© 2010 Nature America, Inc. All rights reserved.
correspondEnce
enzymatic activity3. We targeted residues Asp154, Glu155, Lys136 and
Ser287 based on previous experiments showing increases in stability
and/or enzymatic activity from mutations at these sites2,3. Mutations
at Pro220 were tested based on a report of increased activity with
alterations at this residue5; however, we were unable to find substitutions at Pro220 that increased activity or red-shift. The end result of
this selection process was the variant RLuc/E155G/D162E/A164R/
L165I/M185V/Q235A/S287A, which we denote RLuc7-521.
We used purified protein to assess the bioluminescence emission
spectrum, enzymatic activity and quantum yield of the new variant
(Supplementary Table 1). RLuc7-521’s signal was increased 1.6-fold
on a photons per second per mole basis, and its emission spectra (Fig.
1a) showed a 40-nm red-shift compared to RLuc with both coelenterazine and the substrate analog coelenterazine-v. RLuc7-521’s
quantum yield of 3.9 ± 0.1%, though higher than that of RLuc8.6535 (3.1 ± 0.2%), was lower than that of RLuc (5.3 ± 0.1%), indicating that further improvements may be achievable through additional
mutagenesis. We tested RLuc7-521 as a mammalian reporter gene
in cell culture and observed a nearly twofold improvement in light
output compared to RLuc, and an almost identical intracellular stability (Fig. 1b).
We also tested RLuc7-521 for small-animal imaging by injecting cells transiently transfected with RLuc or RLuc7-521 into mice.
Western analysis of cells before injection showed equivalent levels of
luciferase protein expression between conditions (Supplementary
Fig. 2). Subsequent imaging of the mice showed a 3.3-fold increase
in signal output for RLuc7-521 (Fig. 1c), a reflection of the improved
light output of RLuc7-521 combined with reductions in signal attenuation by tissue for this red-shifted luciferase.
In summary, RLuc7-521 is a seven-mutation variant of RLuc that
shows a green-peaked (521-nm) emission spectrum, an identical
intracellular stability, and at least a twofold increase in signal, with
even greater signal gains in small-animal imaging due to decreased
attenuation of its red-shifted photons. This new variant represents a
direct replacement of Renilla luciferase for reporter-gene applications
as it retains the temporal relationship between gene activation and
luciferase activity while providing greater sensitivity.
Note: Supplementary information is available on the Nature Methods website.
Andreas Markus Loening, Anca Dragulescu-Andrasi &
Sanjiv Sam Gambhir
Molecular Imaging Program at Stanford and Bio-X Program, Departments of
Radiology and Bioengineering, Stanford University, Stanford, California.
e-mail: sgambhir@stanford.edu
ACKNOWLEDGMENTS
This work was supported by the Stanford Medical Scientist Training Program (A.M.L.),
US National Cancer Institute (NCI) CA114747 In Vivo Cellular and Molecular Imaging
Center P50 (S.S.G.), NCI CA119367 Centers of Cancer Nanotechnology Excellence U54
(S.S.G.), NCI R01 CA082214 (S.S.G.) and the NCI Small Animal Imaging Resource
Program. The authors thank B. Rice and T. Troy of Caliper Life Sciences for help with
spectral measurements and Z. Walls and J. Park for experimental assistance.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details accompany the full-text
HTML version of the paper at http://www.nature.com/naturemethods/.
1. Zhao, H. et al. J. Biomed. Opt. 10, 41210 (2005).
2. Loening, A.M., Wu, A.M. & Gambhir, S.S. Nat. Methods 4, 641–643 (2007).
3. Loening, A.M., Fenn, T.D., Wu, A.M. & Gambhir, S.S. Protein Eng. Des. Sel. 19,
391–400 (2006).
4. Loening, A.M., Fenn, T.D. & Gambhir, S.S. J. Mol. Biol. 374, 1017–1028 (2007).
5. Woo, J., Howell, M. & von Arnim, A. Protein Sci. 17, 725–735 (2008).
6 | VOL.7 NO.1 | JANUARY 2010 | nature methods
Restricted ethnic diversity in human
embryonic stem cell lines
To the Editor: Human pluripotent stem cells (hPSCs) have the
capacity to self-renew indefinitely and to differentiate into a wide
array of cell types, which make them a potential source of essentially unlimited quantities of differentiated cells for basic and
clinical research. The tremendous self-renewal of hPSCs might
lead one to conclude that a small number of cell lines would be
sufficient to meet all needs. However, it is becoming increasingly
clear that the genetic background of human cell lines can have
significant effects on experimental results.
Although hundreds of associations between individual alleles and
specific traits or diseases are discovered each year, a full understanding of the relationships between genetic variation and cellular or
organismal phenotype is still remote1. The HapMap project2, along
with many other studies, has made it clear that there are large differences in the allelic frequencies for many single-nucleotide polymorphisms (SNPs) among different ethnicities. Ethnicity can serve
as a proxy for genetic variation to ensure diversity in genetic backgrounds in a study population.
The availability of hPSCs from a variety of ethnic backgrounds
would ensure the generalizability of results as well as increasing
the likelihood that specific alleles or combinations of alleles of
interest will be available for study. This is particularly important
for the use of hPSCs and hPSC-derived cells for drug screening and toxicity studies. Several idiosyncratic drug effects have
been attributed to specific genetic variations3, and the efficacy
and toxicity of numerous drugs are presumed to be influenced by
genetic factors. On the clinical side, hPSCs and their derivatives
are potential sources for cell therapy, and recipients of cell transplants are more likely to find immunologic matches with donors
who share their ethnic background. However, because most hPSC
lines have been generated from de-identified material, the ethnic
backgrounds of the donors are not known.
We determined the ethnic origin of 47 human embryonic stem
cell (hESC) lines (including 42 standard hESC lines and 5 parthenote hESC lines), 5 hiPSC lines and 58 non-pluripotent samples
(19 blood and tissue samples and 39 primary cell lines). Although
this study was not intended to be comprehensive, we included hESC
lines derived in a variety of geographical locations in order to sample
the spectrum of ethnic diversity present in hESC lines in general
(Supplementary Table 1).
Using genome-wide SNP genotyping and Bayesian analysis of
population structure (BAPS; see Supplementary Methods), we
determined that the ethnic origins of the hESC lines included in
this study were quite restricted (Fig. 1, Supplementary Table 1).
The large majority of hESC lines (43 of 47) were of European and
East Asian ethnicity. The diversity of the hiPSC lines and nonpluripotent cells is also shown (Fig. 1, Supplementary Fig. 1). We
note that the location of derivation of the hESC lines is not necessarily indicative of their ethnic origin. For instance, four of the five
hESC lines described in one landmark publication4 were derived
in the United States (Wisconsin) but reportedly5 came from blastocysts transported from Israel by collaborators. Our results on
three of these lines indicate that they show a genetic profile similar to those of people from the region around Adygea (WA01),
Tuscany (WA09) and Tuscany/Palestine (WA07), with indication of
correspondEnce
4
2
2
3
3
4
2
4
2
© 2010 Nature America, Inc. All rights reserved.
2
Ethnic origin of hESC line
Geographic location of source institution for hESC line
Ethnic origin of hiPSC line
Geographic location of source institution for hiPSC line
Admixed sample
Figure 1 | hESC and hiPSC ethnic origins and sites of derivation. Map of the geographic location of the source institution (the Geographic Origin in
Supplementary Table 1) and the Ethnic Origin (the Closest Reference Population in Supplementary Table 1) for 52 pluripotent cell lines. Map for
non-pluripotent samples is in Supplementary Fig. 1.
regional admixture (Supplementary Table 1). This predominance
of Southern European and Middle Eastern ancestry is typical of
Israel, but not typical of Wisconsin, where the population is overwhelmingly of Northern European extraction (US Census Bureau,
Decennial Census of Population, 2000). Thus, it will be important
to use methods such as those described here to accurately identify
the ethnicity of other hESC lines.
We propose that our methods can be used to determine the
ethnicity of existing hPSC lines and to prescreen primary cell
lines in order to target under-represented groups for derivation
of new hPSC lines. We have demonstrated the utility of the latter
approach by generating, to our knowledge, the first Yoruba hiPSC
line, TFDF1iPSC10, which was reprogrammed from the primary
dermal fibroblast line TFDF1 (Supplementary Table 1). In fact,
given that hiPSCs can be generated from hematopoietic stem cells
(HSCs) isolated from human cord blood6, existing cord blood
banks could become a valuable source of ethnically diverse cells
for reprogramming into hiPSCs.
Louise C Laurent1–3,15, Caroline M Nievergelt4,15,
Candace Lynch2,3, Eyitayo Fakunle2,3, Julie V Harness5,
Uli Schmidt6, Vasiliy Galat7,8, Andrew L Laslett9–11,
Timo Otonkoski12,13, Hans S Keirstead5, Andrew Schork4,
Hyun-Sook Park14 & Jeanne F Loring2
1Department of Reproductive Medicine, University of California San Diego, La
Jolla, California, USA. 2Department of Chemical Physiology and 3Center for
Regenerative Medicine, The Scripps Research Institute, La Jolla, California, USA.
4Department of
Psychiatry, University of California San Diego, La Jolla, California,
USA. 5Reeve-Irvine Research Center, Sue and Bill Gross Research Center,
University of California Irvine, Irvine, California, USA. 6Stem Cell Laboratory,
Sydney IVF, Sydney, New South Wales, Australia. 7Department of Pathology and
8iPS and Human Stem Cell Core Facility, Children’s Memorial Research Center,
Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
9Commonwealth Scientific and Industrial Research Organisation (CSIRO)
Molecular and Health Technologies, Clayton, Victoria, Australia. 10Australian Stem
Cell Centre and 11Department of Anatomy and Developmental Biology, Monash
University, Clayton, Victoria, Australia. 12Department of Molecular Neurology,
Biomedicum Stem Cell Center and 13Children’s Hospital, University of Helsinki,
Helsinki, Finland. 14Modern Cell and Tissue Technologies Core, Inc., Seoul, Korea.
15These authors contributed equally to this work.
ACKNOWLEDGMENTS
We would like to thank A. Robins (Novocell, Inc.), J.-C. Izpisua Belmonte (The
Salk Institute and Center of Regenerative Medicine in Barcelona), P. Schwartz
(Children’s Hospital Orange County), N.O. Schmidt (Universitatsklinikum HamburgEppendorf), J. Chun (The Scripps Research Institute), D. Chen (Univ. California,
Irvine), J. Shen (ScienCell Research Laboratories, Inc.) and the US National
Institute of Child Health and Human Development (NICHD) Brain and Tissue Bank
for Developmental Disorders for their generous contributions of samples for this
study. We also thank H. Tran (The Scripps Research Institute), G. Zhou (CSIRO
Molecular and Health Technologies) and H. Chy (CSIRO Molecular and Health
Technologies) for their contributions to the project. L.C.L. is supported by an
NICHD WRHR K12 award and California Institute for Regenerative Medicine (CIRM)
RN2-00931-1. This project was supported by CIRM RT1-01108-1.
1. Frazer, K.A., Murray, S.S., Schork, N.J. & Topol, E.J. Nat. Rev. Genet. 10,
241–251 (2009).
2. The International HapMap Project. Nature 426, 789–796 (2003).
3. Link, E. et al. N. Engl. J. Med. 359, 789–799 (2008).
4. Thomson, J.A. et al. Science 282, 1145–1147 (1998).
5. Vogel, G. Science 295, 1818–1820 (2002).
6. Giorgetti, A. et al. Cell Stem Cell 5, 353–357 (2009).
nature methods | VOL.7 NO.1 | JANUARY 2010 | 7