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Date: 02/03/2012
Misidentification of human cell lines: Science vs. Policy
Yvonne A. Reid, PhD
Manager, Scientist, Cell Biology Program
CELL Culture 2012, San Diego, CA
Outline
• History of misidentified cell lines
• Responsibilities of stakeholders
• STR as the ‘gold standard’ for human
cell line identification
• STR profile testing
• Steps to reduce misidentified cell
lines
2
1952: HeLa - First human cancer cell line was derived
3
George Gey, Mary Kubicek Johns Hopkins University
Hospital, Baltimore, MD
HeLa cell line (ATCC® CCL-2™)
derived from a glandular cervical
cancer
HeLa – Henrietta Lacks
31 year-old mother of 4 children,
Roanoke, VA.
Gey, GO et al. Cancer Res. 12:264, 1952
1950s: Primitive tissue culture practices lead to cross-contamination
• No laminar flow hoods
• No plastics
• No commercial media
•beef embryo extracts
•human cord blood
•chick plasma
4
1959: Proposal for standardized collection of animal cell lines
5
• 1959: NCI proposes standardized
collection of animal cell lines in an
effort to reduce widespread
contamination and misidentification
among cell lines used in research
• 1962: American Type Culture
Collection (ATCC) appointed as
repository for the storage,
authentication and distribution of
animal cell lines. Cell Biology
Collection was established.
Georgetown, DC,
1956
Rockville, MD, 1964
Manassas, VA, 1998
1967 and 1968: Stanley Gartler describes intraspecies cross-contamination
6
Isoenzyme Analysis
Glucose-6-phosphate dehydrogenase (G6PD)
Name Description ATCC catalog
no.
Origin G6PD variant
HeLa Cervical adenocarcinoma; human ATCC®CCL-2™ African Type A (fast)
KB Oral epidermoid carcinoma, human
ATCC®CCL-17™
Caucasian Type A (fast)
HEp-2 Larynx epidermoid carcinoma,
human
ATCC®CCL-23™
Caucasian
Type A (fast)
Chang
liver
Liver, human ATCC®CCL-13™ Caucasian Type A (fast)
Int-407 Embryonic intestine; human ATCC®CCL-6™ Caucasian
Type A (fast)
Type A (fast)
Type B (slow)
Origin
Gartler SM, NCI Monogr. 26:176, 1967; Gartler, SM, Nature 217:750, 1968
Conclusion: 90% (18/20) human cell lines are ‘HeLa’, later confirmed
by karyotyping and DNA fingerprinting analyses
1981: Walter Nelson-Rees describes interspecies cross- contamination
Actual
(43/466 (9.2%))
Purported
(62 Laboratories)
Dog Horse, Human, Mink, Mouse
Hamster Mouse, Human, Marmoset, Rat
Mongoose Human
Human Gibbon
Mink Human
Monkey Horse, Human
Mouse Human
Rabbit Dog
Rat Chicken, Human, Mink, Monkey
Nelson-Rees, WA, et al. Science 212,446, 1981
7
1980 – 2003 Interspecies and Intraspecies cross-contamination
Cellular cross-contamination
Year No. % Type of
contam.
Technology Reference
1984 275 35% Interspecies Karyotyping Hukku, B. et al. Eukaryotic cell
culture. Plenum Press, 1984
1999 252 18% Intraspecies STR profiling Drexler, HG et al. Leukemia
13:1999.
2003 550 15% Intraspecies STR profiling Drexler, HG et al. Leukemia
17:2003
“Less than 50% of researchers regularly verify the identities of their cell
lines using standard methods such as DNA fingerprinting by STR
analysis”
Buehring, G.C., et al. (2004) In Vitro Cell Dev Biol 40:211
8
2004 – 2010: Cellular cross-contamination
persists …
9
Year Title of article Reference
2004 LCC15-MB cells are MDA-MB-435: a review of
misidentified breast and prostate cell lines..
Clin Exp Metastasis. 21(6):535,
2004.
2007 MDA-MB-435: The Questionable Use of a
Melanoma Cell Line as a Model for Human
Breast Cancer is Ongoing
Cancer Biology & Therapy 6:9,
1355, 2007.
2008
Deoxyribonucleic Acid Profiling Analysis of 40
Human Thyroid Cancer Cell Lines Reveals
Cross-Contamination Resulting in Cell Line
Redundancy and Misidentification.
J Clin Endocrinol Metab.
93(11):4331, 2008.
2009
Genetic Profiling Reveals Cross-Contamination
and Misidentification of 6 Adenoid Cystic
Carcinoma Cell Lines: ACC2, ACC3, ACCM,
ACCNS, ACCS and CAC2.
PLoS one. 4(6):e6040, 2009
2010 Verification and Unmasking of Widely Used
Human Esophageal Adenocarcinoma Cell
Lines.
JNCI. 102(4):271, 2010
Boonstra, J.J., et al. (2010) JNCI.102(4):271
Impact of cellular contamination on research
Misidentification of frequently used esophageal adenocarcinoma cell lines
(EAC) Purported STR confirmed (ATCC STRProfile database)
SEG-1 Esophageal
adenocarcinoma cell line H460 (ATCC® HTB-177™)
Lung carcinoma (large
cell lung cancer)
BIC-1 Esophageal
adenocarcinoma cell line SW620 (ATCC® CCL-227™)
Colorectal
adenocarcinoma
SK-GT-5 Esophageal
adenocarcinoma cell line SK-GT-2
Gastric fundus
carcinoma
Experimental results based on contaminated cell lines …
• Clinical trail recruiting EAC patients
• 100 scientific publications
• At least 3 NIH cancer research grants
• 11 US patents
10
Consequences of cellular contamination
• Loss of cell line
• Loss of time and money
• Misinformation in the public domain
• Discordant or irreproducible results
• Private embarrassment /public humiliation
11
The problem of misidentified cell lines
• Misidentified cell lines use is widespread
• Problem not readily recognized by …
• scientists
• reviewers of journals
• editors
• funding agencies
• Institutionalized ignorance; apathy
12
“Cases of Mistaken Identity”
“For decades, biologists working with contaminated or misidentified cell
lines have wasted time and money and produced spurious results;
journals and funding agencies say it’s not their job to solve this
problem”
Response:
“It is hard for me to fathom that the researchers themselves are willing
to ignore this risk (misidentified cell line) that jeopardizes their work and
are not themselves screaming for ways to ensure that they have pure
cell lines for their research.”
Rhitu Chatterjee. Cases of Mistaken Identity (2007) Science
15:928
Michael T. Hamilton, Fire/Rescue Battalion, Chief, Montgomery
County Fire and Rescue Service (MCFRS). Science online 2007.
13
2007: Eradication of cross-contaminated cell lines: call for action
Nardone, R. (2007) Cell Biol Toxicol 23:367.
Stakeholders have a responsibility to prevent and reduce use of
misidentified cell lines
14
2007: Open Letter by Roland Nardone
• Open letter to Michael Leavitt; Secretary of US Dept. Health
• Response by NIH: NIH Notice Number: NOT-OD-08-017, Nov. 28,
2007 – encourages cell line authentication
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-08-017.html
15
Editors of journals are requesting
authentication of cell lines
…as a prerequisite for publication
16
2009: Establishment of an international consensus standard for authentication of human cell lines ASN-0002 - Authentication of Human Cell Lines:
Standardization of STR Profiling
Chaired by John RW Masters, University College of London and
Co-chaired by Yvonne A. Reid, ATCC
Barallon, R. et al. (2010) In Vitro Cell Dev Biol Anim 46:727
January 25, 2012: Final action by ANSI
February 2, 2012: Published date
17
ASN-0002 - Authentication of human cell lines: standardization of STR profiling
• The standard describes a consistent, inexpensive and
universally applicable method for authenticating new and
established cell lines and their criteria for use.
• Section of the standard is modeled after the Scientific
Working Group on DNA Analysis Methods (SWGDAM)
interpretation guidelines of the forensic community.
• Peak amplitude
• Use of controls
• Allele designation
• Data interpretation
• STR database as part of the NCBI BioSample Database; to
contain registered cell lines with STR profiles (under
development).
18
“Evidence suggests that up to a third of established tumour cell lines
being used in scientific and medical research is affected by inter- or
intra-species cross-contamination, or have been wrongly identified,
thereby rendering many of the conclusions doubtful if not completely
invalid.” Lancet Oncology, Contamination of cell lines – a conspiracy of silence Vol.
2, July 2001, p. 393
Misidentification of cell lines
19
STR profiling for speciation and detecting
cellular cross-contamination
Intraspecies identification (within species; human) • STR analysis: variation in the number of tandem repeats
• HLA typing: variation in human leukocyte antigen gene
• SNP analysis: variation in single nucleotide – polymorphism
Interspecies identification (between species) • Isoenzyme analysis: post-translational modification of enzymes
• COI analysis: amplification of mitochondrial cytochrome C oxidase I
gene
• Karyotyping: differences in metaphase chromosome numbers for each
species
©2011 American Type Culture Collection (ATCC) 20
Technologies Power of discrimination
Isoenzyme (G6PD) 2
(fast type A and slow type B)
Karyotyping (G-banding) 100s
HLA typing 1,000s
STR analysis 100,000,000s
Identification of human cell lines
©2011 American Type Culture Collection (ATCC) 21
Short Tandem Repeat (STR) analysis for intraspecies identification of human cell line
22
DNA location Degree of
repetition Number of loci Repeat unit length
Satellite DNA
(centromere) 103 to 107 1 to 2 2 to several thousand bp
Minisatellite DNA
(telomere)
2 to several
hundred Many thousands 9 to 100 bp
Microsatellite DNA
(STRs); randomly
scattered
5 to about a
hundred 104 to 105 1 to 6 bp
STR profiling a method for cell line authentication!
Properties of STRs for DNA profiling
23
Locus name Chromosome
location Repeat motif
No.
repeating
units
No. alleles
observed
D16S539 16q24-gtr GATA 5-15 10
D7S820 7q11.21-22 GATA 6-15 22
D13S317 13q22-q31 TATC 5-15 14
D5S818 5p21-q31 AGAT 7-16 10
CSF1PO 5q33.3-34 TAGA 6-16 15
TPOX 2p23-pter GAAT 6-13 20
vWA 12p23-pter [TCTA]
[TCTG] 10-24 28
THO1 11p15.5 TCAT 3-14 20
Amelogenin Gender determination (not STR marker)
Power of discrimination 1:1.2 x 10E8
Butler, J.M. Forensic DNA Typing, 2001
Advantages of STR analysis
• Target sequence consists of microsatellite DNA
• Typically use 1-2 ng DNA
• 1 to 2 fragments; discrete alleles allow digital record of data
• Highly variable within populations; highly informative
24
Advantages of STR analysis
• Banding pattern is reproducible
• PCR amplifiable, high throughput
• Small size range allows multiplexing
• Allelic ladders simplify interpretation
• Small product size compatible with
degraded DNA
• Rapid processing is attainable
25
Outline of STR profiling procedure
26
Extract DNA
Spot onto FTA® paper
PCR amplified sample PowerPlexv1.2 System)
Resolve PCR fragments (Capillary electrophoresis)
Size PCR Fragments (GeneScan software)
Convert PCR fragment sizes to
alleles (Genotyper software)
Create reference database
• Curate
• Global comparisons
27
STR polymorphism
8
TATC TAGA
homozygous heterozygous
9,10
Gender is important for identification (amelogenin gene)
male
female
AMELX AMELY
AMELX gene contains a 6 bp deletion in the intron 1
28
Human cell line identification: STR analysis
K562
WS1
2 unrelated cell lines (separate individuals, female in origin)
D5S818 D13S317 D7S820 D16S539 vWA THO1 Amel. TPOX CSF1PO
K562 11, 12 8 9, 11 11, 12 16 9.3 X 8, 9 9, 10
WS1 13 12 9, 10 10, 11 17, 18 8, 10 X 8, 9 10, 13
29
Human cell line identification: STR analysis
2 related cell lines (same individual; male in origin)
HAAE-2
aortic artery
HFAE-2
femoral artery
D5S818 D13S317 D7S820 D16S539 vWA THO1 Amel. TPOX CSF1PO
HAAE-2 12,13 11,12 8,10 12,13 14,18 7,9 X,Y 10,11 10,11
HFAE-2 12,13 11,12 8,10 12,13 14,18 7,9 X,Y 10,11 10,11
30
STR analysis used to monitoring genomic stability
Donor
Token
(Pre-MCB)
Distribution
(WCB)
Seed
(MCB)
31
Case study 1: cellular cross-contamination
SK-OV-3
Ovary
SK-OV-3 +
cell line X
32
Case study 2: gender misidentification
Y p
ain
t
Human cell line purported to be of female origin
ST
R a
naly
sis
G-banding
33
Are your cells REALLY what you think they are?
• Getting cell lines from colleague down the hall
• Continuous culturing of working cell banks
• Use of feeder cells
• Mislabeling of culture flasks
• Working with multiple cell lines concurrently
Common sources of cellular contamination
34
Performing STR analysis
• Gene sequencer
• Thermocycler
• Primer kits from manufactures (e.g., Promega)
• STR database of human cell lines
• Experienced technicians
35
Interpreting STR data
36
• Validation of procedure
• Setting of analytical threshold required for interpretation of
results.
• Use of appropriate controls (positive and negative).
• Ability to evaluate internal lane size standards and allelic ladders.
• Appropriate assignment of allele to appropriate peaks or bands.
• Ability to determine appropriate peak height or peak threshold.
• Ability to detect artifacts, i.e. stutter peaks, dye blobs, dye pull-
ups, microvariants, off-ladder alleles, etc.
Criteria for determining quality STR profile
analysis for reliable and interpretable results
Case study 3: complexities of STR patterns
100 pg
template
5 pg
template
DNA Size (bp)
100 pg
template
5 pg
template
DNA Size (bp)
37
LOH or allele
drop-out?
vWA or THO1?
Off ladder allele
Services for STR typing of cell lines
• Cell Banks
• Paternity testing labs
• Universities
• Core labs
ATCC®CRL-2123™, mIMCD-3,
kidney, grown on Matrigel™
38
STR testing results
Result No. of
samples %
Mixture 4 4
Non-human 2 2
Misidentified 4 4
Unique (no match in ATCC
database) 30 31
Exact match to expected 40 41
Similar/related to expected 17 18
TOTAL 97 100
39
• Good documentation
• Highly trained technicians
• Good aseptic techniques
• Use one reservoir of medium per
cell line
• Aliquot stock solutions/reagents
Steps for reducing cellular contamination
ATCC®HTB-174™, NCI-H441, human
papillary adenocarcinoma differentiated
under air-liquid interface conditions
40
• Label flasks (name of cell line, passage number, date of
transfer (use barcoded flasks when available)
• Work with one cell line at a time in biological safety
cabinet
• Clean biological safety cabinet between each cell line
• Allow a minimum of 5 minutes between each cell line
Steps for reducing cellular contamination
ATCC® CCL-2™; Hela, cervical carcinoma. Scanning EM
of cultured HeLa cell undergoing apoptosis. 41
• Quarantine “dirty” cell line from
“clean” cell line
• Manageable work load (reduce
accidents)
• Clean laboratory (reduce bioburden)
• Legible handwriting (printed labels)
Steps for reducing cellular contamination
IPSC colony, on mouse feeder cells,
derived from ATCC® CCL-65™, turner
syndrome fibroblasts, expressing
OCT4, SOX2, KLF4 and cMYC.
42
• Monitor for cell line identity and characteristics
contamination, routinely
• Use seed stock (create master stocks)
• Create “good” working environment
• Review and approve laboratory notebook
• Obtain cell line from a reputable source
Steps for reducing cellular contamination
43
ATCC® CRL-1730™, HUVEC expressing CD34
