HEMATOPOIESISAND STEM CELLS
*Andre Larochelle,1*Michael Savona,2,3Michael Wiggins,3StephanieAnderson,3Brian Ichwan,1Keyvan Keyvanfar,1
Sean J. Morrison,2and Cynthia E. Dunbar1
1National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD;2Howard Hughes Medical Institute, Department of Internal Medicine,
Center for Stem Cell Biology, and Life Sciences Institute, University of Michigan,AnnArbor, MI; and3Department of Hematology and Oncology, SanAntonio
Military Medical Center, SanAntonio, TX
Various combinations of antibodies di-
rected to cell surface markers have been
used to isolate human and rhesus ma-
caque hematopoietic stem cells (HSCs).
These protocols result in poor enrich-
ment or require multiple complex steps.
Recently, a simple phenotype for HSCs
based on cell surface markers from the
signaling lymphocyte activation mol-
ecule (SLAM) family of receptors has
been reported in the mouse. We exam-
ined the possibility of using the SLAM
markers to facilitate the isolation of
highly enriched populations of HSCs in
humans and rhesus macaques. We iso-
lated SLAM (CD150?CD48?) and non-
SLAM (not CD150?CD48?) cells from
human umbilical cord blood CD34?cells
as well as from human and rhesus ma-
caque mobilized peripheral blood CD34?
cells and compared their ability to form
immune-deficient (nonobese diabetic/
severe combined immunodeficiency/
interleukin-2 ?c receptornull, NSG) mice.
We found that the CD34?SLAM popula-
tion contributed equally or less to colony
formation in vitro and to long-term re-
constitution in NSG mice compared with
the CD34?non-SLAM population. Thus,
SLAM family markers do not permit the
same degree of HSC enrichment in hu-
mans and rhesus macaques as in mice.
The search for hematopoietic stem cell (HSC)-specific surface
markers has been a central question for functional stem cell
studies and for the development of clinical applications, includ-
ing transplantation and gene therapy. In mice, using a complex
combination of positive and negative selection for 10 to
12 surface markers (Lin?Sca-1?c-kit?Thy-1lo), 1 in 4.9 cells
provides long-term reconstitution after intravenous injection.1
However, the use of these complex sets of markers, alone or in
combination with the established isolation scheme based on
Hoechst dye efflux (side population),2is incompatible with in
situ histologic analyses.
Recently, a simple and broadly applicable method to isolate
mouse HSCs has been developed based on expression of cell
surface markers, which are members of the signaling lympho-
cyte activation molecule (SLAM) family, including CD150,
4.8 CD150?CD48?bone marrow (BM) cells provides long-
term, multilineage reconstitution in recipient mice, similar to the
enrichment obtained in the complex Lin?Sca-1?c-kit?Thy-1lo
population.1This same strategy has been successfully applied to
enrich HSCs from mouse cyclophosphamide/granulocyte-
colony stimulating factor (G-CSF)–mobilized cells,3mouse
fetal liver,4as well as from the BM of various strains of mice5
and older mice.3Recent data examining the overlap between
SLAM family member expression with the Hoechst dye efflux
side population in conjunction with canonical HSC cell surface
markers have confirmed the potential of CD150?selection for
mouse HSC enrichment.6Although it has been suggested that
some HSC activity may also be present in the CD150?cell
fraction of mouse cells,6data from multiple groups indicate that
there is little or no long-term HSC activity in the CD150?
fraction of mouse hematopoietic cells.1,3,4,7-10
In humans, the CD34 cell surface marker is largely used in
clinical applications for isolation of HSCs and progenitor cells,
and as a predictor of graft HSC content in transplants. The
combination CD34?CD38?provides further enrichment (1 in
600 to 1 in 3500),11but the heterogeneity of this population
precludes studies requiring levels of purity achieved in mouse
models, such as comparing gene expression profiles or in situ
histologic analyses. Given the use of SLAM receptors for
identifying long-term repopulating HSCs in mice,1,3,4,7-10we
hypothesized that this strategy may similarly facilitate the
isolation of highly enriched populations of HSCs in humans and
nonhuman primates. In this study, we isolated SLAM
(CD150?CD48?) and non-SLAM (not CD150?CD48?) cells
from human umbilical cord blood (UCB) cells as well as human
and primate cytokine mobilized peripheral blood (MPB) cells
and compared their ability to form colonies in vitro and to
reconstitute immune-deficient (nonobese diabetic/severe combined
Submitted March 26, 2009; accepted November 29, 2010. Prepublished online
as Blood First Edition paper, December 16, 2010; DOI 10.1182/blood-2009-03-
*A.L. and M.S. contributed equally to this study.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by TheAmerican Society of Hematology
1550BLOOD, 3 FEBRUARY 2011?VOLUME 117, NUMBER 5
For personal use only.on October 26, 2015. by guest
Collection of hematopoietic cells from healthy donors and from
Human MPB CD34?cells were obtained from 3 healthy volunteers (donor
1, 25-year-old woman; donor 2, 21-year-old man; and donor 3, 24-year-old
man) after informed consent in accordance with the Declaration of
Helsinki, under an institutional review board-approved clinical protocol.
The donors received 5 days of G-CSF (filgrastim; Amgen) 10 ?g/kg and
underwent leukapheresis and CD34?cell enrichment as previously de-
scribed.12The CD34?cells were cryopreserved before isolation of SLAM
(CD150?CD48?) and non-SLAM (not CD150?CD48?) cells.
Human UCB samples were obtained from the National Heart, Lung and
Blood Institute Biologic Specimen and Data Repository Coordinating
Center. CD34?cells were collected from 8 pooled UCB samples. Each
cryopreserved UCB unit was thawed at 37°C and washed in normal saline
buffer containing 5% dextran (Hospira) and 2.5% human serum albumin
(Baxter Healthcare). Cells were resuspended in normal saline containing
1% human serum albumin, pooled, and CD34?cells were enriched as
previously described12before isolation of SLAM and non-SLAM cells.
The rhesus macaque (Macaca mulatta) used in these studies (RQ6566,
4-year-old female) was housed and handled in accordance to guidelines
outlined in a protocol approved by the Animal Care and Use Committee of
the National Heart, Lung and Blood Institute. Stem cell mobilization and
apheresis were as described.13The CD34?cells had a purity of 90% and
were used without prior cryopreservation for isolation of SLAM and
Isolation of SLAM and non-SLAM cells
Human and rhesus macaque SLAM and non-SLAM cells were isolated
using human anti-CD150-fluorescein isothiocyanate (clone A12, eBio-
science) and anti-CD48-RPE (clone MEM102, AbD Serotec) monoclonal
antibodies. Cell sorting was performed using a FACSVantage cell sorter
(BD Biosciences). Gates were defined strictly using isotype controls to
eliminate any potential overlap between CD150?and CD150?cells.
NSG mouse repopulation assay
Sublethally irradiated (300 cGy) 8-week-old NSG mice were transplanted
by tail-vein injection with 1 ? 103, 1 ? 104, 1 ? 105, or 1 ? 106human
SLAM cells, non-SLAM cells, or unfractionated CD34?cells. Mice
transplanted with less than 1 ? 105cells also received 1 ? 105CD34?
carrier cells (99.2% purity). At week 8 after transplantation, mice were
killed and BM mononuclear cells were harvested from both femurs and
tibias. Aliquots were stained with human CD45-phycoerythrin (PE),
CD20-PE, CD3-PE, CD36-PE, and CD34-PE antibodies (all from BD
Biosciences) and analyzed on a Coulter FC500 analyzer (Beckman Coulter)
for detection of human cell engraftment.
For colony-forming unit (CFU) assays, 2 ? 105transplanted mouse BM
Stem CellTechnologies) at 37°C in 5% CO2. Colonies of more than 50 cells
were scored after 10 to 14 days of incubation.
Results and discussion
Cell surface receptors of the SLAM family, including CD150,
CD244, and CD48, were found to be differentially expressed
among mouse HSCs (CD150?CD244?CD48?) and more restricted
progenitors (CD150?CD244?CD48?).1Using CFU assays and
transplantation studies in NSG mice, we investigated whether
SLAM markers could also be used to distinguish human progeni-
tors and more primitive repopulating cells (referred to as human
SCID repopulating cells [hSRCs]11).
we omitted the CD244 marker and tested the in vivo repopulating
and colony-forming potential of CD150?CD48?cells (SLAM
cells) compared with non-SLAM cells, which included all viable
cells outside the SLAM gate (not CD150?CD48?). These
2 populations were first sorted from human G-CSF-MPB cells
derived from 3 normal volunteers and pre-enriched in CD34?cells
(99% purity) to facilitate sorting of the rare CD150?CD48?cells.
Using stringent gates defined with isotype controls, we found that
0.4% to 1.1% of human CD34?cells were CD150?CD48?
(Figure 1A). Variable percentages of the SLAM population were in
the CD38?fraction (donor 1, 8%; donor 2, 94%; and donor 3,
87%). No significant numbers of SLAM cells were detected in the
CD34?fraction in any donor (Figure 1B). Therefore, the pre-
(Figure 1E-F), and rhesus macaque MPB cells (Figure 1G-H), indicat-
ing a consistent pattern of CD150 and CD48 expression in various
In CFU assays, SLAM, non-SLAM, and unfractionated CD34?
cell populations obtained from human G-CSF MPB and human
UCB gave rise to colonies of similar size, phenotype, and
frequency (supplemental Figure 1A and supplemental Figure 1B,
respectively, available on the Blood Web site; see the Supplemental
Materials link at the top of the online article) after 10 to 14 days in
culture. When rhesus macaque-mobilized CD34?cells were used
in CFU assays, the size, phenotype, and number of colonies
obtained from SLAM and non-SLAM populations were also
similar to the CFUs derived from unfractionated CD34?cells
(supplemental Figure 1C).
Surprisingly, both SLAM and non-SLAM populations derived
from human MPB CD34?cells from 3 independent donors had in
vivo repopulating potential after intravenous transplantation in
NSG mice, and levels of human cell engraftment based on CD45
cell surface expression were similar to those obtained with
unfractionated CD34?cells (Figure 2A-E). The human SCID
repopulating cell activity in each fraction had a comparable ability
to repopulate mice with multiple hematopoietic lineages (data not
shown). CD34?SLAM cells derived from human UCB provided
only minimal contribution to long-term hematopoietic reconstitu-
tion in NSG mice compared with non-SLAM CD34?cells and
unfractionated CD34?cells (Figure 2F-J). Rhesus SCID repopulat-
ing cells were also detected in both SLAM and non-SLAM
populations derived from MPB CD34?cells, and levels of
engraftment were similar to those obtained after transplantation of
unfractionated CD34?cells (supplemental Figure 2). These data
confirm that both human and rhesus macaque primitive repopulat-
ing cells from various hematopoietic sources are partially con-
tained within the SLAM fraction of cells, but SCID repopulating
cells cannot be isolated based only on SLAM family markers.
To rule out the possibility that these results could be explained
by the existence of other post-translationally modified forms of
CD150 present on human HSCs not recognized by the antibodies
used in this study, we performed real-time reverse-transcriptase
polymerase chain reaction to assess CD150 expression on human
MPB (n ? 3) and UCB (n ? 3) CD34?CD38?cells. CD150
expression was 10-fold lower in this population compared with
unfractionated mononuclear cells. This contrasts with our ability to
detect enrichment of CD150 RNA in mouse HSCs. In addition,
SLAM MARKERS DO NOT PURIFY HUMAN HSCs 1551BLOOD, 3 FEBRUARY 2011?VOLUME 117, NUMBER 5
For personal use only.on October 26, 2015. by guest
using flow cytometry, we were unable to detect increased staining
on human CD34?CD38?cells using multiple CD150 antibodies
(data not shown).
In contrast to mouse studies, our data in humans and rhesus
macaques using UCB and MPB cells indicate that SCID repopulat-
ing cells and progenitor cells can be found in both SLAM and
Figure 1. Cell sorting experiments of SLAM (CD150?CD48?) and non-SLAM (not CD150?CD48?) populations. Human G-CSF MPB CD34?cells (A) and CD34?cells
(B). Human UCB CD34?cells (C) and CD34?cells (D). Human BM CD34?cells (E) and CD34?cells (F). Rhesus macaque MPB CD34?cells (G) and CD34?cells (H). No
significant numbers of SLAM cells are detected in the CD34?population from any hematopoietic source.
1552 LAROCHELLE et al BLOOD, 3 FEBRUARY 2011?VOLUME 117, NUMBER 5
For personal use only. on October 26, 2015. by guest
non-SLAM populations based on the available antibodies against
human and rhesus macaque CD150 and CD48. Therefore, these
combinations of antibodies against SLAM markers are not suitable
for purification or in situ histologic analyses of HSCs in large
mammals. The results derived from the functional studies pre-
sented here correlate with results of recently published flow
cytometry analyses showing that the patterns of expression of the
SLAM family receptors differ between mice and humans.14Sintes
et al14hypothesized, based on expression patterns, that the most
primitive human repopulating cells may be predominantly
CD150?CD48?, an observation contrasting with findings in mice1
where HSCs are enriched in the CD150?CD48?population. This
Figure 2. Summary of human cell engraftment based on CD45 cell surface expression in the BM of nonobese diabetic/severe combined immunodeficiency/
interleukin-2 ?c receptornullmice transplanted with SLAM (CD150?CD48?), non-SLAM (not CD150?CD48?), or unfractionated CD34?cells. Representative flow
cytometry analysis after transplantation of 1 ? 105SLAM (A), non-SLAM (B), or unfractionated human CD34?cells (C) derived from G-CSF MPB. (D) Control mice were
injected intravenously with an equal volume of saline. (E) Summary of human cell engraftment after transplantation of G-CSF MPB cells from 3 independent donors in NSG
mice. Each symbol represents one mouse, and the horizontal lines indicate the mean levels of human cell engraftment (n ? 121 mice). Representative flow cytometry
experiment after transplantation of 1 ? 104SLAM (F), non-SLAM (G), or unfractionated human CD34?cells (H) derived from 8 pooled cord blood samples. (I) Control animals
were transplanted with an equal volume of saline. (J) Summary of human cell engraftment after transplantation of cord blood derived cells in NSG mice (n ? 28 mice).
SLAM MARKERS DO NOT PURIFY HUMAN HSCs 1553BLOOD, 3 FEBRUARY 2011?VOLUME 117, NUMBER 5
For personal use only. on October 26, 2015. by guest
observation is not uncommon and is evocative of the CD34 surface
marker expression detected on the majority of human HSCs but
absent on quiescent mouse HSCs.15-17It is also reminiscent of the
absence of the differentiation marker CD38 on human HSCs11,18-21
and its presence on mouse HSCs.22-26Additional transplantation
studies will be useful to further characterize the repopulating
potential of the CD150?CD48?population in large mammals.
The authors thank D. Stroncek, J. Procter, M. Sabatino, and S.
Leitman for providing human CD34?cells; Sue Ellen Frodigh and
Quyen Chau for processing UCB samples; R. Donahue, M.
Metzger, and A. Krouse for mobilization and apheresis of rhesus
macaques; D. Adams, M. White, and the University of Michigan
Flow Cytometry Core Facility for support; and the animal core
facility staff at the National Institutes of Health and the University
of Michigan for excellent animal care.
This work was supported in part by the intramural research
program of the National Heart, Lung and Blood Institute of the
National Institutes of Health and in part by the Howard Hughes
Medical Institute and by a Michigan Institute for Clinical and
Health Research Pilot Grant.
tal procedures and analyzed the data; A.L. wrote the manuscript;
M.S. contributed to the editing; M.W., S.A., and B.I. performed
some of the transplantation studies; K.K. sorted the various cell
populations; and C.E.D. and S.J.M. designed the experiments and
edited the manuscript.
Conflict-of-interest disclosure: S.J.M. is an author on a patent
for the use of SLAM family markers for the isolation of mamma-
lian HSCs. The remaining authors declare no competing financial
Correspondence: Cynthia E. Dunbar, National Heart, Lung and
Blood Institute, National Institutes of Health, Bldg 10 CRC,
Rm 4-5132, 10 Center Dr, MSC-1202, Bethesda, MD 20892;
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1554 LAROCHELLE et alBLOOD, 3 FEBRUARY 2011?VOLUME 117, NUMBER 5
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online December 16, 2010
2011 117: 1550-1554
Keyvanfar, Sean J. Morrison and Cynthia E. Dunbar
Andre Larochelle, Michael Savona, Michael Wiggins, Stephanie Anderson, Brian Ichwan, Keyvan
purified based only on SLAM family markers
Human and rhesus macaque hematopoietic stem cells cannot be
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