EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

Current State of Human Embryonic Stem Cell Research: An

Overview of Cell Lines and Their Use in Experimental Work
a

ANKE GUHR,a ANDREAS KURTZ,a KELLEY FRIEDGEN,b PETER LOSER

a

Robert Koch Institute, Berlin, Germany; bArnold & Porter, LLP, Washington, DC, USA

Key Words. Human embryonic stem cells Registry Published work Embryonic stem cell lines

ABSTRACT
Research in human embryonic stem cells (hESCs) is a rapidly developing scientific field. In this study we collect and
evaluate a thorough body of data on the current number of
publicly disclosed hESC lines and the extent and impact of
scientific work involving the use of these cells. These data

contribute to the substantiation of the discussion on the

current status of hESC research, provide a basis for the
analysis of the status of such research, and uncover further
needs in terms of registration, banking, standardization, and
tracing. STEM CELLS 2006;24:21872191

INTRODUCTION

terization of only 43.2% (179, of which 171 are in Englishlanguage journals) of these cell lines have been published in
peer-reviewed journals, and the use of an additional 6% (n
25) has been published without detailed characterization data in
peer-reviewed journals. Consequently, 49.2% of all cell lines
have been published so far in scientific journals (Fig. 1B).
Publication in a peer-reviewed journal provides some information about the hESC-like characteristics, but it does not provide
absolute certainty on their quality. Especially in cases where
multiple cell lines are described in a single study, characterization data are only shown for selected cell lines. Seventy-one
different hESC lines are listed in the NIH Stem Cell Registry. Of
these, only 22 are currently available to researchers. Of the
remaining 49 hESC lines, only three have been characterized
with respect to their stem cell nature and have been published in
a peer-reviewed journal; two additional cell lines were published but have been withdrawn by the providers. Although the
vast majority of hESC lines were derived using classic cultivation in the presence of feeder cells, 32 were derived under
conditions free of animal cells and media containing animalderived serum. However, it has to be taken into account that this
is not equivalent to animal-free or xerofree conditions, as
stated by some authors, since serum replacement used in some
of these derivations may also contain animal-derived compounds. Twenty-seven hESC lines harbor defined genetic defects characteristic for distinct inheritable genetic disorders, and
eight cell lines have been shown to have an abnormal karyotype.
In addition to the 414 cell lines, 14 hESC lines have been
clonally derived from existing hESC lines, some of which are
listed in the NIH registry. Almost no information is publicly
available on 106 of the 144 cell lines provided by the Repro-

Since human embryonic stem cell (hESC) lines were first derived in 1998 [1], these cells have been in high demand as
objects of research. The ability of hESCs to reproduce almost
limitlessly and to differentiate into many, if not all, cell types of
the human body has generated an enormous amount of scientific
interest. These unique capabilities provide a means of exploring
many promising lines of research, which are likely to reveal a
deeper understanding of human cellular biology and which may
lead to potential cures for many diseases [2, 3]. On the other
hand, considerable controversy has arisen regarding this type of
research, because derivation of hESCs requires destruction of
early human embryos [4]. Consequently, national legislation
regulating research involving hESCs varies widely across many
countries [5]. The present paper offers information that can
provide needed substance to the debate on hESC research by
presenting comprehensive data on the number of currently existing hESC lines and on the actual extent of experimental work
undertaken with these cells and published as of December 2005
worldwide, based on the exploration of verifiable public
sources.
Figure 1A provides an overview of the number of hESC
lines currently derived and in existence according to published
data available from various sources. In total, information on 414
human ES cell lines was available. According to data published
as of January 1, 2006, hESC lines have been established in at
least 20 countries. Although the number of existing hESC lines
is quite impressive, only limited data on characterization of
these cell lines are publicly available. Currently it is not clear
whether all lines are indeed pluripotent hESC lines. According
to our database searches, derivation and at least partial charac-

Correspondence: Peter Loser, Ph.D., Robert Koch Institute, Seestrasse 10, D-13353 Berlin, Germany. Telephone: 49-(0)30-4547-3127; Fax:
49-(0)30-4547-3206; e-mail: loeserp@rki.de Received January 26, 2005; accepted for publication June 10, 2006; first published online in
STEM CELLS EXPRESS June 15, 2006; available online without subscription through the open access option. AlphaMed Press 10665099/2006/$20.00/0 doi: 10.1634/stemcells.2006-0053

STEM CELLS 2006;24:21872191 www.StemCells.com

2188

Figure 1. Derived hESC lines (as of January 1, 2006). Data were

extracted from the NIH registry, from work published in scientific
journals listed in the PubMed database, and from information either
available online, presented at conferences, or provided in press releases.
To date, only a portion of these cell lines have been published in
scientific journals. No detailed information on the geographic origin of
the 144 cell lines derived at the Reproductive Genetics Institute and
distributed by STEMRIDE International Ltd. is publicly available from
sources used in this study (marked as unknown). hESC lines derived at
ES Cell International were assigned to Australia. Detailed information
on the hESC lines is shown in supplemental online table. (A): Total
number of cell lines sorted by country of origin. (B): Cell lines described or used in work published in peer-reviewed journals. Abbreviation: hESC, human embryonic stem cell.

ductive Genetics Institute in Chicago and available from Stemride International Ltd. Remarkably, there were four times more
new hESC lines published in peer-reviewed journals in 2005
(n 88) than obtainable (available) from the NIH (n 22). A
detailed list of currently existing and publicly known hESC lines
(including their respective references) is provided as supplemental online Data.
We next wished to determine the number of scientific publications reporting on derivation and characterization of hESC
lines or their experimental use. To cover all relevant papers on
experimental use of hESCs, we performed searches of the
PubMed database with no restriction to a publication category
(such as journal article) introduced into the search criteria.

Current State of hESC Research

Figure 2. Overview of published work reporting on experimental use

of hESCs. Searches of the PubMed database were performed as described in the supplemental online methods, and results were evaluated
manually to exclude false-positive hits. (A): Number of papers extracted
from the PubMed database by the described search string and the
number of research papers reporting on derivation and/or experimental
use of hESC lines. Data were sorted by publication year. Advance online
publications available as of December 31, 2005, have been included.
(B): Number of publications describing derivation and/or experimental
use of hESC lines sorted by location of corresponding authors. Abbreviation: hESC, human embryonic stem cell.

Although such restrictions do not reliably exclude papers without relevance for our study, this practice often results in the
exclusion of relevant publications. Therefore, the broader search
criteria reported in the online supporting material were applied.
The searches resulted in more than 2,500 hits over the period
spanning from January 1, 1998 to December 31, 2005. These
hits were manually evaluated to exclude those articles in which
hESCs were not used experimentally (e.g., reviews, tutorials,
news, comments, work on mouse ES cells, etc.). Practical work
using human embryonic carcinoma or embryonic germ cells but
not hESCs was omitted, as was work in which hESC-derived
materials (e.g., RNA or cDNA) but not hESCs were used. We
found a total of 315 research papers describing derivation and/or
experimental use of hESC lines that had been published (including online publication ahead of print) through December 31,

Guhr, Kurtz, Friedgen et al.

2189

Table 1. Most frequently cited papers reporting on derivation of or experimental work with hESCs and published from 1998 to 2004

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Top stem cell papers published between 1998 and 2004

Times
cited

Thomson JA et al. Science 1998;282:1145.

Reubinoff BE et al. Nat Biotechnol 2000;18:399.
Kehat I et al. J Clin Invest 2001;108:407.
Zhang SC et al. Nat Biotechnol 2001;19:1129.
Xu C et al. Nat Biotechnol 2001;19:971.
Amit M et al. Dev Biol 2000;227:271. Assady S. et al. Diabetes 2001;50:1691.
Reubinoff BE et al. Nat Biotechnol 2001;19:1134.
Itskovitz-Eldor J et al. Mol Med 2000;6:88.
Kaufman DS et al. Proc Natl Acad Sci U S A 2001;98:10716.
Schuldiner M et al. Proc Natl Acad Sci U S A 2000;97:11307.
Levenberg S et al. Proc Natl Acad Sci U S A 2002;99:4391.
Richards M et al. Nat Biotechnol 2002;20:933.
Carpenter MK et al. Exp Neurol 2001;172:383. Sato N et al. Nat Med 2004;10:55.
Xu C et al. Circ Res 2002;91:501.
Rajagopal J et al. Science 2003;299:363.
Schuldiner M et al. Brain Res 2001;913:201.
Cowan CA et al. N Engl J Med 2004;350:1353. Drukker M et al. Proc Natl Acad Sci U S A 2002;99:9864.
Xu RH et al. Nat Biotechnol 2002;20:1261.
Mummery C. et al. Circulation 2003;107:2733.
Eiges R et al. Curr Biol 2001;11:514. Zwaka TP Thomson JA Nat Biotechnol 2003;21:319.

1,217
450
252
248
224
211
188
172
158
149
134
109
107
104
103
97
79
77
73
72

Determination of citation frequencies was performed on November 16, 2005, using the Science Citation Index.

2005. Most of these research papers came from groups in the

United States, followed by Israel, the U.K., and South Korea
(Fig. 2). Interestingly, the number of scientific reviews and
papers on ethical or legal aspects of hESC research by far
exceeded the number of original publications describing experimental work conducted with these cells, suggesting that discussion about hESC research has outpaced actual research activities and continues to do so. According to the list of impact
factors of 2004 (Institute for Scientific Information, Thomson
Scientific, Philadelphia, http://www.thomson.com/scientific/scientific.jsp), the average impact factor for journals that have been
publishing experimental work with hESCs was 6.03, indicating
that an outstanding interest exists for this kind of research.
Approximately 28% of published research focused on differentiation of hESCs into specialized cell or tissue types, with a
noticeable emphasis on neural, heart, and blood cells. A comparable number of papers (approximately 27.5%) dealt with the
molecular characterization of hESCs, including signal transduction, gene expression patterns, or early differentiation. Another
33% of papers described derivations of hESC lines or the
establishment of improved culture conditions. In Table 1, the
top 20 publications in the field (cited most frequently since the
establishment of the first stem cell line in 1998) are listed. Table
2 gives an overview of the leading journals in the field of
experimental hESC research with respect to the number of
published research papers. The complete list of publications
detected by our method is available as online supporting material. It is worth noting that according to information provided in
the papers, at least 29% (n 92) of all studies and 54% (n
68) of the U.S. studies were conducted with at least partial
financial support from the NIH.
We have also determined the frequency at which specific
hESC lines have been used in published research. Information
on the hESC line used was available from 91.4% of studies. In
total, 681 uses of hESC lines have been reported in these
www.StemCells.com

Table 2. Top journals with respect to publication of

experimental work involving human embryonic stem cell (hESC)
lines
Journal
Stem Cells
Nat Biotechnol
Stem Cells Dev
Reprod Biomed Online
Proc Natl Acad Sci U S A
Hum Reprod
Blood
Biol Reprod
Biotechnol Bioeng
Cloning Stem Cells
Dev Dyn

Articles

73
16
15
13
12
10
9
6
6
6
6

23.17
5.08
4.76
4.13
3.81
3.17
2.86
1.90
1.90
1.90
1.90

Given are the numbers of publications reporting on derivation of

hESC lines or their experimental use for the period from
November 6, 1998 to December 31, 2005.

studies. Notably, 210 cell lines (50.7%) have never been described or used in published experimental work so far, and 150
cell lines (36.2%) were only described or used in a single
scientific report. Of the remaining 54 hESC lines (13.1%), only
15 (3.6%) have been used in more than 10 studies published in
the period investigated (Table 3). Among these, the cell line H9
and its clonally derived derivates were used most frequently in
work (16.1% of all uses), followed by cell line H1 and its clonal
derivate H1.1 (13.6%). Our data show that NIH-approved cell
lines have been used in the majority of studies published
through 2005. This might be due to the good knowledge of these
cell lines, to the high proportion of research funding by NIH in
the field, or to the fact that use of NIH-approved cell lines has
been in accordance with regulation in other countries, such as
Germany. A considerable portion of work published in 2005
(161 uses/derivations; 43.5% of all published hESC uses in

Current State of hESC Research

2190

Table 3. Most frequently used human embryonic stem cell (hESC) lines
Top hESC lines
Work published 19982005
H9b
H1c
H7
BG01
HES-3
HES-2
HSF-6
BG02
HES-1
Miz- hES1
H13
H14
HES-4
Work published in 2004
H9b
H1c
BG01
H7
HSF-6
HES-3
HES-4
H13
BG02
Work published in 2005d
H1c
H9b
H7
HES-3
BG01
HES-2
HSF-6
BG02
Miz-hES1
SNUhES3
SA002e
AS034f
Miz-hES4

Use in published work

Providera

WA09
WA01
WA07
BG01
ES03
ES02
UC06
BG02
ES01
MI01
WA13
WA14
ES04

110
93
37
26
23
18
16
16
13
12
11
11
11

WiCell Research Institute

WiCell Research Institute
WiCell Research Institute
BresaGen, Inc.
ES Cell International
ES Cell International
University of California, San Francisco
BresaGen, Inc.
ES Cell International
MizMedi Hospital, Seoul National University
WiCell Research Institute
WiCell Research Institute
ES Cell International

WA09
WA01
BG01
WA07
UC06
ES03
ES04
WA13
BG02

34
27
11
9
7
6
5
5
5

WiCell Research Institute

WiCell Research Institute
BresaGen, Inc.
WiCell Research Institute
University of California, San Francisco
ES Cell International
ES Cell International
WiCell Research Institute
BresaGen, Inc., Athens, Georgia

WA01
WA09
WA07
ES03
BG01
ES02
UC06
BG02
MI01
Not listed
SA02
Not listed
Not listed

51
44
19
14
13
10
9
9
8
8
8
6
5

WiCell Research Institute

WiCell Research Institute
WiCell Research Institute
ES Cell International
BresaGen, Inc., Athens, Georgia
ES Cell International
University of California, San Francisco
BresaGen, Inc., Athens, Georgia
MizMedi Hospital, Seoul National University
Seoul National University
Cellartis AB
Cellartis AB
MizMedi Hospital, Seoul National University

NIH code

Shown are the numbers of publications that report on derivation of the particular hESC lines or their experimental use. Note that in
many reports more than one cell line was used. Numbers are given for the total period investigated (Nov. 6, 1998, to Dec. 31, 2005)
(top), for 2004 (middle), and for 2005 (bottom).
a
Providers: WiCell Research Institute, Madison, WI, http://www.wicell.org; BresaGen, Inc., Athens, GA; University of California, San
Francisco, San Francisco, CA; ES Cell International, Singapore, http://www.escellinternational.com; MizMedi Hospital, Seoul National
University, Seoul, South Korea, http://www.useoul.edu; Cellartis AB, Gothenburg, Sweden, http://www.cellartis.com.
b
Including clonally derived cell lines H9.1 and H9.2 (NIH code GE91 and GE92, respectively).
c
Including clonally derived cell line H1.1.
d
Including work published online ahead of print in 2005.
e
Including clonally derived cell line AS002.5.
f
Including clonally derived cell line AS 034.1.

2005) reported on derivation of novel hESC lines or use of

hESC lines not registered at the NIH (supplemental online Fig.
1). However, in most cases, the non-NIH-approved cell lines
were used only in a single study (97 of 161 uses/derivations;
60.2% of reports on use/derivation of novel cell lines in 2005).
This clearly suggests that an increasing number of researchers
are establishing and using their own hESC lines. Although there
is a need for new and easily accessible hESC lines derived under
animal-free conditions, increased use of a multitude of hESC
lines in research might diminish the comparability of results.
Availability of few well-characterized and easily accessible

hESC lines derived under animal-free conditions and provided

by specialized institutions, such as stem cell banks, might be an
alternative for research.
While this paper was under review, Owen-Smith and McCormick [6] published a study of experimental work performed
by the end of 2004 and using hESCs. As a basis for their study,
they used those reports that cited the first derivation of hESCs
by Thomson et al. [1] 1998. Although some of their data is in
good agreement with our findings, there are also some notable
differences. For example, we found evidence for 91 ES cell lines
published by the end of 2004 in peer-reviewed papers, whereas

Guhr, Kurtz, Friedgen et al.

Owen-Smith and McCormick [6] reported 70 hESC lines. This
might be due to differences in the initial search method and to
the different databases used for the studies. For example, 18.5%
(58) of the papers detected by our search method did not cite the
work of Thomson et al. [1]. In addition, whereas Owen-Smith
and McCormick [6] conclude that U.S. research started to lag
behind international hESC research in the last years, our data
revealed that 43% (n 40) of hESC papers published in 2004
came from U.S. groups. Similarly, in 2005, 38% (n 62) of
hESC papers were published by researchers located in the
United States. These divergent findings are probably due to the
fact that international collaborations of U.S. groups have been
marked as collaborative research by Owen-Smith and McCormick [6]. Because there is an ongoing globalization of hESC
research, we considered the localization of the corresponding
authors laboratory as more appropriate for assigning a study on
hESCs to a country.
In summary, we provide data on the current state of experimental research involving human embryonic stem cells. Although the completeness of this review is contingent upon the

REFERENCES
1

Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell

lines derived from human blastocysts. Science 1998;282:11451147.

Pera MF, Trounson AO. Human embryonic stem cells: prospects for
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Liew CG, Moore H, Ruban L et al. Human embryonic stem cells:

Possibilities for human cell transplantation. Ann Med 2005;37:521532.

2191

limitations of the search methodology, incomplete information

in some published articles, and the lack of accepted registry and
tracing mechanisms for hESC lines, we provide evidence that
hESC research is a field that has been developing rapidly,
especially within the last 3 years. Although the number of
published hESC lines has markedly increased within the last 3
years, most published research has been performed with cell
lines derived before the end of 2001. However, the growing
number of a variety of well-characterized new hESC lines
partially harboring defined genetic defects or more suitable for
future clinical applications all but guarantees that an increasing
number of hESC research laboratories will begin using these
lines in the near future.

ACKNOWLEDGMENTS
A.G. and A.K. contributed equally to this work.

DISCLOSURES
The authors indicate no potential conflicts of interest.

de Wert G, Mummery C. Human embryonic stem cells: research, ethics

and policy. Hum Reprod 2003;18:672 682.

Knowles LP. A regulatory patchwork human ES cell research oversight.

Nat Biotechnol 2004;22:157163.

Owen-Smith J, McCormick J. An international gap in human ES cell

research. Nat Biotechnol 2006;24:391392.

See www.StemCells.com for supplemental material available online.

www.StemCells.com