolve a possible contamination or loss of culture flasks.

Setting Up a Master Bank After the thawing of the two ampules, a sample of the cell culture should be withdrawn to carry out a post-thawing microbiological test and a cell viability test and the remaining cells should be subcultured until once again 16 times the initial population is reached (BP10). At this time, when the culture is amplified enough, a sample of the colonies (i.e., those contained in a culture flask) should be taken to perform the same analyses done in the BP5 in the premaster bank that assure the

microbiological and cellular quality of the cells. The 14 left over should be frozen and so obtaining 14 ampules from which, the Master Bank should keep 11 frozen ampules (minimum 10), one should be included in the archive (BP10), another should be given to the depositor and the last one will be used to set up the distribution bank (Fig. 3. Modified version of UK stem cell bank flowchart 11).

Setting Up a Distribution Bank After thawing an ampule, the routine post-thawing microbiological and viability tests should be performed. As only one ampule will be thawed, the flow cytometry analysis of cell

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Fig. 3. Setting up a master bank. (Modified version of UK stem cell bank flowchart; 11.)

viability will be avoided. The cells will be subcultured until their population is multiplied by 16 (BP15) when new microbiological and prefreezing viability tests will be performed. As in previous cases, part of the resulting cells will be used to carry out karyotype analysis and cross-contamination tests and a basic characterization including undifferentiated state of cells in culture and in vitro differentiation potential (Fig. 4. Modified version of UK stem cell bank flowchart 11). As per usual, most of the remaining cells will be frozen to generate 13 frozen ampules: 12 for the distribution bank and 1 for the archive because in this case, one culture flask will be saved and expanded until

obtaining enough material to carry out a full cell line characterization (extension of distribution bank, Fig. 5. Modified version of UK stem cell bank flowchart 11). This will initially include all the microbiological viability, phenotypic, genetic, and crosscontamination analysis already performed in the premaster and master banks, that will be carried out every five passages of the culture, plus some additional studies that will be carry out only once in order to determine the specific gene expression profiling of the line and its vivo differentiation capacity. The characteristic gene expression profile of the cell line could be achieved by whole genome microarrays hibridization in combination

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Fig. 4. Setting up a distribution bank. (Modified version of UK stem cell bank flowchart; 11.)

with miRNAs profiling. The in vivo differentiation ability may be confirmed by a test based on the inoculation of hESCs in inmunodeficient mice and subsequent monitoring of teratoma formation. Moreover, complementary studies as assays of different culture conditions in order to achieve a standardization of them should be carried out. At this stage, samples of genomic DNA and total RNA may also be obtained in order to set up DNA and RNA banks. We should continue to carry out passages and the cells should be frozen at passages BP20, BP25, and BP30. As previously mentioned, every five subcultures the routine control analysis

of contamination, viability, genetic, and phenotypic stability will be carried out.

Microbiological Control in Stem Cell Banks Contamination from the laboratory environment can be monitorized by routine screening for bacteria, fungi, yeast, and mycoplasma by means of the guidelines of European Pharmacopoeia tests (17–19). Specific tests for the detection of bacteria, yeast, and fungi should be used as part of a routine and regular quality control screening procedure in the premaster, master, and distribution banks.

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Fig. 5. Extension of distribution bank. (Modified version of UK stem cell bank flowchart; 11.)

Stem cell banks must assure the quality, the traceability and the safety of the cell cultures and these aims are particularly important in the avoidance of transmissible diseases (20). This should be achieved with close attention to the standardization of processes and the implementation of quality control programmes and methods which reflect current best practices. The origin of the cell lines can have an important effect on the quality since the cell lines recently imported to the laboratory constitute the greatest source of contamination depending on their culture history and past exposure to microorganisms. Thus providers of cell lines should be able to provide details of passage history and appropriate testing (21). Once the cell lines have been obtained from a reliable source it is important at the

earliest stage to establish a premaster bank, and apply appropriate tests to rule out microbiological contaminants. Stem cell banks should screen all processed cells for serious human and animal pathogens and assure that no contaminants are introduced in the banking procedures, including storage. Obviously both serious human or animal pathogens present a risk to other processed tissues or cells and staff, as well as in the future, to the recipient patients. Samples from the cell cultures and their products should be inoculated in either liquid or onto solid growth media. These inoculated media may be incubated at different temperatures reflecting conditions for human pathogen culture and environmental microorganisms with lower growth temperature optima in microbiological culture incubators depending on

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124 ___________________________________________________________________________________________________Nieto et al. the specific testing standard used. For mycoplasma testing, a variety of tests are available like culture detection, indirect DNA staining (Hoechst 33258), polymerase chain reaction (PCR), and the Gen-probe MTC-NI detection system. It is usually recommended to use at least two techniques for testing cell banks to ensure optimum sensitivity and specificity (20). In addition to mycoplasma there are numerous types of microorganisms that may contaminate and persist in cell cultures and go undetected without specific isolation methods. In order to isolate such microorganisms very different culture and conditions should be required for their detection and it should not be possible to cover all types of potential contamination. Nevertheless, it is wise to remain aware of the potential for rare contamination of this type and to be prepared to attempt to isolate such microorganisms should unusual observations arise that could be explained by such contaminants. With respect to viral contamination of cell cultures, some lines may contain endogenous viruses and may be contaminated with exogenous viruses, and may secrete viral particles or express viral antigens on their surface, but it is not possible to assert absolute absence of viral contaminants. Cell banks should have a panel of tests to detect serious pathogens, other endogenous viruses and adventitious viruses. This panel of tests usually includes electronic microscopy, reverse transcriptase detection (as a general test for retroviruses), in vivo and in vitro tests for infectious virus, tests to induce an antibody response in animals (e.g., mouse antibody production tests, rat antibody production tests) and other specific tests to find human, bovine, porcine, or rodent viruses depending on the origin of the biological products used in the cultures (22). There is a common supplement (e.g., bovine serum) or murine feeder cells that are necessary for cell culture and may be contaminated with viruses of animal origin that are susceptible to being transmitted to the recipients. New guidelines describing the screening of bovine serum before its use in the manufacturing of a human biological product have recently been introduced (23). Serum should not contain any detectable bacteria, fungi, or mycoplasmas. The virus-testing list should include known bovine pathogens. Also, since the European regulatory agencies (24) have identified a risk assessment for transmissible spongiform encephalopathies in all products derived from rumiants there is an urgent need for tests for the agents of transmissible spongiform encephalopathies such as the Creutzfeldt–Jakob disease (25,26). Embryonic stem cell lines derived with mouse feeders as well as those derived with human feeders can transmit infectious microorganisms to the recipient (27). The present regulations require the screening of cell and tissue products from donors using tests for a wide spectrum of virus, which cause serious human illnesses such as HIV1/2, hepatitis A, B, C and E, hCMV, and HTLV-I/II, and these would apply to testing for human feeder cells for clinical use (28–30). However, in banks of feeder cells for hESC culture the need for such tests would have to be reviewed on a case by case basis. Furthermore, some of these agents have been implicated in human cancer (31,32). Regarding nonviral agents, recommended tests for human donors include serology for donor antibody to Treponema pallidum (28) and in the US testing for donors of reproductive cells and tissue to Neisseria gonorrhoeae and Chlamydia trachomatis are also recommended and would be recommended as tests

for human feeder cells (25). Some pathogenic agents have a marked variation in their geographical distribution thus the geographical source of feeder cells should be considered when establishing safety testing regimes for feeder cell banks. It is clear that before using human cell products, they should remain under quarantine and be stored in a separate “in process” nitrogen storage vessel until it is determined whether these products are acceptable, based on the results of quality control and the screening tests performed by accredited laboratories for the relevant contaminats.

Control for Assessment of Human Embryonic Stem Cells Quality and Characteristics During the Banking Viability, Cell Cycle Analysis, and Apoptosis Viability assays measure the proportion of viable cells following freezing or thawing cells in the banking process. Viability tests should be done before freezing cells and after thawing cells in order to know the cell recovery rate and the good management of the protocols (premaster, master, and distribution banks). Tests are based on a breakdown in membrane integrity that is determined by the uptake of a dye to which cells are normally impermeable (e.g., trypan blue or propidium iodide [PI]) or the release of a dye normally taken up and retained by viable cells (e.g., diacetyl fluorescein; 33). Trypan blue assay is a short-term, and easy test of cell viability that can give an approximate estimation of the proportion of dead cells. However, the assay with PI provides a more accurate approach to the number of cells that are alive because it is a flow cytometry technique that avoids the human mistake in the cell count. The intercalating properties of PI, a DNA-binding fluorochrome, are used to determine the viability owing to cell membrane permeability of dead cells. DNA analysis should be done using flow cytometry (34). Measurement of DNA content permits monitoring cell proliferation by meaning of the number of S phase cells and also detects the presence of abnormal DNAcontent. Abnormal hypodiploid cultures have a DNAindex (DI) less than one and abnormal hyperdiploid cultures have a DI greater than one, showing abnormal peaks in the histogram of DNA content. Flow cytometry is also used to measure apoptosis in a suspension of hESCs. Early apoptotic event results in exposure of phospohatidylserine on the outer surface of the plasma membrane. Annexin V, in presence of calcium ions, exhibits a high affinity for binding selectively to phosphatidylserine which makes annexin V a powerful and selective tool to detect apoptotic cells. By combining annexin conjugated with a fluorocrome (e.g., fluorescein isothiocyanate [FITC]) and PI three different cell phenotypes can be established: viable cells are unlabeled, early apoptotic cells are labeled annexin V+ IP– and late apoptotic or necrotic cells are labeled annexin V+ IP– (35).

Markers of Pluripotency and Differentiation Human ES cells are morphologically characterized by the formation of round and flat colonies with a high nuclear-tocytoplasmic ratio and one or more prominent nucleoli in each cell. However a complete characterization of hESCs requires an analysis of typical stem cell markers, karyotyping and differentiation potential under in vitro and in vivo conditions that must be maintained throughout the extended culture.

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Embryonic Stem Cell Bank _____________________________________________________________________________________125 Like human embryonal carcinoma cell lines, hESCs express high levels of surface markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 but do not express SSEA-1 (36,37). Furthermore, hES express high levels of alkaline phosphatase and Oct-4, a transcription factor that plays an important role in the maintenance of an undifferentiated fate (38). The cell surface antigen expressions should be assessed by immunocytochemistry and flow cytometry in the premaster, master, and distribution banks. A complementary approach to characterize the undifferentiated state of cells is to determine by quantitative or semiquantitative reverse transcription-PCR, the expression level of genes specifically associated with the stem phenotype (39,40). Among the multiple possible stem cell markers, some of the most widely used are: Oct3/4, Sox2, and Rex1. Other useful markers could be the telomerase associated factors TERF1 and TERF2, UTF-1 gene, and FGFR4.

The M-fluorescent in situ hybridization allows the paint of the chromosomes with different colors to determine the origin of duplication and detecting crytic rearrangements. Comparative genomic hybridation (CGH) is a technique that uses DNAfrom the cells instead of using a standard karyotype for chromosomal analysis (48). This can be useful to detect genetic changes (gain or lose of DNA) when only DNA is available.

Cross-Contamination

A full characterization of hESCs includes a demostration of the ability of hESCs to differentiate into all embryonic germ layers in vitro and in vivo (41). In vitro, when hESCs are cultured in suspension, spontaneously create embryoid bodies (EBs; event cystic EBs) that contain derivatives of the somatic cells found in the adult organism (42–44). This test should be performed in the premaster, master and distribution banks demonstrating by immunocytochemistry the presence of β-tubulin, α-fetoprotein, and muscle actin as markers of ectoderm, endoderm, and mesoderm, respectively. The differentiation process of the culture may also be monitored by reverse transcription PCR technique using genes specifically expressed during the commitment of cells in the three embryonic germ layers (39). The developmental potential of the cells should be examined in vivo using the teratoma formation. This experiment should be carried out in the distribution bank. The hESCs form teratomas following injection into immunocompromised mice. The tumors include tissues of the three embryonic germ layers such as cartilage tissue, smooth muscle, stratified epithelium, and connective tissue (45).

The importance of the DNA fingerprinting technique in the quality control of cell lines to assess the absence of crosscontamination, is increasingly recognized owing to the advantages that it has in comparison to the standard methods of isoenzyme analysis and cytogenetics (49). In the quality control of cell lines, DNA fingerprinting offers a unique way of identifying cell lines and their cross-contamination by a single test. This technique identifies a cell line by visualizing the structure of the repetitive component of genomic DNA, which is extremely variable between individuals. The hypervariable regions of DNA, which are abundant and dispersed throughout the genome, are made up of tandem repeats of a core nucleotide sequence. Two types of tandem repeats exist and are classified based on the numbers of nucleotides in each core repeat (50,51). Minisatellites, also known as variable number of tandem repeat (VNTR) loci, have core repeats of 8 to more than 80 bp. Each VNTR locus has between two to several hundred core repeats on each allele (50). Microsatellites, also known as short tandem repeat (STR) loci, have core repeats of 2–7 bp. STR usually have between 3 and 20 tandem core repeats in each allele but in some cases may have 100 repeats or more (51). Both VNTRs and STRs are useful for DNA fingerprinting testing because they are inherited in a mendelian fashion, that is, an individual receives one allele from each parent. The two alleles can have the same number of tandem repeats, making the individual homozygous at that locus, or a different number of repeats, making the individual heterozygous at that locus. Arange of fingerprinting methods is available, but they generally fall into two groups: singlelocus andmultilocus methods.

Cytogenetic Analysis

Gene Expression Profile

Karyotype analysis should be performed in premaster, master, and distribution banks (in the latter it should be carried out every five passages). Draper et al. (16) observed karyotypic changes involving the gain of chromosome 17q in three independent hESC lines and a gain of chromosome 12 occasionally that may provide useful clues to the genes that permit the adaptation of cells to in vitro culture conditions, limit differentiation and that favour self-renewal. Some authors attributed the karyotypic changes to the use of the enzymatic methods for dissociating and passaging colonies suggesting that mechanical cutting might minimize karyotypic changes (46). For all of these, hESCs deposited in the bank would have to be routinely karyotyped even if the detected genomic changes have little consequence for the behavior of derivate cells that would eventually be used for therapy. The G-bands are demonstrated using Wright or Giemsa staining after a treatment of hESCs with an antimitotic (33). Fluorescent in situ hybridization technique is employed to detect and characterize chromosomal abnormalities that are not routinely delineated with standard banding studies (47).

In addition to the quality assurance of the cell line, some complementary assays should be carried out for a complete characterization of hESCs. Gene expression profiling can help to establish small differences among cell lines that can underlie their particular properties and potential (52). Those differences are due, both to the origin of the line and to the culture conditions used. The high-density DNA microarray analysis is a high-throughput methodology that allows a large-scale gene expression profiling from a limited amount of starting material (53). In addition to the use of complete genome microarrays, the characterization of the expression profile of a specific subset of molecules known as microRNAs (54) can be very useful for the classification of cell lines as has been demonstrated for tumors (55). These assays may be performed in the master bank, at the same time as the differentiation potential of the cell line is established.

Differentiation Potential

DNA and RNA Banks The DNA and RNA banks are facilities that can be offered by the stem cell bank to those groups who request genomic

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126 ___________________________________________________________________________________________________Nieto et al. DNA (i.e., for amplification of specific genes to be cloned and expressed in heterologous systems) or total RNA (i.e., to perform gene expression analysis) from specific cell lines. The setting up of these nucleic acid banks will be delayed until the postdistribution bank owing to the high number of cells necessary to obtain enough material.

Acknowledgments

26. 26

27. 27

This work is supported by the Fundación Progreso y Salud and the Hospital Universitario Virgen de las Nieves (Servicio Andaluz de Salud). We thank Dr. Glyn Stacey from the UK Stem Cells Bank for the information about stem cell banks. 28. 28

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Stem Cell Reviews ♦ Volume 2, 2006