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Keywords:

  • Human embryonic stem cell;
  • Karyotype;
  • Characterization;
  • Mitochondrial sequencing;
  • Methylation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Measures of Identity
  5. Measures of Stability
  6. Measures of Heterogeneity and Differentiation Ability
  7. Reference Standards
  8. Government Regulatory Agency Standards
  9. Summary
  10. Acknowledgements

As of August 2005, 22 human embryonic stem cell (hESC) lines listed on the National Institutes of Health (NIH) hESC Registry were being distributed to investigators. At a June 2005 meeting of NIH-supported hESC researchers, we proposed that a set of shared standards should be available in order to characterize the cells unambiguously in multiple laboratories. Here, we elaborate such a plan to identify a set of standard methods and to initiate collaborative efforts to validate the standards. The standard assays we propose should be comprehensive enough to ensure that hESC banks can provide a consistent and reliable product for NIH researchers, and inexpensive enough that individual laboratories can afford to use at least some of the methods routinely in their laboratories. We expect that as data accumulate and standards evolve, a core set of tests will become the norm for routine assessment of hESC cultures and that these tests will lay the groundwork for clinical applications of these cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Measures of Identity
  5. Measures of Stability
  6. Measures of Heterogeneity and Differentiation Ability
  7. Reference Standards
  8. Government Regulatory Agency Standards
  9. Summary
  10. Acknowledgements

Currently, 22 human embryonic stem cell (hESC) lines are in distribution under the auspices of the National Institutes of Health (NIH), and about 20 others are eligible for NIH funding but are not widely available to researchers. At least 200 other lines have been derived since the August 9, 2001 deadline for NIH funding eligibility (data from http://www.stemcellcommunity.org). There is considerable concern among scientists and the public about which cell lines, if any, are likely to be safe and efficacious for therapeutic use, especially for approaches that use ESC derivatives for cell replacement in degenerative disease. A comprehensive study comparing all of the cell lines would be the ideal solution to this issue, but there are tremendous challenges to be overcome in order to accomplish this goal. The regulations that govern hESCs vary greatly among individual countries. In the U.S., while it is legal to work on any hESC lines, federal funding can be used only for the pre–August 9, 2001 lines. Even when funding is available, no single laboratory has the resources to actively maintain more than a handful of lines at one time. Through collaborations among several laboratories, there have been reports comparing some of the basic cell properties of multiple lines. These reports suggested that the hESC lines that have been studied are similar in their expression of certain cell surface antigens and mRNA or protein markers. While not all markers have been tested in all lines, a group of characteristics is generally accepted as typical of hESCs: the ability to self-renew and differentiate into ectoderm, endoderm, and mesoderm in vitro and in vivo; expression of transcription factors such as POU5F1/Oct3/4 and Nanog; and expression of embryonal carcinoma antigens, including glycolipids such as stage-specific embryonic antigen (SSEA) 3 and the tumor rejection antigen (TRA [“Trafalgar”]) antigens. Interestingly, the majority of these characteristics were identified before hESCs had been isolated, as markers of teratocarcinoma cell lines. More extensive analysis of some of the hESCs has also been performed using gene expression analysis methods, including whole-genome microarray, expressed sequence tag (EST) scan, serial analysis of gene expression (SAGE), and massively parallel signature sequencing (MPSS). There are also efforts under way to identify all of the major proteins produced by ESCs and their derivatives.

An overview of the studies to date suggests that it might be possible to define a unique molecular signature that would serve as a gold standard for undifferentiated hESCs. However, because so many ESC lines have not yet been well characterized, it is imperative that we not jump to the conclusion that we have already reached this goal, or are even close to it. Despite the apparent overall similarity among lines, important differences have been reported. These include differences in growth rates and differentiation, differences in methylation patterns and karyotypic stability, allelic differences, and changes associated with long-term propagation in culture. Table 1 summarizes possible sources of variation that are likely to lead to significant differences in the properties of different lines. Some differences may be intrinsic to the origin of the cells, but even the same cell line can vary considerably because of different culture conditions used by different laboratories. These differences are magnified when the pluripotent cells are induced to differentiate, because the starting population and the method used to differentiate the cells both profoundly affect the character of the differentiated population. The standards we propose will aid in the development of standard culture conditions that should reduce the variation due to differences in technique.

The importance of comparing lines to assess which ones are optimal for therapy and concerns about undocumented diversity in ESC lines used for research have sparked numerous initiatives to develop a set of standards that can be applied universally to all lines. The International Stem Cell Forum (http://www.stemcellforum.org) has developed a multinational cell characterization initiative led by Dr. Peter Andrews to examine the cell surface characteristics, gene expression, microbial status, and other traits of a diverse group of 75 ESC lines. The Characterization Unit at the NIH is developing standardized conditions for the growth and propagation of all NIH-approved lines with the hope that other investigators will follow suit and examine newer lines in a similar fashion. The proposed NIH-sponsored Stem Cell Bank will be charged with developing standard culture conditions to maintain lines for federally funded researchers. In the meantime, the National Institute on Aging (NIA) (Rao lab) and the American Type Culture Collection (ATCC; Manassas, VA, http://www.atcc.org) have initiated a parallel (complementary) analysis of NIH hESC Registry lines (NIA) and newer lines (ATCC) to develop a common dataset of parameters that will allow comparison of lines. Similarly, a collaboration between our laboratory (Loring) at the Burnham Institute for Medical Research (La Jolla, CA) and Illumina, Inc. (San Diego, http://www.illumina.com) is using Illumina's low-cost whole-genome arrays to generate a database of gene expression in multiple ESC lines and has offered to perform such an analysis on all available cell lines. The U.S. Food and Drug Administration (FDA) and Invitrogen Corporation (Carlsbad, CA, http://www.invitrogen.com) have developed focused gene expression arrays that can be used to assess the state of hESC lines and to monitor the degree of differentiation and contamination of cultures. Our laboratory (Rao), BresaGen (Bresagen Division of Novocell, Inc., Irvine, CA), and the ATCC have described cell lines that can be used as controls to allow comparisons between NIH hESC Registry and unapproved lines.

Developing inexpensive, reliable technology that can be used in routine screens is a challenge, but an equally great challenge is to coordinate multiple investigators to supply cells, reach a consensus about markers, and freely share their data. Historically, in the U.S., it has been the role of the federal government to establish standards, host datasets, and develop and enforce guidelines. However, in the case of hESC research, the dual-track policy, the current patent situation, and the fragmented nature of the field has limited the U.S. federal government to a facilitative, rather than a leadership, role. In June 2005, at a meeting of investigators supported by the NIH to distribute hESC lines (R24 awards) and to train new investigators (T15 grants), we discussed the need for a set of standard assays that would allow comparison of hESCs across different laboratories.

Inspired by those discussions, we are making a first attempt to obtain a consensus among hESC researchers, proposing a set of tests that could serve to provide unambiguous data on the state of the cells. We have divided these analyses by the issues they address (summarized in Table 2) and suggest that such tests should be performed on a set schedule and that different subsets need to be performed when banking cells and when using cells for routine experiments in individual laboratories. We also suggest that there is a need for a reference standard cell line that can be used as a control in each laboratory to normalize data across different samples and different laboratories.

Measures of Identity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Measures of Identity
  5. Measures of Stability
  6. Measures of Heterogeneity and Differentiation Ability
  7. Reference Standards
  8. Government Regulatory Agency Standards
  9. Summary
  10. Acknowledgements

Many laboratories maintain more than one ESC line, and there is a real possibility of mix-ups in even the most rigorous labs. Most hESC lines appear similar to each other in their morphology and growth characteristics, and it will be important to develop unambiguous markers of the identity of a cell line to prevent errors in identifying or labeling a particular cell type. Many strategies exist to unambiguously type cell populations. Single tandem repeat (STR) genotyping takes advantage of naturally occurring amplifications that occur within short tandem repeats in different individuals. STR is a historically reliable method that is routinely used in genetic linkage studies and for paternity identification. The polymerase chain reaction (PCR)–based technology can be developed in most labs or can be performed by service laboratories. Human leukocyte antigen (HLA) typing using PCR is commonly used for tissue typing for bone marrow transplantation and is available as a service in most large hospitals. More global typing methods such as single nucleotide polymorphism (SNP) genotyping can provide a detailed fingerprint of any particular cell line. Assays that detect thousands of SNP variants in each sample have the added advantage of providing a wealth of additional information that may be collected to provide data on allelic variability or bias among a large number of hESC lines. For purposes of identification, these analyses would need to be performed only once for each line to establish a profile, and the information should be made freely available to researchers via a Web-based database. Individual researchers could perform the analyses periodically if there is any chance of cross-contamination. All of these methods are relatively inexpensive compared with the other costs of running a stem cell lab; even the most expensive, SNP genotyping, costs less than $1,000 for more than 100,000 SNP genotypes on some platforms. It is important to note that SNP maps of ESCs should be generated using the same technology that underlies the databases being created for the International HapMap Project (http://www.hapmap.org), to provide access to the huge HapMap dataset that may allow a researcher to glean additional information about the properties of any particular line. The predominant genotyping platforms for the HapMap Project are Illumina's Beadarrays (Illumina, Inc., San Diego, http://illumina.com) and Perlegen's use (Perlegen Sciences, Inc., Mountain View, CA, http://www.perlegen.com) of Affymetrix's genotyping technology (Affymetrix, Santa Clara, CA, http://www.affymetrix.com).

Measures of Stability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Measures of Identity
  5. Measures of Stability
  6. Measures of Heterogeneity and Differentiation Ability
  7. Reference Standards
  8. Government Regulatory Agency Standards
  9. Summary
  10. Acknowledgements

Unlike the autologous cell therapy approaches using somatic stem cells, such as hematopoietic stem cells, that require minimal manipulation and limited propagation, hESC therapy will require propagation to obtain large numbers of cells, followed by differentiation into an appropriate phenotype. The population of desired cells will then need to be harvested away from contaminating undifferentiated ESCs, feeder cells, and other inappropriate differentiated cell populations. A critical requirement for these procedures to work will be the ability to maintain the ESCs as a stable self-renewing population that retains the ability to differentiate into the cell type required. It is equally important to develop a set of quality control procedures that will allow rapid assessment of the state of the cells without using too many cells. Table 3 lists proposed measures that could detect alterations that may compromise the value of hESC lines in a clinical setting. Besides using markers associated with undifferentiated cells (such as POU5F1/Oct3/4), we suggest monitoring other factors that would be expected to degrade the quality of cells and alter their differentiation potential in unpredictable ways. These characteristics include telomerase activity (which is high in undifferentiated cells), mitochondrial metabolism, genomic stability, and markers of epigenetic change such as DNA methylation and histone modifications. Gross genetic changes, such as loss or gain of chromosomes and large trans-locations, are easy to detect by karyotyping the cells at regular intervals, an inexpensive procedure (about $300) that is performed as a service by many hospitals. The best resolution obtainable by cytogenetic methods, however, is about 10 Mb, and to detect smaller changes, SNP and CNP (copy number polymorphism) mapping gives 30-kb resolution. Histone modification and DNA methylation are the most commonly measured epigenetic changes; both types of analysis are labor-intensive now, but array-based methods in development have the potential to allow inexpensive assessment of the methylation status of hundreds of regulatory elements.

Measures of Heterogeneity and Differentiation Ability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Measures of Identity
  5. Measures of Stability
  6. Measures of Heterogeneity and Differentiation Ability
  7. Reference Standards
  8. Government Regulatory Agency Standards
  9. Summary
  10. Acknowledgements

The analyses described above allow assessment of the overall self-renewal ability of the cells and their stability over time in culture. However, because of their delicate balance between pluripotence and differentiation, hESCs cannot be maintained in an entirely homogeneous state, even in the most careful laboratories. Even the same cell line cultured in what are apparently identical conditions accumulates different stochastic changes. Different cell lines will differ because of allelic differences in adaptation in culture. Adding to this issue, different laboratories may have different criteria about the degree of acceptable differentiation or degree of feeder contamination. It is therefore important in setting standards to assess the expression of multiple markers of the undifferentiated ESC state as well as the presence or absence of markers of differentiation.

Because most heterogeneity is due to differentiation, the methods we describe in Table 4 encompass both detecting spontaneous differentiation and assessing pluripotence. The most accessible method for most laboratories is immunocytochemistry. Antibodies made to epitopes from embryonal carcinoma cell lines, such as the SSEA and TRA antibodies, are already in wide use to characterize hESCs, and novel epitopes continue to be identified. Similarly, there are multiple reports of PCR primers and quantitative PCR assays that can be used to detect genes that are popular markers of hESCs and differentiated products. Focused filter arrays each representing about 100 genes can be used to perform relatively inexpensive transcript analyses, and several laboratories have used larger scale gene expression methods, ranging from microarrays to MPSS. Other molecular methods, such as micro-RNA profiling and sophisticated proteomic analysis, are in progress in a few labs. The ultimate tests of pluripotence are biological assays for differentiation, such as electrophysiological analysis of neuron and muscle, and histological analysis of teratomas derived from transplanted hESCs. There are several issues that must be addressed in the use of any of these assays. First, there is not yet a consensus about what markers to use; markers that are supposed to be limited to undifferentiated cells usually also mark other cell types, and similarly, markers believed to appear only upon differentiation into a specific cell type are found in other types of cells. Second, exactly what is detected differs from assay to assay. For example, the SSEA antibodies recognize glycolipid epitopes; other antibodies used in immunocytochemistry may cross-react with multiple proteins, and array methods detect mRNA transcripts. Finally, the limits of detection for each assay must be considered; most methods are not quantitative, making it difficult for unbiased reporting of the data in a form that can be universally accessed.

Given this diversity in assay read-out, we recommend that to achieve a consensus, the list of standard markers be identified by national and international consortia. These markers would include reagents such as antibodies and PCR primers, along with clear methods about how to use them and how to interpret the results. This approach may require hands-on international training courses, many of which are already funded by the NIH and other funding agencies.

Reference Standards

  1. Top of page
  2. Abstract
  3. Introduction
  4. Measures of Identity
  5. Measures of Stability
  6. Measures of Heterogeneity and Differentiation Ability
  7. Reference Standards
  8. Government Regulatory Agency Standards
  9. Summary
  10. Acknowledgements

To enhance comparisons across laboratories it will be important to be able not only to run identical tests but also to develop reference standards against which results could be compared. Several possibilities exist. For gene expression studies, for example, one could develop a publicly accessible database of pooled average expression levels from 10–20 hESC lines and differentiated populations derived from them. We have begun to establish a database of gene expression information using cell lines from multiple laboratories. While such databases are useful for gene expression, they are not appropriate for other methods, such as FACS (fluorescence-activated cell sorting) and immunocytochemistry, and they do not allow a laboratory to troubleshoot their own results for technical problems.

An alternative strategy would be to use a readily available cell line that could be cultured and tested in researchers' laboratories. The International Stem Cell Initiative has adopted a reference teratocarcinoma line for some of its assays, which will allow collaborators to compare their results to this reference standard. The issue in this case is to choose which cell line is most appropriate for use as a reference standard. Table 5 highlights the advantages and disadvantages of three types of cell lines: a normal hESC line, a karyotypically abnormal hESC line, and a teratocarcinoma cell line.

While publishing a dataset of 10–20 lines is a reasonable plan, it is a strategy that is difficult to implement. One has to first obtain a consensus on what methodology is reasonable, identify which set of lines will be used as standards, and then identify a group or agency that will fund such an effort and enforce such a standard. In the past, in the U.S. the NIH has taken the lead in such discussions. In the case of hESCs, however, policy does not permit comparison between pre– (funded by federal funds) and post–August 9, 2001 lines, and it will require groups other than the NIH to step forward to try and obtain a consensus. Using a readily available cell line, on the other hand, is much easier to consider. Individual laboratories can determine whether they wish to participate, and essentially a de facto standard line can develop should enough groups in the field deem it critical.

Government Regulatory Agency Standards

  1. Top of page
  2. Abstract
  3. Introduction
  4. Measures of Identity
  5. Measures of Stability
  6. Measures of Heterogeneity and Differentiation Ability
  7. Reference Standards
  8. Government Regulatory Agency Standards
  9. Summary
  10. Acknowledgements

Governmental regulatory agencies that regulate cell therapy are formulating policy specific to hESCs. The FDA in 2002 determined that, unlike autologous hematopoietic stem cells therapies, hESC therapies will be covered under regulations that require the submission of an Investigational New Drug (IND) application. An important component of an IND application is the CMC (chemical manufacturing controls) section, which defines the manufacture of a product, including basic tests, SOPs (standard operating procedures), and evidence that the process can be performed under GMP (good manufacturing practice) principles. Thus, scientists who hope to develop ESC therapies for use in the U.S. need to develop general standards that can be discussed with the FDA. Issues of concern for the FDA are summarized in Table 6; a common set of standard characterization methods will benefit both research and translational studies.

General Guidelines for hESC Culture

Many researchers begin culturing hESCs in collaboration with an experienced colleague or after completing an NIH-sponsored intensive course or private training offered by the providers of the cells. There is no substitute for mentored training, but we want to offer some guidelines for development of standards that will help both experienced and new investigators avoid common problems. After determining what cell lines to use, researchers will want to obtain historical information about the cells and acquire laboratory protocols. Table 7 is a list of steps that we think are critical to successful experimentation with hESCs. The reference cell line should be obtained at the same time as the hESCs, so that it can be used to develop protocols that are at least as good as those used by other researchers in the field. Before culturing the hESCs, researchers need to institute a process for testing and validating media and reagents and plan a regimen of testing that will ensure reliability and reproducibility of the experiments performed with the cells. Most important is to develop a stock or bank of frozen cells that can be used over the course of months or years of experiments. This will ensure that experiments be performed with the same batch of cells. The bank prepared in the laboratory should be tested to confirm its identity and degree of differentiation and its karyotypic stability. We recommend that when new stocks are being generated, the cells be checked by standard methods every fifth or tenth passage. This will allow investigators to compare data from one laboratory with those of another and allow each researcher to know if the cell line in use today is the same as the one he or she started with.

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Measures of Identity
  5. Measures of Stability
  6. Measures of Heterogeneity and Differentiation Ability
  7. Reference Standards
  8. Government Regulatory Agency Standards
  9. Summary
  10. Acknowledgements

As more and more hESC lines are reported, it has become critical that researchers agree to a core set of standards that can be used for comparisons across laboratories and cell lines. These consensus standards will serve as quality control measures for judging the validity of experimental results, and using a common reference standard will ensure fidelity of the comparisons made. If a common set of tests can be defined, the considerable synergy that results will speed development of the multiple uses that hESCs promise and help avoid some of the missteps that hamper the development of a new technology.

Table Table 1.. Potential sources of variation among human embryonic stem cell lines
Differences due to origin of cell lines

Genomic diversity

Stage of blastocyst at derivation

Conditions of early culture (feeder layer, culture conditions)

Imprinting and X-inactivation

Differences arising over time in culture

Genetic changes (loss or gain of specific sequences)

General and specific epigenetic changes (DNA methylation, histone acetylation)

Differences due to mosaicism in cultures

Partial or terminal differentiation of subpopulations within cultures

Variation among epigenetic and genetic changes

Table Table 2.. Issues for embryonic stem cell (ESC) standardization
How do ESCs differ from each other?
  • Measures of identity

Do ESCs change after time in culture?
  • Measures of long-term stability

Are all ESCs equally pluripotent?
  • Measures of ability to differentiate

Can ESCs be safe enough to use for cell therapy?
  • Government regulatory agency standards

Table Table 3.. Measures of stability
Markers associated with self-renewal
Telomerase activity
Mitochondrial stability
  • Standard sequencing methods

  • Sequencing by microarray

Genomic stability
  • Karyotype

  • Fluorescent in situ hybridization (FISH)

  • Single nucleotide polymorphism (SNP) genotyping

  • Spectral karyotyping (SKY)

  • Comparative genome hybridization (CGH)

Epigenetic stability
  • Methylation changes

  • Histone modification

  • X chromosome inactivation

Table Table 4.. Measures of heterogeneity and differentiation ability
Immunocytochemistry and immunoblot
Fluorescence-activated cell sorting (FACS) analysis
Gene expression
  • Reverse transcription–polymerase chain reaction (RT-PCR) and quantitative RT-PCR

  • Array hybridization methods

  • Massively parallel signature sequencing (MPSS)

Micro-RNA profiling
Proteomic analysis methods (two-dimensional polyacrylamide gel electrophoresis [PAGE], mass spectrometry, surface enhanced laser desorption and ionization [SELDI])
Functional assays
Teratoma analysis
Table Table 5.. Cell lines for use in reference standards
  1. a

    Abbreviations: ATCC, American Type Culture Collection (Manassas, VA, http://www.atcc.org); ESC, embryonic stem cell; hESC, human embryonic stem cell; MPSS, massively parallel signature sequencing.

 AdvantagesDisadvantages
Consensus standard ESC line
  • Cell type is the same as those being tested

  • Accumulated data may be used for preclinical studies

  • Deciding which single line should be standard, a National Institutes of Health hESC Registry line or a more recent derivative

  • Culture requirements are complex

  • Instability in culture

  • High cost of acquiring cells

  • U.S. patent blocks free distribution of normal hESCs in the U.S.

Karyotypically abnormal hESC variant (such as BG01v)
  • Distributed by ATCC (low cost)

  • Extensive history and documentation

  • Array and MPSS baseline data available

  • Not covered by U.S. patent on hESCs

  • Culture conditions are relatively complex

  • Grows only slightly faster than normal cells

  • Maintenance expensive

  • Possible intellectual property restrictions by commercial providers

Human embryonal carcinoma line (such as NTERA2)
  • Distributed by ATCC (low cost)

  • Simple culture methods

  • Extensive history and documentation

  • Array and MPSS baseline data available

  • No patent issues for researchers

  • Grows much faster than hESCs

  • Retains germ cell markers as well as ESC markers

  • May not have complete differentiation repertoire

  • Aneuploidy prevents germ cell differentiation studies

Table Table 6.. U.S. Food and Drug Administration chemistry manufacturing and control standards
Donor and consent documentation
Source testing
Standard operating procedures
Scale-up/process can be performed under good manufacturing practice (GMP) conditions
General viral, xeno, and quality testing
Lot-to-lot variability
Potency and efficacy
Table Table 7.. Guidelines for establishing a human embryonic stem cell culture facility
Obtain reference standard cell line
Prepare stock of 40–50 vials
Characterize stock (measures of identity, heterogeneity, sterility [mycoplasma])
Begin experiments (use same passage for replicate experiments)
Test every fifth or tenth passage of stock (measures of stability, heterogeneity, identity if more than one line is cultured)
Share and compare results of standard assays with other laboratories

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Measures of Identity
  5. Measures of Stability
  6. Measures of Heterogeneity and Differentiation Ability
  7. Reference Standards
  8. Government Regulatory Agency Standards
  9. Summary
  10. Acknowledgements

This work was supported by the NIH, the NIA, the Robert Packard Center for ALS Research, the CNS Foundation, the Alzheimer's Association, and the Burnham Institute for Medical Research. We thank all members of our laboratories for stimulating discussions. M.S.R. acknowledges the contributions of Dr. S. Rao that made undertaking this project possible. J.F.L. thanks Dr. D. L. Barker for advice and support.

Disclosures

J.F.L. holds stock in Illumina, Inc. At the time of this publication, M.S.R. will be an employee of Invitrogen Corp.